Consulting Editor, Circulation Research (2015 - Present)
Editorial Board, Circulation Research (2014 - 2015)
Guest Editor, Current Treatment Options in Cardiovascular Disease (2013 - Present)
Member, American Heart Association National Research Committee, Stem Cell Research Subgroup (2013 - Present)
Member, American Heart Association National Stem Cell Therapy Writing Group (2012 - Present)
Assistant Professor of Medicine, Stanford University, School of Medicine (2012 - Present)
Organizing Committee, NIH/NHLBI Cardiovascular Regenerative Medicine Symposium (2011 - Present)
Associate Editor, BMC Cardiovascular Disease (2011 - Present)
Editorial Board, Frontiers in Pharmacotherapy of Cardiovascular Disorders (2010 - Present)
Research Administration Advisory Committee Member, Massachusetts General Hospital (2010 - 2012)
Assistant Physician, Massachusetts General Hospital (2009 - 2012)
Assistant Professor of Medicine, Harvard Medical School (2009 - 2012)
Editorial Board, World Journal of Stem Cell (2009 - Present)
Editorial Board, Clinical Medicine Insights: Cardiology (2007 - Present)
Director, Mouse Microinjection Core, Massachusetts General Hospital (2007 - 2012)
Instructor in Medicine, Harvard Medical School (2006 - 2009)
Honors & Awards
NIH Director's Pioneer Award, National Institutes of Health, Office of the Director (2014-2019)
David Lawrence Stein Award, American Heart Association-Western Affiliate (2014)
NIH Director's New Innovator Award, National Institutes of Health, Office of the Director (2008-2013)
Endowed Faculty Scholar, Child Health Research Institute/ Lucile Packard Foundation for Children's Health (2013-2018)
Seed Grant Award (Co-Recipient with Dr. Beth Pruitt), Stanford Cardiovascular Institute (2013-2014)
SPARK Research Award, Division of Cardiology, Massachusetts General Hospital (2010-2011)
Fellow, American College of Cardiology (2010)
Progenitor Cell Biology Consortium, Co-Principal Investigator, NIH/NHLBI (2009-2016)
Seed Grant Recipient, Harvard Stem Cell Institute (2008-2010)
Young Investigator Competitive Award in Cardiovascular Medicine, GlaxoSmithKline Education and Research Foundation (2007-2009)
de Gunzburg Family Scholar, Massachusetts General Hospital (2006)
Abstract of Distinction, Research Symposium - Massachusetts General Hospital (2005)
K08 Mentored Clinical Scientist Award, NIH/NHLBI (2005-2011)
NIH/NHLBI Scholarship, Keystone Symposium on Molecular Mechanism of Cardiac Disease and Regeneration (2005)
Career Development Award in Cardiovascular Medicine, American College of Cardiology Foundation/Pfizer (2004-2007)
ACCF/Bristol Meyers Travel Award, American College of Cardiology (2002)
Merck/ACC Young Investigator Award - 2nd Place, American College of Cardiology (2001)
Henry Christian Award for Research Excellence, American Federation for Medical Research (1999)
Experimental Pathologist-in-Training, American Society for Investigative Pathology (1998)
Award for Academic Excellence and Achievement, American Society of Clinical Pathologists (1996, 1997)
Tau Beta Pi, Stanford University School of Engineering (1992)
Terman Award, Stanford University School of Engineering (1992)
President's Scholar, Stanford University (1989)
Research Fellowship, Boston Children's Hospital/Harvard Medical School, Stem Cell Biology (2006)
Board Certification, Cardiovascular Medicine, ABIM (2005)
Fellowship, Massachusetts General Hospital/Harvard Medical School, Cardiovascular Medicine (2004)
Board Certification, Internal Medicine, ABIM (2003)
Residency, Duke University Hospital, Internal Medicine (2001)
MD, Duke University School of Medicine, Medicine (1999)
PhD, Duke University School of Arts and Sciences, Pathology (1998)
BS, Stanford University, Mechanical Engineering (1992)
BS, Stanford University, Biological Science (1992)
Community and International Work
National Asian Pacific American Medical Student Association
Opportunities for Student Involvement
Sean M. Wu. "United States Patent 13/552975 Methods and compounds for reducing intracellular lipid storage", Massachusetts General Hospital, Jul 20, 2011
Current Research and Scholarly Interests
My lab seeks to identify mechanisms regulating cardiac lineage commitment during embryonic development and the biology of cardiac progenitor cells in development and disease. We believe that by understanding the transcriptional and epigenetic basis of cardiomyocyte growth and differentiation, we can identify the most effective ways to repair diseased adult hearts. We employ mouse and human embryonic and induced pluripotent stem cells as well as rodents as our in vivo models for investigation.
