Current Research and Scholarly Interests
The Quertermous laboratory employs two basic approaches of study to better understand the genetic basis of atherosclerotic heart disease. One approach uses basic molecular biology methodology, primarily working with cellular and genetic mouse models, and is focused on the recently identified apelin-APJ pathway. A second approach employs the power of modern human genetics. Informative cohorts have been collected that allow investigation of risk factors such as hypertension and insulin resistance as well as coronary heart disease. Initial studies have employed the candidate gene approach, and more recently whole genome association studies, to identify allelic variation that is associated with risk factor and disease susceptibility.
Independent Studies (6)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
Postdoctoral Faculty Sponsor
Ivan Carcamo Oribe, Dharini Dhinakaran Iyer, Maurizio Morri, Manabu Nagao, Christoph Olivier, Milos Pjanic, Robert Wirka, Quanyi Zhao
Postdoctoral Research Mentor
Dharini Dhinakaran Iyer, Maurizio Morri, Manabu Nagao, Christoph Olivier, Milos Pjanic, Robert Wirka, Quanyi Zhao
- Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci PLOS GENETICS 2015; 11 (5)
Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap.
2015; 11 (5)
Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including "vascular disease," "disorder of artery," and "occlusion of artery," as well as disease-related cellular functions including "cellular movement" and "cellular growth and proliferation." In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.
View details for DOI 10.1371/journal.pgen.1005155
View details for PubMedID 26020946
Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene
JOURNAL OF CLINICAL INVESTIGATION
2015; 125 (4): 1739-1751
Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.
View details for DOI 10.1172/JCI74592
View details for Web of Science ID 000352248600037
View details for PubMedID 25798622
New genetic loci link adipose and insulin biology to body fat distribution.
2015; 518 (7538): 187-196
Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.
View details for DOI 10.1038/nature14132
View details for PubMedID 25673412
View details for PubMedCentralID PMC4338562
- Dissecting the causal genetic mechanisms of coronary heart disease. Current atherosclerosis reports 2014; 16 (5): 406-?
- Clinical interpretation and implications of whole-genome sequencing. JAMA : the journal of the American Medical Association 2014; 311 (10): 1035-1045
- Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation. PLoS genetics 2014; 10 (3)
- Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of TCF21 Expression at the 6q23.2 Coronary Heart Disease Locus PLOS GENETICS 2013; 9 (7)
Apelin-APJ Signaling Is a Critical Regulator of Endothelial MEF2 Activation in Cardiovascular Development
2013; 113 (1): 22-31
The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish.This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets.We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells.Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.
View details for DOI 10.1161/CIRCRESAHA.113.301324
View details for Web of Science ID 000320985200011
View details for PubMedID 23603510
View details for PubMedCentralID PMC3739451
- Loss of CDKN2B Promotes p53-Dependent Smooth Muscle Cell Apoptosis and Aneurysm Formation ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 2013; 33 (1): E1-?
- Large-scale association analysis identifies new risk loci for coronary artery disease NATURE GENETICS 2013; 45 (1): 25-U52
CDKN2B Regulates Cell Fate Decisions in Human Vascular Smooth Muscle Cells
LIPPINCOTT WILLIAMS & WILKINS. 2010
View details for Web of Science ID 000208231602363
Hundreds of variants clustered in genomic loci and biological pathways affect human height
2010; 467 (7317): 832-838
Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.
View details for DOI 10.1038/nature09410
View details for Web of Science ID 000282898700065
View details for PubMedID 20881960
Biological, clinical and population relevance of 95 loci for blood lipids
2010; 466 (7307): 707-713
Plasma concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with plasma lipids in >100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P < 5 x 10(-8)), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (for example, CYP7A1, NPC1L1 and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and have an impact on lipid traits in three non-European populations (East Asians, South Asians and African Americans). Our results identify several novel loci associated with plasma lipids that are also associated with CAD. Finally, we validated three of the novel genes-GALNT2, PPP1R3B and TTC39B-with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD.
View details for DOI 10.1038/nature09270
View details for Web of Science ID 000280562500029
View details for PubMedID 20686565
Upregulation of the apelin-APJ pathway promotes neointima formation in the carotid ligation model in mouse
2010; 87 (1): 156-165
To investigate apelin-APJ (angiotensin receptor-like 1) signalling in vascular remodelling, we have examined the pathophysiological response to carotid ligation in apelin knockout mice.Apelin null animals compared with wild-type mice had significantly decreased neointimal lesion area (1.17 +/- 0.17 vs. 3.33 +/- 1.04 x 10(4) microm(2), P < 0.05) and intima/media ratio (0.81 +/- 0.23 vs. 1.49 +/- 0.44, P < 0.05), averaged over four sites 0.5-2 mm from the ligation. Exogenous apelin infusion rescued the apelin-KO phenotype, promoting neointima formation in the null animals. Apelin null animals showed decreased smooth muscle positive area in the neointima (82.3 +/- 2.4 vs. 63.9 +/- 8.4, P < 0.05), and a smaller percentage BrdU positive cells in the neointima and media (11.06 +/- 1.00 vs. 6.53 +/- 0.86, P < 0.05). Apelin mRNA expression increased initially (5.2-fold, P < 0.01) followed by increased apelin receptor expression (10.1-fold, P < 0.05) in the ligated artery. Cytochemistry studies localized apelin expression to luminal endothelial cells and apelin receptor upregulation to smooth muscle cells (SMC) in the media and neointima. In vitro experiments with cultured rat aortic SMC revealed that apelin stimulation increased migration. In contrast to the increased expression of apelin and apelin receptor in carotid remodelling, expression was not upregulated in the apoE high fat model, and correlated with the known disease-inhibitory effect in this model.These data suggest that increased apelin receptor expression by SMC provides a paracrine pathway in injured vessels that allows endothelial-derived apelin to stimulate their division and migration into the neointima.
View details for DOI 10.1093/cvr/cvq052
View details for Web of Science ID 000278690000021
View details for PubMedID 20176814
Genome-wide meta-analyses identify multiple loci associated with smoking behavior
2010; 42 (5): 441-U134
Consistent but indirect evidence has implicated genetic factors in smoking behavior. We report meta-analyses of several smoking phenotypes within cohorts of the Tobacco and Genetics Consortium (n = 74,053). We also partnered with the European Network of Genetic and Genomic Epidemiology (ENGAGE) and Oxford-GlaxoSmithKline (Ox-GSK) consortia to follow up the 15 most significant regions (n > 140,000). We identified three loci associated with number of cigarettes smoked per day. The strongest association was a synonymous 15q25 SNP in the nicotinic receptor gene CHRNA3 (rs1051730[A], beta = 1.03, standard error (s.e.) = 0.053, P = 2.8 x 10(-73)). Two 10q25 SNPs (rs1329650[G], beta = 0.367, s.e. = 0.059, P = 5.7 x 10(-10); and rs1028936[A], beta = 0.446, s.e. = 0.074, P = 1.3 x 10(-9)) and one 9q13 SNP in EGLN2 (rs3733829[G], beta = 0.333, s.e. = 0.058, P = 1.0 x 10(-8)) also exceeded genome-wide significance for cigarettes per day. For smoking initiation, eight SNPs exceeded genome-wide significance, with the strongest association at a nonsynonymous SNP in BDNF on chromosome 11 (rs6265[C], odds ratio (OR) = 1.06, 95% confidence interval (Cl) 1.04-1.08, P = 1.8 x 10(-8)). One SNP located near DBH on chromosome 9 (rs3025343[G], OR = 1.12, 95% Cl 1.08-1.18, P = 3.6 x 10(-8)) was significantly associated with smoking cessation.
View details for DOI 10.1038/ng.571
View details for Web of Science ID 000277179500017
View details for PubMedID 20418890
View details for PubMedCentralID PMC2914600
Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis
JOURNAL OF CLINICAL INVESTIGATION
2008; 118 (10): 3343-3354
Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.
View details for DOI 10.1172/JCI34871
View details for Web of Science ID 000259828600016
View details for PubMedID 18769630
Network analysis of human in-stent restenosis
2006; 114 (24): 2644-2654
Recent successes in the treatment of in-stent restenosis (ISR) by drug-eluting stents belie the challenges still faced in certain lesions and patient groups. We analyzed human coronary atheroma in de novo and restenotic disease to identify targets of therapy that might avoid these limitations.We recruited 89 patients who underwent coronary atherectomy for de novo atherosclerosis (n=55) or in-stent restenosis (ISR) of a bare metal stent (n=34). Samples were fixed for histology, and gene expression was assessed with a dual-dye 22,000 oligonucleotide microarray. Histological analysis revealed significantly greater cellularity and significantly fewer inflammatory infiltrates and lipid pools in the ISR group. Gene ontology analysis demonstrated the prominence of cell proliferation programs in ISR and inflammation/immune programs in de novo restenosis. Network analysis, which combines semantic mining of the published literature with the expression signature of ISR, revealed gene expression modules suggested as candidates for selective inhibition of restenotic disease. Two modules are presented in more detail, the procollagen type 1 alpha2 gene and the ADAM17/tumor necrosis factor-alpha converting enzyme gene. We tested our contention that this method is capable of identifying successful targets of therapy by comparing mean significance scores for networks generated from subsets of the published literature containing the terms "sirolimus" or "paclitaxel." In addition, we generated 2 large networks with sirolimus and paclitaxel at their centers. Both analyses revealed higher mean values for sirolimus, suggesting that this agent has a broader suppressive action against ISR than paclitaxel.Comprehensive histological and gene network analysis of human ISR reveals potential targets for directed abrogation of restenotic disease and recapitulates the results of clinical trials of existing agents.
View details for DOI 10.1161/CIRCULATIONAHA.106.637025
View details for Web of Science ID 000243477800015
View details for PubMedID 17145989
Proteomic profiles of serum inflammatory markers accurately predict atherosclerosis in mice
2006; 25 (2): 194-202
At a population level, inflammatory markers have been shown to predict outcome and response to therapy in patients with atherosclerotic cardiovascular disease. However, current markers are not sufficiently sensitive or specific to provide clinical utility for managing individual patients. We hypothesize that measurement of multiple circulating disease-related inflammatory factors will be more informative, allowing the early identification of vascular wall disease activity. We have investigated whether protein microarray-based abundance measurements of circulating proteins can predict the severity of atherosclerotic disease. Using a longitudinal experimental design with apolipoprotein E-deficient mice and control C57Bl/6J and C3H/HeJ wild-type mice, we measured the time-related serum protein expression of 30 inflammatory markers using a protein microarray. We were able to identify a subset of proteins that classify and predict the severity of atherosclerotic disease with a high level of accuracy. The time-specific vascular expression of these markers was verified by showing that their gene expression in the mouse aorta correlated closely to the temporal pattern of serum protein levels. In conclusion, these data suggest that quantification of multiple disease-related inflammatory proteins can provide a more sensitive and specific methodology for assessing atherosclerotic disease activity in humans, and identify candidate biomarkers for such studies.
View details for DOI 10.1152/physiolgenomics.00240.2005
View details for Web of Science ID 000236791300002
View details for PubMedID 16418319
Signature patterns of gene expression in mouse atherosclerosis and their correlation to human coronary disease
2005; 22 (2): 213-226
The propensity for developing atherosclerosis is dependent on underlying genetic risk and varies as a function of age and exposure to environmental risk factors. Employing three mouse models with different disease susceptibility, two diets, and a longitudinal experimental design, it was possible to manipulate each of these factors to focus analysis on genes most likely to have a specific disease-related function. To identify differences in longitudinal gene expression patterns of atherosclerosis, we have developed and employed a statistical algorithm that relies on generalized regression and permutation analysis. Comprehensive annotation of the array with ontology and pathway terms has allowed rigorous identification of molecular and biological processes that underlie disease pathophysiology. The repertoire of atherosclerosis-related immunomodulatory genes has been extended, and additional fundamental pathways have been identified. This highly disease-specific group of mouse genes was combined with an extensive human coronary artery data set to identify a shared group of genes differentially regulated among atherosclerotic tissues from different species and different vascular beds. A small core subset of these differentially regulated genes was sufficient to accurately classify various stages of the disease in mouse. The same gene subset was also found to accurately classify human coronary lesion severity. In addition, this classifier gene set was able to distinguish with high accuracy atherectomy specimens from native coronary artery disease vs. those collected from in-stent restenosis lesions, thus identifying molecular differences between these two processes. These studies significantly focus efforts aimed at identifying central gene regulatory pathways that mediate atherosclerotic disease, and the identification of classification gene sets offers unique insights into potential diagnostic and therapeutic strategies in atherosclerotic disease.
View details for DOI 10.1152/physiolgenomics.00001.2005
View details for Web of Science ID 000230987900011
View details for PubMedID 15870398
Genetic structure, self-identified race/ethnicity, and confounding in case-control association studies
AMERICAN JOURNAL OF HUMAN GENETICS
2005; 76 (2): 268-275
We have analyzed genetic data for 326 microsatellite markers that were typed uniformly in a large multiethnic population-based sample of individuals as part of a study of the genetics of hypertension (Family Blood Pressure Program). Subjects identified themselves as belonging to one of four major racial/ethnic groups (white, African American, East Asian, and Hispanic) and were recruited from 15 different geographic locales within the United States and Taiwan. Genetic cluster analysis of the microsatellite markers produced four major clusters, which showed near-perfect correspondence with the four self-reported race/ethnicity categories. Of 3,636 subjects of varying race/ethnicity, only 5 (0.14%) showed genetic cluster membership different from their self-identified race/ethnicity. On the other hand, we detected only modest genetic differentiation between different current geographic locales within each race/ethnicity group. Thus, ancient geographic ancestry, which is highly correlated with self-identified race/ethnicity--as opposed to current residence--is the major determinant of genetic structure in the U.S. population. Implications of this genetic structure for case-control association studies are discussed.
View details for Web of Science ID 000226215100012
View details for PubMedID 15625622
The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo
2005; 65 (1): 73-82
The endogenous peptide apelin is differentially regulated in cardiovascular disease but the nature of its role in cardiac function remains unclear.We investigated the functional relevance of this peptide using ECG and respiration gated magnetic resonance imaging, conductance catheter pressure-volume hemodynamic measurements, and echocardiography in vivo. In addition, we carried out histology and immunohistochemistry to assess cardiac hypertrophy and to localize apelin and APJ in the adult and embryonic mouse heart.Intraperitoneal injection of apelin (300 microg/kg) resulted in a decrease in left ventricular end diastolic area (pre: 0.122+/-0.007; post: 0.104+/-0.005 cm(2), p=0.006) and an increase in heart rate (pre: 537+/-20; post: 559+/-19 beats per minute, p=0.03). Hemodynamic measurements revealed a marked increase in ventricular elastance (pre: 3.7+/-0.9; post: 6.5+/-1.4 mm Hg/RVU, p=0.018) and preload recruitable stroke work (pre: 27.4+/-8.0; post: 51.8+/-3.1, p=0.059) with little change in diastolic parameters following acute infusion of apelin. Chronic infusion (2 mg/kg/day) resulted in significant increases in the velocity of circumferential shortening (baseline: 5.36+/-0.401; 14 days: 6.85+/-0.358 circ/s, p=0.049) and cardiac output (baseline: 0.142+/-0.019; 14 days: 0.25+/-0.019 l/min, p=0.001) as determined by 15 MHz echocardiography. Post-mortem corrected heart weights were not different between apelin and saline groups (p=0.5) and histology revealed no evidence of cellular hypertrophy in the apelin group (nuclei per unit area, p=0.9). Immunohistochemistry studies revealed APJ staining of myocardial cells in all regions of the adult mouse heart. Antibody staining, as well as quantitative real time polymerase chain reaction identified expression of both APJ and apelin in embryonic myocardium as early as embryonic day 13.5.Apelin reduces left ventricular preload and afterload and increases contractile reserve without evidence of hypertrophy. These results associate apelin with a positive hemodynamic profile and suggest it as an attractive target for pharmacotherapy in the setting of heart failure.
View details for DOI 10.1016/j.cardiores.2004.08.018
View details for Web of Science ID 000226477600011
View details for PubMedID 15621035
View details for PubMedCentralID PMC2517138
The effects of the renin-angiotensin-aldosterone system gene polymorphisms on insulin resistance in hypertensive families
JOURNAL OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM
2012; 13 (4): 446-454
The hereditability of insulin resistance has been demonstrated in both familial and twin studies. The effects of renin-angiotensin-aldosterone system gene polymorphisms on insulin resistance remain inconclusive.This is a sibling-based association study. Polymorphisms of renin-angiotensin-aldosterone system genes were examined in 1113 hypertension and 676 normotension siblings from Chinese and Japanese hypertensive families. The generalized estimation equations method was used to compare the differences in metabolic variables between hypertension and normotensive siblings.For the G-6A polymorphism of AGT, GG siblings had lower 2-h insulin than siblings carrying the A allele (p=0.006). Siblings with different variants of the angiotensin II type 1 receptor A1166C had no difference in metabolic variables. Siblings carrying the D allele of the angiotensin converting enzyme gene had higher levels of fasting glucose, fasting insulin, area under the curve of insulin levels and the homeostasis model assessment of insulin resistance than II siblings (all p<0.05). Lower levels of fasting glucose and 2-h glucose were observed in siblings with the T allele than their CC homozygotes for the C-344T polymorphism of CYP11B2 (p<0.05). Siblings carrying three high-risk genotypes of the angiotensin converting enzyme, angiotensinogen and CYP11B2 had higher fasting glucose level than siblings carrying no high-risk genotypes (p=0.011).Our comprehensive analysis of renin-angiotensin-aldosterone system gene polymorphisms demonstrates that the angiotensin converting enzyme and CYP11B2 gene polymorphisms are associated with insulin resistance in hypertensive families.
View details for DOI 10.1177/1470320312438790
View details for Web of Science ID 000311304100006
View details for PubMedID 22419662
Common ALDH2 genetic variants predict development of hypertension in the SAPPHIRe prospective cohort: Gene-environmental interaction with alcohol consumption
BMC CARDIOVASCULAR DISORDERS
Genetic variants near/within the ALDH2 gene encoding the mitochondrial aldehyde dehydrogenase 2 have been associated with blood pressure and hypertension in several case-control association studies in East Asian populations.Three common tag single nucleotide polymorphisms (tagSNP) in the ALDH2 gene were genotyped in 1,134 subjects of Chinese origin from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family cohort. We examined whether the ALDH2 SNP genotypes predicted the development of hypertension in the prospective SAPPHIRe cohort.Over an average follow-up period of 5.7 years, carriers homozygous for the rs2238152 T allele in the ALDH2 gene were more likely to progress to hypertension than were non-carriers (hazard ratio [HR], 2.88, 95% confidence interval [CI], 1.06-7.84, P = 0.03), corresponding to a population attributable risk of ~7.1%. The risk associated with the rs2238152 T allele were strongest in heavy/moderate alcohol drinkers and was reduced in non-drinkers, indicating an interaction between ALDH2 genetic variants and alcohol intake on the risk of hypertension (P for interaction = 0.04). The risk allele was associated with significantly lower ALDH2 gene expression levels in human adipose tissue.ALDH2 genetic variants were associated with progression to hypertension in a prospective Chinese cohort. The association was modified by alcohol consumption.
View details for DOI 10.1186/1471-2261-12-58
View details for Web of Science ID 000308988000001
View details for PubMedID 22839215
View details for PubMedCentralID PMC3476438
Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells
2012; 7 (6)
Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors.
View details for DOI 10.1371/journal.pone.0038328
View details for Web of Science ID 000305339900024
View details for PubMedID 22675543
View details for PubMedCentralID PMC3365885
FGD5 Mediates Proangiogenic Action of Vascular Endothelial Growth Factor in Human Vascular Endothelial Cells
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2012; 32 (4): 988-U316
Vascular endothelial growth factor (VEGF) exerts proangiogenic action and induces activation of a variety of proangiogenic signaling pathways, including the Rho family small G proteins. However, regulators of the Rho family small G proteins in vascular endothelial cells (ECs) are poorly understood. Here we attempted to clarify the expression, subcellular localization, downstream effectors, and proangiogenic role of FGD5, a member of the FGD family of guanine nucleotide exchange factors.FGD5 was shown to be selectively expressed in cultured human vascular ECs. Immunofluorescence microscopy showed that the signal for FGD5 was observed at peripheral membrane ruffles and perinuclear regions in human umbilical vein ECs. Overexpression of FGD5 increased Cdc42 activity, whereas knockdown of FGD5 by small interfering RNAs inhibited the VEGF-induced activation of Cdc42 and extracellular signal-regulated kinase. VEGF-promoted capillary-like network formation, permeability, directional movement, and proliferation of human umbilical vein ECs and the reorientation of the Golgi complex during directional cell movement were attenuated by knockdown of FGD5.This study provides the first demonstration of expression, subcellular localization, and function of FGD5 in vascular ECs. The results suggest that FGD5 regulates proangiogenic action of VEGF in vascular ECs, including network formation, permeability, directional movement, and proliferation.
View details for DOI 10.1161/ATVBAHA.111.244004
View details for Web of Science ID 000301672300023
View details for PubMedID 22328776
Replication of genome-wide association signals of type 2 diabetes in Han Chinese in a prospective cohort
2012; 76 (3): 365-372
A recent genome-wide association study for type 2 diabetes in Han Chinese identified several novel genetic variants. We investigated their associations with quantitative measures to explore the mechanism by which these variants influence glucose homoeostasis. We also examined whether these variants predict progression to diabetes in a large prospective family based Chinese cohort. Five single nucleotide polymorphisms (SNPs) near the protein tyrosine phosphatase, receptor type, D (PTPRD), SRR, MAF/WWOX, and KCNQ1 genes were genotyped in 1138 subjects of Chinese origin from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance study. At baseline, the risk-conferring rs7192960 C allele near the MAF/WWOX genes was associated with lower homoeostasis model assessment of β-cell (HOMA-β) (P = 0·01) and second-phase insulin response in oral glucose tolerance test (OGTT) (P = 0·04). The risk-conferring rs2237897 C alleles in the KCNQ1 gene were associated with higher fasting glucose (P = 0·009), lower HOMA-β (P = 0·03), and lower first-phase insulin response in OGTT (P = 0·03). Over an average follow-up period of 5·43 years, participants with the risk-conferring rs17584499 TT genotype in the PTPRD gene were more likely to progress from nondiabetes to diabetes than were noncarriers (hazard ratio: 8·82, P = 4 × 10(-5) ). The risk-conferring T allele in the PTPRD gene was associated with greater increase in homoeostasis model assessment of insulin resistance (HOMA-IR) (P = 0·04) over time. PTPRD gene expression in human adipose tissues was negatively associated with fasting insulin levels and HOMA-IR. Genetic variants near the KCNQ1 and MAF/WWOX genes are associated with reduced insulin secretion. The PTPRD genetic variant appears to be associated with progression to diabetes in Han Chinese, most likely through increased insulin resistance.
View details for DOI 10.1111/j.1365-2265.2011.04175.x
View details for Web of Science ID 000299946000009
View details for PubMedID 21767287
The angiogenic factor Dell prevents apoptosis of endothelial cells through integrin binding
2012; 151 (2): 296-305
Del1 is a secreted protein that is expressed in the endothelium during development and can stimulate angiogenesis through integrin binding and signaling. We were interested in the specific effects of del1 on endothelial cell biology to gain insight into its biologic role during angiogenesis.Primary endothelial cells were treated with a variety of inducers of apoptosis and anoikis followed by assays for numbers of apoptotic cells, and harvest of total protein for immunoblot analysis.Del1 prevented endothelial cell apoptosis in response to TNFα/IFNγ, etoposide, and anoikis, but had no effect on proliferation. The anti-apoptotic effect was mediated specifically through binding of integrin αvβ3 by the RGD motif. FAK/ERK and Akt signaling were both necessary to mediate the anti-apoptotic effect of Del1 with the exception of anoikis, which required only Akt activation.Del1 has been previously shown to promote vascular smooth muscle cell adhesion, migration, and proliferation. We demonstrate here that Del1 prevented apoptosis of endothelial cells in cell culture through integrin binding without any effect on proliferation.
View details for DOI 10.1016/j.surg.2011.07.013
View details for Web of Science ID 000299607800019
View details for PubMedID 21893328
In Vivo Bioluminescence Imaging of Inducible Nitric Oxide Synthase Gene Expression in Vascular Inflammation
MOLECULAR IMAGING AND BIOLOGY
2011; 13 (6): 1061-1066
Inflammation plays a critical role in atherosclerosis and is associated with upregulation of inducible nitric oxide synthase (iNOS). We studied bioluminescence imaging (BLI) to track iNOS gene expression in a murine model of vascular inflammation.Macrophage-rich vascular lesions were induced by carotid ligation plus high-fat diet and streptozotocin-induced diabetes in 18 iNOS-luc reporter mice. In vivo iNOS expression was imaged serially by BLI over 14 days, followed by in situ BLI and histology.BLI signal from ligated carotids increased over 14 days (9.7 ± 4.4 × 10(3 ) vs. 4.4 ± 1.7 × 10(3) photons/s/cm(2)/sr at baseline, p < 0.001 vs. baseline, p < 0.05 vs. sham controls). Histology confirmed substantial macrophage infiltration, with iNOS and luciferase expression, only in ligated left carotid arteries and not controls.BLI allows in vivo detection of iNOS expression in murine carotid lesions and may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models.
View details for DOI 10.1007/s11307-010-0451-5
View details for Web of Science ID 000296794400002
View details for PubMedID 21057879
In vivo Molecular MRI of Cell Survival and Teratoma Formation Following Embryonic Stem Cell Transplantation Into the Injured Murine Myocardium
MAGNETIC RESONANCE IN MEDICINE
2011; 66 (5): 1374-1381
Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long-term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO-conjugated monoclonal antibody against these antigens (SPIO-MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO-MAb. The human and mouse ESCs were transduced with the RG (ESC-RGs) and transplanted into the peri-infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO-MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC-derived teratoma formation in the injured murine myocardium.
View details for DOI 10.1002/mrm.22929
View details for Web of Science ID 000296389800019
View details for PubMedID 21604295
Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk
2011; 478 (7367): 103-109
Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention.
View details for DOI 10.1038/nature10405
View details for Web of Science ID 000295575400043
View details for PubMedID 21909115
Cardiac pressure overload hypertrophy is differentially regulated by beta-adrenergic receptor subtypes
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2011; 301 (4): H1461-H1470
In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.
View details for DOI 10.1152/ajpheart.00453.2010
View details for Web of Science ID 000295360100028
View details for PubMedID 21705675
View details for PubMedCentralID PMC3197363
Racial variation in lipoprotein-associated phospholipase A(2) in older adults
BMC CARDIOVASCULAR DISORDERS
Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is a predictor of cardiovascular events that has been shown to vary with race. The objective of this study was to examine factors associated with this racial variation.We measured Lp-PLA₂ mass and activity in 714 healthy older adults with no clinical coronary heart disease and not taking dyslipidemia medication. We evaluated the association between race and Lp-PLA₂ mass and activity levels after adjustment for various covariates using multivariable linear regression. These covariates included age, sex, diabetes, hypertension, body mass index, lipid measurements, C-reactive protein, smoking status, physical activity, diet, income, and education level. We further examined genetic covariates that included three single nucleotide polymorphisms shown to be associated with Lp-PLA₂ activity levels.The mean age was 66 years. Whites had the highest Lp-PLA₂ mass and activity levels, followed by Hispanics and Asians, and then African-Americans; in age and sex adjusted analyses, these differences were significant for each non-White race as compared to Whites (p < 0.0001). For example, African-Americans were predicted to have a 55.0 ng/ml lower Lp-PLA₂ mass and 24.7 nmol/ml-min lower activity, compared with Whites, independent of age and sex (p < 0.0001). After adjustment for all covariates, race remained significantly correlated with Lp-PLA₂ mass and activity levels (p < 0.001) with African-Americans having 44.8 ng/ml lower Lp-PLA₂ mass and 17.3 nmol/ml-min lower activity compared with Whites (p < 0.0001).Biological, lifestyle, demographic, and select genetic factors do not appear to explain variations in Lp-PLA₂ mass and activity levels between Whites and non-Whites, suggesting that Lp-PLA₂ mass and activity levels may need to be interpreted differently for various races.
View details for DOI 10.1186/1471-2261-11-38
View details for Web of Science ID 000293271500001
View details for PubMedID 21714927
MicroRNA-26a Is a Novel Regulator of Vascular Smooth Muscle Cell Function
JOURNAL OF CELLULAR PHYSIOLOGY
2011; 226 (4): 1035-1043
Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-β-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-β pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.
View details for DOI 10.1002/jcp.22422
View details for Web of Science ID 000287258800019
View details for PubMedID 20857419
A Bivariate Genome-Wide Approach to Metabolic Syndrome STAMPEED Consortium
2011; 60 (4): 1329-1339
OBJECTIVE The metabolic syndrome (MetS) is defined as concomitant disorders of lipid and glucose metabolism, central obesity, and high blood pressure, with an increased risk of type 2 diabetes and cardiovascular disease. This study tests whether common genetic variants with pleiotropic effects account for some of the correlated architecture among five metabolic phenotypes that define MetS. RESEARCH DESIGN AND METHODS Seven studies of the STAMPEED consortium, comprising 22,161 participants of European ancestry, underwent genome-wide association analyses of metabolic traits using a panel of ∼2.5 million imputed single nucleotide polymorphisms (SNPs). Phenotypes were defined by the National Cholesterol Education Program (NCEP) criteria for MetS in pairwise combinations. Individuals exceeding the NCEP thresholds for both traits of a pair were considered affected. RESULTS Twenty-nine common variants were associated with MetS or a pair of traits. Variants in the genes LPL, CETP, APOA5 (and its cluster), GCKR (and its cluster), LIPC, TRIB1, LOC100128354/MTNR1B, ABCB11, and LOC100129150 were further tested for their association with individual qualitative and quantitative traits. None of the 16 top SNPs (one per gene) associated simultaneously with more than two individual traits. Of them 11 variants showed nominal associations with MetS per se. The effects of 16 top SNPs on the quantitative traits were relatively small, together explaining from ∼9% of the variance in triglycerides, 5.8% of high-density lipoprotein cholesterol, 3.6% of fasting glucose, and 1.4% of systolic blood pressure. CONCLUSIONS Qualitative and quantitative pleiotropic tests on pairs of traits indicate that a small portion of the covariation in these traits can be explained by the reported common genetic variants.
View details for DOI 10.2337/db10-1011
View details for Web of Science ID 000289496100029
View details for PubMedID 21386085
View details for PubMedCentralID PMC3064107
Distribution of the number of false discoveries in large-scale family-based association testing with application to the association between PTPN1 and hypertension and obesity
2011; 129 (4): 425-432
We present a model-free approach to the study of the number of false discoveries for large-scale simultaneous family-based association tests (FBATs) in which the set of discoveries is decided by applying a threshold to the test statistics. When the association between a set of markers in a candidate gene and a group of phenotypes is studied by a class of FBATs, we indicate that a joint null hypothesis distribution for these statistics can be obtained by the fundamental statistical method of conditioning on sufficient statistics for the null hypothesis. Based on the joint null distribution of these statistics, we can obtain the distribution of the number of false discoveries for the set of discoveries defined by a threshold; the size of this set is referred to as its tail count. Simulation studies are presented to demonstrate that the conditional, not the unconditional, distribution of the tail count is appropriate for the study of false discoveries. The usefulness of this approach is illustrated by re-examining the association between PTPN1 and a group of blood-pressure-related phenotypes reported by Olivier et al. (Hum Mol Genet 13:1885-1892, 2004); our results refine and reinforce this association.
View details for DOI 10.1007/s00439-010-0936-y
View details for Web of Science ID 000289275200008
View details for PubMedID 21188419
Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease
2011; 43 (4): 333-U153
We performed a meta-analysis of 14 genome-wide association studies of coronary artery disease (CAD) comprising 22,233 individuals with CAD (cases) and 64,762 controls of European descent followed by genotyping of top association signals in 56,682 additional individuals. This analysis identified 13 loci newly associated with CAD at P < 5 × 10⁻⁸ and confirmed the association of 10 of 12 previously reported CAD loci. The 13 new loci showed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6% to 17% increase in the risk of CAD per allele. Notably, only three of the new loci showed significant association with traditional CAD risk factors and the majority lie in gene regions not previously implicated in the pathogenesis of CAD. Finally, five of the new CAD risk loci appear to have pleiotropic effects, showing strong association with various other human diseases or traits.
View details for DOI 10.1038/ng.784
View details for Web of Science ID 000288903700013
View details for PubMedID 21378990
Disruption of the Apelin-APJ System Worsens Hypoxia-Induced Pulmonary Hypertension
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2011; 31 (4): 814-U212
The G-protein-coupled receptor APJ and its ligand apelin are highly expressed in the pulmonary vasculature, but their function in this vascular bed is unclear. We hypothesized that disruption of apelin signaling would lead to worsening of the vascular remodeling associated with pulmonary hypertension (PH).We found that apelin-null mice developed more severe PH compared with wild-type mice when exposed to chronic hypoxia. Micro-computed tomography of the pulmonary arteries demonstrated significant pruning of the microvasculature in the apelin-null mice. Apelin-null mice had a significant reduction of serum nitrate levels. This was secondary to downregulation of endothelial nitric oxide synthase (eNOS), which was associated with reduced expression of Kruppel-like factor 2 (KLF2), a known regulator of eNOS expression. In vitro knockdown studies targeting apelin in human pulmonary artery endothelial cells demonstrated decreased eNOS and KLF2 expression, as well as impaired phosphorylation of AMP-activated kinase and eNOS. Moreover, serum apelin levels of patients with PH were significantly lower than those of controls.These data demonstrate that disruption of apelin signaling can exacerbate PH mediated by decreased activation of AMP-activated kinase and eNOS, and they identify this pathway as a potentially important therapeutic target for treatment of this refractory human disease.
View details for DOI 10.1161/ATVBAHA.110.219980
View details for Web of Science ID 000288437800017
View details for PubMedID 21233449
Apelin Decreases Lipolysis via G(q), G(i), and AMPK-Dependent Mechanisms
2011; 152 (1): 59-68
The release of free fatty acids (FFAs) from adipocytes (i.e. lipolysis) is increased in obesity and is a contributory factor to the development of insulin resistance. A recently identified adipokine, apelin, is up-regulated in states of obesity. Although apelin is secreted by adipocytes, its functions in them remain largely unknown. To determine whether apelin affects lipolysis, FFA, glycerol, and leptin levels, as well as abdominal adiposity, were measured at baseline and after reintroduction of exogenous apelin in apelin-null mice. To examine apelin's effects in vitro, isoproterenol-induced FFA/glycerol release, and hormone-sensitive lipase (HSL) and acetyl CoA carboxylase phosphorylation were investigated in 3T3-L1 cells and isolated wild-type adipocytes. Serum FFA, glycerol, and leptin concentrations, as well as abdominal adiposity, were significantly increased in apelin-null vs. wild-type mice; these changes were ameliorated in response to exogenous apelin. Apelin also reduced isoproterenol-induced FFA release in adipocytes isolated from wild-type but not APJ-null mice. In 3T3-L1 cells and isolated adipocytes, apelin attenuated isoproterenol-induced FFA/glycerol release. Apelin's inhibition was reversed by pertussis toxin, the G(q) inhibitor glycoprotein antagonist 2A, and the AMP-activated protein kinase inhibitors compound C and dorsomorphin. Apelin increased HSL phosphorylation at Ser-565 and also abrogated isoproterenol-induced HSL phosphorylation at Ser-563. Notably, apelin increased acetyl CoA carboxylase phosphorylation, suggesting AMPK activation. In conclusion, apelin negatively regulates lipolysis. Its actions may be mediated by pathways involving G(q), G(i), and AMP-activated protein kinase.
View details for DOI 10.1210/en.2010-0576
View details for Web of Science ID 000285573000008
View details for PubMedID 21047945
Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies
2011; 377 (9763): 383-392
We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis.We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12,393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644).In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10(-13)). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10(-9)). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction.Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD.The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.
View details for DOI 10.1016/S0140-6736(10)61996-4
View details for Web of Science ID 000287337000028
View details for PubMedID 21239051
Chromatin Remodeling Pathways in Smooth Muscle Cell Differentiation, and Evidence for an Integral Role for p300
2010; 5 (12)
Phenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity.
View details for DOI 10.1371/journal.pone.0014301
View details for Web of Science ID 000285246900013
View details for PubMedID 21179216
Lack of Association Between the Trp719Arg Polymorphism in Kinesin-Like Protein-6 and Coronary Artery Disease in 19 Case-Control Studies
JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
2010; 56 (19): 1552-1563
We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD).Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with noncarriers.The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports.A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups.The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study.
View details for DOI 10.1016/j.jacc.2010.06.022
View details for Web of Science ID 000283538000005
View details for PubMedID 20933357
View details for PubMedCentralID PMC3084526
Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index
2010; 42 (11): 937-U53
Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.
View details for DOI 10.1038/ng.686
View details for Web of Science ID 000283540500010
View details for PubMedID 20935630
Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution
2010; 42 (11): 949-U160
Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10⁻⁹ to P = 1.8 × 10⁻⁴⁰) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10⁻³ to P = 1.2 × 10⁻¹³). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.
View details for DOI 10.1038/ng.685
View details for Web of Science ID 000283540500011
View details for PubMedID 20935629
Sex-specific genetic architecture of human fatness in Chinese: the SAPPHIRe Study
2010; 128 (5): 501-513
To dissect the genetic architecture of sexual dimorphism in obesity-related traits, we evaluated the sex-genotype interaction, sex-specific heritability and genome-wide linkages for seven measurements related to obesity. A total of 1,365 non-diabetic Chinese subjects from the family study of the Stanford Asia-Pacific Program of Hypertension and Insulin Resistance were used to search for quantitative trait loci (QTLs) responsible for the obesity-related traits. Pleiotropy and co-incidence effects from the QTLs were also examined using the bivariate linkage approach. We found that sex-specific differences in heritability and the genotype-sex interaction effects were substantially significant for most of these traits. Several QTLs with strong linkage evidence were identified after incorporating genotype by sex (G × S) interactions into the linkage mapping, including one QTL for hip circumference [maximum LOD score (MLS) = 4.22, empirical p = 0.000033] and two QTLs: for BMI on chromosome 12q with MLS 3.37 (empirical p = 0.0043) and 3.10 (empirical p = 0.0054). Sex-specific analyses demonstrated that these linkage signals all resulted from females rather than males. Most of these QTLs for obesity-related traits replicated the findings in other ethnic groups. Bivariate linkage analyses showed several obesity traits were influenced by a common set of QTLs. All regions with linkage signals were observed in one gender, but not in the whole sample, suggesting the genetic architecture of obesity-related traits does differ by gender. These findings are useful for further identification of the liability genes for these phenotypes through candidate genes or genome-wide association analysis.
View details for DOI 10.1007/s00439-010-0877-5
View details for Web of Science ID 000283094100004
View details for PubMedID 20725740
Design of the Coronary ARtery DIsease Genome-Wide Replication And Meta-Analysis (CARDIoGRAM) Study A Genome-Wide Association Meta-analysis Involving More Than 22 000 Cases and 60 000 Controls
2010; 3 (5): 475-U186
Recent genome-wide association studies (GWAS) of myocardial infarction (MI) and other forms of coronary artery disease (CAD) have led to the discovery of at least 13 genetic loci. In addition to the effect size, power to detect associations is largely driven by sample size. Therefore, to maximize the chance of finding novel susceptibility loci for CAD and MI, the Coronary ARtery DIsease Genome-wide Replication And Meta-analysis (CARDIoGRAM) consortium was formed.CARDIoGRAM combines data from all published and several unpublished GWAS in individuals with European ancestry; includes >22 000 cases with CAD, MI, or both and >60 000 controls; and unifies samples from the Atherosclerotic Disease VAscular functioN and genetiC Epidemiology study, CADomics, Cohorts for Heart and Aging Research in Genomic Epidemiology, deCODE, the German Myocardial Infarction Family Studies I, II, and III, Ludwigshafen Risk and Cardiovascular Heath Study/AtheroRemo, MedStar, Myocardial Infarction Genetics Consortium, Ottawa Heart Genomics Study, PennCath, and the Wellcome Trust Case Control Consortium. Genotyping was carried out on Affymetrix or Illumina platforms followed by imputation of genotypes in most studies. On average, 2.2 million single nucleotide polymorphisms were generated per study. The results from each study are combined using meta-analysis. As proof of principle, we meta-analyzed risk variants at 9p21 and found that rs1333049 confers a 29% increase in risk for MI per copy (P=2×10⁻²⁰).CARDIoGRAM is poised to contribute to our understanding of the role of common genetic variation on risk for CAD and MI.
View details for DOI 10.1161/CIRCGENETICS.109.899443
View details for Web of Science ID 000283163100012
View details for PubMedID 20923989
Endothelial cell-selective adhesion molecule modulates atherosclerosis through plaque angiogenesis and monocyte-endothelial interaction
2010; 80 (2): 179-187
Endothelial cell-selective adhesion molecule (ESAM) is a new member of the immunoglobulin superfamily, which is expressed in vascular endothelial cells. Previous studies have demonstrated that ESAM regulates angiogenesis, endothelial permeability, and leukocyte transmigration. However, little is known concerning the role of ESAM in atherosclerosis. In this study, we assessed the effects of ESAM inactivation on atherosclerosis in mice. ESAM-/- mice were bred with apoE-/- mice to generate double knockout mice, and the aortic lesion size of apoE-/- and ESAM-/-apoE-/- mice was compared histologically. Although plasma cholesterol levels were higher in ESAM-/-apoE-/- mice, the lesion size was markedly smaller than in apoE-/- mice. ESAM-/-apoE-/- mice exhibited a decrease in the number of vasa vasorum and macrophages in the vessel wall. In vitro adhesion assays showed that THP-1 cells, which did not express ESAM, bound to the ESAM-coated culture plates, suggesting that ESAM may interact with heterophilic ligand(s) on monocytes. Moreover, downregulation of ESAM by siRNA in the endothelial monolayer diminished transendothelial migration of THP-1 cells. In conclusion, ESAM inactivation can reduce susceptibility to atherosclerosis by inhibiting plaque neovascularization and macrophage infiltration into the atheroma.
View details for DOI 10.1016/j.mvr.2010.04.005
View details for Web of Science ID 000281107100001
View details for PubMedID 20406651
Impact of Combined Deficiency of Hepatic Lipase and Endothelial Lipase on the Metabolism of Both High-Density Lipoproteins and Apolipoprotein B-Containing Lipoproteins
2010; 107 (3): 357-U90
Hepatic lipase (HL) and endothelial lipase (EL) are extracellular lipases that both hydrolyze triglycerides and phospholipids and display potentially overlapping or complementary roles in lipoprotein metabolism.We sought to dissect the overlapping roles of HL and EL by generating mice deficient in both HL and EL (HL/EL-dko) for comparison with single HL-knockout (ko) and EL-ko mice, as well as wild-type mice.Reproduction and viability of the HL/EL-dko mice were impaired compared with the single-knockout mice. The plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol, and phospholipids in the HL/EL-dko mice were markedly higher than those in the single-knockout mice. Most notably, the HL/EL-dko mice exhibited an unexpected substantial increase in small low-density lipoproteins. Kinetic studies with [(3)H]cholesteryl ether-labeled very-low-density lipoproteins demonstrated that the HL/EL-dko mice accumulated counts in the smallest low-density lipoprotein-sized fractions, as assessed by size exclusion chromatography, suggesting that it arises from lipolysis of very-low-density lipoproteins. HDL from all 3 lipase knockout models had an increased cholesterol efflux capacity but reduced clearance of HDL cholesteryl esters versus control mice. Despite their higher HDL cholesterol levels, neither HL-ko, EL-ko, nor HL/EL-dko mice demonstrated an increased rate of macrophage reverse cholesterol transport in vivo.These studies reveal an additive effect of HL and EL on HDL metabolism but not macrophage reverse cholesterol transport in mice and an unexpected redundant role of HL and EL in apolipoprotein B lipoprotein metabolism.
View details for DOI 10.1161/CIRCRESAHA.110.219188
View details for Web of Science ID 000280603000007
View details for PubMedID 20558822
An "Almost Exhaustive" Search-Based Sequential Permutation Method for Detecting Epistasis in Disease Association Studies
2010; 34 (5): 434-443
Due to the complex nature of common diseases, their etiology is likely to involve "uncommon but strong" (UBS) interactive effects--i.e. allelic combinations that are each present in only a small fraction of the patients but associated with high disease risk. However, the identification of such effects using standard methods for testing association can be difficult. In this work, we introduce a method for testing interactions that is particularly powerful in detecting UBS effects. The method consists of two modules--one is a pattern counting algorithm designed for efficiently evaluating the risk significance of each marker combination, and the other is a sequential permutation scheme for multiple testing correction. We demonstrate the work of our method using a candidate gene data set for cardiovascular and coronary diseases with an injected UBS three-locus interaction. In addition, we investigate the power and false rejection properties of our method using data sets simulated from a joint dominance three-locus model that gives rise to UBS interactive effects. The results show that our method can be much more powerful than standard approaches such as trend test and multifactor dimensionality reduction for detecting UBS interactions.
View details for DOI 10.1002/gepi.20496
View details for Web of Science ID 000280349600007
View details for PubMedID 20583286
Role of endothelial cell-selective adhesion molecule in hematogeneous metastasis
2010; 80 (1): 133-141
The spread of malignant cells from a localized tumor is thought to be directly related to the number of microvessels in the tumor. The endothelial cell-selective adhesion molecule (ESAM) is a member of the immunoglobulin superfamily that mediates homophilic interactions between endothelial cells. Previous studies have indicated that ESAM regulates angiogenesis in the primary tumor growth and endothelial permeability. In this study, we aimed to further elucidate the role of ESAM in tumor metastasis through angiogenic processes. ESAM expression was higher in hypervascular metastatic tumor tissues than in normal tissues in human lungs. Cell culture studies found that conditioned medium from B16F10 melanoma cells increased ESAM expression in endothelial cells and promoted endothelial migration and tube formation. The B16F10 medium-induced endothelial migration and tube formation were significantly attenuated when ESAM was downregulated by siRNA transfection. Intravenous injection of B16F10 cells into ESAM+/+ and ESAM-/- mice for comparison of metastatic potential resulted in the number of metastatic lung nodules in ESAM-/- mice being 83% lower than of those in ESAM+/+ mice. The microvascular density in the tumor was also lower in ESAM-/- than in ESAM+/+ mice. These findings indicate that ESAM regulates tumor metastasis through endothelial cell migration and tube formation in metastatic nodules. Inhibition of ESAM may therefore inhibit tumor metastasis by inhibiting the angiogenic processes.
View details for DOI 10.1016/j.mvr.2010.02.006
View details for Web of Science ID 000278950700019
View details for PubMedID 20153339
Detailed Physiologic Characterization Reveals Diverse Mechanisms for Novel Genetic Loci Regulating Glucose and Insulin Metabolism in Humans
59th Annual Meeting of the American-Society-of-Human-Genetics
AMER DIABETES ASSOC. 2010: 1266–75
OBJECTIVE Recent genome-wide association studies have revealed loci associated with glucose and insulin-related traits. We aimed to characterize 19 such loci using detailed measures of insulin processing, secretion, and sensitivity to help elucidate their role in regulation of glucose control, insulin secretion and/or action. RESEARCH DESIGN AND METHODS We investigated associations of loci identified by the Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) with circulating proinsulin, measures of insulin secretion and sensitivity from oral glucose tolerance tests (OGTTs), euglycemic clamps, insulin suppression tests, or frequently sampled intravenous glucose tolerance tests in nondiabetic humans (n = 29,084). RESULTS The glucose-raising allele in MADD was associated with abnormal insulin processing (a dramatic effect on higher proinsulin levels, but no association with insulinogenic index) at extremely persuasive levels of statistical significance (P = 2.1 x 10(-71)). Defects in insulin processing and insulin secretion were seen in glucose-raising allele carriers at TCF7L2, SCL30A8, GIPR, and C2CD4B. Abnormalities in early insulin secretion were suggested in glucose-raising allele carriers at MTNR1B, GCK, FADS1, DGKB, and PROX1 (lower insulinogenic index; no association with proinsulin or insulin sensitivity). Two loci previously associated with fasting insulin (GCKR and IGF1) were associated with OGTT-derived insulin sensitivity indices in a consistent direction. CONCLUSIONS Genetic loci identified through their effect on hyperglycemia and/or hyperinsulinemia demonstrate considerable heterogeneity in associations with measures of insulin processing, secretion, and sensitivity. Our findings emphasize the importance of detailed physiological characterization of such loci for improved understanding of pathways associated with alterations in glucose homeostasis and eventually type 2 diabetes.
View details for DOI 10.2337/DB09-1568
View details for Web of Science ID 000277554700019
View details for PubMedID 20185807
Persistent Donor Cell Gene Expression among Human Induced Pluripotent Stem Cells Contributes to Differences with Human Embryonic Stem Cells
2010; 5 (2)
Human induced pluripotent stem cells (hiPSCs) generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs), as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.
View details for DOI 10.1371/journal.pone.0008975
View details for Web of Science ID 000274209700007
View details for PubMedID 20126639
View details for PubMedCentralID PMC2813859
Apelin is necessary for the maintenance of insulin sensitivity
AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM
2010; 298 (1): E59-E67
The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.
View details for DOI 10.1152/ajpendo.00385.2009
View details for Web of Science ID 000272793700007
View details for PubMedID 19861585
Ontogeny of apelin and its receptor in the rodent gastrointestinal tract
2009; 158 (1-3): 32-39
Apelin is the endogenous ligand for the APJ receptor and both apelin and APJ are expressed in the gastrointestinal (GI) tract. The aim of this study was to define ontogeny of apelin and APJ in the developing rodent GI tract by measuring expression levels and characterizing abundance and cellular localization at an embryonic stage (E18.5 or E21), two postnatal stages (P4, P16) and in the adult. Apelin and APJ mRNA levels were measured by real time RT-PCR, apelin and APJ-containing cells were identified by immunohistochemical (IHC) staining. Gastric, duodenal and colonic apelin and APJ mRNA levels were highest at birth and declined postnatally. In the postnatal rat stomach, few apelin peptide-containing cells were identified, the density of gastric apelin-containing cells increased progressively after weaning and into adulthood. A robust APJ immunostaining was observed postnatally in the epithelium, intestinal goblet cells and in smooth muscle cells. In the adult rat, APJ immunostaining in the surface epithelium and goblet cells decreased markedly. During the early postnatal period, in an apelin-deficient mouse, APJ expression and immunostaining in the gut were reduced suggesting that apelin regulates APJ. Together, our data support a role for the apelin-APJ system in the regulation of smooth muscle, epithelial and goblet cell function in the GI tract.
View details for DOI 10.1016/j.regpep.2009.07.016
View details for Web of Science ID 000271556100006
View details for PubMedID 19660504
Endogenous regulation of cardiovascular function by apelin-APJ
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2009; 297 (5): H1904-H1913
Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.
View details for DOI 10.1152/ajpheart.00686.2009
View details for Web of Science ID 000271143400045
View details for PubMedID 19767528
View details for PubMedCentralID PMC2781363
Endothelial cell specific adhesion molecule (ESAM) localizes to platelet-platelet contacts and regulates thrombus formation in vivo
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
2009; 7 (11): 1886-1896
In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown.To determine whether ESAM has a role in thrombus formation.We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM(-/-) mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM(-/-) platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, alpha(IIb)beta(3) activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets.These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.
View details for DOI 10.1111/j.1538-7836.2009.03606.x
View details for Web of Science ID 000271032900020
View details for PubMedID 19740102
Admixture mapping of quantitative trait loci for blood lipids in African-Americans
HUMAN MOLECULAR GENETICS
2009; 18 (11): 2091-2098
Blood lipid levels, including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG), are highly heritable traits and major risk factors for atherosclerotic cardiovascular disease (CVD). Using individual ancestry estimates at marker locations across the genome, we present a novel quantitative admixture mapping analysis of all three lipid traits in a large sample of African-Americans from the Family Blood Pressure Program. Regression analysis was performed with both total and marker-location-specific European ancestry as explanatory variables, along with demographic covariates. Robust permutation analysis was used to assess statistical significance. Overall European ancestry was significantly correlated with HDL-C (negatively) and TG (positively), but not with LDL-C. We found strong evidence for a novel locus underlying HDL-C on chromosome 8q, which correlated negatively with European ancestry (P = .0014); the same location also showed positive correlation of European ancestry with TG levels. A region on chromosome 14q also showed significant negative correlation between HDL-C levels and European ancestry. On chromosome 15q, a suggestive negative correlation of European ancestry with TG and positive correlation with HDL-C was observed. Results with LDL-C were less significant overall. We also found significant evidence for genome-wide ancestry effects underlying the joint distribution of HDL-C and TG, not fully explained by the locus on chromosome 8. Our results are consistent with a genetic contribution to and may explain the healthier HDL-C and TG profiles found in Blacks versus Whites. The identified regions provide locations for follow-up studies of genetic variants underlying lipid variation in African-Americans and possibly other populations.
View details for DOI 10.1093/hmg/ddp122
View details for Web of Science ID 000265951600018
View details for PubMedID 19304782
Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (20): 8227-8232
Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirus-mediated signal sequence trap method, we identified a previously undescribed gene, termed ARIA (apoptosis regulator through modulating IAP expression), which regulates endothelial apoptosis and angiogenesis. ARIA was expressed in blood vessels during mouse embryogenesis, as well as in endothelial cells both in vitro and in vivo. ARIA is a unique protein with no homology to previously reported conserved domain structures. Knockdown of ARIA in HUVECs by using small interfering RNA significantly reduced endothelial apoptosis without affecting either cell migration or proliferation. ARIA knockdown significantly increased inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression, although their mRNA expression was not changed. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the antiapoptotic effect of ARIA knockdown. Using yeast 2-hybrid screening, we identified the interaction of ARIA with 20S proteasome subunit alpha-7. Thereafter, we found that cIAP-1 and cIAP-2 were degraded by proteasomes in endothelial cells under normal condition. Overexpression of ARIA significantly reduced cIAP-1 expression, and this reduction was abolished by proteasomal inhibition in BAECs. Also, knockdown of ARIA demonstrated an effect similar to proteasomal inhibition with respect to not only expression but also subcellular localization of cIAP-1 and cIAP-2. In vivo angiogenesis studied by Matrigel-plug assay, mouse ischemic retinopathy model, and tumor xenograft model was significantly enhanced by ARIA knockdown. Together, our data indicate that ARIA is a unique factor regulating endothelial apoptosis, as well as angiogenesis, presumably through modulating proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.
View details for DOI 10.1073/pnas.0806780106
View details for Web of Science ID 000266209000027
View details for PubMedID 19416853
Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2009; 296 (5): H1329-H1335
Apelin is a potent inodilator with recently described antiatherogenic properties. We hypothesized that apelin might also attenuate abdominal aortic aneurysm (AAA) formation by limiting disease-related vascular wall inflammation. C57BL/6 mice implanted with osmotic pumps filled with apelin or saline were treated with pancreatic elastase to create infrarenal AAAs. Mice were euthanized for aortic PCR analysis or followed ultrasonographically and then euthanized for histological analysis. The cellular expression of inflammatory cytokines and chemokines in response to apelin was also assessed in cultured macrophages, smooth muscle cells, and fibroblasts. Apelin treatment resulted in diminished AAA formation, with a 47% reduction in maximal cross-sectional area (0.74 vs. 1.39 mm(2), P < 0.03) and a 57% reduction in macrophage infiltrate (113 vs. 261.3 cells/high-power field, P < 0.0001) relative to the saline-treated group. Apelin infusion was also associated with significantly reduced aortic macrophage colony-stimulating factor expression and decreased monocyte chemattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-6, and tumor necrosis factor (TNF)-alpha mean mRNA levels. Apelin stimulation of cultured macrophages significantly reduced MCP-1 and TNF-alpha mRNA levels relative to baseline (2.03- and 1.89-fold reduction, P < 0.03, respectively) but did not affect intimal adhesion molecule expression or medial or adventitial cell cytokine production. Apelin significantly reduces aneurysm formation in the elastase model of human AAA disease. The mechanism appears to be decreased macrophage burden, perhaps related to an apelin-mediated decrease in proinflammatory cytokine and chemokine activation.
View details for DOI 10.1152/ajpheart.01341.2008
View details for Web of Science ID 000265659100020
View details for PubMedID 19304942
Insulin resistance independently predicts the progression of coronary artery calcification
AMERICAN HEART JOURNAL
2009; 157 (5): 939-945
Change in coronary artery calcification is a surrogate marker of subclinical coronary artery disease (CAD). In the only large prospective study, CAD risk factors predicted progression of coronary artery calcium (CAC).We measured CAC at enrollment and after 24 months in a community-based sample of 869 healthy adults aged 60 to 72 years who were free of clinical CAD. We assessed predictors of the progression of CAC using univariate and multivariate models after square root transformation of the Agatston scores. Predictors tested included age, sex, race/ethnicity, smoking status, body mass index, family history of CAD, C-reactive protein and several measures of diabetes, insulin levels, blood pressure, and lipids.The mean age of the cohort was 66 years, and 62% were male. The median CAC at entry was 38.6 Agatston units and increased to 53.3 Agatston units over 24 months (P < .01). The CAC progression was associated with white race, diabetes, dyslipidemia, hypertension, lower diastolic blood pressure, and higher pulse pressure. After controlling for these variables, higher fasting insulin levels independently predicted CAC progression.Insulin resistance, in addition to the traditional cardiac risk factors, independently predicts progression of CAC in a community-based population without clinical CAD.
View details for DOI 10.1016/j.ahj.2009.02.006
View details for Web of Science ID 000265987800031
View details for PubMedID 19376325
Targeted inactivation of endothelial lipase attenuates lung allergic inflammation through raising plasma HDL level and inhibiting eosinophil infiltration
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
2009; 296 (4): L594-L602
Endothelial lipase (EL) is a novel phospholipase that determines plasma high-density lipoprotein cholesterol (HDL-C) levels. We have investigated the role of HDL-C in lung allergic inflammation by using EL knockout (EL-KO) mice that are high in HDL-C. EL-KO and wild-type control mice were sensitized and challenged with ovalbumin to evoke eosinophilic inflammation in the lung. EL was expressed in epithelial cells, alveolar type II cells, and endothelial cells in the lung, and its expression was upregulated during inflammation. Concomitant with attenuated hyperresponsiveness of the airway smooth muscles, the number of eosinophils in bronchoalveolar lavage and the expression of VCAM-1 were lower in EL-KO mice than in control mice. HDL reduced cytokine-induced VCAM-1 expression in cultured endothelial cells. When plasma HDL levels were decreased to similar levels in both mouse groups by adenovirus-mediated overexpression of EL, however, eosinophil infiltration was still lower in EL-KO mice. In vitro adhesion assays revealed that EL expression on the cell surface promoted the interaction of eosinophils through the ligand-binding function of EL. In summary, targeted inactivation of EL attenuated allergic inflammation in the lung, and the protective effects in EL-KO mice were associated with high plasma HDL levels, downregulation of VCAM-1, and loss of the direct ligand-binding function of EL. Thus EL is a novel modulator of the progression of allergic asthma.
View details for DOI 10.1152/ajplung.90530.2008
View details for Web of Science ID 000264443000004
View details for PubMedID 19168574
Characterizing the admixed African ancestry of African Americans
2009; 10 (12)
Accurate, high-throughput genotyping allows the fine characterization of genetic ancestry. Here we applied recently developed statistical and computational techniques to the question of African ancestry in African Americans by using data on more than 450,000 single-nucleotide polymorphisms (SNPs) genotyped in 94 Africans of diverse geographic origins included in the HGDP, as well as 136 African Americans and 38 European Americans participating in the Atherosclerotic Disease Vascular Function and Genetic Epidemiology (ADVANCE) study. To focus on African ancestry, we reduced the data to include only those genotypes in each African American determined statistically to be African in origin.From cluster analysis, we found that all the African Americans are admixed in their African components of ancestry, with the majority contributions being from West and West-Central Africa, and only modest variation in these African-ancestry proportions among individuals. Furthermore, by principal components analysis, we found little evidence of genetic structure within the African component of ancestry in African Americans.These results are consistent with historic mating patterns among African Americans that are largely uncorrelated to African ancestral origins, and they cast doubt on the general utility of mtDNA or Y-chromosome markers alone to delineate the full African ancestry of African Americans. Our results also indicate that the genetic architecture of African Americans is distinct from that of Africans, and that the greatest source of potential genetic stratification bias in case-control studies of African Americans derives from the proportion of European ancestry.
View details for DOI 10.1186/gb-2009-10-12-r141
View details for Web of Science ID 000274289000011
View details for PubMedID 20025784
View details for PubMedCentralID PMC2812948
Role of Endothelial Lipase in Plasma HDL levels in a Murine Model of Hypertriglyceridemia
JOURNAL OF ATHEROSCLEROSIS AND THROMBOSIS
2009; 16 (4): 327-338
Hypertriglyceridemia is the most common cause of low plasma high-density lipoprotein cholesterol (HDL-C) levels; however, the correlation between high triglyceride (TG) and low HDL-C remains unclear. Endothelial lipase (EL) is a determinant of plasma HDL levels. We investigated the role of EL in HDL metabolism in a murine model of acute hypertriglyceridemia.To establish TG-dominant hyperlipidemia, EL-/- and wild-type (WT) mice were injected with Poloxamer-407 (P-407, 0.5 g/kg, i.p.). A single injection of P-407 resulted in a marked increase in plasma TG and cholesterol levels together with a decrease in HDL-C levels. Although plasma TG levels were similar in EL-/- and WT mice after P-407 injection, HDL-C levels were 80% higher and the HDL particle size was significantly larger in EL-/- mice than in WT mice. P-407 treatment inhibited plasma lipoprotein lipase activity and EL phospholipase activity, without decreasing their expressions. Adenovirus-mediated overexpression of EL in the liver reduced plasma HDL-C levels in both normo- and hyperlipidemic mice, while overexpression of catalytically inactive EL reduced HDL-C levels in hyperlipidemic mice. Cell culture experiments revealed that both catalytically active and inactive EL promoted cellular HDL uptake to the same extent.EL regulates plasma HDL levels in mice in the normolipidemic as well as the acute hypertriglyceridemic state. EL can modulate plasma HDL-CHOL levels through both its lipolytic and ligand-binding functions in hypertriglyceridemic mice, while lipolytic activity appears to be the main determinant for its effects on HDL metabolism in normolipidemic mice.
View details for Web of Science ID 000269754900003
View details for PubMedID 19672025
The Adhesion Molecule Esam1 Is a Novel Hematopoietic Stem Cell Marker
2009; 27 (3): 653-661
Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.