Independent Studies (11)
- Directed Investigation
BIOE 392 (Spr)
- Directed Reading in Medicine
MED 299 (Aut)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr)
- Early Clinical Experience in Medicine
MED 280 (Win)
- Graduate Research
MED 399 (Aut)
- Graduate Research
STEMREM 399 (Aut, Win, Spr)
- Medical Scholars Research
MED 370 (Aut)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Undergraduate Research
MED 199 (Aut)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr)
- Directed Investigation
Fetal Mammalian Heart Generates a Robust Compensatory Response to Cell Loss.
2015; 132 (2): 109-121
-Heart development is tightly regulated by signaling events acting upon a defined number of progenitor and differentiated cardiac cells. While loss-of-function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell (CPC) or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration following extensive cell loss.-By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of CPC or cardiomyocyte ablation. Remarkably, ablation of up to 60% of CPCs at embryonic day 7.5 was well-tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50-60%) at embryonic day 9.0 could be fully rescued by residual myocytes with no obvious adult cardiac functional deficit. In both ablation models, an increase in cardiomyocyte proliferation rate was detected and accounted for at least some of the rapid recovery of myocardial cellularity and heart size.-Our study defines the threshold for cell loss in the embryonic mammalian heart and reveals a robust cardiomyocyte compensatory response that sustains normal fetal development.
View details for DOI 10.1161/CIRCULATIONAHA.114.011490
View details for PubMedID 25995316
Identification of cardiovascular lineage descendants at single-cell resolution.
2015; 142 (5): 846-857
The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells.
View details for DOI 10.1242/dev.116897
View details for PubMedID 25633351
- Molecular Regulation of Cardiomyocyte Differentiation CIRCULATION RESEARCH 2015; 116 (2): 341-353
- Members Only: hypoxia-induced cell cycle activation in cardiomyocytes. Cell Metabolism 2015; 22: 365-366
Patching up broken hearts: cardiac cell therapy gets a bioengineered boost.
Cell stem cell
2014; 15 (6): 671-673
Preclinical and clinical studies for cardiac cell therapy have only seen moderate success due to poor engraftment and survival of transplanted cells. In this issue of Cell Stem Cell, Ye et al. (2014) employ a growth-factor-loaded fibrin patch and show improved cardiovascular cell survival after cell transplantation into a porcine model of ischemia reperfusion.
View details for DOI 10.1016/j.stem.2014.11.008
View details for PubMedID 25479741
Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral drug screening platform.
2014; 115 (6): 556-566
Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
View details for DOI 10.1161/CIRCRESAHA.115.303810
View details for PubMedID 25015077
Harnessing the potential of induced pluripotent stem cells for regenerative medicine
NATURE CELL BIOLOGY
2011; 13 (5): 497-505
The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.
View details for DOI 10.1038/ncb0511-497
View details for Web of Science ID 000290148700004
View details for PubMedID 21540845
Generation of Functional Ventricular Heart Muscle from Mouse Ventricular Progenitor Cells
2009; 326 (5951): 426-429
The mammalian heart is formed from distinct sets of first and second heart field (FHF and SHF, respectively) progenitors. Although multipotent progenitors have previously been shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. We have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome-wide profiling of coding and noncoding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of limited in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue, representing a combination of tissue engineering and stem cell biology.
View details for DOI 10.1126/science.1177350
View details for Web of Science ID 000270818600053
View details for PubMedID 19833966
Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart
2008; 454 (7200): 109-U5
The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.
View details for DOI 10.1038/nature07060
View details for Web of Science ID 000257308300047
View details for PubMedID 18568026
Origins and fates of cardiovascular progenitor cells
2008; 132 (4): 537-543
Multipotent cardiac progenitor cells are found in the fetal and adult heart of many mammalian species including humans and form as intermediates during the differentiation of embryonic stem cells. Despite similar biological properties, the molecular identities of these different cardiac progenitor cell populations appear to be distinct. Elucidating the origins and lineage relationships of these cell populations will accelerate clinical applications such as drug screening and cell therapy as well as shedding light on the pathogenic mechanisms underlying cardiac diseases.