View details for DOI 10.1634/stemcells.2008-0824
View details for Web of Science ID 000264706900016
View details for PubMedID 19074415
Peroxisome Proliferator-Activated Receptor Gamma Polymorphisms and Coronary Heart Disease
Single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptor gamma (PPARG) gene have been associated with cardiovascular risk factors, particularly obesity and diabetes. We assessed the relationship between 4 PPARG SNPs (C-681G, C-689T, Pro12Ala, and C1431T) and coronary heart disease (CHD) in the PRIME (249 cases/494 controls, only men) and ADVANCE (1,076 cases/805 controls, men or women) studies. In PRIME, homozygote individuals for the minor allele of the PPARG C-689T, Pro12Ala, and C1431T SNPs tended to have a higher risk of CHD than homozygote individuals for the frequent allele (adjusted OR [95% CI] = 3.43 [0.96-12.27], P = .058, 3.41 [0.95-12.22], P = .060 and 5.10 [0.99-26.37], P = .050, resp.). No such association could be detected in ADVANCE. Haplotype distributions were similar in cases and control in both studies. A meta-analysis on the Pro12Ala SNP, based on our data and 11 other published association studies (6,898 CHD cases/11,287 controls), revealed that there was no evidence for a significant association under the dominant model (OR = 0.99 [0.92-1.07], P = .82). However, there was a borderline association under the recessive model (OR = 1.29 [0.99-1.67], P = .06) that became significant when considering men only (OR = 1.73 [1.20-2.48], P = .003). In conclusion, the PPARG Ala12Ala genotype might be associated with a higher CHD risk in men but further confirmation studies are needed.
View details for DOI 10.1155/2009/543746
View details for Web of Science ID 000283415200001
View details for PubMedID 20016803
Del-1, an Endogenous Leukocyte-Endothelial Adhesion Inhibitor, Limits Inflammatory Cell Recruitment
2008; 322 (5904): 1101-1104
Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus-1) as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1 deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/- mice displayed significantly higher neutrophil accumulation in lipopolysaccharide-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1 double-deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.
View details for DOI 10.1126/science.1165218
View details for Web of Science ID 000260867700037
View details for PubMedID 19008446
- Apelin-APJ signaling in retinal angiogenesis ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 2008; 28 (10): 1687-1688
Molecular and physiological characterization of RV remodeling in a murine model of pulmonary stenosis
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2008; 295 (3): H1351-H1368
Right ventricular (RV) dysfunction is a common long-term complication in patients after the repair of congenital heart disease. Previous investigators have examined the cellular and molecular mechanisms of left ventricular (LV) remodeling, but little is known about the stressed RV. Our purpose was to provide a detailed physiological characterization of a model of RV hypertrophy and failure, including RV-LV interaction, and to compare gene alterations between afterloaded RV versus LV. Pulmonary artery constriction was performed in 86 mice. Mice with mild and moderate pulmonary stenosis (PS) developed stable hypertrophy without decompensation. Mice with severe PS developed edema, decreased RV function, and high mortality. Tissue Doppler imaging demonstrated septal dyssynchrony and deleterious RV-LV interaction in the severe PS group. Microarray analysis showed 196 genes with increased expression and 1,114 with decreased expression. Several transcripts were differentially increased in the afterloaded RV but not in the afterloaded LV, including clusterin, neuroblastoma suppression of tumorigenicity 1, Dkk3, Sfrp2, formin binding protein, annexin A7, and lysyl oxidase. We have characterized a murine model of RV hypertrophy and failure, providing a platform for studying the physiological and molecular events of RV remodeling. Although the molecular responses of the RV and LV to afterload stress are mostly concordant, there are several key differences, which may represent targets for RV failure-specific therapy.
View details for DOI 10.1152/ajpheart.91526.2007
View details for Web of Science ID 000258949200055
View details for PubMedID 18586894
View details for PubMedCentralID PMC2544484
Susceptibility locus for clinical and subclinical coronary artery disease at chromosome 9p21 in the multi-ethnic ADVANCE study
HUMAN MOLECULAR GENETICS
2008; 17 (15): 2320-2328
A susceptibility locus for coronary artery disease (CAD) at chromosome 9p21 has recently been reported, which may influence the age of onset of CAD. We sought to replicate these findings among white subjects and to examine whether these results are consistent with other racial/ethnic groups by genotyping three single nucleotide polymorphisms (SNPs) in the risk interval in the Atherosclerotic Disease, Vascular Function, and Genetic Epidemiology (ADVANCE) study. One or more of these SNPs was associated with clinical CAD in whites, U.S. Hispanics and U.S. East Asians. None of the SNPs were associated with CAD in African Americans although the power to detect an odds ratio (OR) in this group equivalent to that seen in whites was only 24-30%. ORs were higher in Hispanics and East Asians and lower in African Americans, but in all groups the 95% confidence intervals overlapped with ORs observed in whites. High-risk alleles were also associated with increased coronary artery calcification in controls and the magnitude of these associations by racial/ethnic group closely mirrored the magnitude observed for clinical CAD. Unexpectedly, we noted significant genotype frequency differences between male and female cases (P = 0.003-0.05). Consequently, men tended towards a recessive and women tended towards a dominant mode of inheritance. Finally, an effect of genotype on the age of onset of CAD was detected but only in men carrying two versus one or no copy of the high-risk allele and presenting with CAD at age >50 years. Further investigations in other populations are needed to confirm or refute our findings.
View details for DOI 10.1093/hmg/ddn132
View details for Web of Science ID 000257788300007
View details for PubMedID 18443000
View details for PubMedCentralID PMC2733811
Absence of evidence for an association between resistin gene variants and insulin resistance in an Asian population with low and high blood pressure
DIABETES RESEARCH AND CLINICAL PRACTICE
2008; 81 (2): 231-237
Although the function of resistin in human biology is unclear, some evidence suggests resistin gene variants influence insulin resistance, and insulin resistance-related hypertension. We searched for associations between common resistin gene variants and factors related to insulin resistance in Asian individuals with high or low blood pressure (BP).Non-diabetic Chinese or Japanese sibling pairs were included if one had extreme hypertension and the other was either hypertensive or hypotensive. Four common, non-coding single nucleotide polymorphisms (SNPs) were identified by sequencing the resistin gene in 24 hypertensive probands. Generalized estimating equations (GEEs)-based regressions were then performed to test for SNP associations using the entire study population (n=1556).Of 72 tests, only one was significant at the 0.05 level; 3.5 significant tests were expected by chance alone. High variability in insulin and triglyceride levels created wide confidence intervals, thus the negative results are not conclusive for these phenotypes. However, the large sample size resulted in narrow confidence intervals for BMI, fasting and 120min post-load glucose, and high and low density lipoprotein cholesterol (LDL-C).Several factors associated with insulin resistance are not likely influenced by the resistin gene in non-diabetic Asian individuals with high and low blood pressure.
View details for DOI 10.1016/j.diabres.2008.04.001
View details for Web of Science ID 000259289400016
View details for PubMedID 18501464
Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality
2008; 3 (7)
Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse.We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes.This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment.
View details for DOI 10.1371/journal.pone.0002609
View details for Web of Science ID 000264065800015
View details for PubMedID 18648539
View details for PubMedCentralID PMC2481284
Increased rate of hair regrowth in mice with constitutive overexpression of Del 1
JOURNAL OF SURGICAL RESEARCH
2008; 146 (1): 73-80
Developmental endothelial locus (Del)1 is a secreted extracellular matrix-associated protein that stimulates angiogenesis through integrin binding and is implicated in vasculogenesis. We hypothesized that increased expression of an angiogenic factor would lead to enhanced wound healing.Transgenic mice had Del1 cloned behind a keratin 14 promoter (K14-Del1) to drive constitutive expression in basal keratinocytes. Transgenic animals and wild-type litter mates underwent excisional wounding or depilation, and tissues were harvested at various time points. Wound healing and hair regrowth were assessed by photography, histology, and immunohistochemistry. For injection experiments, purified Del1 protein was injected in the flanks of wild-type mice with carrier on the contralateral flank as a control. Del1 expression during hair development was performed using transgenic mice with a LacZ cassette introduced downstream from the native promoter.K14-Del1 animals appeared normal and healed excisional wounds normally but demonstrated an increased rate of hair regrowth after wound healing. Using depilation experiments to specifically address hair follicle growth, we found increased hair regrowth was independent of wounding. This was confirmed by injection of purified Del1 protein. During normal hair anagenesis, Del1 is expressed in the root of the hair follicle.Constitutive expression of Del1 in skin does not affect skin vascularity or improve wound healing. Surprisingly, we found the primary effect of constitutive Del1 expression in the basal keratinocytes was increased hair growth following induction of anagenesis. During normal hair anagenesis, we see expression of Del1 in the root of the hair follicle suggesting it may function there to stimulate hair growth.
View details for DOI 10.1016/j.jss.2007.02.024
View details for Web of Science ID 000254798700011
View details for PubMedID 17764695
Common polymorphisms of ALOX5 and ALOX5AP and risk of coronary artery disease
2008; 123 (4): 399-408
Recent human genetic studies suggest that allelic variants of leukotriene pathway genes influence the risk of clinical and subclinical atherosclerosis. We sequenced the promoter, exonic, and splice site regions of ALOX5 and ALOX5AP and then genotyped 7 SNPs in ALOX5 and 6 SNPs in ALOX5AP in 1,552 cases with clinically significant coronary artery disease (CAD) and 1,583 controls from Kaiser Permanente including a subset of participants of the coronary artery risk development in young adults study. A nominally significant association was detected between a promoter SNP in ALOX5 (rs12762303) and CAD in our subset of white/European subjects (adjusted odds ratio per minor allele, log-additive model, 1.32; P = 0.002). In this race/ethnic group, rs12762303 has a minor allele frequency of 15% and is tightly linked to variation at the SP1 variable tandem repeat promoter polymorphism. However, the association between CAD and rs12762303 could not be reproduced in the atherosclerosis risk in communities study (hazard rate ratio per minor allele; 1.08, P = 0.1). Assuming a recessive mode of inheritance, the association was not significant in either population study but our power to detect modest effects was limited. No significant associations were observed between all other SNPs and the risk of CAD. Overall, our findings do not support a link between common allelic variation in or near ALOX5 or ALOX5AP and the risk of CAD. However, additional studies are needed to exclude modest effects of promoter variation in ALOX5 on the risk of CAD assuming a recessive mode of inheritance.
View details for DOI 10.1007/s00439-008-0489-5
View details for Web of Science ID 000254959600008
View details for PubMedID 18369664
A near null variant of 12/15-LOX encoded by a novel SNP in ALOX15 and the risk of coronary artery disease
2008; 198 (1): 136-144
Murine genetic models suggest that function of the 12/15-LOX enzyme promotes atherosclerosis. We tested the hypothesis that exonic and/or promoter single nucleotide polymorphisms (SNPs) in the human 12/15-LOX gene (ALOX15) alter the risk of symptomatic coronary artery disease (CAD).We resequenced ALOX15 and then genotyped a common promoter and a less common novel coding SNP (T560M) in 1809 subjects with CAD and 1734 controls from Kaiser Permanente including a subset of participants of the Coronary Artery Risk Development in Young Adults study. We found no association between the promoter SNP and the risk of CAD. However, heterozygote carriers of the 560M allele had an increased risk of CAD (adjusted OR, 1.62; P=0.02) compared to non-carriers. In vitro studies demonstrated a 20-fold reduction in the catalytic activity of 560M when compared to 560T. We then genotyped T560M in 12,974 participants of the Atherosclerosis Risk in Communities study and similarly found that heterozygote carriers had an increased risk of CAD compared to non-carriers (adjusted HR, 1.31; P=0.06). In both population studies, homozygote carriers were rare and associated with a non-significant decreased risk of CAD compared to non-carriers (adjusted OR, 0.55; P=0.63 and HR, 0.93; P=0.9).A coding SNP in ALOX15 (T560M) results in a near null variant of human 12/15-LOX. Assuming a co-dominant mode of inheritance, this variant does not protect against CAD. Assuming a recessive mode of inheritance, the effect of this mutation remains unclear, but is unlikely to provide a protective effect to the degree suggested by mouse knockout studies.
View details for DOI 10.1016/j.atheroscierosis.2007.09.003
View details for Web of Science ID 000255491800016
View details for PubMedID 17959182
Failure to replicate an association of SNPs in the oxidized LDL receptor gene (OLRI) with CAD
BMC MEDICAL GENETICS
The lectin-like oxidized LDL receptor LOX-1 (encoded by OLR1) is believed to play a key role in atherogenesis and some reports suggest an association of OLR1 polymorphisms with myocardial infarction (MI). We tested whether single nucleotide polymorphisms (SNPs) in OLR1 are associated with clinically significant CAD in the Atherosclerotic Disease, VAscular FuNction, & Geneti C Epidemiology (ADVANCE) study.ADVANCE is a population-based case-control study of subjects receiving care within Kaiser Permanente of Northern California including a subset of participants of the Coronary Artery Risk Development in Young Adults (CARDIA) study. We first resequenced the promoter, exonic, and splice site regions of OLR1 and then genotyped four single nucleotide polymorphisms (SNPs), including a non-synonymous SNP (rs11053646, Lys167Asn) as well as an intronic SNP (rs3736232) previously associated with CAD.In 1,809 cases with clinical CAD and 1,734 controls, the minor allele of the coding SNP was nominally associated with a lower odds ratio (OR) of CAD across all ethnic groups studied (minimally adjusted OR 0.8, P = 0.007; fully adjusted OR 0.8, P = 0.01). The intronic SNP was nominally associated with an increased risk of CAD (minimally adjusted OR 1.12, p = 0.03; fully adjusted OR 1.13, P = 0.03). However, these associations were not replicated in over 13,200 individuals (including 1,470 cases) in the Atherosclerosis Risk in Communities (ARIC) study.Our results do not support the presence of an association between selected common SNPs in OLR1 and the risk of clinical CAD.
View details for DOI 10.1186/1471-2350-9-23
View details for Web of Science ID 000255652400001
View details for PubMedID 18384690
View details for PubMedCentralID PMC2322963
The negative correlation between plasma adiponectin and blood pressure depends on obesity: A family-based association study in SAPPHIRe
AMERICAN JOURNAL OF HYPERTENSION
2008; 21 (4): 471-476
The association between plasma adiponectin level and blood pressure remains inconclusive. Because obese subjects may have different mechanisms to regulate blood pressure, we hypothesized that obesity may be an important modifier. In order to minimize confounding effects from unidentified factors, a family-based design was employed to explore the relationship.A total of 1,048 subjects from 478 Chinese or Japanese families with a mean age of 50.4 +/- 9.0 years were included (the SAPPHIRe (Stanford-Asian Pacific Program in Hypertension and Insulin Resistance) cohort). Blood pressure was recorded automatically and the average of the last two out of three consecutive readings was used in the analysis. A subject with "hypertension" was defined as one with a systolic blood pressure (SBP) > or =140 mm Hg, or a diastolic blood pressure (DBP) > or =90 mm Hg, or who was already on medication for hypertension. Obesity was defined as having a body mass index (BMI) > or =25 kg/m(2). The updated homeostasis model assessment was used for calculating the indices of insulin sensitivity (HOMA2 %S). Fasting plasma adiponectin was determined using radioimmunoassay.Subjects with hypertension had significantly lower plasma adiponectin levels than those without hypertension (5.99 +/- 3.64 microg/ml vs. 6.65 +/- 3.86 microg/ml, P < 0.01). Plasma adiponectin level correlated negatively with hypertension after adjusting for age, sex, and HOMA2%S (odds ratio (OR) 0.94, 95% confidence interval (CI) 0.90-0.98). In subjects without hypertension (n = 349), the plasma adiponectin level correlated negatively with SBP in those who were obese, after adjustment for age, sex, BMI, and HOMA2 %S (beta = -0.58, P = 0.03). The association was not significant in those without obesity.Plasma adiponectin level correlates negatively with hypertension. In subjects without hypertension, the relationship between plasma adiponectin level and SBP depends on the presence of obesity.
View details for DOI 10.1038/ajh.2008.5
View details for Web of Science ID 000254137500025
View details for PubMedID 18369365
In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2008; 294 (1): H88-H98
Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.
View details for DOI 10.1152/ajpheart.00935.2007
View details for Web of Science ID 000252261200013
View details for PubMedID 17906101
View details for PubMedCentralID PMC2570026
A novel platform device for rodent echocardiography.
2008; 49: E1-7
Acquisition of echocardiographic data from rodents is subject to wide variability due to variations in technique. We hypothesize that a dedicated imaging platform can aid in standardization of technique and improve the quality of images obtained. We constructed a device consisting of a boom-mounted steel platform frame (25 x 35 x 3 cm) on which a transparent polyethylene membrane is mounted. The animal is placed onto the membrane and receives continual inhaled anesthesia via an integrated port. The membrane allows for probe positioning from beneath the animal to obtain standard echo-views in left lateral decubitus or prone positions. The frame can be set at any desired angle ranging from 0 to 360 degrees along either the long or short axis. Adult male Sprague-Dawley rats (n = 5) underwent echocardiography (General Electric, Vivid 7, 14 MHz) using the platform. The device allowed for optimal positioning of animals for a variety of standard echocardiographic measurements. Evaluations among all animals showed minimal variability between two different operators and time points. We tested the feasibility of the device for supporting the assessment of cardiac function in a disease model by evaluating a separate cohort of adult male spontaneously hypertensive rats (n = 5) that underwent left anterior descending coronary artery ligation. Serial echocardiography demonstrated statistically significant decreases of fractional shortening and ejection fraction (p < 0.01) 240 days after surgery. Our novel imaging platform allowed for consistent collection of high-quality echocardiographic data from rats. Future studies will focus on improving this technology to allow for standardized high-throughput echocardiographic analysis in small animal models of disease.
View details for PubMedID 18506056
Association of polymorphisms in platelet and hemostasis system genes with acute myocardial infarction
AMERICAN HEART JOURNAL
2007; 154 (6): 1052-1058
Genetic polymorphisms may affect the balance between coagulation and fibrinolysis and thereby affect individual vulnerability to acute myocardial infarction (MI) among patients with underlying coronary atherosclerosis.We enrolled 1375 patients with an initial clinical presentation of coronary disease. We genotyped 49 single nucleotide polymorphisms (SNPs) in 9 coagulation system genes and compared patients who had an initial acute MI with patients who presented with stable exertional angina.An SNP in CD36 (rs3211956) was significantly (P = .04) more common among patients who presented with acute MI (minor allele frequency 10.5%) than patients with stable exertional angina (minor allele frequency 8.0%). This association became marginally significant, however, after adjustment for conventional cardiac risk factors in an additive genetic model (odds ratio 1.34, CI 1.00-1.88, P = .053). An SNP in ITGB3 (Leu59Pro, rs5918) was slightly, but not significantly (P = .083), more common among patients with acute MI (minor allele frequency 14.5%) than among patients with stable exertional angina (minor allele frequency 12.0%). Two linked SNPs in THBD (Ala473Val, rs1042579; and rs3176123) were slightly, but not significantly (P = .079 and 0.052, respectively), less common among patients with acute MI (minor allele frequency 16.1%) than among patients with stable exertional angina (18.7% and 19.0%, respectively).Four SNPs in platelet glycoprotein and hemostatic genes were nominally associated with acute MI rather than stable exertional angina as the initial clinical presentation of coronary artery disease. These findings are suggestive but require independent confirmation in larger studies.
View details for DOI 10.1016/j.ahj.2007.05.021
View details for Web of Science ID 000251396200008
View details for PubMedID 18035074
Polymorphisms in hypoxia inducible factor 1 and the initial clinical presentation of coronary disease
AMERICAN HEART JOURNAL
2007; 154 (6): 1035-1042
Only some patients with coronary artery disease (CAD) develop acute myocardial infarction (MI), and emerging evidence suggests vulnerability to MI varies systematically among patients and may have a genetic component. The goal of this study was to assess whether polymorphisms in genes encoding elements of pathways mediating the response to ischemia affect vulnerability to MI among patients with underlying CAD.We prospectively identified patients at the time of their initial clinical presentation of CAD who had either an acute MI or stable exertional angina. We collected clinical data and genotyped 34 polymorphisms in 6 genes (ANGPT1, HIF1A, THBS1, VEGFA, VEGFC, VEGFR2).The 909 patients with acute MI were significantly more likely than the 466 patients with stable angina to be male, current smokers, and hypertensive, and less likely to be taking beta-blockers or statins. Three polymorphisms in HIF1A (Pro582Ser, rs11549465; rs1087314; and Thr418Ile, rs41508050) were significantly more common in patients who presented with stable exertional angina rather than acute MI, even after statistical adjustment for cardiac risk factors and medications. The HIF-mediated transcriptional activity was significantly lower when HIF1A null fibroblasts were transfected with variant HIF1A alleles than with wild-type HIF1A alleles.Polymorphisms in HIF1A were associated with development of stable exertional angina rather than acute MI as the initial clinical presentation of CAD.
View details for DOI 10.1016/j.ahj.2007.07.042
View details for Web of Science ID 000251396200006
View details for PubMedID 18035072
Matrix metalloproteinase circulating levels, genetic polymorphisms, and susceptibility to acute myocardial infarction among patients with coronary artery disease
AMERICAN HEART JOURNAL
2007; 154 (6): 1043-1051
The aim of this study was to assess systematic differences between patients with acute myocardial infarction (MI) and patients with stable angina in matrix metalloproteinase (MMP) circulating levels and genetic polymorphisms.We identified adults in a large integrated health care delivery system whose initial clinical presentation of coronary disease was either an acute MI or stable exertional angina. A total of 909 patients with acute MI, 466 patients with stable angina, and 1023 healthy older control subjects were genotyped. Serum levels of pro-MMP1, MMP2, MMP3, MMP9, and MMP10 were measured in 199 randomly selected patients from each group.At a median of 15 weeks after initial clinical presentation, higher circulating levels of MMP2 and MMP9 were independently associated with acute MI after statistical adjustment for conventional risk factors, hs-CRP levels, and cardiac medications. By contrast, none of the polymorphisms in MMP1, MMP2, MMP3, MMP9, or MMP10 was significantly associated with either acute MI compared with angina, or with coronary disease compared with controls.Circulating levels of MMP2 and MMP9 are independently associated with development of an acute MI rather than stable angina as the initial clinical presentation of coronary artery disease.
View details for DOI 10.1016/j.ahj.2007.06.042
View details for Web of Science ID 000251396200007
View details for PubMedID 18035073
HIF-1 regulates hypoxia- and insulin-induced expression of apelin in adipocytes
AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM
2007; 293 (6): E1590-E1596
Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O(2) environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1alpha gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CoCl(2)) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl(2)-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1alpha-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia- and insulin-induced apelin expression.
View details for DOI 10.1152/ajpendo.00490.2007
View details for Web of Science ID 000251510200014
View details for PubMedID 17878221
Do plasma biomarkers of coagulation and fibrinolysis differ between patients who have experienced an acute myocardial infarction versus stable exertional angina?
AMERICAN HEART JOURNAL
2007; 154 (6): 1059-1064
Circulating concentrations of proteins associated with coagulation and fibrinolysis may differ between individuals with coronary artery disease (CAD) who develop an acute myocardial infarction (AMI) rather than stable exertional angina.We compared plasma concentrations of fibrinogen, d-dimer, tissue-type plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) between patients whose first clinical manifestation of CAD was an AMI (n = 198) rather than stable exertional angina (n = 199). We also compared plasma concentrations of these proteins between patients with symptomatic CAD (either AMI or stable angina; n = 397) and healthy, control subjects (n = 197) to confirm the sensitivity of these assays to detect epidemiologic associations.At a median of 15 weeks after presentation, patients with AMI had slightly higher d-dimer concentrations than patients with stable angina (P = .057), but were not significantly different in other markers. By contrast, fibrinogen, d-dimer, and tissue-type plasminogen activator were significantly higher (P < .001) and PAI-1 lower in patients with CAD than in healthy control subjects. After statistical adjustment for clinical covariates, cardiac risk factors, medications, and other confounders, fibrinogen, d-dimer, and PAI-1 remained significantly associated with CAD.Selected plasma markers of coagulation and fibrinolysis did not distinguish patients presenting with AMI from those with stable exertional angina.
View details for DOI 10.1016/j.ahj.2007.09.015
View details for Web of Science ID 000251396200009
View details for PubMedID 18035075
Circulating chemokines accurately identify individuals with clinically significant atherosclerotic heart disease
2007; 31 (3): 402-409
Serum inflammatory markers correlate with outcome and response to therapy in subjects with cardiovascular disease. However, current individual markers lack specificity for the diagnosis of coronary artery disease (CAD). We hypothesize that a multimarker proteomic approach measuring serum levels of vascular derived inflammatory biomarkers could reveal a "signature of disease" that can serve as a highly accurate method to assess for the presence of coronary atherosclerosis. We simultaneously measured serum levels of seven chemokines [CXCL10 (IP-10), CCL11 (eotaxin), CCL3 (MIP1 alpha), CCL2 (MCP1), CCL8 (MCP2), CCL7 (MCP3), and CCL13 (MCP4)] in 48 subjects with clinically significant CAD ("cases") and 44 controls from the ADVANCE Study. We applied three classification algorithms to identify the combination of variables that would best predict case-control status and assessed the diagnostic performance of these models with receiver operating characteristic (ROC) curves. The serum levels of six chemokines were significantly higher in cases compared with controls (P < 0.05). All three classification algorithms entered three chemokines in their final model, and only logistic regression selected clinical variables. Logistic regression produced the highest ROC of the three algorithms (AUC = 0.95; SE = 0.03), which was markedly better than the AUC for the logistic regression model of traditional risk factors of CAD without (AUC = 0.67; SE = 0.06) or with CRP (AUC = 0.68; SE = 0.06). A combination of serum levels of multiple chemokines identifies subjects with clinically significant atherosclerotic heart disease with a very high degree of accuracy. These results need to be replicated in larger cross-sectional studies and their prognostic value explored.
View details for DOI 10.1152/physiolgenomics.00104.2007
View details for Web of Science ID 000251780600005
View details for PubMedID 17698927
Frontiers in nephrology: Genomic approaches to understanding the molecular basis of atherosclerosis
JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY
2007; 18 (11): 2853-2862
Atherosclerosis is a complex multicellular disease that is responsible for pathology in various organ systems. The understanding of its initiation and progression has been enhanced in recent years by the application of high-throughput genomic tools such as the microarray. Increasing in genomic coverage, such tools allow a view of the disease unaffected by previous conjecture as to the primary signal of interest. New statistical tools and pathway modeling techniques have established definitively for the first time the central role of inflammation in this process. This article reviews the genomic literature relating to atherosclerosis from cell culture, animal models, and human tissues. In this comparison of these differing approaches, the available data are synthesized to reach a new understanding of the complex interplay between vascular wall and immune system components.
View details for DOI 10.1681/ASN.2007040514
View details for Web of Science ID 000250737600012
View details for PubMedID 17942952
Genetic susceptibility to peripheral arterial disease: A dark corner in vascular biology
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2007; 27 (10): 2068-2078
Peripheral arterial disease (PAD) is characterized by reduced blood flow to the limbs, usually as a consequence of atherosclerosis, and affects approximately 12 million Americans. It is a common cause of cardiovascular morbidity and an independent predictor of cardiovascular mortality. Similar to other atherosclerotic diseases, such as coronary artery disease, PAD is the result of the complex interplay between injurious environmental stimuli and genetic predisposing factors of the host. Genetic susceptibility to PAD is likely contributed by sequence variants in multiple genes, each with modest effects. Although many of these variants probably alter susceptibility both to PAD and to coronary artery disease, it is likely that there exists a set of variants specifically to alter susceptibility to PAD. Despite the prevalence of PAD and its high societal burden, relatively little is known about such genetic variants. This review summarizes our limited present knowledge and gives an overview of recent, more powerful approaches to elucidating the genetic basis of PAD. We discuss the advantages and limitations of genetic studies and highlight the need for collaborative networks of PAD investigators for shedding light on this dark corner of vascular biology.
View details for DOI 10.1161/01.ATV.0000282199.66398.8c
View details for Web of Science ID 000249587000002
View details for PubMedID 17656669
View details for PubMedCentralID PMC4321902
Hepatic proprotein convertases modulate HDL metabolism
2007; 6 (2): 129-136
The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.
View details for DOI 10.1016/j.cmet.2007.07.009
View details for Web of Science ID 000248665500008
View details for PubMedID 17681148
Bivariate genome-wide scan for metabolic phenotypes in non-diabetic Chinese individuals from the stanford, Asia and pacific program of hypertension and insulin resistance family study
2007; 50 (8): 1631-1640
Hypertension, obesity, impaired glucose tolerance and dyslipidaemia are metabolic abnormalities that often cluster together more often than expected by chance alone. Since these metabolic variables are highly heritable and are at least partially genetically determined, the clustering of defects in these traits implies that pleiotropic effects, where a common set of genes influences more than one trait simultaneously, are likely.We conducted bivariate linkage analyses for highly correlated traits, aiming to dissect the genetic architecture affecting these traits, in 411 Chinese families participating in the Stanford Asia-Pacific Program of Hypertension and Insulin Resistance Study.We confirmed the pleiotropic effects of the locus at 37 cM on chromosome 20 on the following pairs: (1) fasting insulin and insulin AUC (empirical p = 0.0006); (2) fasting insulin and homeostasis model assessment of beta cell function (HOMA-beta) (empirical p = 0.0051); and (3) HOMA of insulin resistance (IR) and HOMA-beta (empirical p = 0.0044). In addition, the peak logarithm of the odds (LOD) scores of linkage between a chromosomal locus and a trait for the pair fasting insulin and HOMA-IR rose to 5.10 (equivalent LOD score in univariate analysis, LOD() = 4.01, empirical p = 8.0 x 10(-5)) from 3.67 and 3.42 respectively for these two traits in univariate analysis. Additional significant linkage evidence, not shown in single-trait analysis, was identified at 45 cM on chromosome 16 for the pair 1 h insulin and the AUC for insulin, with a LOD score of 4.29 (or LOD() = 3.27, empirical p = 2.0 x 10(-4)). This new locus is also likely to harbour the common genes regulating these two traits (p = 1.73 x 10(-6)).These data help provide a better understanding of the genomic structure underlying the metabolic syndrome.
View details for DOI 10.1007/s00125-007-0720-2
View details for Web of Science ID 000248225100009
View details for PubMedID 17579830
Endothelial lipase is increased by inflammation and promotes LDL uptake in macrophages
JOURNAL OF ATHEROSCLEROSIS AND THROMBOSIS
2007; 14 (4): 192-201
Endothelial lipase (EL) is a member of the lipoprotein lipase family that regulates HDL metabolism. EL is known to act as a bridging molecule for monocytes or lipoproteins in vascular endothelial cells. We investigated the role and regulatory mechanisms of EL expression in macrophages.Macrophages originating from wild-type (EL+/+) and EL-deficient (EL-/-) mice were stimulated with lipopolysaccharide (LPS). The expression of EL mRNA was evaluated by northern blotting. DiI-LDL was used to measure the uptake of native low-density lipoprotein (nLDL).LPS increased EL mRNA levels by increasing intracellular oxidative stress in the macrophages. LPS did not affect EL expression in macrophages derived from Toll-like receptor 4 (TLR4) gene mutant mice, C3H/HeJ. The uptake of nLDL after LPS-treatment was significantly lower in macrophages from EL-/- mice than those from EL+/+ mice. Simvastatin suppressed the LPS-induced upregulation of EL expression and uptake of nLDL.EL expression is upregulated by LPS via TLR4 and promotes the uptake of nLDL by macrophages. Simvastatin inhibits the LPS-induced up-regulation and uptake in macrophages. Thus, our findings provide a novel role for EL in lipoprotein metabolism and would expand the range of anti-atherogenic effects of statins.
View details for Web of Science ID 000249405200007
View details for PubMedID 17726294
Heritability of left ventricular mass in Japanese families living in Hawaii: the SAPPHIRe Study
JOURNAL OF HYPERTENSION
2007; 25 (5): 985-992
Established determinants of left ventricular (LV) mass explain only a modest fraction of its variability. Family studies to date suggest that a proportion of the unexplained variability can be accounted for by additive polygenic effects. An estimate of this proportion has not been reported previously in an East Asian population. The objective of this study was to estimate the heritability of LV mass in Japanese families living in Hawaii.We analyzed data by components of variance in a sample of 169 hypertensive families (n = 476 subjects) and, separately, in a population-based sample of 256 families (n = 501 subjects) participating in the Honolulu Heart Program.In multivariate models, established predictors of LV mass explained about half the total variance of LV mass. Using SOLAR, our estimates of the narrow sense heritability of LV mass ranged from 42.5% (SE 9.8, P < 0.0001) in our sample of hypertensive families to 60.6% (SE 11.7, P < 0.0001) in our population-based sample of families. Parametric bootstrap analyses confirmed that the inference for each sample was appropriate.Assuming the absence of shared familial environmental effects, close to half of the unexplained variance of LV mass in Japanese subjects living in Hawaii is genetic in nature. This estimate was observed in two independent samples. Therefore, the pursuit of novel genetic determinants of LV mass through either whole genome or candidate gene association studies of this population may be worthwhile. Such studies are certainly feasible.
View details for Web of Science ID 000245741200015
View details for PubMedID 17414662
Apelin and its G protein-coupled receptor regulate cardiac development as well as cardiac function
2007; 12 (3): 319-320
The Apelin pathway has only recently emerged as an important regulator of cardiac and vascular function, mediating adaptation to physiological stress and disease. In this issue of Developmental Cell, experiments in zebrafish convincingly show a critical role for this pathway in myocardial cell specification and heart development.
View details for DOI 10.1016/j.devcel.2007.02.005
View details for Web of Science ID 000245291400001
View details for PubMedID 17336895
Metabolic syndrome and early-onset coronary artery disease - Is the whole greater than its parts?
JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
2006; 48 (9): 1800-1807
We sought to examine the association between the metabolic syndrome (MetS) (defined both by the 2001 National Cholesterol Educational Program Adult Treatment Panel III [ATP-III] definition and the American Heart Association/National Heart, Lung and Blood Institute [AHA/NHLBI] revision incorporating the lower threshold for impaired fasting glucose [IFG]) and early-onset coronary artery disease (CAD).The impact of MetS on premature CAD has not been studied extensively. Lowering the threshold to define the IFG component (from 110 to 100 mg/dl) and the value of the syndrome as a whole versus its individual components are subjects of intense debate.We performed a case-control study with 393 early-onset CAD subjects (acute myocardial infarction, angina with > or =50% stenosis, or coronary revascularization) in men under age 46 years or women under age 56 years and 393 control subjects individually matched for gender, age, and race/ethnicity.By conditional logistic regression, presence of ATP-III MetS without diabetes (adjusted odds ratio [adj-OR] 4.9; 95% confidence interval [CI] 3.4 to 8.0) and with diabetes (adj-OR 8.0, 95% CI 4.39 to 14.6) was a strong independent determinant of early-onset CAD. Using the AHA/NHLBI revision, these ORs became slightly stronger. However, neither definition of MetS remained significantly associated with early-onset CAD in multivariate models adjusting for individual components.The presence of MetS imparts a high risk of early-onset clinical CAD, but the prognostic information associated with the syndrome is not greater than the sum of its parts.
View details for DOI 10.1016/j.jacc.2006.03.070
View details for Web of Science ID 000241804400012
View details for PubMedID 17084253
- Opposing cardiovascular roles for the angiotensin and apelin signaling pathways JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 2006; 41 (5): 778-781
Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization
2006; 99 (4): 424-433
Actin cytoskeleton reorganization is a fundamental process for actin-based cellular functions such as cytokinesis, phagocytosis, and chemotaxis. Regulating actin cytoskeleton reorganization is therefore an attractive approach to control endothelial and inflammatory cells function and to treat cardiovascular diseases. Here, we identified glia maturation factor-gamma (GMFG) as a novel factor in actin cytoskeleton reorganization and is expressed preferentially in microvascular endothelial and inflammatory cells. During mouse embryogenesis, GMFG was expressed predominantly in blood islands of the yolk sac, where endothelial and hematopoietic cells develop simultaneously. In endothelial cells, GMFG was colocalized with F-actin in membrane ruffles and was associated with F-actin assessed by actin co-sedimentation assay. Interestingly, GMFG was phosphorylated at N-terminal serine, and its phosphorylation was enhanced by coexpression of dominant active Rac1 and Cdc42. Furthermore, a pseudophosphorylated form of GMFG (GMFG-S2E) demonstrated higher association with F-actin. Stable expression of GMFG-S2E remarkably enhanced stimulus-responsive lamellipodia and subsequent membrane ruffle formation in HeLa cells presumably through its interaction with Arp2/3 complex. Expression of GMFG enhanced actin-based cellular functions such as migration and tube-formation in endothelial cells. Moreover, we found that GMFG expression was significantly increased in a cardiac ischemia/reperfusion model where inflammation and angiogenesis take place actively. Taken together, our findings define a novel pathway in the regulation of actin-based cellular functions. Regulating GMFG function may provide a novel approach to modulate the pathophysiology of cardiovascular diseases.
View details for DOI 10.1161/.01.RES.0000237662.23539.0b
View details for Web of Science ID 000239829400016
View details for PubMedID 16873721
Molecular signatures determining coronary artery and saphenous vein smooth muscle cell phenotypes - Distinct responses to stimuli
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2006; 26 (5): 1058-1065
Phenotypic differences between vascular smooth muscle cell (VSMC) subtypes lead to diverse pathological processes including atherosclerosis, postangioplasty restenosis and vein graft disease. To better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes, we compared gene expression profiles and functional responses to oxidized low-density lipoprotein (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM).OxLDL and PDGF elicited markedly different functional responses and expression profiles between the 2 SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration and modified gene expression of chemokines (CXCL10, CXCL11 and CXCL12), proinflammatory cytokines (IL-1, IL-6, and IL-18), insulin-like growth factor binding proteins (IGFBPs), and both endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-kappaB and phosphatidylinositol signaling pathways, and upregulated prostaglandin (PG) receptors and synthases. In untreated cells, alpha-chemokines, proinflammatory cytokines, and genes associated with apoptosis, inflammation, and lipid biosynthesis were higher in CASM, whereas some beta-chemokines, metalloproteinase inhibitors, and IGFBPs were higher in SVSM. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. In summary, our results suggest dramatic differences in gene expression patterns and functional responses to OxLDL and PDGF between venous and arterial SMCs, with venous SMCs having stronger proliferative/migratory responses to stimuli but also higher expression of atheroprotective genes at baseline.These results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.
View details for DOI 10.1161/01.ATV.0000208185.16371.97
View details for Web of Science ID 000236942400017
View details for PubMedID 16456091
The impact of data quality on the identification of complex disease genes: experience from the Family Blood Pressure Program
EUROPEAN JOURNAL OF HUMAN GENETICS
2006; 14 (4): 469-477
The application of genome-wide linkage scans to uncover susceptibility loci for complex diseases offers great promise for the risk assessment, treatment, and understanding of these diseases. However, for most published studies, linkage signals are typically modest and vary considerably from one study to another. The multicenter Family Blood Pressure Program has analyzed genome-wide linkage scans of over 12 000 individuals. Based on this experience, we developed a protocol for large linkage studies that reduces two sources of data error: pedigree structure and marker genotyping errors. We then used the linkage signals, before and after data cleaning, to illustrate the impact of missing and erroneous data. A comprehensive error-checking protocol is an important part of complex disease linkage studies and enhances gene mapping. The lack of significant and reproducible linkage findings across studies is, in part, due to data quality.
View details for DOI 10.1038/sj.ejhg.5201582
View details for Web of Science ID 000236202600012
View details for PubMedID 16493446
Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation
2006; 25 (1): 29-38
Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.
View details for DOI 10.1152/physiolgenomics.00254.2005
View details for Web of Science ID 000236722700004
View details for PubMedID 16390873
Statin and beta-blocker therapy and the initial presentation of coronary heart disease
ANNALS OF INTERNAL MEDICINE
2006; 144 (4): 229-238
Coronary atherosclerosis develops slowly over decades but is frequently characterized clinically by sudden unstable episodes. Patients who present with unstable coronary disease, such as acute myocardial infarction, may systematically differ from patients who present with relatively stable coronary disease, such as exertional angina.To examine whether medication use or patient characteristics influence the mode of initial clinical presentation of coronary disease.Case-control study.Large integrated health care delivery system in northern California.Adults whose first clinical presentation of coronary disease was either acute myocardial infarction (n = 916) or stable exertional angina (n = 468).Use of cardiac medications before the event from pharmacy databases and demographic, lifestyle, and clinical characteristics from self-report and clinical and administrative databases.Compared with patients with incident stable exertional angina, patients with incident acute myocardial infarction were more likely to be men, smokers, physically inactive, and hypertensive but were less likely to have a parental history of coronary disease. Patients presenting with myocardial infarction were much less likely to have received statins (19.3% vs. 40.4%; P < 0.001) and beta-blockers (19.0% vs. 47.7%; P < 0.001) than patients presenting with exertional angina. After adjustment for potential confounders, recent use of statins (adjusted odds ratio, 0.45 [95% CI, 0.32 to 0.62]) and beta-blockers (adjusted odds ratio, 0.26 [CI, 0.19 to 0.35]) was associated with lower likelihoods of presenting with an acute myocardial infarction than with stable angina.This observational study did not have information on all possible confounding factors, including use of aspirin therapy.Statin and beta-blocker use was associated with lower odds of presenting with an acute myocardial infarction than with stable angina. Additional studies are needed to confirm that these therapies protect against unstable, higher-risk clinical presentations of coronary disease.
View details for Web of Science ID 000235543100001
View details for PubMedID 16490908
Differences in vascular bed disease susceptibility reflect differences in gene expression response to atherogenic stimuli
2006; 98 (2): 200-208
Atherosclerosis occurs predominantly in arteries and only rarely in veins. The goal of this study was to test whether differences in the molecular responses of venous and arterial endothelial cells (ECs) to atherosclerotic stimuli might contribute to vascular bed differences in susceptibility to atherosclerosis. We compared gene expression profiles of primary cultured ECs from human saphenous vein (SVEC) and coronary artery (CAEC) exposed to atherogenic stimuli. In addition to identifying differentially expressed genes, we applied statistical analysis of gene ontology and pathway annotation terms to identify signaling differences related to cell type and stimulus. Differential gene expression of untreated venous and arterial endothelial cells yielded 285 genes more highly expressed in untreated SVEC (P<0.005 and fold change >1.5). These genes represented various atherosclerosis-related pathways including responses to proliferation, oxidoreductase activity, antiinflammatory responses, cell growth, and hemostasis functions. Moreover, stimulation with oxidized LDL induced dramatically greater gene expression responses in CAEC compared with SVEC, relating to adhesion, proliferation, and apoptosis pathways. In contrast, interleukin 1beta and tumor necrosis factor alpha activated similar gene expression responses in both CAEC and SVEC. The differences in functional response and gene expression were further validated by an in vitro proliferation assay and in vivo immunostaining of alphabeta-crystallin protein. Our results strongly suggest that different inherent gene expression programs in arterial versus venous endothelial cells contribute to differences in atherosclerotic disease susceptibility.
View details for DOI 10.1161/01.RES.0000200738.50997.f2
View details for Web of Science ID 000235064900010
View details for PubMedID 16373601
Dual in vivo magnetic resonance evaluation of magnetically labeled mouse embryonic stem cells and cardiac function at 1.5 T
MAGNETIC RESONANCE IN MEDICINE
2006; 55 (1): 203-209
Cell therapy has demonstrated the potential to restore injured myocardium. A reliable in vivo imaging method to localize transplanted cells and monitor their restorative effects will enable a systematic investigation of this therapeutic modality. The dual MRI capability of imaging both magnetically labeled mouse embryonic stem cells (mESC) and their restorative effects on cardiac function in a murine model of acute myocardial infarction is demonstrated. Serial in vivo MR detection of transplanted mESC and monitoring of the mESC-treated myocardium was conducted over a 4-week period using a 1.5 T clinical scanner. During the 4-week duration, the mESC-treated myocardium demonstrated sustained improvement of the left ventricular (LV) ejection fraction and conservation of LV mass. Furthermore, no significant difference of their restorative effects on the cardiac function was created by the magnetic labeling of mESC. Thus, in vivo MRI enables simultaneous detection of transplanted mESC and their therapeutic effect on the injured myocardium.
View details for DOI 10.1002/mrm.20702
View details for Web of Science ID 000234342800025
View details for PubMedID 16315206
An updated meta-analysis of genome scans for hypertension and blood pressure in the NHLBI Family Blood Pressure Program (FBPP)
AMERICAN JOURNAL OF HYPERTENSION
2006; 19 (1): 122-127
A meta-analysis of the results from a multicenter genome-wide linkage study for hypertension and blood pressure (BP) based on an initial sample of 6,245 individuals was published in 2003. We report here a combined linkage analysis of hypertension and BP using the complete Family Blood Pressure Program (FBPP) dataset, which includes a total of 12,028 genotyped individuals. Genome-wide linkage analyses for hypertension and BP were first performed in each of the studied ethnic group within each network and the results were combined with a meta-analysis using a modified Fisher's method of combining P values. Our meta-analysis of genome scans for the latest FBPP dataset reveals suggestive linkage for hypertension and BP at several regions on the human genome. Strong evidence for linkage at two of these regions, 2p14 and 3p14.1, have also been published in previous meta-analyses, making them good candidate locations for susceptibility variants.
View details for DOI 10.1016/j.amjhyper.2005.07.010
View details for Web of Science ID 000234851700023
View details for PubMedID 16461203
Genome-wide expression dynamics during mouse embryonic development reveal similarities to Drosophila development
2005; 288 (2): 595-611
Gene transcription mediates many vital aspects of mammalian embryonic development. A comprehensive characterization and analysis of the dynamics of gene transcription in the embryo is therefore likely to provide significant insights into the basic mechanisms of this process. We used microarrays to map transcription in the mouse embryo in the important period from embryonic day 8 (e8.0) to postnatal day 1 (p1) during which the bulk of the differentiation and development of organ systems takes place. Analysis of these expression profiles revealed distinct patterns of gene expression which correlate with the differentiation of organs including the nervous system, liver, skin, lungs, and digestive system, among others. Statistical analysis of the data based on Gene Ontology (GO) group annotation showed that specific temporal sequence patterns in gene class utilization across development are very similar to patterns seen during the embryonic development of Drosophila, suggesting conservation of the temporal progression of these processes across 550 million years of evolution. The temporal profiles of gene expression and activation of processes revealed here provide intriguing insights into the mechanisms of mammalian development, embryogenesis, and organogenesis, as well as into the evolution of developmental processes.
View details for DOI 10.1016/j.ydbio.2005.09.036
View details for Web of Science ID 000234455900023
View details for PubMedID 16310180
Overexpression of the Del1 gene causes dendritic branching in the mouse mesentery
ANATOMICAL RECORD PART A-DISCOVERIES IN MOLECULAR CELLULAR AND EVOLUTIONARY BIOLOGY
2005; 287A (2): 1165-1175
In the present study, we established transgenic mice overexpressing Del1, a ligand of integrins, to examine the effect of overexpression of Del1 on vascular morphogenesis. In the wild-type mouse, mesenteric vessels are shaped like rakes consisting of a long stalk and short branches at the periphery. In contrast, those in transgenic mice showed typical dendritic architecture consisting of a few large primary branches with smaller spreading branches. The phenotype of mice overexpressing Del1 suggests the existence of a tissue-specific mechanism for branching morphogenesis in the mesentery.
View details for DOI 10.1002/ar.a.20247
View details for Web of Science ID 000233701700001
Two major QTLs and several others relate to factors of metabolic syndrome in the family blood pressure program
2005; 46 (4): 751-757
Genome-wide variance components linkage analysis was performed on 4 latent factors underlying metabolic syndrome derived from 10 risk factors. The latent factors represent obesity and insulin, blood pressure, lipids and insulin, and central obesity. The metabolic syndrome factor scores were derived in 4 ethnic groups recruited in 3 Networks of the Family Blood Pressure Program: GENOA (blacks, Hispanics, and whites), HyperGEN (blacks and whites), SAPPHIRe (Asians). Heritabilities of metabolic syndrome factors ranged from 66% for obesity and insulin to 11% for blood pressure factor. We observed higher heritabilities for obesity and insulin, and lipids and insulin, whereas those for blood pressure and central obesity were smaller. Linkage analysis detected two major quantitative trait loci. One of them linked to the obesity and insulin factor with a lod score of 3.94 (P=0.00001, marker GATA11A06, D18S53, 41.24 cM) at marker positions linkage (lod 4.71, at 46.84 cM at 1-cM-apart distances linkage), located on chromosome 18p11.21 in GENOA black. The other linked to the blood pressure factor with a lod score of 3.22 (P=0.000059, marker GATA49C09, D17S1290, 82 cM) at marker positions linkage (lod 3.56, at 84.63 cM for 1 cM apart distances linkage) located on chromosome 17q23.1 in Hispanics. These quantitative trait loci, together with 4 additional ones with lod scores >2.5, and 30 additional ones with lod score >1.7, offer hope for dissecting the genetic architecture of metabolic syndrome with beneficial implications for molecular diagnosis, prognosis, and in potential medical intervention.
View details for DOI 10.1161/01.HYP.0000184249.20016.bb
View details for Web of Science ID 000232559000022
View details for PubMedID 16172425
Pathway analysis of coronary atherosclerosis
2005; 23 (1): 103-118
Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease.
View details for DOI 10.1152/physiolgenomics.00101.2005
View details for Web of Science ID 000232065200012
View details for PubMedID 15942018
An evaluation of the metabolic syndrome in a large multi-ethnic study: the Family Blood Pressure Program.
Nutrition & metabolism
2005; 2: 17-?
The Family Blood Pressure Program is an ongoing, NHLBI-sponsored, multi-center program to study the genetic determinants of high blood pressure. The goal of this particular study was to study patterns of metabolic syndrome (MetS) in four ethnic groups: African Americans, Caucasians, Hispanics, and Asians.A major part of participants in three networks GENOA, HyperGEN and SAPPHIRe were recruited mainly through hypertensive probands. MetS was defined as a categorical trait following the National Cholesterol Education Program definition (c-MetS). MetS was also characterized quantitatively through multivariate factor analyses (FA) of 10 risk variables (q-MetS). Logistic regression and frequency tables were used for studying associations among traits.Using the NCEP definition, the Hispanic sample, which by design was enriched for type 2 diabetes (T2D), had a very high prevalence of MetS (73%). In contrast, its prevalence in Chinese was the lowest (17%). In African Americans and Hispanics, c-MetS was more prevalent in women than in men. Association of c-MetS with type 2 diabetes (T2D) was prominent in the Hispanics and African Americans, less pronounced in the Whites and Japanese, (although still significant), and weakest in the Chinese sample. Using FA without rotation, we found that the main factor loaded obesity (OBS) and blood pressure (BP) in African Americans; OBS and insulin (INS) in Hispanics, in Japanese, and in Whites; and OBS alone in Chinese. In Hispanics, Whites, and Japanese, BP loaded as a separate factor. Lipids in combination with INS also loaded in a separate factor. Using FA with Varimax rotation, 4 independent factors were identified: "Obesity-INS," "Blood pressure," "Lipids-INS," and "Central obesity." They explained about 60% of the variance present in the original risk variables.MetS ethnic differences were identified. Ascertaining for hypertension or T2D increased the MetS prevalence in networks compared with the one in the US general population. Obesity was the most prominent risk factor contributing to both c-MetS and q-MetS. INS contributed in two important factors (obesity and lipids). The information imbedded into c-MetS trait /q-MetS factors scores can contribute in future research of the MetS, especially its utilization in the genetic analysis.
View details for PubMedID 16076393
Platelet endothelial aggregation receptor 1 (PEAR1), a novel epidermal growth factor repeat-containing transmembrane receptor, participates in platelet contact-induced activation
JOURNAL OF BIOLOGICAL CHEMISTRY
2005; 280 (26): 24680-24689
The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.
View details for DOI 10.1074/jbc.M413411200
View details for Web of Science ID 000230114000048
View details for PubMedID 15851471
Increased expression of endothelial lipase in rat models of hypertension
2005; 66 (3): 594-600
To gain a better understanding of the involvement of endothelial lipase (EL) in vascular disease, we examined whether the EL expression is regulated in animal models of hypertension.The rat cDNA homologue of EL was identified using reverse transcription-polymerase chain reaction. Cultured rat aortic smooth muscle cells were stimulated with angiotensin II (Ang II) and phorbol 12-myristate 13-acetate (PMA), and EL mRNA expression was analyzed by Northern blotting. EL mRNA levels in tissues from stroke-prone spontaneously hypertensive rats (SHR-SP) and Ang II-induced hypertensive rats were evaluated using RNase protection assays.Rat EL cDNA encoded a protein containing 493 amino acid residues including a signal peptide, and shares 91.9% and 80.9% sequence homology with murine and human EL, respectively. Northern blotting revealed that EL was expressed in a wide range of rat tissues. In cultured rat aortic smooth muscle cells, Ang II and PMA increased EL mRNA levels by 2.9- and 3.3-fold, respectively. In Ang II-induced hypertensive rats, EL expression was upregulated in the aorta, heart, and lung. In SHR-SP, EL expression was upregulated in the aorta and heart.EL expression is increased in rat models of hypertension. Thus, EL might have a role in the local pathophysiology of vascular diseases.
View details for DOI 10.1016/j.cardiores.2005.01.013
View details for Web of Science ID 000229853100021
View details for PubMedID 15914124
Myocardial restoration with embryonic stem cell bioartificial tissue transplantation
JOURNAL OF HEART AND LUNG TRANSPLANTATION
2005; 24 (6): 737-744
The optimal cell-matrix combination for robust and sustained myocardial restoration has not been identified. The present study utilizes embryonic stem cells as the substrate of bioartificial myocardial tissue and evaluates engraftment in, and functional recovery of, the recipient heart.Collagen type I was populated with undifferentiated green fluorescent protein (GFP)-positive mouse embryonic stem cells. An intramural left ventricular pouch was fashioned after ligation of the left anterior descending artery in an athymic nude rat heterotopic heart transplant model. The bioartificial mixture (0.125 ml) was implanted in the infarcted area within the pouch. Echocardiography was performed to assess fractional shortening in: Group I, infarcted rats that received cell-matrix implants; Group II, rats given matrix implant without cells; Group III, rats given no matrix or cells; and Group IV, rats receiving transplanted hearts without ligation (n = 5/group). Hearts were stained for GFP, cardiac markers (connexin-43, alpha-sarcomeric actin), hematoxylin-eosin (H&E) and trichrome.Embryonic stem cells formed stable intramyocardial grafts that were incorporated into the surrounding area without distorting myocardial geometry, thereby preventing ventricular wall thinning (anterior wall thickness was: Group I, 1.4 +/- 0.1 mm; Group II, 1.0 +/- 0.1 mm, Group III, 0.9 +/- 0.2 mm; and Group IV, 1.3 +/- 0.2 mm). The inoculated cells expressed connexin-43 and alpha-sarcomeric actin in vivo. Fractional shortening was better in embryonic stem cell-treated animals (Group I, 21.5 +/- 3.5%; Group II, 12.4 +/- 2.8%; Group III, 8.2 +/- 2.9%; Group IV, 23.2 +/- 4.2%).Embryonic stem cells are an efficient alternative substrate for myocardial tissue engineering and can prevent myocardial wall thinning and improve contractility after implantation into injured myocardium in a 3-dimensional matrix.
View details for DOI 10.1016/j.healun.2004.03.023
View details for Web of Science ID 000229869700014
View details for PubMedID 15949735
An autosomal genome-wide scan for loci linked to pre-diabetic phenotypes in nondiabetic Chinese subjects from the Stanford Asia-Pacific program of hypertension and insulin resistance family study
2005; 54 (4): 1200-1206
Type 2 diabetes is a complex disease involving both genetic and environmental components. Abnormalities in insulin secretion and insulin action usually precede the development of type 2 diabetes and can serve as good quantitative measures for genetic mapping. We therefore undertook an autosomal genomic search to locate the quantitative trait locus (QTL) linked to these traits in 1,365 nondiabetic Chinese subjects from 411 nuclear families. Residuals of these log-transformed quantitative traits were analyzed in multipoint linkage analysis using a variance-components approach. The most significant QTL for fasting insulin, which coincides with the QTL for homeostasis model assessment of insulin resistance, was located at 37 cM on chromosome 20, with a maximum empirical logarithm of odds (LOD) score of 3.01 (empirical P = 0.00006) when adjusted for age, sex, BMI, antihypertensive medications, recruitment centers, and environmental factors. In the same region, a QTL for fasting glucose was identified at 51 cM, with an empirical LOD score of 2.03 (empirical P = 0.0012). There were other loci with maximum empirical LOD scores >or=1.29 located on chromosomes 1q, 2p, 5q, 7p, 9q, 10p, 14q, 18q, and 19q for different diabetes-related traits. These loci may harbor genes that regulate glucose homeostasis either independently or via interactions of the genes within these regions.
View details for Web of Science ID 000228094300035
View details for PubMedID 15793262
Genome-wide linkage scans for fasting glucose, insulin, and insulin resistance in the National Heart, Lung, and Blood Institute Family Blood Pressure Program - Evidence of linkages to chromosome 7q36 and 19q13 from meta-analysis
2005; 54 (3): 909-914
Genome-wide linkage analyses were performed using a multipoint variance components method in eight study groups from four multicenter networks (whites and blacks in GenNet; whites, blacks, and Mexican Americans in GENOA; whites and blacks in HyperGEN; and Asians in SAPPHIRe) that comprise the National Heart, Lung, and Blood Institute Family Blood Pressure Program (FBPP), in order to identify quantitative trait loci (QTLs) influencing fasting glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR). These study populations were enriched with subjects who had elevated blood pressure. Participants fasting <8 h, those with a history of type 2 diabetes, or those on antidiabetic medications were excluded from the current investigation. These three phenotypes were suitably transformed to approximate normal distributions. Each phenotype was adjusted for the effects of age, BMI, and field center separately by sex within each of the eight network ethnicity groups before genetic analysis. A total of 8,664 subjects comprising 5,923 sibpairs from 4,043 families with 365 markers were available for conducting a meta-analysis using a modified Fisher's method of combining the P values from each of the eight scans. Evidence of linkages was found on chromosome 7q36 at 163 cM, with a logarithm of odds (LOD) score of 3.21 for HOMA-IR, and on chromosome 19q13 at 88 cM, with a LOD score of 3.33 for fasting glucose. We also found suggestive linkages (LOD score >/=2.2) on chromosome 7q36 at 163 cM, with LOD scores of 2.31 for fasting glucose and 2.26 for fasting insulin (versus the LOD score of 3.21 for HOMA-IR at this locus). In conclusion, QTLs were identified on chromosomes 7q36 and 19q13 for fasting glucose, insulin, and insulin resistance in large and multiple-ethnicity populations in the FBPP with good replications across several other independent studies for relevant traits. Follow-up dense mapping and association studies are warranted.
View details for Web of Science ID 000227423600042
View details for PubMedID 15734873
Mouse strain-specific differences in vascular wall gene expression and their relationship to vascular disease
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2005; 25 (2): 302-308
Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice.We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis.Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study. To gain insights into the molecular pathways that are differentially activated in strains of mice with varied susceptibility to atherosclerosis, we performed comprehensive transcriptional profiling of their vascular wall. Genes identified through these studies expand the repertoire of factors in disease-related signaling pathways and identify novel candidate genes in atherosclerosis.
View details for DOI 10.1161/011.ATV.0000151372.86863.a5
View details for Web of Science ID 000226594000009
View details for PubMedID 15550693
Admixture mapping for hypertension loci with genome-scan markers
2005; 37 (2): 177-181
Identification of genetic variants that contribute to risk of hypertension is challenging. As a complement to linkage and candidate gene association studies, we carried out admixture mapping using genome-scan microsatellite markers among the African American participants in the US National Heart, Lung, and Blood Institute's Family Blood Pressure Program. This population was assumed to have experienced recent admixture from ancestral groups originating in Africa and Europe. We used a set of unrelated individuals from Nigeria to represent the African ancestral population and used the European Americans in the Family Blood Pressure Program to provide estimates of allele frequencies for the European ancestors. We genotyped a common set of 269 microsatellite markers in the three groups at the same laboratory. The distribution of marker location-specific African ancestry, based on multipoint analysis, was shifted upward in hypertensive cases versus normotensive controls, consistent with linkage to genes conferring susceptibility. This shift was largely due to a small number of loci, including five adjacent markers on chromosome 6q and two on chromosome 21q. These results suggest that chromosome 6q24 and 21q21 may contain genes influencing risk of hypertension in African Americans.
View details for DOI 10.1038/ng1510
View details for Web of Science ID 000226690100025
View details for PubMedID 15665825
Molecular isolation and characterization of a soluble isoform of activated leukocyte cell adhesion molecule that modulates endothelial cell function
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (53): 55315-55323
Cell adhesion molecules regulate a variety of endothelial cell functions such as migration, response to inflammation, and angiogenesis. Recently, activated leukocyte cell adhesion molecule (ALCAM), a member of the Ig superfamily, has been detected in the primitive subsets of hematopoietic cells and endothelial cells during embryogenesis. ALCAM supports the development of hematopoietic cells as well as enhancing capillary tube formation in vitro. Here, we isolated a novel soluble isoform of ALCAM (sALCAM) that is produced via alternative splicing. sALCAM contains the single amino-terminal Ig-like domain of ALCAM and lacks a transmembrane domain. When expressed in cultured cells, sALCAM was properly secreted into the media. Both ALCAM and sALCAM are expressed in a variety of cultured human endothelial cells. Notably, their transcripts were differentially regulated in human microvascular endothelial cells (HMVEC) upon tumor necrosis factor-alpha stimulation. ALCAM significantly enhanced tube formation of endothelial-like yolk sac cells on Matrigel, whereas it inhibited their migration in vitro. sALCAM completely abolished these effects of ALCAM. Furthermore, sALCAM enhanced migration of mock-transfected endothelial-like yolk sac cells that do not express ALCAM, indicating that sALCAM has an independent effect on cell migration in addition to modulating ALCAM function. In addition, sALCAM significantly enhanced migration of HMVEC, whereas it inhibited tube formation of HMVEC on Matrigel. sALCAM demonstrated an ability to bind ALCAM and partially inhibited ALCAM-ALCAM homophilic interactions. Taken together, these data characterize a novel soluble isoform of ALCAM that may have ALCAM-dependent and ALCAM-independent functions, providing further insights regarding the role of this adhesion molecule in the regulation of endothelial cell function.