View details for DOI 10.1016/j.cell.2008.02.002
View details for Web of Science ID 000253817900012
View details for PubMedID 18295570
Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart
2006; 127 (6): 1137-1150
Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.
View details for DOI 10.1016/j.cell.2006.10.028
View details for Web of Science ID 000242991000013
View details for PubMedID 17123591
Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.
; 10 (8): e0135880
In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.
View details for DOI 10.1371/journal.pone.0135880
View details for PubMedID 26323090
- Small RNAs Make Big Impact in Cardiac Repair CIRCULATION RESEARCH 2015; 116 (3): 393-395
Comparing mouse and human pluripotentstem cell derived cardiac cells: both systemshave advantages for pharmacological and toxicological screening.
Journal of pharmacological and toxicological methods
Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10 μM), thapsigargin (1 μM) and ZD7288 (10μM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1 μM). Human cells were also sensitive to the addition of trastuzumab and doxorubicin as well as a low oxygen environment. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.
View details for DOI 10.1016/j.vascn.2015.04.009
View details for PubMedID 25957031
Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation.
Journal of visualized experiments : JoVE
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.
View details for DOI 10.3791/52628
View details for PubMedID 25867738
Pharmacological inhibition of TGFß receptor improves Nkx2.5 cardiomyoblast-mediated regeneration.
2015; 105 (1): 44-54
Our previous study found that A83-01, a small molecule type 1 TGFβ receptor inhibitor, could induce proliferation of postnatal Nkx2.5(+) cardiomyoblasts in vitro and enhance their cardiomyogenic differentiation. The present study addresses whether A83-01 treatment in vivo could increase cardiomyogenesis and improve cardiac function after myocardial infarction through an Nkx2.5(+) cardiomyoblast-dependent process.To determine the effect of A83-01 on the number of Nkx2.5(+) cardiomyoblasts in the heart after myocardial injury, we treated transgenic Nkx2.5 enhancer-GFP reporter mice for 7 days with either A83-01 or DMSO and measured the number of GFP(+) cardiomyoblasts in the heart at 1 week after injury by flow cytometry. To determine the degree of new cardiomyocyte formation after myocardial injury and the effect of A83-01 in this process, we employed inducible Nkx2.5 enhancer-Cre transgenic mice to lineage label postnatal Nkx2.5(+) cardiomyoblasts and their differentiated progenies after myocardial injury. We also examined the cardiac function of each animal by intracardiac haemodynamic measurements. We found that A83-01 treatment significantly increased the number of Nkx2.5(+) cardiomyoblasts at baseline and after myocardial injury, resulting in an increase in newly formed cardiomyocytes. Finally, we showed that A83-01 treatment significantly improved ventricular elastance and stroke work, leading to improved contractility after injury.Pharmacological inhibition of TGFβ signalling improved cardiac function in injured mice and promoted the expansion and cardiomyogenic differentiation of Nkx2.5(+) cardiomyoblasts. Direct modulation of resident cardiomyoblasts in vivo may be a promising strategy to enhance therapeutic cardiac regeneration.
View details for DOI 10.1093/cvr/cvu229
View details for PubMedID 25362681
- Harnessing the induction of cardiomyocyte proliferation for cardiac regenerative medicine. Curr Treat Options Cardio Med 2015; In Press.
- Somatic Cell Reprogramming into Cardiovascular Lineages JOURNAL OF CARDIOVASCULAR PHARMACOLOGY AND THERAPEUTICS 2014; 19 (4): 340-349
Insulin-like growth factor promotes cardiac lineage induction in vitro by selective expansion of early mesoderm.
2014; 32 (6): 1493-1502
A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) towards a cardiac fate is essential for translational applications in disease modeling and therapy. We screened a panel of 44 cytokines/signaling molecules for their ability to enhance Nkx2.5(+) cardiac progenitor cell (CPC) formation during in vitro embryonic stem cell (ESC) differentiation. Treatment of murine ESCs with insulin or insulin-like growth factors (IGF1/2) during early differentiation increased mesodermal cell proliferation and, consequently, CPC formation. Furthermore, we show that downstream mediators of IGF signaling (e.g. phospho-Akt and mTOR) are required for this effect. These data support a novel role for IGF family ligands to expand the developing mesoderm and promote cardiac differentiation. Insulin or IGF treatment could provide an effective strategy to increase the PSC-based generation of CPCs and cardiomyocytes for applications in regenerative medicine. Stem Cells 2014.