View details for DOI 10.1074/jbc.M407776200
View details for Web of Science ID 000225960800043
View details for PubMedID 15496415
Endothelial lipase modulates monocyte adhesion to the vessel wall - A potential role in inflammation
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (52): 54032-54038
Endothelial lipase (EL), a new member of the lipoprotein lipase gene family, plays a central role in high density lipoprotein metabolism. Previous studies indicated that EL is expressed in endothelial cells, macrophages, and smooth muscle cells in atherosclerotic lesions in human coronary arteries. However, the functional role of EL in the local vessel wall remains obscure. In this study, we evaluated the ability of EL to modulate monocyte adhesion to the endothelial cell surface. EL mRNA and protein levels were markedly increased in tissues of the mouse model of inflammation induced by lipopolysaccharide injection. Adhesion assays in vitro revealed that overexpression of EL in COS7 or Pro5 cells enhanced monocyte bindings to the EL-expression cells. Heparin or heparinase treatment inhibited EL-mediated increases of monocyte adhesion in a dose-dependent manner. Moreover, ex vivo adhesion assays revealed that the number of adherent monocytes on aortic strips was significantly increased in EL transgenic mice and decreased in EL knock-out mice as compared with wild-type mice. These results suggest that EL on the endothelial cell surface can promote monocyte adhesion to the vascular endothelium through the interaction with heparan sulfate proteoglycans. Thus, the up-regulation of EL by inflammatory stimuli may be involved in the progression of inflammation.
View details for DOI 10.1074/jbc.M411112200
View details for Web of Science ID 000225793600019
View details for PubMedID 15485805
Genome-wide expression profiling of a cardiac pressure overload model identifies major metabolic and signaling pathway responses
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2004; 37 (6): 1159-1170
Cardiac hypertrophy is a predictor of cardiovascular morbidity and mortality independent of other risk factors. Pressure overload induces the development of left ventricular hypertrophy (LVH) and left atrial enlargement (LAE) in the mammalian heart. To systematically investigate the transcriptional changes, which mediate these processes, we have performed a genome-wide transcriptional profiling of each of the four heart chambers from mice subjected to transverse aortic constriction (TAC). A major new finding of this analysis is that during enlargement the left atrium undergoes radical changes in gene transcription that may play a significant role in pathophysiology. Structural changes in the LA and LV are correlated with significant changes in the transcriptional profile of these chambers, with thousands of differentially expressed known and novel factors. Statistical analysis of the results identified Gene Ontology biological process groups with significant group-wide changes, including angiogenesis, energy pathways, fatty acid oxidation, oxidative phosphorylation, cytoskeletal and matrix reorganization, and G-protein coupled receptor (GPCR) signaling. To facilitate future research, a searchable annotated Internet database has been constructed that allows access to the expression data presented here. Further study of these genes and processes will lead to better understanding of pathways involved in the pathophysiology of the cardiac response to pressure overload.
View details for DOI 10.1016/j.yjmcc.2004.09.003
View details for Web of Science ID 000225905300007
View details for PubMedID 15572046
Transcriptional profiling of in vitro smooth muscle cell differentiation identifies specific patterns of gene and pathway activation
2004; 19 (3): 292-302
Mesodermal and epidermal precursor cells undergo phenotypic changes during differentiation to the smooth muscle cell (SMC) lineage that are relevant to pathophysiological processes in the adult. Molecular mechanisms that underlie lineage determination and terminal differentiation of this cell type have received much attention, but the genetic program that regulates these processes has not been fully defined. Study of SMC differentiation has been facilitated by development of the P19-derived A404 embryonal cell line, which differentiates toward this lineage in the presence of retinoic acid and allows selection for cells adopting a SMC fate through a differentiation-specific drug marker. We sought to define global alterations in gene expression by studying A404 cells during SMC differentiation with oligonucleotide microarray transcriptional profiling. Using an in situ 60-mer array platform with more than 20,000 mouse genes derived from the National Institute on Aging clone set, we identified 2,739 genes that were significantly upregulated after differentiation was completed (false-detection ratio <1). These genes encode numerous markers known to characterize differentiated SMC, as well as many unknown factors. We further characterized the sequential patterns of gene expression during the differentiation time course, particularly for known transcription factor families, providing new insights into the regulation of the differentiation process. Changes in genes associated with specific biological ontology-based pathways were evaluated, and temporal trends were identified for functional pathways. In addition to confirming the utility of the A404 model, our data provide a large-scale perspective of gene regulation during SMC differentiation.
View details for DOI 10.1152/physiolgenomics.00148.2004
View details for Web of Science ID 000225840800007
View details for PubMedID 15340120
Biethnic comparisons of autosomal genomic scan for loci linked to plasma adiponectin in populations of Chinese and Japanese origin
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
2004; 89 (11): 5772-5778
Adiponectin is secreted by adipocytes and is thought to have insulin-sensitizing and antiatherogenic effects. Two previous genome scans for plasma adiponectin have identified different regions for European and Pima Indian populations. We here present multipoint linkage analysis of adiponectin levels using a variance-components model for 1007 siblings (from 360 nuclear families) of Chinese origin and 352 siblings (from 147 nuclear families) of Japanese origin. We found heritability for adiponectin concentrations was 0.70 for Chinese and 0.48 for Japanese. Autosomal genome scan was performed using microsatellite markers span at an interval of approximately 10 cM. Suggestive linkage of adiponectin, after adjusting for age and sex, was found on chromosome 15 at 39 cM (maximal LOD score = 3.19, P = 6.3 x 10(-5)) for Chinese; and on chromosome 18 at 28 cM (maximal LOD score = 2.40, P = 4.4 x 10(-4)) for Japanese. There were tentative loci of weak linkage on chromosomes 3, 18, and 20 in Japanese. We provide novel loci on chromosomes 15, 18, and 20 and confirm a region on chromosome 3 as reported in Pima Indians, which may influence differentially on circulating adiponectin concentrations in Chinese and Japanese populations. Further fine mapping of these regions will help to identify the gene(s) that might affect adiponectin levels.
View details for DOI 10.1210/jc.2004-0640
View details for Web of Science ID 000224946300075
View details for PubMedID 15531541
Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (43): 45085-45092
Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.
View details for DOI 10.1074/jbc.M406360200
View details for Web of Science ID 000224505600101
View details for PubMedID 15304490
Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice
JOURNAL OF LIPID RESEARCH
2004; 45 (9): 1614-1623
Both LPL and HL are synthesized in parenchymal cells, are secreted, and bind to endothelial cells. To learn where endothelial lipase (EL) is synthesized in adult animals, the localization of EL in mouse and rat liver was studied by immunohistochemical analysis. Furthermore, to test whether EL could play a role in atherogenesis, the expression of EL in the aorta and liver of apolipoprotein E knockout (EKO) mice was determined. EL in both mouse and rat liver was colocalized with vascular endothelial cells but not with hepatocytes. In contrast, HL was present in both hepatocytes and endothelial cells. By in situ hybridization, EL mRNA was present only in endothelial cells in liver sections. EL was also present at low levels in aorta of normal mice. We fed EKO mice and wild-type mice a variety of diets and determined EL expression in liver and aorta. EKO mice showed significant expression of EL in aorta. EL expression was lower in the liver of EKO mice than in normal mice. Cholesterol feeding decreased EL in liver of both types of mice. In the aorta, EL was higher in EKO than in wild-type mice, and cholesterol feeding had no effect. Together, these data suggest that EL may be upregulated at the site of atherosclerotic lesions and thus could supply lipids to the area.
View details for DOI 10.1194/jlr.M400069-JLR200
View details for Web of Science ID 000223923500004
View details for PubMedID 15175355
Tree-structured supervised learning and the genetics of hypertension
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (29): 10529-10534
This paper is about an algorithm, FlexTree, for general supervised learning. It extends the binary tree-structured approach (Classification and Regression Trees, CART) although it differs greatly in its selection and combination of predictors. It is particularly applicable to assessing interactions: gene by gene and gene by environment as they bear on complex disease. One model for predisposition to complex disease involves many genes. Of them, most are pure noise; each of the values that is not the prevalent genotype for the minority of genes that contribute to the signal carries a "score." Scores add. Individuals with scores above an unknown threshold are predisposed to the disease. For the additive score problem and simulated data, FlexTree has cross-validated risk better than many cutting-edge technologies to which it was compared when small fractions of candidate genes carry the signal. For the model where only a precise list of aberrant genotypes is predisposing, there is not a systematic pattern of absolute superiority; however, overall, FlexTree seems better than the other technologies. We tried the algorithm on data from 563 Chinese women, 206 hypotensive, 357 hypertensive, with information on ethnicity, menopausal status, insulin-resistant status, and 21 loci. FlexTree and Logic Regression appear better than the others in terms of Bayes risk. However, the differences are not significant in the usual statistical sense.
View details for Web of Science ID 000222842700009
View details for PubMedID 15249660
Developmental endothelial locus-1 (Del-1), a novel angiogenic protein - Its role in ischemia
2004; 109 (10): 1314-1319
Developmentally regulated endothelial locus-1 (Del-1) is an extracellular matrix protein that is expressed by endothelial cells during embryological vascular development. We speculated that Del-1 may be reexpressed in ischemia and may be involved in endogenous angiogenesis.Del-1 protein was detected by immunohistochemistry in murine ischemic hindlimb after femoral artery excision. To determine whether exogenous Del-1 would augment angiogenesis in vivo, Del-1 or vehicle was administered for 3 weeks by intramuscular injection of murine ischemic hindlimbs. Angiogenesis was quantified by gadolinium-MRI perfusion and capillary densitometry. We used a disc angiogenesis system (DAS) to characterize the angiogenic response to vehicle (PBS), Del-1, Del-1 mutant (altered RGD domain), Del-1 minor (truncated discoidin-I-like domain), or basic fibroblast growth factor. After 14 days, the discs were extracted and sectioned to quantify vascular growth by morphometry. Endogenous Del-1 protein expression was increased in ischemic hindlimbs. Administration of Del-1 increased hindlimb vascular flow index and capillary density. In the DAS, Del-1 doubled fibrovascular growth, as did basic fibroblast growth factor. However, angiogenesis was not enhanced by the Del-1 mutant or Del-1 minor proteins.Del-1 is expressed in ischemic tissue. Del-1 stimulates angiogenesis, an effect that is dependent on the RGD motif and a second signaling sequence in the discoidin-I-like domain. Exogenous intramuscular administration of Del-1 significantly enhances angiogenesis in the murine ischemic hindlimb. Del-1 may prove to be a novel therapeutic agent for patients with ischemia.
View details for DOI 10.1161/01.CIR.0000118465.36018.2D
View details for Web of Science ID 000220213600020
View details for PubMedID 14981004
Molecular cloning of nonsecreted endothelial cell-derived lipase isoforms
2004; 83 (1): 24-33
To expand our knowledge of factors involved in lipid metabolism in the blood vessel wall, we have cloned unique molecular isoforms of endothelial cell-derived lipase (EDL) (HGMW-approved symbol/LIPG). One isoform encoded a truncated protein (EDL2a) lacking the first 80 amino acid residues of the previously characterized EDL1a isoform, including the signal peptide. A similar second clone (EDL2b) was identified that lacked not only the first 80 amino acids, but also a 74-amino-acid region that encodes a portion of the lid domain. RT-PCR analysis confirmed expression of EDL2a/2b isoforms in several human tissues and cultured cells, including endothelial cells. Western blot and immunofluorescence studies using stable transfectants revealed that EDL2a and EDL2b were localized in the cytosol, while, EDL1a was secreted into the culture medium. Cell extracts of EDL2a/2b transfectants did not have triglyceride or phospholipase activity. Thus endothelial cells express three EDL isoforms, two of which remain intracellular and do not function as lipases.
View details for DOI 10.1016/S0888-7543(03)00181-2
View details for Web of Science ID 000187775700004
View details for PubMedID 14667806
Physical inactivity is an important lifestyle determinant of insulin resistance in hypertensive patients
2004; 13 (6): 355-361
The purpose of the study is to assess the relative impact of lifestyle factors including physical inactivity, cigarette smoking, and alcohol intake on insulin resistance in hypertensive patients. In total, 872 hypertensive patients, of Chinese and Japanese origin, from the Stanford Asia and Pacific Program for Hypertension and Insulin Resistance were included for the current analysis. Homeostasis model assessment for insulin resistance (HOMA-IR) and the insulin sensitivity index ISI0,120 were chosen as surrogate measures of insulin resistance. Standardized interview-administered questionnaires were used to obtain information on demographic and lifestyle characteristics. The sedentary hypertensive patients were more insulin resistant than the non-sedentary hypertensive. There were significant differences in Log (HOMA-IR) (0.06-unit increases, p < 0.01) and Log (ISI0,120) (0.05-unit decreases, p < 0.01) between sedentary and non-sedentary hypertensive patients after controlling demographic variables. There were no differences in insulin sensitivity in subjects with different smoking status. Neither smoking nor alcohol intake was persistently associated with insulin resistance in the analysis. Our results suggest that physical inactivity is an important lifestyle determinant of insulin resistance in hypertensive patients. The influences of smoking and alcohol intake on insulin resistance are less significant than physical inactivity in hypertensive subjects.
View details for DOI 10.1080/08037050410004828
View details for Web of Science ID 000225937200007
View details for PubMedID 15771220
Transcriptional profiling of the heart reveals chamber-specific gene expression patterns
2003; 93 (12): 1193-1201
Cardiac chamber-specific gene expression is critical for the normal development and function of the heart. To investigate the genetic basis of cardiac anatomical specialization, we have undertaken a nearly genome-wide transcriptional profiling of the four heart chambers and the interventricular septum. Rigorous statistical analysis has allowed the identification of known and novel members of gene families that are felt to be important in cardiac development and function, including LIM proteins, homeobox proteins, wnt and T-box pathway proteins, as well as structural proteins like actins and myosins. In addition, these studies have allowed the identification of thousands of additional differentially expressed genes, for which there is little structural or functional information. Clustering of genes with known and unknown functions provides insights into signaling pathways that are essential for development and maintenance of chamber-specific features. To facilitate future research in this area, a searchable internet database has been constructed that allows study of the chamber-specific expression of any gene represented on this comprehensive microarray. It is anticipated that further study of genes identified through this effort will provide insights into the specialization of heart chamber tissues, and their specific roles in cardiac development, aging, and disease.
View details for DOI 10.1161/01.RES.0000103171.42654.DD
View details for Web of Science ID 000187183500007
View details for PubMedID 14576202
Comparison of developmental endothelial locus-1 angiogenic factor with vascular endothelial growth factor in a porcine model of cardiac ischemia
ANNALS OF THORACIC SURGERY
2003; 76 (4): 1246-1251
This study compared the angiogenic effects of developmental endothelial locus-1 (DEL-1), vascular endothelial growth factor (VEGF), as well as the negative control, beta-galactosidase (beta-gal), in a porcine model of cardiac ischemia.Twenty pigs underwent left circumflex artery occlusions. After 3 weeks, the animals received myocardial injections of adenovirus expressing beta-gal (n=6), DEL-1 (n=7), or VEGF (n=7). At 7 weeks, animals were assessed for both function and coronary flow and compared with baseline measurements.Regional wall motion index and global ejection fraction showed deterioration in function in the beta-gal group and no change in the VEGF and DEL-1 groups between the treatment and harvest time points. Preload recruitable stroke work suggested functional improvement in the VEGF group (35.8 +/- 8.6 vs 56.4 +/- 17.8, p = 0.033). The increase in the DEL-1 group was not statistically significant (27.3 +/- 9.8 vs, 40.2 +/- 19.4, p = 0.067). The beta-gal group exhibited minimal change (30.7 +/- 14.8 vs 35.9 +/- 12.1, p = 0.96). Regional blood flow as assessed by fluorescent microspheres was improved under stress conditions in the VEGF group (1.00 +/- 0.15 vs 1.15 +/-0.22, p = 0.03).Treatment with VEGF led to a modest improvement in regional blood flow and cardiac function in previously ischemic myocardial tissue.
View details for Web of Science ID 000185717900061
View details for PubMedID 14530019
Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction
2003; 108 (12): 1432-1439
Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin-angiotensin receptor-like 1 (APJ) signaling has ever been described.We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein-coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction.The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.
View details for DOI 10.1161/01.CIR.0000091235.94914.75
View details for Web of Science ID 000185467400005
View details for PubMedID 12963638
Targeted disruption of endothelial cell-selective adhesion molecule inhibits angiogenic processes in vitro and in vivo
JOURNAL OF BIOLOGICAL CHEMISTRY
2003; 278 (36): 34598-34604
Endothelial cell-selective adhesion molecule (ESAM) is a member of the immunoglobulin receptor family that mediates homophilic interactions between endothelial cells. To address potential in vivo angiogenic functions of this molecule, mice lacking ESAM (ESAM-/-) were generated by gene-targeted deletion. ESAM-/- mice did not show overt morphological defects in the vasculature. To evaluate the role of ESAM in pathological angiogenesis, wild type (WT) and ESAM-/- mice were injected with melanoma and Lewis lung carcinoma cells. By 14 days after injection, tumor volumes of B16F10 and LL/2 in ESAM-/- mice were 48 and 37% smaller, respectively, compared with WT mice. Vascular density of the tumors, as determined by CD31 staining, was also decreased in the ESAM null animals. Matrigel plug assays showed less neovascularization in ESAM-/- mice than in WT mice. ESAM-/- endothelial cells exhibited less in vitro tube formation and decreased migration in response to basic fibroblast growth factor when compared with WT cells, and endothelial-like yolk sac cells engineered to overexpress ESAM showed accelerated tube formation in vitro. These in vitro and in vivo studies suggest that ESAM has a redundant functional role in physiological angiogenesis but serves a unique and essential role in pathological angiogenic processes such as tumor growth.
View details for DOI 10.1074/jbc.M304890200
View details for Web of Science ID 000185047500121
View details for PubMedID 12819200
Neovascularization of ischemic tissues by gene delivery of the extracellular matrix protein Del-1
JOURNAL OF CLINICAL INVESTIGATION
2003; 112 (1): 30-41
The ECM protein Del-1 is one of several novel ECM proteins that accumulate around angiogenic blood vessels in embryonic and tumor tissue and promote angiogenesis in the absence of exogenous growth factors. Del-1 expressed in mouse or rabbit ischemic hind-limb muscle by gene transfer rapidly promotes new blood vessel formation and restores muscle function. This angiogenic ECM protein initiates angiogenesis by binding to integrin alphavbeta5 on resting endothelium, thereby resulting in expression of the transcription factor Hox D3 and integrin alphavbeta3. Hox D3 converts resting endothelium to angiogenic endothelium by inducing expression of proangiogenic molecules such as integrin alphavbeta3. These findings provide evidence for an angiogenic switch that can be initiated in the absence of exogenous growth factors and indicate that the angiogenic matrix protein Del-1 may be a useful tool for the therapy of ischemic disease.
View details for DOI 10.1172/JCI200317034
View details for Web of Science ID 000183926700008
View details for PubMedID 12840057
Genetic epistasis of adiponectin and PPAR gamma 2 genotypes in modulation of insulin sensitivity: a family-based association study
2003; 46 (7): 977-983
Genetic interactions in modulating the phenotypes of a complex trait, such as insulin sensitivity, were usually taken for granted. However, this has not been commonly shown. Previous studies have suggested that both PPARgamma2 and adiponectin genes could influence insulin sensitivity. Therefore it is likely that they could modulate insulin sensitivity through gene to gene interactions.We genotyped 1793 subjects of Chinese and Japanese descendents from 601 hypertensive families recruited in Sapphire study for a T94G in the adiponectin gene exon 2 and the PPARgamma2 Pro12Ala polymorphisms. Serum insulin concentrations and insulin resistance index (HOMA(IR)) were used as the markers of insulin sensitivity.We found that the T allele of adiponectin gene was associated with a higher Ins60 and higher area under curve of insulin (AUCi) in OGTT utilizing all subjects in a mixed model that corrected for family effects. Important interactions between adiponectin and PPARgamma2 genotypes were found in fasting insulin concentrations (Ins0), insulin concentrations at 2-h (Ins120) in OGTT and insulin resistance index (HOMA(IR)). The main effects of the PPARgamma2 genotypes were in the plasma glucose concentrations in OGTT. In contrast, the main effects of adiponectin genotypes were in every insulin variable, including Ins0, Ins60, Ins120, AUCi and HOMA(IR). The subjects carrying the adiponectin G allele and the PPARgamma2 Ala12 allele seemed to be more insulin sensitive.These results showed that adiponectin is a genetic factor associated with insulin sensitivity. Interactions with PPARgamma2 genotypes modified this association.
View details for DOI 10.1007/s00125-003-1136-2
View details for Web of Science ID 000184644300014
View details for PubMedID 12827242
Immunohistochemical localization of endothelial cell-derived lipase in atherosclerotic human coronary arteries
2003; 58 (3): 647-654
A novel lipoprotein lipase (LPL)-like gene, endothelial cell-derived lipase (EDL), was recently cloned from vascular endothelial cells. The presence of LPL in the vascular wall has been implicated in the progression of atherosclerosis through the bridging function between lipoprotein particles and matrix proteoglycans to enhance lipoprotein uptake into the vascular wall. The aim of this study was to investigate the local expression of EDL in human coronary arteries.Human coronary arterial specimens from 10 autopsied cases were examined by immunohistochemistry with polyclonal antibodies against specific synthetic EDL peptides. Immunohistochemical analysis revealed that EDL was expressed in endothelial cells and medial smooth muscle cells in non-atherosclerotic coronary arteries. In addition, EDL was expressed in infiltrating cells within atheromatous plaques as well as endothelial and smooth muscle cells. Double labeling immunofluorescence confirmed EDL positive-cells were endothelial cells, smooth muscle cells and macrophages. EDL immunoreactivity was also detected in neovasculature within atheromatous plaques in atherosclerotic coronary arteries.These results suggest that EDL may have unique functional roles in the pathogenesis of coronary artery diseases such as atherosclerosis as well as in lipid metabolism in the vessel wall.
View details for DOI 10.1016/S0008-6363(03)00287-6
View details for Web of Science ID 000183651300019
View details for PubMedID 12798438
Identification of endothelial cell genes by combined database mining and microarray analysis
2003; 13 (3): 249-262
Vascular endothelial cells maintain the interface between the systemic circulation and soft tissues and mediate critical processes such as inflammation in a vascular bed-selective fashion. To expand our understanding of the genetic pathways that underlie these specific functions, we have focused on the identification of novel genes that are differentially expressed in all endothelial cells, as well as restricted groups of this cell type. Virtual subtraction was conducted employing gene expression data deposited in public databases and 384 genes identified. These genes were spotted on custom microarrays, along with 288 genes identified through subtraction cloning from TGF-beta-stimulated endothelial cells. Arrays were evaluated with RNA samples representing endothelial cells cultured from four vascular sources and five non-endothelial cell types. These studies identified 64 pan-endothelial markers that were differentially expressed with at least a threefold difference (range 3- to 55-fold). In addition, differences in gene expression profiles among endothelial cells from different vascular beds were identified. Validation of these findings was performed by RNA blot expression studies, and a number of the novel genes were shown to be expressed under angiogenic conditions in the developing mouse embryo. The combined tools of database mining and transcriptional profiling thus provide expanded knowledge of endothelial cell gene expression and endothelial cell biology.
View details for DOI 10.1152/physiolgenomics.00186.2002
View details for Web of Science ID 000182853000008
View details for PubMedID 12644598
FGF-1 enhanced cardiogenesis in differentiating embryonal carcinoma cell cultures, which was opposite to the effect of FGF-2
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2003; 35 (4): 421-425
To investigate the effect of fibroblast growth factors (FGFs) on cellular differentiation, we employed a multipotent mouse embryonal carcinoma cell line, P19, which differentiates into cardiac muscle, skeletal muscle and neural cells in the presence of the appropriate concentrations of retinoic acid (RA). Under conditions appropriate for cardiac muscle differentiation, the expression of FGF-1 was significantly enhanced before any tissue-specific gene was induced. In contrast, up-regulation of the FGF-2 gene was observed with skeletal muscle-inducing concentrations of RA. Exogenous FGF-1, under skeletal muscle-inducing conditions, suppressed the expression of marker genes for skeletal muscle and induced cardiac alpha myosin heavy chain (alphaMHC) gene with up-regulation of bone morphogenetic protein-4 (BMP-4) and GATA-4. Unlike FGF-1, exogenous FGF-2 promoted skeletal muscle differentiation. These results indicate that FGF-1 and FGF-2 play different roles in P19 cell differentiation induced by RA.
View details for DOI 10.1016/S0022-2828(03)00019-1
View details for Web of Science ID 000182715200011
View details for PubMedID 12689822
Sagittal abdominal diameter is associated with insulin sensitivity in Chinese hypertensive patients and their siblings
JOURNAL OF HUMAN HYPERTENSION
2003; 17 (3): 193-198
The purpose of the study is to observe the relation between anthropometric measurements, focusing on sagittal abdominal diameter (SAD), and insulin sensitivity indices in Chinese hypertensive patients and their siblings. In total, 907 participants, 537 hypertensive and 370 nonhypertensive, from 311 Taiwanese families were drawn from the Stanford Asia and Pacific Program for Hypertension and Insulin Resistance for the study. The participants received anthropometric measurements and 75-g oral glucose tolerance tests after an overnight fast. Fasting insulin, homeostasis model assessment for insulin resistance (HOMA-IR), and the insulin sensitivity index ISI(0,120) were chosen as surrogate measures of insulin sensitivity. In addition to Pearson and partial correlations, we used generalized estimating equations (GEEs) to examine the association between anthropometric measurements and insulin sensitivity indices. A small deviance in the GEEs indicates the goodness of model fit, irrespective of the independence among variables. The hypertensive patients were older in age, wider in waist circumference (WC), larger in body mass index (BMI) and SAD, and more insulin resistant than the nonhypertensive counterparts. The logarithmic transformation of fasting insulin, HOMA-IR, and ISI(0,120) significantly correlated with SAD, WC, and BMI before and after adjustments for age and sex. The deviances of SAD in the GEEs were similar to those of WC in all subjects, while BMI had smaller deviances than SAD and WC in the hypertensive patients. Our results suggest that the performance of SAD in predicting insulin sensitivity is comparable with WC in Chinese hypertensive patients and their siblings. BMI, however, seems to have better association with insulin sensitivity than SAD and WC in the patients with hypertension.
View details for DOI 10.1038/sj.jhh.1001532
View details for Web of Science ID 000182115100007
View details for PubMedID 12624610
Endothelial lipase is a major determinant of HDL level
JOURNAL OF CLINICAL INVESTIGATION
2003; 111 (3): 347-355
A new member of the lipase gene family, initially termed endothelial lipase (gene nomenclature, LIPG; protein, EL), is expressed in a variety of different tissues, suggesting a general role in lipid metabolism. To assess the hypothesis that EL plays a physiological role in lipoprotein metabolism in vivo, we have used gene targeting of the native murine locus and transgenic introduction of the human LIPG locus in mice to modulate the level of EL expression. Evaluation of these alleles in a C57Bl/6 background revealed an inverse relationship between HDL cholesterol level and EL expression. Fasting plasma HDL cholesterol was increased by 57% in LIPG(-/-) mice and 25% in LIPG(+/-) mice and was decreased by 19% in LIPG transgenic mice as compared with syngeneic controls. Detailed analysis of lipoprotein particle composition indicated that this increase was due primarily to an increased number of HDL particles. Phospholipase assays indicated that EL is a primary contributor to phospholipase activity in mouse. These data indicate that expression levels of this novel lipase have a significant effect on lipoprotein metabolism.
View details for DOI 10.1172/JCI200316306
View details for Web of Science ID 000180811400012
View details for PubMedID 12569160
Use of high throughput genomic tools for the study of endothelial cell biology.
Lymphatic research and biology
2003; 1 (2): 133-145
The endothelium is an active, dynamic and heterogeneous organ. It lines the vessels in every organ system and regulates diverse and important biological functions. Over the past several years researchers have gained enormous insights into endothelial cell function in physiological processes such as coagulation and vascular reactivity, and pathophysiological disease states such as inflammation and atherosclerosis. Despite our expanding knowledge of endothelial cell biology, the molecular mechanisms underlying these functions remain largely unknown. The newly developed high throughput genomic tools and accompanying analytical methods provide powerful approaches for identifying new endothelial cell genes and characterizing their role in health and disease. Here, we review some of the recent genomics and proteomic advances that are providing new methodologies for endothelial cell and vascular biology research.
View details for PubMedID 15624421
Therapeutic lymphangiogenesis with human recombinant VEGF-C.
2002; 16 (14): 1985-1987
Chronic regional impairments of the lymphatic circulation often lead to striking architectural abnormalities in the lymphedematous tissues. Lymphedema is a common, disabling disease that currently lacks a cure. Vascular endothelial growth factors C and D mediate lymphangiogenesis through the VEGFR-3 receptor on lymphatic endothelia. The purpose of this study was to investigate the therapeutic potential for lymphangiogenesis with VEGF-C. We developed a rabbit ear model to simulate human chronic postsurgical lymphatic insufficiency. Successful, sustained surgical ablation of the ear lymphatics was confirmed by water displacement volumetry. After complete healing, the experimental animals (n=8) received a single, s.c. 100 microg dose of VEGF-C in the operated ear; controls (n=8) received normal saline. Radionuclide lymphoscintigraphy was performed to quantitate lymphatic function. Immunohistochemistry (IHC) was performed 7-8 days following treatment. After VEGF-C, there was a quantifiable amelioration of lymphatic function. IHC confirmed a significant increase in lymphatic vascularity, along with reversal of the intense tissue hypercellularity of untreated lymphedema. This study confirms the capacity of a single dose of VEGF-C to induce therapeutic lymphangiogenesis in acquired lymphedema. In addition to improving lymphatic function and vascularity, VEGF-C can apparently reverse the abnormalities in tissue architecture that accompany chronic lymphatic insufficiency.
View details for PubMedID 12397087
Endothelial lipase: a new lipase on the block
JOURNAL OF LIPID RESEARCH
2002; 43 (11): 1763-1769
Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.
View details for DOI 10.1197/jlr.R200011-JLR200
View details for Web of Science ID 000179294500001
View details for PubMedID 12401876
Inducible and selective transgene expression in murine vascular endothelium
2002; 11 (2): 99-107
We have developed a system utilizing the murine Tie2 promoter/enhancer coupled with the "tetracycline-on" regulatory elements to create a model that allows regulated and selective expression of a beta-galactosidase (betaGal) reporter transgene in the adult murine vascular endothelium. Two independent lines of viable and fertile mice were characterized, and they exhibit minimal betaGal expression under basal conditions. In response to exogenous doxycycline (Dox), selective expression of betaGal was demonstrated in the vascular endothelium of all tissues examined. En face analyses of the aorta and its principle branches indicate that the vast majority of lumenal endothelial cells express the transgene. Inducible betaGal expression also extends to the endocardium and the microvasculature of all organs. There is no evidence of specific transgene expression in nonendothelial cell types. Induction of the betaGal was effectively achieved after 3 days of oral Dox treatment and persisted for over 3 mo with continuous administration. This model can now be widely applied to study the role of specific genes in the phenotype of adult murine vasculature.
View details for DOI 10.1152/physiolgenomics.00059.2002
View details for Web of Science ID 000178892800008
View details for PubMedID 12388791
The embryonic angiogenic factor Del1 accelerates tumor growth by enhancing vascular formation
2002; 64 (1): 148-161
Del1 is a unique alpha v beta 3 integrin ligand that is produced by endothelial cells, and thus provides an autocrine signaling pathway in this cell type. It is expressed transiently in the embryo and mediates cell attachment, migration, and activation of cytoplasmic signaling molecules in focal contacts. Del1 also activates angiogenesis in the chick chorioallantoic membrane assay. Reexpression of this embryonic signaling molecule has now been documented in naturally occurring human tumors, where it is expressed by both tumor cells and angiogenic endothelial cells, suggesting that Del1 is important in mediating angiogenesis under pathophysiological conditions in the adult. To investigate the role of Del1 in tumor growth and angiogenesis, human 143B osteosarcoma cells and murine Lewis lung carcinoma cells were engineered to express Del1 and compared to control transfectants for their ability to produce tumors in nude or syngeneic mice, respectively. Del1 expressing tumors showed a two- to fourfold increase in capillary density and an accelerated rate of growth. Expression of Del1 also correlated with a decrease in apoptosis in tumor cells in vivo. Taken together, these data suggest that Del1 acts as an angiogenic factor in the context of solid tumor formation and that this increase in vascularization accelerates tumor growth through decreased apoptosis.
View details for DOI 10.1006/mvre.2002.2414
View details for Web of Science ID 000176631000017
View details for PubMedID 12074641
Del1 mediates VSMC adhesion, migration, and proliferation through interaction with integrin alpha v beta(3)
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2002; 282 (5): H1924-H1932
Del1 is a matrix protein transiently expressed by embryonic endothelial cells. It was recently demonstrated that vascular endothelial cells adhere and interact with Del1 through alpha(v)beta(3)- integrins, providing an autocrine angiogenic signaling pathway in this cell type. To determine whether Del1 might signal to other cell types in the vessel wall in a paracrine fashion, studies were conducted with vascular smooth muscle cells (VSMC). Del1 promoted adhesion and migration of VSMC in a dose-dependent fashion. These functions were mediated through alpha(v)beta(3)-integrins, as the vitronectin receptor inhibitory peptide containing penacillamine (PCN) arginine-glycine-aspartic acid (PCN-RGD) and an antibody specific for the alpha(v)beta(3)-integrin specifically blocked both adhesion and migration. Adhesion of VSMC to Del1 was associated with organization of actin filaments and formation of focal contacts enriched in vinculin and alpha(v)beta(3). Furthermore, Del1 supported VSMC proliferation at least in part by inhibiting these cells from undergoing apoptosis. These data, in conjunction with evidence that Del1 expression is reactivated in vascular injury, suggest that Del1 may have a paracrine role in vessel wall development and remodeling.
View details for DOI 10.1152/ajpheart.00921.2001
View details for Web of Science ID 000175011700043
View details for PubMedID 11959660
Identification of an octamer element required for in vivo expression of the TIE1 gene in endothelial cells
2001; 360: 23-29
TIE1, an endothelial-cell-specific tyrosine kinase receptor, is required for the survival and growth of microvascular endothelial cells during the capillary sprouting phase of vascular development. To investigate the molecular mechanisms that regulate the expression of TIE1 in the endothelium, we analysed transgenic mouse embryos carrying wild-type or mutant TIE1 promoter/LacZ constructs. Our data indicate that an upstream DNA octamer element (5'-ATGCAAAT-3') is required for the in vivo expression of TIE1 in embryonic endothelial cells. Transgenic embryos carrying the wild-type TIE1 promoter (-466 to +78 bp) fused to LacZ and spanning the octamer element demonstrate endothelial-cell-specific expression of the reporter transgene. Point mutations introduced within the octamer element result in a significant decrease of endothelial LacZ expression, suggesting that the octamer site functions as a positive regulator for TIE1 gene expression in endothelial cells. DNA-protein binding studies show that the octamer element exhibits an endothelial-cell-specific pattern of binding via interaction with endothelial-cell-restricted factor(s). Our findings suggest an important role for the octamer element in regulating the expression of the TIE1 receptor in the embryonic endothelium and suggest a common mechanism for the regulation of the angiogenic and cell-specific TIE1 and TIE2 genes during vascular development.
View details for Web of Science ID 000172466900003
View details for PubMedID 11695988
NHLBI workshop report: endothelial cell phenotypes in heart, lung, and blood diseases.
American journal of physiology. Cell physiology
2001; 281 (5): C1422-33
Endothelium critically regulates systemic and pulmonary vascular function, playing a central role in hemostasis, inflammation, vasoregulation, angiogenesis, and vascular growth. Indeed, the endothelium integrates signals originating in the circulation with those in the vessel wall to coordinate vascular function. This highly metabolic role differs significantly from the historic view of endothelium, in which it was considered to be merely an inert barrier. New lines of evidence may further change our understanding of endothelium, in regard to both its origin and function. Embryological studies suggest that the endothelium arises from different sites, including angiogenesis of endothelium from macrovascular segments and vasculogenesis of endothelium from microcirculatory segments. These findings suggest an inherent phenotypic distinction between endothelial populations based on their developmental origin. Similarly, diverse environmental cues influence endothelial cell phenotype, critical to not only normal function but also the function of a diseased vessel. Consequently, an improved understanding of site-specific endothelial cell function is essential, particularly with consideration to environmental stimuli present both in the healthy vessel and in development of vasculopathic disease states. The need to examine endothelial cell phenotypes in the context of vascular function served as the basis for a recent workshop sponsored by the National Heart, Lung, and Blood Institute (NHLBI). This report is a synopsis of pertinent topics that were discussed, and future goals and research opportunities identified by the participants of the workshop are presented.
View details for PubMedID 11600404
Vezf1/DB1 is an endothelial cell-specific transcription factor that regulates expression of the endothelia-1 promoter
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (42): 39197-39205
Coordinated gene regulation within the vascular endothelium is required for normal cardiovascular patterning during development and for vascular homeostasis during adulthood, yet little is known about the mechanisms that regulate endothelial transcriptional events. Vascular endothelial zinc finger 1 (Vezf1)/DB1 is a recently identified zinc finger-containing protein that is expressed specifically within endothelial cells during development. In this report, we demonstrate that Vezf1/DB1 is a nuclear localizing protein that potently and specifically activates transcription mediated by the human endothelin-1 promoter, in a Tax-independent manner, in transient transfection assays. Using a combination of deletion mutagenesis and electrophoretic mobility shift assays, a novel Vezf1/DB1-responsive element was localized to a 6-base pair (bp) motif, ACCCCC, located 47 bp upstream of the endothelin-1 transcription start site. Recombinant Vezf1/DB1 also bound to this sequence, and a 2-bp mutation in this element abolished Vezf1/DB1 responsiveness by the endothelin-1 promoter. Vezf1/DB1 could be identified with a specific antibody in nuclear complexes from endothelial cells that bound to this element. Regulation of endothelin-1 promoter activity by Vezf1/DB1 provides a mechanism for endothelin-1 expression in the vascular endothelium during development and to maintain vascular tone; Vezf1/DB1 itself is a candidate transcription factor for modifying endothelial cell phenotypes in order to appropriately assemble and maintain the cardiovascular system.
View details for Web of Science ID 000171673200114
View details for PubMedID 11504723
Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (19): 16223-16231
To gain fundamental information regarding the molecular basis of endothelial cell adhesive interactions during vascular formation, we have cloned and characterized a unique cell adhesion molecule. This molecule, named endothelial cell-selective adhesion molecule (ESAM), is a new member of the immunoglobulin superfamily. The conceptual protein encoded by cDNA clones consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Northern blot analysis showed ESAM to be selectively expressed in cultured human and murine vascular endothelial cells and revealed high level expression in lung and heart and low level expression in kidney and skin. In situ hybridization analysis indicated that ESAM is primarily expressed in the developing vasculature of the embryo in an endothelial cell-restricted pattern. Epitope-tagged ESAM was shown to co-localize with cadherins and catenins in cell-cell junctions. In aggregation assays employing ESAM-expressing Chinese hamster ovary cells, this novel molecule was shown to mediate cell-cell adhesion through homophilic interactions. The endothelial cell-selective expression of this immunoglobulin-like adhesion molecule coupled with its in vitro functional profile strongly suggests a role in cell-cell interactions that is critical for vascular development or function.
View details for Web of Science ID 000168623100088
View details for PubMedID 11279107
Production of a genomic DNA library.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2001; Chapter 5: Unit5 7-?
The Basic Protocol describes the strategy of producing a genomic DNA library. A number of small-scale ligations are performed using a set amount of vector and varying amounts of insert. Test ligations are transformed into bacteria (plasmid vectors) or packaged and plated on host bacteria (lambda and cosmid vectors). The number of clones in the different ligations is compared, and the optimum ratio of vector to insert is indicated by the ligation with the most recombinant clones. A large-scale ligation is then set up using this optimum ratio. This protocol employs a bacteriophage vector; however, cosmid or plasmid vectors can be used with minor modifications.
View details for DOI 10.1002/0471142727.mb0507s13
View details for PubMedID 18265251
Plating and transferring bacteriophage libraries.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2001; Chapter 6: Unit6 1-?
The usefulness of a recombinant phage library depends on the ability to screen a large number of phage and identify the clone that carries the DNA sequence of interest. This unit presents a protocol in which phage are allowed to multiply in host bacteria in a thin layer of agarose on regular bacterial plates. When nitrocellulose is applied to the agarose, phage particles and unpackaged DNA adsorb to the filter to produce a replica of the plate surface. If the agarose surface is not excessively wet, there will be little spreading of the phage on the filter. Subsequent treatment of the filter with sodium hydroxide destroys the phage particles and denatures the phage DNA which then binds to the nitrocellulose. Neutralization of the filters is required to maintain the integrity of the nitrocellulose. Hybridization of these filters to a DNA or RNA probe will identify the location of the phage plaque of interest, which can then be recovered from the plate.
View details for DOI 10.1002/0471142727.mb0601s34
View details for PubMedID 18265254
Purification of bacteriophage clones.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2001; Chapter 6: Unit6 5-?
Careful purification of the clone of interest away from contaminating phage is required before growth and characterization of the clone can proceed. It is common for a "purified" clone to be contaminated by a second phage, leading to confusing results and wasted time. Several rounds of purification should be performed even if the phage appears pure as early as the secondary screening stage. In this unit, phage plates are correctly oriented to the autoradiograph film, and a region that should contain the clone of interest is sampled by toothpicking each phage plaque onto secondary plates containing a lawn of host cells. Alternatively, a plug of agarose can be taken from the primary plate, placed in suspension medium, and this solution used to plate a small secondary library. Plaques on the secondary plates are transferred to nitrocellulose filters, hybridized to a 32P-labeled probe, and an isolated positive plaque is picked, diluted in suspension medium, and regrown. This process is repeated until the desired plaque is purified.
View details for DOI 10.1002/0471142727.mb0605s13
View details for PubMedID 18265260
Size fractionation using sucrose gradients.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2001; Chapter 5: Unit5 3-?
DNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.
View details for DOI 10.1002/0471142727.mb0503s00
View details for PubMedID 18265247
Size fractionation using agarose gels.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2001; Chapter 5: Unit5 4-?
In the Basic Protocol, digested genomic DNA is size fractionated on a slab agarose gel, and the appropriate region of the gel is defined by Southern blot analysis (for subgenomic libraries) or by size (for genomic libraries) and the DNA is eluted. An Alternate Protocol describes electrophoresis using a Bull's-eye apparatus. Digested genomic DNA is loaded onto a large preparative circular agarose gel in this apparatus, the DNA fragments are electrophoresed toward the center of the gel and then eluted. Fragments leaving the gel are pooled and constitute a fraction. Fractions containing the gene of interest are identified by Southern blotting of a small aliquot of alternate fractions. This procedure allows the purification of large amounts of size-fractionated DNA that is particularly well suited for genomic library construction and normally allows creation of large numbers of recombinant clones.
View details for DOI 10.1002/0471142727.mb0504s34
View details for PubMedID 18265248
Regulated expression of endothelial cell-derived lipase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2000; 272 (1): 90-93
A lipoprotein lipase-like gene was recently cloned from endothelial cells. In vitro functional experiments have suggested that this endothelial-derived lipase (EDL) has phospholipase activity, and preliminary in vivo studies have suggested a role in the regulation of high-density lipoprotein metabolism. To investigate local control of lipase activity and lipid metabolism in the blood vessel wall, we have examined the regulation of EDL expression in cultured human umbilical vein and coronary artery endothelial cells. EDL mRNA levels were upregulated in both cell types by inflammatory cytokines implicated in vascular disease etiology, including TNF-alpha and IL-1beta. In addition, both fluid shear stress and cyclic stretch were found to increase the EDL mRNA levels in these cultured cells. This highly regulated expression of EDL in vascular endothelial cells suggests that this recently identified lipase is intricately involved in modulating vessel wall lipid metabolism and may play a role in vascular diseases such as atherosclerosis.
View details for Web of Science ID 000087378900015
View details for PubMedID 10872808
Cardiovascular overexpression of transforming growth factor-beta(1) causes abnormal yolk sac vasculogenesis and early embryonic death
2000; 86 (10): 1024-1030
Transforming growth factor-beta(1) (TGF-beta(1)) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-beta(1) expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-beta(1) in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-beta(1) is expressed from the SM22alpha promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22alpha-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22alpha-beta-galactosidase transgene. SM22alpha- beta-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22alpha-TGF-beta(1) transgenic mice died before E11.5. SM22alpha-TGF-beta(1) transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-beta(1) from the SM22alpha promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-beta(1) expression in adults may require a conditional transgenic approach. Moreover, because the SM22alpha promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22alpha promoter to drive expression of "vasculoactive" transgenes may be particularly likely to cause embryonic death.
View details for Web of Science ID 000087431300005
View details for PubMedID 10827131
Endothelial cell-specific regulation of the murine endothelin-1 gene
17th Scientific Meeting of the International-Society-of-Hypertension
LIPPINCOTT WILLIAMS & WILKINS. 2000: S7–S11
Endothelin-1 (ET-1) plays an important role in the development, physiology and pathophysiology of the cardiovascular system in mammals. ET-1 is mainly expressed in endothelial cells thus making it an attractive model for the study of transcriptional regulation in this cell type. We have previously reported that expression of the human ET-1 gene is positively regulated by a cooperative interaction between GATA-2 and AP-1 transcription factors in cultured endothelial cells, however these factors are not sufficient to mediate cell type-specific expression. In vivo transcription studies of the murine ET-1 gene have demonstrated the presence of important cell-specific DNA elements in the 5.9 kb region upstream of the transcription initiation site. Using reporter gene transfection, site-directed mutagenesis and DNA-protein binding studies of the 5.9 kb region, we have identified a tripartite DNA element that positively regulates the expression of ET-1 specifically in cultured endothelial cells. This complex enhancer element demonstrates an endothelial cell-specific pattern of binding, suggesting that it interacts with cell-restricted regulatory factors. These findings provide important insights into the mechanisms that mediate the expression of ET-1 in the endothelium and a basis for future transgenic and cloning studies aimed at identifying the endothelial cell-specific binding site and transcription factor(s).
View details for Web of Science ID 000165288900003
View details for PubMedID 10976773
Octamer-dependent in vivo expression of the endothelial cell-specific TIE2 gene
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (29): 20376-20383
The TIE2 gene, also known as TEK, encodes a tyrosine kinase receptor that is required for the normal development of the vascular system during embryogenesis. TIE2 is specifically expressed in endothelial cells; however, the transcriptional mechanisms that regulate this highly restricted pattern of expression remain unknown. Here we demonstrate that a consensus octamer element located in the 5'-flanking region of TIE2 is required for normal expression in embryonic endothelial cells. Transgenic embryos carrying a TIE2/LacZ construct spanning 2.1 kilobases of upstream regulatory sequences exhibit expression of the reporter transgene specifically in endothelial cells. Site-directed mutagenesis of a consensus octamer element located in this region results in the loss of enhancer activity and significantly impairs the endothelial expression of the reporter transgene. Consistent with the in vivo data, in vitro DNA-protein binding studies show that the consensus octamer element displays an endothelial cell-specific pattern of binding, suggesting an interaction with a protein complex consisting of Oct1 and an endothelial cell-restricted cofactor. These data identify a novel role for the octamer element as an essential regulator of TIE2 expression, define the first known transcriptional pathway that mediates the expression of a developmental endothelial cell gene, and provide insights into the transcriptional mechanisms that regulate development of the vasculature during embryogenesis.
View details for Web of Science ID 000081438300054
View details for PubMedID 10400661
Cloning of a unique lipase from endothelial cells extends the lipase gene family
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (20): 14170-14175
A new lipoprotein lipase-like gene has been cloned from endothelial cells through a subtraction methodology aimed at characterizing genes that are expressed with in vitro differentiation of this cell type. The conceptual endothelial cell-derived lipase protein contains 500 amino acids, including an 18-amino acid hydrophobic signal sequence, and is 44% identical to lipoprotein lipase and 41% identical to hepatic lipase. Comparison of primary sequence to that of lipoprotein and hepatic lipase reveals conservation of the serine, aspartic acid, and histidine catalytic residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein endothelial cells, human coronary artery endothelial cells, and murine endothelial-like yolk sac cells by Northern blot. In addition, Northern blot and in situ hybridization analysis revealed expression of the endothelial-derived lipase in placenta, liver, lung, ovary, thyroid gland, and testis. A c-Myc-tagged protein secreted from transfected COS7 cells had phospholipase A1 activity but no triglyceride lipase activity. Its tissue-restricted pattern of expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions in lipoprotein metabolism and in vascular disease.
View details for Web of Science ID 000080322200064
View details for PubMedID 10318835
Del1 induces integrin signaling and angiogenesis by ligation of alpha V beta 3
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (16): 11101-11109
Del1 is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. Del1 is expressed in an endothelial cell-restricted pattern during early development. In studies reported here, recombinant baculovirus Del1 protein was shown to promote alphavbeta3-dependent endothelial cell attachment and migration. Attachment of endothelial cells to Del1 was associated with clustering of alphavbeta3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes. These events were shown to be associated with phosphorylation of proteins in the focal complexes, including the time-dependent phosphorylation of p125(FAK), MAPK, and Shc. When recombinant Del1 was evaluated in an in ovo chick chorioallantoic membrane assay, it was found to have potent angiogenic activity. This angiogenic activity was inhibited by a monoclonal antibody directed against alphavbeta3, and an RAD mutant Del1 protein was inactive. Thus Del1 provides a unique autocrine angiogenic pathway for the embryonic endothelium, and this function is mediated in part by productive ligation of integrin alphavbeta3.
View details for Web of Science ID 000079751900062
View details for PubMedID 10196194
Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries
MECHANISMS OF DEVELOPMENT
1998; 73 (1): 33-43
The basic helix-loop-helix (bHLH) class of transcription factors have been linked to a variety of cellular differentiation processes, including myogenesis, neurogenesis and hematopoiesis. Here we report the cloning of a new member of this family of factors, capsulin. Capsulin was shown to be expressed as early as 9.5 days of mouse development, with expression in mesodermal cells that are progenitors of the epicardium and the coronary arteries. At later stages of development, expression is seen in mesenchymal cells that are closely associated with the epithelium of the developing lung, gut and kidney. In the proepicardial organ, and in the organs where it is expressed in later development, capsulin is expressed in cells that give will give rise to smooth muscle. Given the likely expression of capsulin in smooth muscle cell progenitors, and significant sequence similarity through the bHLH domain, capsulin may be a functional ortholog of a Drosophila gene that is expressed in cells that give rise to the longitudinal visceral muscle. Capsulin alone or in combination with other bHLH proteins, was shown to function as a transcription factor by its ability to transactivate both a synthetic and a native promoter, each of which contains multiple E-boxes. These studies extend the growing family of bHLH factors that are expressed in the early mesoderm, and suggest that capsulin may have a functional role in development of the coronary vasculature and organs containing epithelial lined tubular structures.
View details for Web of Science ID 000073567200003
View details for PubMedID 9545526
Regional variability in preproEndothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension
JOURNAL OF CLINICAL INVESTIGATION
1998; 101 (6): 1389-1397
We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.
View details for Web of Science ID 000072754900023
View details for PubMedID 9502781
Functional analysis of the endothelial cell-specific Tie2/Tek promoter identifies unique protein-binding elements
1998; 330: 335-343
To investigate the molecular basis of endothelial cell-specific gene expression, we have examined the DNA sequences and the cognate DNA-binding proteins that mediate transcription of the murine tie2/tek gene. Reporter transfection experiments conformed with earlier findings in transgenic mice, indicating that the upstream promoter of Tie2/Tek is capable of activating transcription in an endothelial cell-specific fashion. These experiments have also allowed the identification of a single upstream inhibitory region (region I) and two positive regulatory regions (regions U and A) in the proximal promoter. Electrophoretic mobility-shift assays have allowed further characterization of three novel DNA-binding sequences associated with these regions and have provided preliminary characterization of the protein factors binding to these elements. Two of the elements (U and A) confer increased transcription on a heterologous promoter, with element U functioning in an endothelial-cell-selective manner. By employing embryonic endothelial-like yolk sac cells in parallel with adult-derived endothelial cells, we have identified differences in functional activity and protein binding that may reflect mechanisms for specifying developmental regulation of tie2/tek expression. Further study of the DNA and protein elements characterized in these experiments is likely to provide new insight into the molecular basis of developmental- and cell-specific gene expression in the endothelium.
View details for Web of Science ID 000072441800048
View details for PubMedID 9461528
Cloning and characterization of developmental endothelial locus-1: An embryonic endothelial cell protein that binds the alpha v beta 3 integrin receptor
GENES & DEVELOPMENT
1998; 12 (1): 21-33
We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the alphavbeta3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the alphavbeta3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.
View details for Web of Science ID 000071455400004
View details for PubMedID 9420328
Identification of an endothelial cell-specific regulatory region in the murine endothelin-1 gene
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (51): 32613-32622
Endothelin-1 is a 21-amino acid peptide first characterized as a potent vasoactive compound synthesized by endothelial cells. Because of its high level cell-restricted pattern of expression, we have employed this gene as a model for investigating the DNA and protein elements that mediate endothelial cell-specific gene expression. In this study we have identified a complex positive regulatory region located at base pairs -364 to -320 in the murine endothelin-1 gene. This region consists of three functionally dependent elements, ETE-C, ETE-D, and ETE-E, which are all required for full activity. When a 43-base pair fragment containing these three elements was employed in heterologous promoter experiments, this sequence was capable of increasing transcriptional activity in an endothelial cell-specific fashion. None of the elements contains a recognized consensus sequence known to bind transcriptional regulatory proteins in higher eukaryotes; however, each element does appear to mediate protein binding. The combination of all three elements promotes binding of a protein complex that is endothelial cell-specific. This is the first evidence for an endothelial cell-specific DNA regulatory element and cognate binding proteins.
View details for Web of Science ID 000071108000096
View details for PubMedID 9405477
Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension
JOURNAL OF CLINICAL INVESTIGATION
1997; 99 (6): 1380-1389
The human endothelin-1 (ET-1) gene under the control of its natural promoter was transferred into the germline of mice. The transgene was expressed predominantly in the brain, lung, and kidney. Transgene expression was associated with a pathological phenotype manifested by signs such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis leading to a progressive decrease in glomerular filtration rate. This pathology developed in spite of only slightly elevated plasma and tissue ET-1 concentrations. Blood pressure was not affected even after the development of an impaired glomerular filtration rate. Therefore, these transgenic lines provide a new blood pressure-independent animal model of ET-1-induced renal pathology leading to renal fibrosis and fatal kidney disease.
View details for Web of Science ID A1997WQ61300032
View details for PubMedID 9077548
Functional activity of the CFTR Cl- channel in human myocardium
HEART AND VESSELS
1997; 12 (6): 255-261
The cyclic AMP (cAMP)-dependent chloride channel in the heart has been identified in various species as the cystic fibrosis transmembrane conductance regulator (CFTR). Although functional expression of the channel in the human atrium has been reported, we could not induce any cAMP-dependent chloride conductance in the atrial cells even with maximal cAMP stimulation, whereas the conductance could be induced in rabbit ventricular cells. To clarify the discrepancy between the results, we examined the level of CFTR mRNA expression in both conductance-positive (human colonic epithelium and rabbit ventricle) and -negative (human atrium) tissues. Total RNA samples prepared from these tissues were subjected to the reverse transcription-polymerase chain reaction (RT-PCR). While CFTR transcripts were amplified from the conductance-positive samples, no amplified products could be detected from the conductance-negative sample. A nested PCR performed on the RT-PCR products of the conductance-negative sample resulted in successful amplification of the transcripts, indicating that the level of the CFTR mRNA expression in human atrium is extremely low compared with that in colonic epithelium and rabbit ventricle. The same molecular results were observed in human ventricular tissues. A nucleotide sequencing of the amplified transcripts showed that exon 5 of the CFTR gene was not alternatively spliced in human atrium and ventricle, and both the exon 5 spliced and unspliced isoforms were expressed in rabbit ventricle, unlike the findings of previous reports. Our data suggest that the amount of CFTR expressed in human myocardium might be physiologically insufficient to activate detectable cAMP-dependent chloride conductance.
View details for Web of Science ID 000076649800001
View details for PubMedID 9860191
Role of endothelin in a rabbit model of acute myocardial infarction: Effects of receptor antagonists
JOURNAL OF CARDIOVASCULAR PHARMACOLOGY
1996; 28 (6): 774-783
The role of endothelin (ET) in acute myocardial infarction and proarrhythmic potential was investigated in a rabbit model. One group of rabbits underwent 30 min of circumflex occlusion and 3 h of reperfusion with measurements of myocardial blood flow and myocardial levels of ET-1 messenger RNA (mRNA). In a second group, the systemic and coronary effects of exogenous ET were studied in animals pretreated with either saline, FR139317, an ETA-receptor antagonist, or PD145065, an ETA-and ETB-receptor antagonist. In a third study, animals undergoing 30 min of circumflex occlusion followed by 48 h of reperfusion were treated with exogenous ET-1, FR139317, PD145065, or saline. Arrhythmias were recorded and infarct size measured at 48 h. These studies revealed that ischemia and reperfusion was followed by a progressive microcirculatory failure ("no-reflow phenomenon") in rabbits. This was associated with a 2.6-fold elevation in levels of myocardial ET-1 mRNA in the ischemic zone in comparison to the nonischemic zone (p = 0.04). Exogenous ET-1 caused elevation in coronary and systemic vascular resistance that was significantly blocked by antagonism of the ETA receptor. In rabbits subjected to myocardial ischemia and reperfusion, ET-1 infusion led to a higher incidence of ventricular arrhythmias, whereas ET-receptor antagonism with PD145065 significantly reduced ventricular arrhythmias. Exogenous ET-1 and FR139317 failed to alter infarct size (AN) of the area at risk (AR) compared with control [AN/AR(%) was 46 +/- 8, 55 +/- 9, and 47 +/- 7, respectively]. However, PD145065 significantly decreased AN/AR (22 +/- 7; p < or = 0.02). The increased production of ET-1, resulting from increased levels of mRNA after reperfusion, may contribute to the no-reflow phenomenon. Although the vasoconstrictor effects of ET-1 can be blocked by ETA-receptor antagonism alone, only blockade of both the ETA and ETB receptors significantly reduced infarct size. These data suggest that production of ET increases in the heart during ischemia and is deleterious to the reperfused myocardium.
View details for Web of Science ID A1996VX27900007
View details for PubMedID 8961075
Endogenous endothelin-1 mediates cardiac hypertrophy and switching of myosin heavy chain gene expression in rat ventricular myocardium
JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
1996; 27 (5): 1286-1291
We investigated the role of endogenous endothelin-1 in the development of cardiac hypertrophy in vivo under pressure overload conditions.Endothelin-1, a potent vasoconstrictor peptide, has recently been shown to act as a growth factor of myocardial cells in culture.We examined the effect of an endothelin-A receptor antagonist (FR139317) on the development of right ventricular hypertrophy in rats with monocrotaline-induced pulmonary hypertension. Three groups of rats were studied: those given monocrotaline alone or monocrotaline plus FR139317 and those given vehicle alone (control group).The ratio of right ventricular systolic pressure to aortic systolic pressure was similarly elevated in rats treated with monocrotaline and monocrotaline plus FR139317. The right ventricular/left ventricular weight ratio was increased in monocrotaline-treated rats but lower in rats treated with monocrotaline plus FR139317 than in those treated with monocrotaline alone (p < 0.01). As a biochemical marker of hypertrophy, the isoform ratio of beta-myosin heavy chain protein was determined for the right ventricular tissue samples. This ratio was increased in all monocrotaline-treated rats but was lower (p < 0.01) in rats given monocrotaline plus FR139317 than in those given monocrotaline alone. The isoform ratio of beta-myosin heavy chain messenger ribonucleic acid quantitated by S1 nuclease mapping also was lower (p < 0.025) in rats receiving monocrotaline plus FR139317 than in those receiving monocrotaline alone.These data suggest that blocking the action of endothelin-1 with a receptor antagonist ameliorates cardiac hypertrophy in this model system, and that this action is not mediated by ameliorating hemodynamic changes.
View details for Web of Science ID A1996UD65100047
View details for PubMedID 8609357
Genomic organization and chromosomal localization of the gene TCF15 encoding the early mesodermal basic helix-loop-helix factor bHLH-EC2
1995; 30 (3): 598-601
bHLH-EC2 is a recently characterized member of a growing family of basic helix-loop-helix transcription factors. This family includes bHLH factors such as twist, which appear to be primarily involved in early mesodermal differentiation, and bHLH factors such as TAL-1, which have been characterized through their association with chromosomal breakpoints associated with T-cell leukemias. To provide for studies aimed at understanding the genetic regulation of bHLH-EC2, we have characterized the organization of this gene and conducted preliminary studies of the transcriptional activity of the upstream promoter region. The mouse bHLH-EC2 gene was found to consist of two exons separated by a 5-kb intron, an organization pattern similar to the mouse twist gene. The transcription initiation site was identified by RNase protection assay and primer extension analysis. Linked promoter-reporter gene transfection experiments in cultured cells indicated that while the identified upstream sequence can function to promote transcription, it does not function in a cell-specific fashion. To investigate the possible association of bHLH-EC2 with hematological malignancy, the chromosomal location of this gene in the human was mapped by fluorescence in situ hybridization and assigned to chromosome band 20p13.
View details for Web of Science ID A1995TN85800025
View details for PubMedID 8825648
MOUSE COL18A1 IS EXPRESSED IN A TISSUE-SPECIFIC MANNER AS 3 ALTERNATIVE VARIANTS AND IS LOCALIZED IN BASEMENT-MEMBRANE ZONES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1995; 92 (19): 8763-8767
We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.
View details for Web of Science ID A1995RU75900047
View details for PubMedID 7568013
DIRECTED ENDOTHELIAL DIFFERENTIATION OF CULTURED EMBRYONIC YOLK-SAC CELLS IN-VIVO PROVIDES A NOVEL CELL-BASED SYSTEM FOR GENE-THERAPY
1995; 13 (5): 541-547
Cultured murine yolk sac cells transfected with the cytomegalovirus immediate early promoter/human growth hormone (CMVIE-hGH) fusion gene, expressing high levels of hGH in culture, and suspended in Matrigel were subcutaneously (s.c.) injected into experimental mice. The injected cells were shown to form discrete vesicular structures within the Matrigel implant, suggesting directed differentiation of the embryonic yolk sac cells into endothelial tissue. Human growth hormone radioimmune assay of these mice showed sustained physiologically significant levels of hGH in their serum for beyond four months. These results confirmed that long-term cultured murine embryonic yolk sac cells can be induced to differentiate into endothelial cells both in vivo and in vitro and suggested a novel approach to the delivery to the circulation of therapeutic proteins for the treatment of inherited and acquired diseases.