View details for DOI 10.1002/stem.1660
View details for PubMedID 24496962
Telocytes in human heart valves
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
2014; 18 (5): 759-765
Valve interstitial cells (VICs) are responsible for maintaining the structural integrity and dynamic behaviour of the valve. Telocytes (TCs), a peculiar type of interstitial cells, have been recently identified by Popescu's group in epicardium, myocardium and endocardium (visit www.telocytes.com). The presence of TCs has been identified in atria, ventricles and many other tissues and organ, but not yet in heart valves. We used transmission electron microscopy and immunofluorescence methods (double labelling for CD34 and c-kit, or vimentin, or PDGF Receptor-β) to provide evidence for the existence of TCs in human heart valves, including mitral valve, tricuspid valve and aortic valve. TCs are found in both apex and base of heart valves, with a similar density of 27-28 cells/mm(2) in mitral valve, tricuspid valve and aortic valve. Since TCs are known for the participation in regeneration or repair biological processes, it remains to be determined how TCs contributes to the valve attempts to re-establish normal structure and function following injury, especially a complex junction was found between TCs and a putative stem (progenitor) cell.
View details for DOI 10.1111/jcmm.12285
View details for Web of Science ID 000335861500002
View details for PubMedID 24674389
Myeloid zinc finger 1 (mzf1) differentially modulates murine cardiogenesis by interacting with an nkx2.5 cardiac enhancer.
2014; 9 (12)
Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations.
View details for DOI 10.1371/journal.pone.0113775
View details for PubMedID 25436607
- Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening STEM CELL RESEARCH & THERAPY 2013; 4
Screening drug-induced arrhythmia events using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.
2013; 128 (11): S3-13
Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
View details for DOI 10.1161/CIRCULATIONAHA.112.000570
View details for PubMedID 24030418
Meta-Analysis of Stem Cell Therapy in Chronic Ischemic Cardiomyopathy
AMERICAN JOURNAL OF CARDIOLOGY
2013; 112 (2): 217-225
Studies investigating bone marrow stem cell therapy (BMSCT) in patients with chronic ischemic cardiomyopathy have yielded mixed results. A meta-analysis of randomized controlled trials of BMSCT in patients with chronic ischemic cardiomyopathy was undertaken to assess its efficacy and the best route of administration. The MEDLINE, Embase, Cumulative Index to Nursing & Allied Health Literature, and Cochrane Library databases were searched through April 2012 for randomized controlled trials that investigated the impact of BMSCT and its route of administration on left ventricular (LV) function in patients with chronic ischemic cardiomyopathy and systolic dysfunction. Of the 226 reports identified, 10 randomized controlled trials including 519 patients (average LV ejection fraction [LVEF] at baseline 32 ± 7%) were included in the analysis. On the basis of a random-effects model, BMSCT improved the LVEF at 6 months by 4.48% (95% confidence interval [CI] 2.43% to 6.53%, p = 0.0001). A greater improvement in the LVEF was seen with intramyocardial injection compared with intracoronary infusion (5.13% [95% CI 3.17% to 7.10%], p <0.00001, vs 2.32% [95% CI -2.06% to 6.70%], p = 0.30). Overall, there were reductions in LV end-systolic volume of -20.64 ml (95% CI -33.21 to -8.07, p = 0.001) and LV end-diastolic volume of -16.71 ml (95% CI -31.36 to -2.06, p = 0.03). In conclusion, stem cell therapy may improve LVEF and favorably remodel the heart in patients with chronic ischemic cardiomyopathy. On the basis of current limited data, intramyocardial injection may be superior to intracoronary infusion in patients with LV systolic dysfunction.