View details for Web of Science ID A1995RX36500011
View details for PubMedID 8528104
COOPERATIVE INTERACTION OF GATA-2 AND AP1 REGULATES TRANSCRIPTION OF THE ENDOTHELIN-1 GENE
MOLECULAR AND CELLULAR BIOLOGY
1995; 15 (8): 4225-4231
Endothelin-1 (ET-1) is a 21-amino-acid vasoactive peptide initially characterized as a product of endothelial cells. Reporter gene transfection experiments have indicated that a GATA site and an AP1 site are essential for ET-1 promoter function in endothelial cells, and GATA-2 appears to be the active GATA factor which regulates ET-1 expression. To look for interactions between AP1 and GATA-2, transactivation experiments were performed with expression vectors encoding c-Jun, c-Fos, and GATA-2. Cooperativity between the AP1 complex and GATA-2 was observed as a synergistic increase in transcriptional activity of the ET-1 reporter plasmid. In addition, AP1 was able to potentiate the action of GATA-2 on reporter constructs lacking a functional AP1 site. In a similar fashion, GATA-2 was able to potentiate the action of AP1 despite deletion of the GATA site. Experiments with GATA-1 and GATA-3 expression vectors provided evidence that this capacity to interact with AP1 may be a characteristic of all GATA family members. Biochemical evidence for AP1-GATA interaction was provided by immunoprecipitation experiments. A GATA-2-specific antiserum was shown to immunoprecipitate in vitro-synthesized Jun and Fos protein from reticulocyte lysate. Also, antisera directed against Jun and Fos were able to immunoprecipitate from nuclear extracts a GATA-binding protein, indicating the association of AP1 and GATA proteins in vivo.
View details for Web of Science ID A1995RJ77900026
View details for PubMedID 7623817
REGIONAL AND MATURATION-ASSOCIATED EXPRESSION OF ENDOTHELIN-2 IN RAT GASTROINTESTINAL-TRACT
JOURNAL OF HISTOCHEMISTRY CYTOCHEMISTRY
1995; 43 (2): 203-209
Endothelin 2 (ET2), also referred to as vasoactive intestinal contractor peptide, is a member of a family of vasoactive peptides. ET2 is a potent constrictor of intestinal smooth muscle, and the mRNA that encodes it has been detected in murine intestinal extracts. To further investigate the potential physiological roles of ET2, we characterized the cellular distribution of ET2 gene expression in adult rat gastrointestinal tract. Using an RNAse protection assay, an overall proximal to distal gradient of increasing ET2 gene expression was observed from stomach to colon. In situ hybridization studies confirmed this finding and demonstrated ET2 mRNA localized in lamina propria stromal cells. Moreover, ET2 gene expression in stromal cells increased from crypt to villous tip. The results demonstrate that ET2 is produced by stromal cells in villi throughout the intestine. Increased ET2 gene expression at the villous tip is associated with more mature overlying epithelial cells, suggesting a possible role for this vasoactive peptide in intestinal epithelial differentiation or secretory activity.
View details for Web of Science ID A1995QC78200009
View details for PubMedID 7822776
THE IMMUNOREACTIVE REGION IN A NOVEL AUTOANTIGEN CONTAINS A NUCLEAR-LOCALIZATION SEQUENCE
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
1994; 72 (3): 380-389
Antibodies in the serum of patients with autoimmune diseases have been used to identify human autoantigens. Because autoantibodies often recognize active sites within corresponding protein antigens, autoantibodies have facilitated the functional characterization of these polypeptides. In the present study, serum from a patient with Sjögren's syndrome was used to identify a novel autoantigen which was designated Ge-1. Using the patient's serum, a 4.8-kb cDNA encoding Ge-1 was identified. Fragments of the cDNA were ligated into prokaryotic expression vectors, expressed in Escherichia coli, and used to produce recombinant Ge-1 fusion proteins. Fusion proteins containing different portions of Ge-1 were used to identify a 58 amino acid immunoreactive region within the protein. This immunoreactive region contained the protein's putative nuclear localization sequence (NLS). To demonstrate that the immunoreactive region was capable of functioning as a NLS, a eukaryotic expression plasmid was constructed to encode the immunoreactive region fused to the cytoplasmic protein, chicken muscle pyruvate kinase. After transfection of this plasmid into COS-1 cells, the fusion protein was detected in the nucleus. The presence of the NLS motif within the immunoreactive region of Ge-1 and other nuclear autoantigens suggests that the NLS may be a target of human autoantibodies.
View details for Web of Science ID A1994PC72800013
View details for PubMedID 7520377
RESIDENT RESEARCH AWARD - PULMONARY HYPOXIA INCREASES ENDOTHELIN-1 GENE-EXPRESSION IN SHEEP
JOURNAL OF SURGICAL RESEARCH
1994; 57 (2): 280-283
The hypoxic pulmonary vasoconstrictor response (HPVR) is a physiologic mechanism for directing pulmonary blood flow to nonhypoxic regions of the lung. The mechanism of this response remains unclear. To investigate the role of endothelin-1 (ET-1), a potent vasoconstrictor produced by vascular endothelium, in HPVR an in vivo model of alveolar hypoxia was developed. When one lung in an anesthetized sheep was made hypoxic, the static ET-1 mRNA levels in lung tissue increased in proportion to the observed decrease in pulmonary blood flow (Qp) to that lung. With reversal of hypoxia, Qp and ET-1 levels returned to baseline. This relationship between alveolar hypoxia and ET-1 mRNA levels suggests a role for ET-1 in the local pulmonary response to hypoxia.
View details for Web of Science ID A1994PA25300009
CLONING AND CHARACTERIZATION OF A BASIC HELIX-LOOP-HELIX PROTEIN EXPRESSED IN EARLY MESODERM AND THE DEVELOPING SOMITES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (15): 7066-7070
Basic helix-loop-helix (bHLH) heterodimer protein complexes regulate transcription of genes during the processes of differentiation and development. To study the molecular basis of early mesodermal differentiation, we sought to identify bHLH proteins from cells of mesodermal origin. By using an interaction cloning strategy with a radiolabled recombinant bHLH protein, E12, a clone encoding a potential heterodimer partner was isolated from an endothelial cell library. This gene (bHLH-EC2) is most homologous to Twist but shares similarity within the bHLH domain with TAL1 and other members of this family. bHLH-EC2 is expressed in cultured endothelial cells and in embryonic stem cell, erythroleukemia, and muscle cell lines in a differentiation-dependent manner. In situ hybridization studies of mouse embryos reveal that bHLH-EC2 is expressed throughout the primitive mesoderm as early as 7.5 days postcoitum. Expression then becomes restricted to the paraxial mesoderm and to the dermamyotome of the developing somite. Expression of bHLH-EC2 in cells destined to become myoblasts thus predates expression of myogenic bHLH factors. bHLH-EC2 is expressed in early endothelial and hematopoietic cells in vivo, as shown by RNA studies of embryonic yolk sac and cultured cells derived from yolk sac explants. These findings suggest that bHLH-EC2 plays a role in the development of multiple cell types derived from the primitive mesoderm.
View details for Web of Science ID A1994NY34800066
View details for PubMedID 8041747
COSEGREGATION OF THE ENDOTHELIN-3 LOCUS WITH BLOOD-PRESSURE AND RELATIVE HEART-WEIGHT IN INBRED DAHL RATS
JOURNAL OF HYPERTENSION
1994; 12 (6): 643-651
To determine whether the endothelin-1 or endothelin-3 genes are genetically linked with blood pressure and relative heart weight in segregating rat populations, in the context of an elevated dietary sodium chloride intake.Endothelin-1 and endothelin-3 genotypes of rats in segregating populations, derived from crosses of Dahl salt-sensitive (SS/Jr) rats with contrasting inbred strains, including Lewis rats, spontaneously hypertensive rats and Dahl salt-resistant (SR/Jr) rats, were determined using restriction fragment length polymorphisms. Segregating populations were fed a high (8%)-sodium chloride diet. Linkage of genotype with blood pressure or relative heart weight was determined by analysis of variance. Chromosomal location of the rat endothelin-3 gene was determined by genotyping a panel of recombinant inbred strains.Two alleles for the endothelin-1 gene and three alleles for the endothelin-3 gene were identified. The endothelin-1 locus did not cosegregate with blood pressure or relative heart weight. The endothelin-3 locus cosegregated with blood pressure and relative heart weight in an SS/Jr x F1 (SS/Jr x SR/Jr) population, but not in populations containing a higher percentage of genes from the SR/Jr strain. The endothelin-3 and seminal vesicle protein-1 loci were linked and located on rat chromosome 3.The endothelin-3 gene is, or is linked to, a locus on chromosome 3 that regulates blood pressure and relative heart weight in inbred Dahl rats, and these effects were strongly dependent on the genetic background.
View details for Web of Science ID A1994NV92600004
View details for PubMedID 7963489
EFFECT OF HEAVY-CHAIN SIGNAL PEPTIDE MUTATIONS AND NH(2)-TERMINAL CHAIN-LENGTH ON BINDING OF ANTIDIGOXIN ANTIBODIES
JOURNAL OF BIOLOGICAL CHEMISTRY
1993; 268 (31): 23000-23007
In certain instances, antibody variable region mutations outside of the antigen-combining site influence antigen binding. We reported previously that a heavy chain mutation (Ser-94-->Arg) decreased binding of the anti-digoxin antibody 40-150, whereas an additional signal peptide mutation at the -2 position (Gln-->Pro) causing NH2-terminal 2-residue truncation partially restored binding. To assess the combined effects on binding of two seemingly distant mutations, we constructed signal peptide mutations and NH2-terminal deletions in the presence of Ser-94 and Arg-94. Deletions of one to three amino acids had little effect on binding for Ser-94 mutants, whereas 2-residue truncations produced directly or by signal peptide mutation increased affinity approximately 40-fold for Arg-94 mutants. These observations are consistent with the reported computer-generated model of antibody 40-150. Introduction of Pro at the signal peptide -3 position in 40-150 resulted in cleavage at alternative sites, with varying effects on affinity. Introduction of Pro at -2 into the anti-digoxin antibody 26-10 resulted, unexpectedly, in expression of heavy chains with 3 extra NH2-terminal residues, causing an approximately 100-fold reduction in affinity. Thus, both extensions and deletions of the heavy chain amino terminus can enhance or reduce antigen binding, depending on the structural context of specific antigen combining sites.
View details for Web of Science ID A1993MF51500010
View details for PubMedID 8226814
STRUCTURAL ORGANIZATION AND CHROMOSOMAL ASSIGNMENT OF THE GENE ENCODING THE HUMAN HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH-FACTOR DIPHTHERIA-TOXIN RECEPTOR
1993; 32 (31): 7932-7938
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently identified, potent smooth muscle cell mitogen of macrophage origin. It is expressed in a highly regulated fashion in vascular endothelial and smooth muscle cells, indicating a potentially important role for this gene in atherosclerosis. In addition, the HB-EGF precursor has recently been found to function as a receptor for diphtheria toxin. Using an HB-EGF cDNA probe, we cloned the human gene encoding HB-EGF. The HB-EGF gene contains six exons and five intervening sequences spanning 14 kb of DNA. By primer extension and S1 nuclease analysis, we located a major transcription start site (corresponding to an A residue) 14 bp beyond the 5' end of the HB-EGF cDNA. There were no TATAAA or CCAAT consensus sequences upstream of the transcription start site. The density of primer extension bands generated by RNA from endothelial cells treated with tumor necrosis factor-alpha (TNF-alpha) was 10 times higher than that of bands generated by the control, indicating that TNF-alpha increased the level of HB-EGF mRNA. Using transient reporter gene transfection experiments, we show that 2.0 kb of HB-EGF 5'-flanking sequence has promoter activity in bovine aortic endothelial cells. By analysis of DNA isolated from human-mouse somatic hybrid cell lines, we assign the HB-EGF gene to chromosome 5. By functional study, chromosome 5 has been associated with diphtheria toxin susceptibility.
View details for Web of Science ID A1993LV43600014
View details for PubMedID 8347598
HIGH-LEVEL EXPRESSION OF ANTIBODY-PLASMINOGEN ACTIVATOR FUSION PROTEINS IN HYBRIDOMA CELLS
1993; 69 (2): 221-229
We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).
View details for Web of Science ID A1993KP50200006
View details for PubMedID 8446952
INDUCTION OF HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH-FACTOR MESSENGER-RNA BY PHORBOL ESTER AND ANGIOTENSIN-II IN RAT AORTIC SMOOTH-MUSCLE CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1992; 267 (34): 24892-24896
To determine whether the gene encoding the recently identified heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle cell (SMC) mitogen of macrophage origin, is transcribed and regulated in vascular SMC, we isolated cDNA clones encoding rat HB-EGF from a macrophage library. Using the rat HB-EGF cDNA as a probe for RNA blot analysis, we detected low levels of HB-EGF mRNA in rat aortic SMC in culture. However, 20 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 10(-6) M angiotensin II (AII) induced a marked increase in HB-EGF mRNA levels in rat aortic SMC (11- and 4.6-fold, respectively) that was both dose- and time-dependent. In response to TPA and AII, HB-EGF mRNA levels increased rapidly, peaked at 2 h, and returned to base line at 7 h. This effect of AII on HB-EGF induction was specific, as evidenced by the fact that it could be completely blocked by the AII antagonist saralasin. This is the first demonstration that HB-EGF is transcribed and regulated in SMC. The inducible transcription of this potent SMC mitogen gene in vascular SMC suggests that HB-EGF may have an important autocrine role in the proliferation of SMC in vascular diseases such as atherosclerosis and hypertension.
View details for Web of Science ID A1992KA26300109
View details for PubMedID 1447224
Cloning and expression of a cDNA encoding human endothelium-derived relating factor/nitric oxide synthase.
journal of biological chemistry
1992; 267 (31): 22694-?
View details for PubMedID 1385404
CLONING AND EXPRESSION OF A CDNA-ENCODING HUMAN ENDOTHELIUM-DERIVED RELAXING FACTOR NITRIC-OXIDE SYNTHASE
JOURNAL OF BIOLOGICAL CHEMISTRY
1992; 267 (21): 14519-14522
Nitric oxide, which accounts for the biological activity of endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). We report here the cloning and functional expression of a cDNA encoding human endothelial NOS. Oligonucleotides corresponding to amino acid sequences shared by cytochrome P450 reductase and the recently identified brain NOS were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS-related protein. This partial cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS. The translated human protein is 1294 amino acids long and shares 52% of its amino acid sequence with brain NOS. Using RNA blot hybridization, abundant endothelial NOS mRNA was detected in unstimulated human umbilical vein endothelial cells. To determine the functional activity of the endothelial protein, we ligated the cDNA into an expression vector and transfected it into NIH3T3 cells. Cells expressing this cDNA contained abundant NADPH diaphorase activity, a histochemical marker for NOS. In co-culture assays, nitric oxide production by transfected cells increased guanylate cyclase activity in reporter rat fetal lung fibroblasts. In addition, NOS-catalyzed conversion of arginine to citrulline in transfected cells was significantly increased by A23187, a calcium ionophore. Isolation of a cDNA encoding a calcium-regulated, constitutively expressed human endothelial NOS, capable of producing EDRF in blood vessels, will accelerate the characterization of the role of this enzyme in normal and abnormal endothelial regulation of vascular tone.
View details for Web of Science ID A1992JF08800002
View details for PubMedID 1378832
GENETIC-REGULATION OF ENDOTHELIN-1 IN VASCULAR ENDOTHELIAL-CELLS
TRENDS IN CARDIOVASCULAR MEDICINE
1992; 2 (4): 129-133
Endothelin-1 (ET-1) is a potent vasoconstrictor and smooth muscle cell mitogen synthesized and secreted by endothelial cells. This vasoactive peptide is genetically regulated by many of the cytokines, hormones, and physical forces that are involved in vascular disease processes. Transcriptional regulation of the ET-1 gene depends upon the cis-acting elements of the ET-1 promoter and their interaction with the protooncogene products Fos and Jun as well as other DNA-binding proteins.
View details for Web of Science ID A1992JC09600002
View details for PubMedID 21239258
TUMOR-NECROSIS-FACTOR INCREASES TRANSCRIPTION OF THE HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH-FACTOR GENE IN VASCULAR ENDOTHELIAL-CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1992; 267 (14): 9467-9469
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently identified, potent vascular smooth muscle cell (SMC) mitogen of macrophage origin. To determine whether this gene is transcribed and regulated in vascular endothelial cells, we measured HB-EGF mRNA levels in human umbilical vein endothelial cells (HUVEC) by RNA blot analysis with an HB-EGF cDNA probe. The base-line level of HB-EFG mRNA in HUVEC in culture was low. However, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta markedly increased HB-EGF mRNA levels in HUVEC by 12- and 7-fold, respectively, and induction of the gene by TNF-alpha was both dose- and time-dependent. In response to TNF-alpha, HB-EGF mRNA levels quickly increased and peaked at 1 h, indicating that HB-EGF belongs to the family of immediate early genes. In nuclear run-off experiments, TNF-alpha increased the rate of HB-EGF gene transcription by 3.2-fold. To our knowledge this is the first demonstration that the HB-EGF gene is transcribed in vascular endothelial cells. The inducible transcription of this potent SMC mitogen gene in endothelial cells suggests that HB-EGF may have an important role in the pathogenesis of atherosclerosis.
View details for Web of Science ID A1992HT96500005
View details for PubMedID 1577791
STIMULATION OF ENDOTHELIN-1 GENE-EXPRESSION BY INSULIN IN ENDOTHELIAL-CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1991; 266 (34): 23251-23256
The present study characterized the regulation of the genetic expression of the vasoactive peptide endothelin-1 (ET-1) by insulin in bovine aortic endothelial cells. By RNA blot analysis, insulin (1.67 x 10(-8) M) increased ET-1 mRNA levels by 2.3-fold over the basal within 10 min and attained a maximum (5.3-fold increase) in 2 h. Dose-response studies showed that a maximum effect of insulin was reached at 1.67 x 10(-8) M although a significant increase can be observed at 1.66 x 10(-9) M. Radioligand receptor studies indicated that the affinity constant for insulin receptors on endothelial cells correlated closely with the dose response observed for ET-1 mRNA. The ET-1 mRNA half-life was estimated with actinomycin D studies to be 20 min in control cells and was not affected by insulin treatment. Moreover, the effects of phorbol 12-myristate 13-acetate (PMA) and insulin were additive in the induction of ET-1 gene expression. When protein kinase C in the bovine aortic endothelial cells was down-regulated by preincubation with 8 x 10(-7) M PMA for 24 or 48 h, insulin was still able to increase ET-1 mRNA levels whereas PMA was ineffective. Using a chloramphenicol acetyltransferase (CAT) fusion plasmid containing the CAT gene and the 5'-flanking region of the ET-1 gene (Lee, M. E., Bloch, K. D., Clifford, J. A., and Quertermous, T. (1990) J. Biol. Chem. 265, 10446-10450), we observed that 1.67 x 10(-8) M insulin increased CAT enzyme activity and mRNA levels. The insulin dose-response curve observed for CAT activity correlated with that observed for ET-1 mRNA levels. These results suggest that insulin stimulates expression of the ET-1 gene at the transcriptional level via its own receptors. This effect is mediated mostly through a protein kinase C-independent pathway, suggesting the existence of an insulin-responsive element in the ET-1 gene 5'-flanking sequence.
View details for Web of Science ID A1991GT48300072
View details for PubMedID 1744120
A RECOMBINANT CHIMERIC PLASMINOGEN-ACTIVATOR WITH HIGH-AFFINITY FOR FIBRIN HAS INCREASED THROMBOLYTIC POTENCY INVITRO AND INVIVO
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1991; 88 (22): 10337-10341
A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either beta-globin or mouse immunoglobulin. This modification resulted in a greater than 100-fold improvement in the level of protein expression. The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis. Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.
View details for Web of Science ID A1991GP89500088
View details for PubMedID 1946453
REGULATION OF ENDOTHELIN-1 GENE-EXPRESSION BY FOS AND JUN
JOURNAL OF BIOLOGICAL CHEMISTRY
1991; 266 (28): 19034-19039
The endothelin peptides constitute a family of potent vasoconstrictor molecules. Endothelin-1 (ET1) is secreted by vascular endothelial cells and may have a role in the regulation of vascular tone. To better understand the function of ET1, we have investigated the transcriptional regulation of the ET1 gene. Utilizing reporter gene transfection experiments, we have previously identified two promoter regions, located at base pairs -148 to -117 (Region A) and -117 to -98 (Region B) of the ET1 gene. Both regions are necessary for high level ET1 transcription in endothelial cells. A nuclear protein binding to the GATA motif in Region A has been identified and proven to be necessary for expression of the ET1 gene. However, the cis-acting sequences and their cognate binding proteins for Region B have not been investigated. To identify protein binding motifs in Region B we performed DNase I footprinting and gel mobility shift assays using a DNA fragment encoding base pairs -204 to -94 of the ET1 gene. Results from these studies indicated that the AP1 consensus sequence (GTGACTAA) in Region B as the only protein-binding motif. Site-directed mutagenesis of the ET1 AP1 site resulted in a 30-fold reduction in promoter activity, establishing the functional significance of this sequence. Additional experiments investigated the role of Jun and Fos in ET1 transcription. By employing antisera to Jun and Fos in gel mobility shift assays, both of these proteins were identified as endothelial cell nuclear proteins binding to the ET1 AP1 sequence. In trans-activation experiments, we showed that cotransfection of c-fos and c-jun expression plasmids markedly increased the transcription rate of chloramphenicol acetyltransferase reporter plasmids containing three synthetic ET1 AP1 sites. Taken together, these data indicate the importance of the AP1 recognition sequence, and the role of Fos and Jun proteins in the regulation of ET1 gene transcription.
View details for Web of Science ID A1991GJ47200096
View details for PubMedID 1918021
CLONING OF THE GATA-BINDING PROTEIN THAT REGULATES ENDOTHELIN-1 GENE-EXPRESSION IN ENDOTHELIAL-CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1991; 266 (24): 16188-16192
Previously, we have identified two regions (A and B) of the endothelin-1 promoter that are important for the expression of this gene in cultured vascular endothelial cells. The cis-acting sequence in one of these regions (Region A) includes the core binding motif GATA, raising the possibility that this region of DNA mediates binding of a member of the GATA-binding protein family. In this report, we describe the use of polymerase chain reaction in conjunction with cDNA cloning to characterize the GATA-binding protein expressed in endothelial cells. The nucleotide sequence of endothelial cell cDNA clones is highly homologous to that of the chicken GATA-2 (NF-E1b) gene, indicating that our clones encode the human GATA-2 gene transcript. By RNA blot analysis, this gene is expressed in cultured cell lines derived from a number of different tissues. Transactivation experiments utilizing human GATA-2 eukaryotic expression vectors indicate that the GATA-2 protein interacts with the endothelin-1 GATA sequence to increase transcription of reporter genes in both BAEC and HeLa cells. These data provide the first evidence for a non-erythroid target gene regulated by GATA-2 and indicate that GATA-2 may have a more broad role in transcriptional regulation than the erythroid-specific GATA-1 protein.
View details for Web of Science ID A1991GB97700098
View details for PubMedID 1714909
EFFECT OF CHEMICAL CONJUGATION OF RECOMBINANT SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR WITH MONOCLONAL ANTIPLATELET ANTIBODIES ON PLATELET-AGGREGATION AND ON PLASMA CLOT LYSIS INVITRO AND INVIVO
1991; 78 (4): 1005-1018
The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u-PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin-labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu-PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).
View details for Web of Science ID A1991GB20800018
View details for PubMedID 1831057
CDNA CLONING AND CHROMOSOMAL ASSIGNMENT OF THE ENDOTHELIN-2 GENE - VASOACTIVE INTESTINAL CONTRACTOR PEPTIDE IS RAT ENDOTHELIN-2
1991; 10 (1): 236-242
Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.
View details for Web of Science ID A1991FK40800030
View details for PubMedID 1840558
HYBRID MOLECULES - INSIGHTS INTO PLASMINOGEN-ACTIVATOR FUNCTION
MOLECULAR BIOLOGY & MEDICINE
1991; 8 (2): 245-255
Hybrid molecules containing the catalytic domain of either tissue plasminogen activator (tPA) or single chain urokinase-type plasminogen activator (scuPA), and the fibrin binding domain of a murine antifibrin monoclonal antibody were constructed using either cDNA or genomic DNA encoding the plasminogen activator and genomic DNA encoding antifibrin monoclonal antibody 59D8. In order to optimize expression of these fusion proteins in hybridoma cells, we compared plasminogen activator 3' UT domains (which decrease mRNA stability) with immunoglobulin and beta globin 3' UT domains (which increase mRNA stability). The presence of the plasminogen activator 3' UT domain resulted in approximately tenfold lower steady-state mRNA levels, and 300 to 500-fold lower levels of expressed functional protein. The initial goal of these studies was to increase the fibrinolytic potency and selectivity of tPA or scuPA. Fusion proteins comprising an antifibrin antibody domain and the catalytic domain of either tPA or scuPA were expressed and shown to have very different properties. The fusion protein that comprised the Fab portion of an antifibrin antibody and the catalytic domain of tPA, while displaying antigen binding properties indistinguishable from those of the parent antibody and amidolytic activity similar to that of tPA, was not more efficient than tPA in an in vitro clot lysis assay. In contrast, it had been shown that tPA chemically coupled to the same antibody was four- to sixfold more efficient in fibrinolysis both in vitro and in vivo. A recombinant scuPA-antifibrin antibody hybrid, however, was sixfold more potent than scuPA in vitro and 20-fold more potent in a rabbit thrombolysis model. An explanation for this apparent discrepancy may relate to the requirement for stimulation by fibrin in order for tPA to achieve its maximal catalytic activity, a property that was demonstrated to have been lost in the antifibrin-tPA fusion protein. In contrast, the activity of urokinase is independent of the presence of fibrin. This may explain the greater success achieved in enhancing catalytic activity in the urokinase-antifibrin fusion protein. It is of additional interest that fibrin or soluble fibrin fragments stimulate the catalytic activity of both tPA and the isolated tPA B chain, demonstrating that at least part of the enhanced catalytic activity of tPA observed in the presence of fibrin is independent of fibrin binding either by the tPA kringles or finger domain (or any heavy chain domain). These data indicate that it is possible to construct recombinant hybrid molecules in which both plasminogen activator catalytic function and antibody binding are preserved.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1991HG66400009
View details for PubMedID 1806766
POLYMERASE CHAIN-REACTION CLONING OF L-TYPE CALCIUM-CHANNEL SEQUENCES FROM THE HEART AND THE BRAIN
1990; 274 (1-2): 207-213
The sequences of the highly conserved S4 regions of voltage-sensitive ion channels were used to design oligonucleotide primers for the polymerase chain reaction. Specific fragments of the cDNA encoding L-type calcium channels from the heart, brain, and skeletal muscle were amplified and cloned. The nucleotide sequences of the cardiac and brain calcium channels obtained are identical over this region, and share 78% homology with the skeletal muscle calcium channel. Comparison of the predicted amino acid sequences of our clones with those of other calcium channels reveals unexpected patterns of conservation which suggest alternative exon use.
View details for Web of Science ID A1990EK67600051
View details for PubMedID 1701401
EXPRESSION OF THE POTENT VASOCONSTRICTOR ENDOTHELIN IN THE HUMAN CENTRAL-NERVOUS-SYSTEM
JOURNAL OF CLINICAL INVESTIGATION
1990; 86 (1): 141-147
Endothelin is a potent vasoconstrictive peptide initially characterized as a product of endothelial cells. To examine the potential role of endothelin as a neuropeptide, we studied its distribution in the human central nervous system. RNA blot hybridization provided evidence of endothelin gene transcription in a variety of functional regions of the brain. In situ hybridization confirmed the widespread pattern of endothelin transcription and indicated that the highest density of cells containing endothelin mRNA is in the hypothalamus. This technique localized endothelin transcription to cells of the nervous system as well as the vascular endothelium. Immunocytochemical studies detected endothelin immunoreactivity in neurons, providing evidence of the synthesis of the peptide in this cell type and confirming that endothelin is a neuropeptide. Although the prominent expression of endothelin in the hypothalamus may indicate a central vasoregulatory role for the peptide, the widespread distribution of endothelin in neurons in other areas of the brain implies a more fundamental role in the regulation of nervous system function.
View details for Web of Science ID A1990DN42300021
View details for PubMedID 2195059
FUNCTIONAL-ANALYSIS OF THE ENDOTHELIN-1 GENE PROMOTER - EVIDENCE FOR AN ENDOTHELIAL CELL-SPECIFIC CIS-ACTING SEQUENCE
JOURNAL OF BIOLOGICAL CHEMISTRY
1990; 265 (18): 10446-10450
Endothelin-1 (ET-1) is a peptide synthesized by endothelial cells both in culture and in vivo. ET-1 induces contraction of smooth muscle cells and stimulates growth in a variety of mesenchymal cell types. We have previously characterized the genomic organization of the ET-1 gene and described its chromosomal localization and promoter region sequence. In this report, we describe the use of fusion plasmids containing ET-1 5'-flanking sequence and the chloramphenicol acetyltransferase gene to identify cis-acting sequences that direct transcription of the ET-1 gene. When transfected into bovine aortic endothelial cells, constructs containing 143 base pairs of ET-1 5'-flanking sequence allowed maximal transcription, whereas constructs containing 129 base pairs of sequence had 40-fold lower rates of transcription. A synthetic DNA fragment encoding the region delineated by these deletion mutants was found to have a positive effect on transcription when placed in either orientation upstream of short inactive ET-1 promoter constructs. However, this increase in transcription was noted only when a second region containing an AP1 consensus sequence was also included in the constructs. In experiments with a heterologous promoter and a 119-base pair DNA fragment containing these two functional regions, this 119-base pair sequence acted in a positive and endothelial cell-specific fashion. Taken together, these data localize cis-acting sequences important in determining the rate and tissue specificity of ET-1 gene transcription and should allow the study of protein-DNA interactions which mediate transcription of this gene in endothelial cells.
View details for Web of Science ID A1990DK85100045
View details for PubMedID 2191950
ANTIBODY-TARGETED THROMBOLYTIC AGENTS (REPRINTED FROM SCIENCE, 1989, VOL 243, PG 51-56)
JAPANESE CIRCULATION JOURNAL-ENGLISH EDITION
1990; 54 (4): 345-353
Plasminogen activator therapy for acute myocardial infarction has become standard medical practice. Bleeding complications, however, limit the utility of the currently available agents. This article reviews how the tools of immunology, molecular biology and protein engineering are being used to develop safer and more effective plasminogen activators.
View details for Web of Science ID A1990DW52500001
View details for PubMedID 2204740
BINDING OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR WITH HUMAN ENDOTHELIAL-CELL MONOLAYERS - CHARACTERIZATION OF THE HIGH-AFFINITY INTERACTION WITH PLASMINOGEN-ACTIVATOR INHIBITOR-1
JOURNAL OF BIOLOGICAL CHEMISTRY
1990; 265 (5): 2569-2575
The formation and release of covalent complexes between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) limits the application of equilibrium radioligand binding analysis to characterize the interaction between t-PA and human umbilical vein endothelial cell (HUVEC) monolayers. To avoid this difficulty, we used a recombinant mutant of t-PA, S478A rt-PA, in which alanine has been substituted for the active-site serine. Although the mutant is incapable of covalently reacting with PAI-1, 125I-labeled S478A rt-PA binding to HUVEC monolayers is specific and reversible and is characterized by a high affinity (Kd of 1.5 nM) and a large number of sites (1.5 x 10(6)/cell). This binding was shown to occur through noncovalent interaction with PAI-1 in the HUVEC monolayer by the fact that a monoclonal anti-PAI-1 antibody (MA-7D4) completely blocked S478A rt-PA binding. Two solution-phase assays with recombinant PAI-1 (rPAI-1) confirmed this noncovalent interaction: complexes between 125I-S478A rt-PA and rPAI-1 could be isolated by immunoprecipitation with anti-PAI-1 antibodies, and S478A rt-PA competed with rt-PA for inactivation by rPAI-1. In contrast diisopropylphosphate rt-PA (in which the active site serine is chemically modified) showed minimal binding to HUVEC monolayers, as a result of impaired interaction with PAI-1, in the two assays. Thus, both wild-type rt-PA and S478A rt-PA interact with the HUVEC monolayer through PAI-1. With rt-PA this results in the formation of covalent rt-PA.PAI-1 complexes that are released from the monolayer into the supernatant. With S478A rt-PA this results in the formation of noncovalent complexes that remain associated with the HUVEC monolayer, thereby identifying a large pool of reactive PAI-1 molecules in the monolayer.