View details for DOI 10.1016/j.amjcard.2013.03.021
View details for Web of Science ID 000322206500013
View details for PubMedID 23623290
A83-01, a TGF beta RI inhibitor, can proliferate adult cardiac progenitor cells and improve cardiac contractility of myocardial infarcted mice
ACTA PHARMACOLOGICA SINICA
2013; 34: 57-57
View details for Web of Science ID 000322051500249
- Autophagy - the friendly fire in endothelial cell regeneration. Focus on "Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions" AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 2013; 304 (7): C614-C616
- At a Crossroad Cell Therapy for Cardiac Repair CIRCULATION RESEARCH 2013; 112 (6): 884-890
Essential and Unexpected Role of Yin Yang 1 to Promote Mesodermal Cardiac Differentiation
2013; 112 (6): 900-U104
Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective: To identify novel regulators of mesodermal cardiac lineage commitment.We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell-derived CPCs. From 32 candidate transcription factors screened, we found that Yin Yang 1 (YY1), a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays, in vivo chromatin immunoprecipitation, and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by embryonic stem cell-based assays in which we showed that the overexpression of YY1 enhanced the cardiogenic differentiation of embryonic stem cells into CPCs.These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.
View details for DOI 10.1161/CIRCRESAHA.113.259259
View details for Web of Science ID 000316189900007
View details for PubMedID 23307821
- Of fish and men: clonal lineage analysis identifies divergence in myocardial development. Circulation research 2013; 112 (4): 583-585
iPS Cell Modeling of Cardiometabolic Diseases
JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH
2013; 6 (1): 46-53
Cardiometabolic diseases encompass simple monogenic enzyme deficiencies with well-established pathogenesis and clinical outcomes to complex polygenic diseases such as the cardiometabolic syndrome. The limited availability of relevant human cell types such as cardiomyocytes has hampered our ability to adequately model and study pathways or drugs relevant to these diseases in the heart. The recent discovery of induced pluripotent stem (iPS) cell technology now offers a powerful opportunity to establish translational platforms for cardiac disease modeling, drug discovery, and pre-clinical testing. In this article, we discuss the excitement and challenges of modeling cardiometabolic diseases using iPS cell and their potential to revolutionize translational research.
View details for DOI 10.1007/s12265-012-9413-4
View details for Web of Science ID 000313657700006
View details for PubMedID 23070616
Early cardiac development: a view from stem cells to embryos
2012; 96 (3): 352-362
From the 1920s, early cardiac development has been studied in chick and, later, in mouse embryos in order to understand the first cell fate decisions that drive specification and determination of the endocardium, myocardium, and epicardium. More recently, mouse and human embryonic stem cells (ESCs) have demonstrated faithful recapitulation of early cardiogenesis and have contributed significantly to this research over the past few decades. Derived almost 15 years ago, human ESCs have provided a unique developmental model for understanding the genetic and epigenetic regulation of early human cardiogenesis. Here, we review the biological concepts underlying cell fate decisions during early cardiogenesis in model organisms and ESCs. We draw upon both pioneering and recent studies and highlight the continued role for in vitro stem cells in cardiac developmental biology.
View details for DOI 10.1093/cvr/cvs270
View details for Web of Science ID 000311306800006
View details for PubMedID 22893679
- Reprogramming the Beat Kicking It Up a Notch CIRCULATION 2012; 126 (9): 1009-1011
Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5
2012; 111 (1): 50-55
Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown.To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT).We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes.Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.
View details for DOI 10.1161/CIRCRESAHA.112.270264
View details for Web of Science ID 000306061700012
View details for PubMedID 22581928
Small molecule regulators of postnatal Nkx2.5 cardiomyoblast proliferation and differentiation
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
2012; 16 (5): 961-965
While recent data have supported the capacity for a neonatal heart to undergo cardiomyogenesis, it is unclear whether these new cardiomyocytes arise from an immature cardiomyoblast population or from the division of mature cardiomyocytes. By following the expression of enhanced Green Fluorescent Protein (eGFP) in an Nkx2.5 enhancer-eGFP transgenic mice, we have identified a population of immature cells that can undergo cardiomyogenic as well as smooth muscle cell differentiation in the neonatal heart. Here, we examined growth factors and small molecule regulators that potentially regulate the proliferation and cardiomyogenic versus smooth muscle cell differentiation of neonatal Nkx2.5-GFP (+) cells in vitro. We found that A83-01 (A83), an inhibitor of TGF-βRI, was able to induce an expansion of neonatal Nkx2.5-eGFP (+) cells. In addition, the ability of A83 to expand eGFP (+) cells in culture was dependent on signalling from the mitogen-activated protein kinase kinase (MEK) as treatment with a MEK inhibitor, PD0325901, abolished this effect. On the other hand, activation of neonatal Nkx2.5-eGFP (+) cells with TGF-β1, but not activin A nor BMP2, led to smooth muscle cell differentiation, an effect that can be reversed by treatment with A83. In summary, small molecule inhibition of TGF-β signalling may be a promising strategy to induce the expansion of a rare population of postnatal cardiomyoblasts.