View details for Web of Science ID A1990CM84800030
View details for PubMedID 2105930
CDNA CLONING AND CHROMOSOMAL ASSIGNMENT OF THE GENE ENCODING ENDOTHELIN-3
JOURNAL OF BIOLOGICAL CHEMISTRY
1989; 264 (30): 18156-18161
The vasoactive peptide endothelin 1 (ET1) is encoded by a well characterized gene located on human chromosome 6. Recently, two human genomic fragments were isolated which potentially encode related vasoconstrictor peptides, endothelin 2 (ET2) and endothelin 3 (ET3) (Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., and Masaki, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867). Inoue et al. were unable to detect transcripts of the ET2 and ET3 genes and observed ET1 gene expression exclusively in endothelial cells. In this study, we document transcription of the ET3 gene by isolating from a hypothalamic cDNA library DNA clones complementary to human ET3 mRNA. ET3 mRNA encodes a 238-amino acid precursor that includes ET3 and a 15-amino acid homologous segment, the ET3-like sequence. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, we assigned the ET3 gene to human chromosome 20. The ET3 and ET1 genes are, therefore, not genetically linked. RNA blot hybridization with restriction fragments derived from cDNAs revealed that the ET3 and ET1 genes are both expressed in lung, pancreas, and spleen. Cultured endothelial cells and cardiac tissues express the ET1 but not the ET3 gene. Observations that genes encoding endothelin-related peptides are expressed in a variety of human tissues suggest that these peptides may participate in complex vasoregulatory mechanisms.
View details for Web of Science ID A1989AV75800081
View details for PubMedID 2509452
MODULATION OF MESSENGER-RNA LEVELS FOR URINARY-TYPE AND TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR-1 AND INHIBITOR-2 IN HUMAN-FIBROBLASTS BY INTERLEUKIN 1
JOURNAL OF IMMUNOLOGY
1989; 143 (3): 890-895
Plasminogen activators and their inhibitors are thought to play an important role in the regulation of a variety of pathologic processes including inflammation and wound healing. IL-1 is one inflammatory mediator which has been shown to increase release of plasminogen activator (PA) Ag and activity by mesenchymal cells such as chondrocytes and synoviocytes. We have found that rIL-1 beta induces a rapid and significant accumulation of both tissue-and urinary-type plasminogen activator (t-PA and u-PA) mRNA and type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2) mRNA in MRC-5 fetal lung fibroblasts. An SV40 transformed fibroblast cell line, XP12RO, showed an identical response of PAI-1 and t-PA message levels but revealed no change in PAI-2 or u-PA mRNA levels with rIL-1 beta stimulation. Treatment with the transcriptional inhibitor actinomycin D blocked accumulation of t-PA, u-PA, PAI-1, and PAI-2 mRNA, suggesting that RNA synthesis is required for accumulation of all four transcripts. Cycloheximide (CHX) treatment altered the rate of PAI-1 and t-PA mRNA accumulation, but both were able to increase in the absence of protein synthesis. CHX blocked the rIL-1 beta-induced increase in PAI-2 mRNA levels normally observed at 8 h, indicating that protein synthesis is required for this response to IL-1. The increase in u-PA message level was augmented in a synergistic fashion by CHX. These data for PAI-2 and u-PA provide evidence for short-lived proteins which act either to modulate transcription of these genes or regulate mRNA stability. Thus plasminogen activators and their inhibitors are regulated in a positive and complex fashion in the fibroblast by IL-1, suggesting an important role for these molecules and this cell type in the response to inflammation.
View details for Web of Science ID A1989AG04500020
View details for PubMedID 2501387
THROMBIN REGULATION OF MESSENGER-RNA LEVELS OF TISSUE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR-1 IN CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS
1989; 74 (1): 222-228
Cultured human umbilical vein endothelial cells release tissue plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in response to alpha thrombin stimulation. In order to study the mechanisms of thrombin stimulation, we measured changes in levels of mRNA for t-PA and PAI-1 following exposure of endothelial cells to 3 U/mL alpha thrombin. Alpha thrombin causes a significant and time-dependent increase in the mRNA levels of both t-PA and PAI-1. Catalytically inactivated diisofluorophosphate (DIP) treated thrombin and alpha thrombin pretreated with hirudin do not alter t-PA and PAI-1 mRNA levels. We conclude that the increased secretion of t-PA and PAI-1 by human umbilical vein endothelial cells in response to alpha thrombin is mediated at least partially through an increase in mRNA levels. In addition, an active thrombin catalytic site is required for these increases in mRNA to occur.
View details for Web of Science ID A1989AE78100031
View details for PubMedID 2502201
STRUCTURAL ORGANIZATION AND CHROMOSOMAL ASSIGNMENT OF THE GENE ENCODING ENDOTHELIN
JOURNAL OF BIOLOGICAL CHEMISTRY
1989; 264 (18): 10851-10857
Endothelin is a 21-amino acid vasoconstrictor synthesized and secreted by vascular endothelial cells. The human peptide is derived from a 212-amino acid precursor, preproendothelin. A nearly full length clone containing DNA complementary to human preproendothelin mRNA was isolated, and its nucleotide sequence was determined. Using this cDNA as a probe, the genomic organization of the human endothelin gene was determined and the promoter region delineated. The gene contains five exons and four intervening sequences. Nucleotide sequences encoding endothelin are contained within the second exon, and the third exon specifies a portion of preproendothelin that is homologous to endothelin. The second and third exons may represent descendants of a common progenitor exon. The 3'-untranslated portion of the gene contains a 250-base pair region that is highly conserved between human and porcine genomes and may have an important role in endothelin mRNA stability. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, the endothelin gene was assigned to human chromosome 6.
View details for Web of Science ID A1989AB64300079
View details for PubMedID 2659594
VARIABILITY IN MESSENGER-RNA LEVELS IN HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS OF DIFFERENT LINEAGE AND TIME IN CULTURE
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY
1989; 25 (3): 289-292
Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plasminogen activator inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression in cultured endothelium.
View details for Web of Science ID A1989T933200011
View details for PubMedID 2538412
- PLASMINOGEN ACTIVATORS - THE OLD AND THE NEW CIRCULATION 1989; 79 (2): 217-224
INNOVATIVE APPROACHES TO PLASMINOGEN-ACTIVATOR THERAPY
1989; 243 (4887): 51-56
Plasminogen activator therapy for acute myocardial infarction has become standard medical practice. Bleeding complications, however, limit the utility of the currently available agents. This article reviews how the tools of molecular biology and protein engineering are being used to develop safer and more effective plasminogen activators.
View details for Web of Science ID A1989R646900027
View details for PubMedID 2492113
RECOMBINANT ANTIBODIES POSSESSING NOVEL EFFECTOR FUNCTIONS
METHODS IN ENZYMOLOGY
1989; 178: 515-527
Our method for constructing an antifibrin antibody-t-PA chimeric protein can be adapted to form other bifunctional, antibody-targeted proteins. Once an appropriate targeting antibody is obtained, the investigator can derive the heavy chain loss variant cell lines and clone the functional heavy chain rearrangement transcribed by the hybridoma. Other useful reagents include antisera directed against mouse Fab and antisera against whatever effector component is to be combined with the antibody. These are helpful during the screening of transfectants and the characterization of the secreted fusion protein, and they allow for protein purification by affinity chromatography. An assay of the functional activity of the effector domain is also desirable. The apparent retention of enzymatic activity and substrate specificity in our antibody-targeted plasminogen activator hybrid demonstrates that even complex molecules with strict folding requirements and multiple intrachain disulfide bonds can be used to form hybrid recombinant proteins. We have documented by electrophoretic transfer blotting that the heavy chain-t-PA fusion protein is secreted in association with light chain in the form of a 180-kDa dimer. The heavy chains appear to be attached by disulfide bonds at the hinge region, as is the case with the heavy chains of natural immunoglobulins. Our method can be adapted to various uses. More or less of the antibody constant region could be employed, depending on the desired geometry and the immunologic interactions mediated by the Fc domain. We have made a recombinant fusion peptide containing an additional 100 constant region amino acids but found that its targeting and catalytic abilities did not differ from those of the smaller molecule. Recent reports indicate that it is possible to express an antibody Fv that has full antigen recognition and binding properties; such small immunoglobulins could minimize potential immunogenicity while affording full targeting capability. The use of a human constant region sequence may also provide a less immunogenic molecule, and, by transferring the complementarity-determining regions of the monoclonal antibody into human variable region sequence, it may be possible to completely "humanize" an antibody-directed chimeric protein. The application of these and other innovative approaches should soon make antibodies an attractive means of targeting a wide range of molecules, both in scientific investigation and in medical therapy.
View details for Web of Science ID A1989CX43700035
View details for PubMedID 2513468
STRUCTURAL INTEGRITY OF THE GLYCOPROTEIN-IIB AND GLYCOPROTEIN-IIIA GENES IN GLANZMANN THROMBASTHENIA PATIENTS FROM ISRAEL
1988; 72 (5): 1833-1836
Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full-length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.
View details for Web of Science ID A1988Q745100059
View details for PubMedID 3179450
NONLINKAGE OF THE T-CELL RECEPTOR ALPHA-GENE, BETA-GENE, AND GAMMA-GENE TO SYSTEMIC LUPUS-ERYTHEMATOSUS IN MULTIPLEX FAMILIES
ARTHRITIS AND RHEUMATISM
1988; 31 (11): 1371-1376
To test the possibility that T cell antigen receptor (TcR) genes are linked to the genes involved in the pathogenesis of systemic lupus erythematosus (SLE), genomic DNA restriction fragment length polymorphisms were studied, using the Southern blot technique, in 5 families with multiple members with SLE, 14 unrelated SLE patients, and 14 normal controls. Polymorphic patterns were detected with probes for all 3 TcR chains, but there was no significant difference in the distribution of the restriction fragment length polymorphism pattern among the patients, the relatives, and the controls. Furthermore, in the families with at least 2 individuals with the disease, each of the 3 TcR chain genes did not cosegregate with the disease. We conclude that TcR alpha, beta, and gamma chain genes are not likely to be linked to genes related to SLE.
View details for Web of Science ID A1988Q973200005
View details for PubMedID 2903748
- INCREASING SELECTIVITY OF PLASMINOGEN ACTIVATORS WITH ANTIBODIES CLINICAL RESEARCH 1988; 36 (5): 501-506
T-CELL RECEPTOR GENE REARRANGEMENT AND EXPRESSION IN HUMAN NATURAL-KILLER CELLS - NATURAL-KILLER ACTIVITY IS NOT DEPENDENT ON THE REARRANGEMENT AND EXPRESSION OF T-CELL RECEPTOR ALPHA-GENES, BETA-GENES, OR GAMMA-GENES
1988; 27 (4): 231-238
To test the hypothesis that the T-cell receptor (Tcr) gamma gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16+ lymphocytosis were analyzed for rearrangements and expression of the human Tcr alpha, beta, and gamma genes. Two of the clones displayed distinct rearrangements of their Tcr beta and gamma genes and expressed mature Tcr alpha, beta, and gamma RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gamma gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr alpha and beta gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.
View details for Web of Science ID A1988M164700001
LEFT-VENTRICULAR EJECTION FRACTION - PHYSICIAN ESTIMATES COMPARED WITH GATED BLOOD POOL SCAN MEASUREMENTS
ARCHIVES OF INTERNAL MEDICINE
1988; 148 (4): 882-885
Gated blood pool scanning (GBPS) is an expensive, frequently used test to assess the left ventricular ejection fraction (LVEF). To determine whether a simpler method of evaluating LVEFs was reliable, we compared the LVEFs derived by GBPS with those estimated in a cardiologist's examination in 125 hospitalized patients. Of the physician estimates, 56% were accurate to within 7.5%, while 17% were underestimates and 27% were overestimates. The variables that were most predictive of reduced LVEF included cardiomegaly and pulmonary venous congestion on chest roentgenogram and S3 gallop, hypotension, and sustained left ventricular apex beat on examination. Prior hypertension was correlated with an increased LVEF. Variables associated with physician error in estimating the LVEF included a history of hypertension, bronchodilator therapy, and right bundle-branch block seen on the electrocardiogram. These data suggest that although qualitatively accurate estimates of the LVEF can sometimes be made on the basis of clinical findings, GBPS should be performed when management decisions hinge on a precise knowledge of this value.
View details for Web of Science ID A1988M903000017
View details for PubMedID 3355308
CONSTRUCTION AND EXPRESSION OF A RECOMBINANT ANTIBODY-TARGETED PLASMINOGEN-ACTIVATOR
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1987; 84 (19): 6904-6908
Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin beta chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent than t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, we have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, we have produced a hybrid protein capable of high-affinity fibrin binding and plasminogen activation.
View details for Web of Science ID A1987K314300062
View details for PubMedID 3116546
MEASURING THE HUMAN T-CELL RECEPTOR GAMMA-CHAIN LOCUS
1987; 237 (4819): 1217-1219
The human T cell receptor gamma locus, including eleven variable-region, five joining-region, and two constant-region segments, is contained in 160 kilobases. During T cell somatic development these genes undergo rearrangement by deletion of the sequences separating the variable and joining regions. The molecular map of this locus was completely defined by deletion mapping and restriction mapping. Restriction fragments were resolved by standard agarose electrophoresis and field inversion electrophoresis. These studies demonstrate that the deletions in this locus, which occur during the formation of a functional T cell receptor gamma-chain gene, range from 50 to 145 kilobases in length. These studies also provide a structural basis for understanding the development of the gamma-chain peptide repertoire, and extends the potential of the emerging pulsed-field electrophoretic technology.
View details for Web of Science ID A1987J838900048
View details for PubMedID 3498213
PROVOCATIVE PATTERN OF REARRANGEMENTS OF THE GENES FOR THE GAMMA-CHAIN AND BETA-CHAIN OF THE T-CELL RECEPTOR IN HUMAN LEUKEMIAS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1987; 84 (13): 4547-4551
To examine the distribution of rearrangements of the gamma- and beta-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, we analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged gamma- and beta-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had beta-chain gene rearrangements, and 41% (24/58) had gamma-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The gamma-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA+ ALL patients, while the beta-chain genes were not rearranged in any of the 7 cALLA- ALL patients examined but were rearranged in 32% (18/56) of the cALLA+ patients. Neither TCR gene was found to be rearranged in acute nonlymphoid leukemia patients (0/12) or in patients with B-cell (surface immunoglobulin-positive) leukemia (0/3). Of the 44 cALLA+ patients in which a direct comparison of gamma- and beta-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only gamma-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. beta-Chain rearrangements have not been found in the absence of gamma-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements. A provocative finding was that only a small percentage (11%) of the patients with cALLA- B precursor cell ALLs had rearranged TCR genes, while 50% of the cALLA+ leukemia patients had at least gamma-chain rearrangement, raising a question as to whether indeed cALLA- cells are precursors to cALLA+ cells. Interestingly, 18% (2/11) of the cytoplasmic immunoglobulin (cIg)-positive cALLA+ (pre-B) ALLs involved TCR gene rearrangements, compared to 60% (21/35) of the cIg-negative cases, suggesting the possibility that the majority of functional B cells are derived from the cALLA+ pool that contains immunoglobulin but not TCR gene rearrangements.
View details for Web of Science ID A1987J070000042
View details for PubMedID 2955409
HUMAN T-CELL-GAMMA CHAIN JOINING REGIONS AND T-CELL DEVELOPMENT
JOURNAL OF IMMUNOLOGY
1987; 138 (8): 2687-2690
We have cloned and sequenced two homologous J regions of the T cell gamma gene and localized one 5' of each constant region. They have been numbered J gamma 1.1 and J gamma 2.1; other J regions have been renamed in a similar manner. Southern blot analysis suggests that there is comparatively more rearrangement to these J regions in thymocytes than peripheral blood T cells. We propose that either there is gamma-chain gene rearrangement first to J gamma 1.1 or 2.1 and then to J gamma 1.3 or 2.3, or there is selected cell death of thymic T cells bearing a J gamma 1.1 or 2.1 rearrangement.
View details for Web of Science ID A1987G768100047
View details for PubMedID 2951442
COMPARISON OF T-CELL RECEPTOR GENE REARRANGEMENTS IN PATIENTS WITH LARGE ANTIGRANULOCYTES T-CELL LEUKEMIA AND FELTYS SYNDROME
JOURNAL OF IMMUNOLOGY
1987; 138 (6): 1724-1729
Felty's syndrome (FS) refers to the occurrence of rheumatoid arthritis, splenomegaly, and neutropenia. A subset of these patients has recently been described with a chronic T cell leukemia of large granular lymphocytes (LGCL). To examine the spectrum of lymphocyte abnormalities in FS and LGCL, we examined phenotypic and genotypic properties of lymphocytes from eight FS patients. In two of these FS patients, we observed an elevated proportion of T cells with an unusual phenotype (CD3+/Leu-7+/Leu-8-/CR3+) (46 +/- 5% of mononuclear cells). The FS lymphocytes had large granular morphology on Wright-Giemsa stain and were active in antibody-dependent cellular cytotoxic activity. This phenotype, morphology, and activity was similar to LGCL patients except that the latter T cells additionally expressed the Fc-IgG receptor recognized by monoclonal antibody Leu-11 (CD 15). In the remaining six FS patients, the proportion of CD3+/Leu-7+/CR 3+ T cells was only 10 +/- 8%, which was not significantly different from age-matched normal subjects (6.6 +/- 2.2%). To determine the clonality of T lymphocytes in FS and LGCL, we examined DNA for rearrangements of the T cell antigen receptor beta-chain (Ti beta) and gamma-chain (Ti gamma) genes by using Southern blotting techniques. We found a clonal rearrangement of the Ti beta 1 and Ti gamma genes in both LGCL patients. In contrast, no clonal rearrangements of Ti beta or Ti gamma genes were detected in lymphocytes from the FS patients. These results indicate that FS patients are heterogeneous in their phenotype and that one subset exhibits polyclonal expansion of an unusual lymphocyte subset.
View details for Web of Science ID A1987G388300012
View details for PubMedID 3102595
T-CELL RECEPTOR-CD3 COMPLEX DURING EARLY T-CELL DIFFERENTIATION - ANALYSIS OF IMMATURE T-CELL ACUTE LYMPHOBLASTIC LEUKEMIAS (T-ALL) AT DNA, RNA, AND CELL-MEMBRANE LEVEL
JOURNAL OF IMMUNOLOGY
1987; 138 (4): 1260-1269
T cell acute lymphoblastic leukemias (T-ALL) can be regarded as the malignant counterparts of cells in various T cell differentiation stages. To study the expression of the human T cell receptor (TcR)-CD3 complex during the early stages of T cell differentiation, we have analyzed 22 T-ALL at the cell membrane level and the DNA level and 12 of them at the RNA level. According to their immunologic phenotype, the T-ALL could be divided into three main groups: 10 immature T-ALL (CD1-/CD3-), seven common thymocytic T-ALL (CD1+/CD3-or+), and five mature T-ALL (CD1-/CD3+). Among the 10 immature T-ALL three appeared to express the immunologic phenotype of the putative prothymocyte (TdT+/HLA-DR+/CD7+/CD2+/CD5-/CD1-/CD3-), whereas the other seven T-ALL appeared to be immature thymocytic (TdT+/HLA-DR-/CD7+/CD2+/CD5+/CD1-/CD3-). Transcripts of the CD3-delta and CD3-epsilon genes were present in all CD3- and CD3+ T-ALL tested, including prothymocytic T-ALL. However, prothymocytic T-ALL had germline TcR-beta genes and were not rearranged to the characterized TcR-gamma joining regions. The presence of CD3 transcripts and absence of TcR gene rearrangements in prothymocytic T-ALL supports their immature T cell character. Two immature thymocytic T-ALL also had germline TcR-gamma genes and one of them had germline TcR-beta genes. In all other T-ALL the TcR-gamma and TcR-beta genes were rearranged. The presumptive functional 1.3-kilobase TcR-beta transcripts were detected in the majority of T-ALL with rearranged TcR-beta genes. Distinct levels of TcR-gamma transcripts appeared to be present only in some thymocytic T-ALL, i.e., some immature thymocytic T-ALL and common thymocytic T-ALL. TcR-alpha mRNA could only be detected in CD3+ mature T-ALL, but was absent in all CD3+ common thymocytic T-ALL tested. Our data indicate that CD3 gene transcription is one of the earliest events during T cell differentiation and already occurs in prothymocytes. The TcR-gamma and TcR-beta genes rearrange early during thymocytic differentiation and can subsequently be transcribed. High levels of TcR-gamma gene transcription may only occur in a part of the T cells during thymic differentiation, while TcR-beta gene transcription continues during further differentiation. TcR-alpha gene transcription may be the final step in the production of the complete set of TcR and CD3 proteins, resulting in the expression of the TcR alpha beta-CD3 complex at the cell surface of mature T cells.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1987F948200043
View details for PubMedID 3100631
IMMUNOGLOBULIN AND T-CELL RECEPTOR GENE REARRANGEMENT AND EXPRESSION IN HUMAN LYMPHOID LEUKEMIA-CELLS AT DIFFERENT STAGES OF MATURATION
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1986; 83 (22): 8759-8763
The use of probes to genes (IG and TCRB) encoding immunoglobulins (IG) and the beta chain of the T-cell antigen receptor (TCRB), respectively, have become a sensitive means to assess clonality and lineage in lymphoid malignancies. It has become apparent that some individual cases show rearrangements of both IG and TCRB genes. In an attempt to more accurately define cell lineage we have analyzed cells from patients with B- or T-cell leukemia (n = 26) at various stages of maturation with probes to two additional TCR genes, TCRG and TCRA (encoding the TCR gamma and alpha chains, respectively), as well as the IG heavy chain joining region (IGHJ) and TCRB genes. On Southern blot analysis, the mature T-cell leukemia cells studied had rearranged TCRG and TCRB while IGHJ remained as in the germ line. The mature B-cell leukemia cells studied had rearranged IGHJ with germ-line TCRG and TCRB. These data suggest that, in the majority of more mature leukemias, cells have rearranged IG or TCR genes but not both. In contrast, cells from five of nine precursor B-cell leukemia patients and cell lines from one of four precursor T-cell leukemia patients had rearranged both IGHJ and TCR genes. TCRG and TCRB mRNAs were expressed in the cells of precursor T- but not B-cell leukemia patients studied. The spectrum of leukemia cells studied within the T-cell series permitted an assessment of the order of TCR gene rearrangements. Two of 13 patients had cells with germ-line TCRG and TCRB, 2 patients had cells with rearranged TCRG alone, and the remainder had cells with rearranged TCRG and TCRB. TCRG and TCRB mRNAs were expressed in precursor T-cell leukemia cells, whereas TCRB and TCRA were expressed in mature T-cell leukemia cells. These results parallel observations from mouse studies on gene expression and support the view of a hierarchy of TCR gene rearrangements in T-lymphocyte ontogeny. TCRG genes are rearranged first, subsequently TCRB genes are rearranged, followed by TCRA gene activation.
View details for Web of Science ID A1986E906700068
View details for PubMedID 3464980
HUMAN T-CELL GAMMA-CHAIN GENE REARRANGEMENTS IN ACUTE LYMPHOID AND NONLYMPHOID LEUKEMIA - COMPARISON WITH THE T-CELL RECEPTOR BETA-CHAIN GENE
JOURNAL OF IMMUNOLOGY
1986; 137 (6): 2043-2049
Rearrangement of germ-line genes coding for T and B cell antigen receptor molecules is an early event in lymphoid development which eventually leads to the generation of clonal diversity in receptor-positive lymphocytes. Three T cell-associated rearranging genes have been described. Two, T alpha and T beta, code for the two polypeptide chains that form the T cell receptor heterodimer. The function of the third gene, the gamma-gene (T gamma), is not known. To learn more about the behavior of T gamma during lymphoid ontogeny, we compared rearrangement of T gamma and T beta genes in leukemic cells arrested at varied stages of lymphoid and myeloid development. We analyzed 38 fresh cell lines and 15 established cell lines from a total of 53 leukemic patients. Cells were immunophenotyped with a panel of monoclonal antibodies recognizing T-, B-, or myeloid-associated surface markers. Sixteen T-lineage cases were studied; 15 displayed both T beta and T gamma rearrangements. The exception (germ-line for T beta and T gamma) was an immature CD2(T11)+, CD3(T3)-, CD7(3A1)+, CD1(T6)+, CD5(T101)+ phenotype. Fourteen non-T non-B leukemias were analyzed; eight were germ-line for both T beta and T gamma, four had rearrangements involving both T beta and T gamma, and two were germ-line for T beta and rearranged to T gamma. Four cases with acute biphenotypic leukemia were studied; two had rearrangements of T beta and T gamma, and two were germ-line for both genes. Cells from nonlymphocytic leukemias were studied in 19 cases. All were found to be germ-line for both T beta and T gamma. Fifty-one of 53 genomic DNA samples were concordant for T gamma and T beta rearrangement. These results indicate that rearrangement of T gamma can occur in leukemic cells of B cell as well as T cell precursor origin, as has been reported previously for T beta.
View details for Web of Science ID A1986D956900047
View details for PubMedID 3489046
HUMAN T-CELL GAMMA-GENES CONTAIN N-SEGMENTS AND HAVE MARKED JUNCTIONAL VARIABILITY
1986; 322 (6075): 184-187
The gamma-chain genes are encoded by immunoglobulin-like gene segments in germline DNA which rearrange during the somatic development of T cells to form an active gene. The protein produced by these genes has not been identified and the diversity of the proteins that the genes can express has not been determined. We expect that the diversity of expressed gamma-chains is produced by the same three mechanisms that produce diversity of other immunoglobulin-like genes: (1) germline variable (V) and joining (J) region repertoires; (2) somatic mutation; and (3) junctional diversity. To define the contribution of each of these mechanisms to the generation of gamma-chain diversity, several gamma-chain complementary clones and rearranged gamma-chain genes have been characterized. Most of these clones seem to encode a defective gamma-chain, the variable- and constant-region portions being joined such that they would not be translated in the same reading frame. Here we report that the germline J-region diversity of the human T-cell gamma-chain is very limited and that somatic mutation does not contribute to the diversity of the gamma-chains encoded by the cloned segments. However, the junctional diversity of these gamma-chain genes is extensive. We suggest that N sequences (template-independent sequences) have been inserted enzymatically into all of the gamma-chain genes characterized.
View details for Web of Science ID A1986D143800069
View details for PubMedID 3453106
T-CELL RECEPTOR GENE REARRANGEMENTS DEFINE A MONOCLONAL T-CELL PROLIFERATION IN PATIENTS WITH T-CELL LYMPHOCYTOSIS AND CYTOPENIA
1986; 67 (4): 914-918
We have used probes from the T cell receptor beta and gamma chain loci to investigate the clonality of T lymphocytes in eight patients with T cell lymphocytosis and cytopenia (TCLC). This syndrome, which is strongly associated with rheumatoid arthritis, is characterized by peripheral blood and bone marrow lymphocytosis and neutropenia, red cell aplasia, or both. By means of T cell monoclonal antibodies and flow cytometry, T lymphocytes from patients with this syndrome have been shown to have characteristic immunologic features. Investigators have disagreed as to whether the syndrome represents a T cell malignancy or a more benign immunologic disorder. DNA from five of five patients with symptomatic "classic" T cell lymphocytosis with cytopenia demonstrated unique rearrangements of the T cell receptor beta chain locus, whereas neither of two patients with atypical features showed rearrangement. In addition, we found evidence for gamma chain rearrangement in those DNAs with clonal beta chain rearrangement. We thus postulate that the classic form of this syndrome is associated with a monoclonal proliferation of T cells. Its potential relationship to T cell chronic lymphocytic leukemia is discussed.
View details for Web of Science ID A1986A807600011
View details for PubMedID 3485459
CLONING AND SEQUENCE-ANALYSIS OF COMPLEMENTARY-DNA ENCODING AN ABERRANTLY REARRANGED HUMAN T-CELL GAMMA-CHAIN
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1986; 83 (8): 2619-2623
Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation.
View details for Web of Science ID A1986C010700071
View details for PubMedID 3458221
SPECTRUM OF CONDITIONS INITIALLY SUGGESTING ACUTE AORTIC DISSECTION BUT WITH NEGATIVE AORTOGRAMS
AMERICAN JOURNAL OF CARDIOLOGY
1986; 57 (4): 322-326
Between 1963 and 1983, 55 patients presented to our hospital with a clinical picture that suggested aortic dissection but with aortograms that were interpreted as negative for that entity. In 4 patients, the aortographic findings subsequently proved to be false negative. The remaining 51 patients had the following diagnoses: myocardial infarction in 9 patients; aortic regurgitation in 5; thoracic nondissecting aneurysm in 4; musculoskeletal pain in 4; mediastinal tumor in 4; pericarditis in 3; acute coronary insufficiency in 3; cholecystitis in 2; miscellaneous in 3; and unknown in 14. The clinical features in these patients were compared with those of 125 patients with true aortic dissection. Three features were significantly more prevalent in patients with than without dissection: prior systemic hypertension, pain for 24 hours or less, and migratory pain. Patients without dissection were younger than those with distal dissection and had significantly less systemic hypertension, posterior thoracic pain and migratory pain. Patients without dissection had significantly less frequent congestive heart failure, pulse deficits and aortic regurgitation, and more frequent hypertension and pain for more than 24 hours than patients with proximal dissection. This study defines the actual differential diagnosis of aortic dissection at our hospital, the frequency of false-negative aortographic findings and contrasts the clinical features of patients with and without dissection.
View details for Web of Science ID A1986AYX4500024
View details for PubMedID 3946223
HUMAN T-CELL GAMMA-CHAIN GENES - ORGANIZATION, DIVERSITY, AND REARRANGEMENT
1986; 231 (4735): 252-255
The human T-cell gamma chain genes have been characterized in an attempt to better understand their role in immune response. These immunoglobulin-like genes are encoded in the genome in variable, joining, and constant segments. The human gamma genes include at least six variable region genes, two joining segments, and two constant-region genes in germline DNA. Variable and joining segments recombine during the development of T cells to form rearranged genes. The diversity of human gamma genes produced by this recombinational mechanism is greater than that produced by the murine genome but is more limited than that of other immunoglobulin-like genes.
View details for Web of Science ID A1986AXH2600025
View details for PubMedID 3079918
PACEMAKER FAILURE RESULTING FROM RADIATION-DAMAGE
1983; 148 (1): 257-258
The authors present a case of radiation-induced pacemaker failure. After 2,000 rad (20 Gy) of photon irradiation for metastatic bronchogenic carcinoma, the pulse generator circuitry failed, producing a "runaway" rhythm. This suggests that present pacemaker circuitry may be more susceptible to irradiation than previously believed, and that even modest radiation doses can induce life-threatening arrhythmias.
View details for Web of Science ID A1983QV03900053
View details for PubMedID 6856846