View details for DOI 10.1111/j.1582-4934.2011.01513.x
View details for Web of Science ID 000303239500002
View details for PubMedID 22212626
- Putting the Pieces Together: Stem Cells and The Quest to Heal A Broken Heart CardioSource World News 2012; 12: 22-27
Induced pluripotent stem cell modeling of complex genetic diseases.
Drug discovery today. Disease models
2012; 9 (4): e147-e152
The study of complex disease genetics by genome-wide association studies (GWAS) has led to hundreds of genomic loci associated with disease traits in humans. However, the functional consequences of most loci are largely undefined. We discuss here the potential for human induced pluripotent stem (iPS) cells to bridge the gap between genetic variant and mechanisms of complex disease. We also highlight specific diseases and the roadblocks that must be overcome before iPS cell technology can be widely adopted for complex disease modeling.
View details for DOI 10.1016/j.ddmod.2012.04.002
View details for PubMedID 23690830
- A Brief Primer on the Development of the Heart Heart Failure, 2nd Ed. 2012: Chapter 1
Epigenetic mechanisms in cardiac development and disease
ACTA BIOCHIMICA ET BIOPHYSICA SINICA
2012; 44 (1): 92-102
During mammalian development, cardiac specification and ultimately lineage commitment to a specific cardiac cell type is accomplished by the action of specific transcription factors (TFs) and their meticulous control on an epigenetic level. In this review, we detail how cardiac-specific TFs function in concert with nucleosome remodeling and histone-modifying enzymes to regulate a diverse network of genes required for processes such as cell growth and proliferation, or epithelial to mesenchymal transition (EMT), for instance. We provide examples of how several cardiac TFs, such as Nkx2.5, WHSC1, Tbx5, and Tbx1, which are associated with developmental and congenital heart defects, are required for the recruitment of histone modifiers, such as Jarid2, p300, and Ash2l, and components of ATP-dependent remodeling enzymes like Brg1, Baf60c, and Baf180. Binding of these TFs to their respective sites at cardiac genes coincides with a distinct pattern of histone marks, indicating that the precise regulation of cardiac gene networks is orchestrated by interactions between TFs and epigenetic modifiers. Furthermore, we speculate that an epigenetic signature, comprised of TF occupancy, histone modifications, and overall chromatin organization, is an underlying mechanism that governs cardiac morphogenesis and disease.
View details for DOI 10.1093/abbs/gmr090
View details for Web of Science ID 000298386700010
View details for PubMedID 22194017
Reprogramming of mouse, rat, pig, and human fibroblasts into iPS cells.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2012; Chapter 23: Unit 23 15-?
The induction of pluripotency in somatic cells by transcription-factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically modified large animal models or potentially transplantable donor tissues or organs. This unit describes the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts.
View details for DOI 10.1002/0471142727.mb2315s97
View details for PubMedID 22237859
Developmental and Regenerative Biology of Multipotent Cardiovascular Progenitor Cells
2011; 108 (3): 353-364
Our limited ability to improve the survival of patients with heart failure is attributable, in part, to the inability of the mammalian heart to meaningfully regenerate itself. The recent identification of distinct families of multipotent cardiovascular progenitor cells from endogenous, as well as exogenous, sources, such as embryonic and induced pluripotent stem cells, has raised much hope that therapeutic manipulation of these cells may lead to regression of many forms of cardiovascular disease. Although the exact source and cell type remains to be clarified, our greater understanding of the scientific underpinning behind developmental cardiovascular progenitor cell biology has helped to clarify the origin and properties of diverse cells with putative cardiogenic potential. In this review, we highlight recent advances in the understanding of cardiovascular progenitor cell biology from embryogenesis to adulthood and their implications for therapeutic cardiac regeneration. We believe that a detailed understanding of cardiogenesis will inform future applications of cardiovascular progenitor cells in heart failure therapy and regenerative medicine.
View details for DOI 10.1161/CIRCRESAHA.110.227066
View details for Web of Science ID 000286930500012
View details for PubMedID 21293007
- Regenerative strategies for cardiac disease In: Stem Cells and Regenerative Medicine. Humana Press. 2011; 1: 579-593
Origin of Cardiac Progenitor Cells in the Developing and Postnatal Heart
JOURNAL OF CELLULAR PHYSIOLOGY
2010; 225 (2): 321-325
The mammalian heart lacks the capacity to replace the large numbers of cardiomyocytes lost due to cardiac injury. Several different cell-based routes to myocardial regeneration have been explored, including transplantation of cardiac progenitors and cardiomyocytes into injured myocardium. As seen with cell-based therapies in other solid organ systems, inherent limitations, such as host immune response, cell death and long-term graft instability have hampered meaningful cardiac regeneration. An understanding of the cell biology of cardiac progenitors, including their developmental origin, lineage markers, renewal pathways, differentiation triggers, microenvironmental niche, and mechanisms of homing and migration to the site of injury, will enable further refinement of therapeutic strategies to enhance clinically meaningful cardiac repair.
View details for DOI 10.1002/jcp.22281
View details for Web of Science ID 000283003400007
View details for PubMedID 20568226
Isolation and functional characterization of pluripotent stem cell-derived cardiac progenitor cells.
Current protocols in stem cell biology
2010; Chapter 1: Unit 1F 10-?
The use of transgenic markers in pluripotent stem cells allows the facile isolation of transient cell populations that appear at certain phases of embryonic development. Here, we describe a procedure for deriving cardiac progenitors from mouse pluripotent stem cells carrying a GFP reporter under the control of an Nkx2.5 enhancer sequence. The cells are propagated under standard conditions and are differentiated using the hanging-droplet method with medium optimized for commitment to the cardiac lineage. Cardiac progenitors are isolated from the differentiation culture using fluorescence-activated cell sorting (FACS) and can be cultured further for functional characterization and experimentation. The protocols described here can be applied to both embryonic and induced pluripotent stem cells and can easily be adapted to transgenic lines carrying other cardiac cell lineage reporters.
View details for DOI 10.1002/9780470151808.sc01f10s14
View details for PubMedID 20814937
Promises and pitfalls in cell replacement therapy for heart failure.
Drug discovery today. Disease mechanisms
2010; 7 (2): e109-e115
Symptomatic heart failure is a complex clinical syndrome with a poor prognosis. Many efforts have been made to develop new therapeutic strategies to improve prognosis associated with heart failure. In this context, different stem cell populations for cardiac regenerative therapy have been examined recently. Here we discuss the potential strategies for using stem cells in cardiac regenerative therapy and the barriers that remain before an effective cell-based cardiac regenerative therapy can be employed clinically.
View details for PubMedID 21180399
The integrative aspects of cardiac physiology and their implications for cell-based therapy
ANALYSIS OF CARDIAC DEVELOPMENT: FROM EMBRYO TO OLD AGE
2010; 1188: 7-14
Cardiac development is characterized by a complex interplay of chemical, mechanical, and electrical forces, which together contribute to the proper formation of the heart muscle. In adult myocardium, cardiomyocytes are elongated, well-coupled by gap junctions, and organized in spatially well-defined muscle fibers. This specific tissue architecture affects electromechanical activation and global cardiac function. Since the adult heart has only limited capacity for repair after injury, a significant loss of myocardial tissue often leads to impaired cardiac function. Recent efforts to transplant autologous cells to counteract this cardiomyocyte loss have resulted in marginal functional improvement and no evidence of myocyte regeneration. In order to achieve durable therapeutic efficiency, the transplanted cells will need to not only be cardiomyogenic, but also functionally integrate with host myocardial tissue and thereby contribute to both structural and functional restoration.
View details for DOI 10.1111/j.1749-6632.2009.05077.x
View details for Web of Science ID 000277731600002
View details for PubMedID 20201880
- Integrated aspects of cardiac cell therapy. Ann. N.Y. Acad. Sci. 2010; 1188: 7-14
- Cardiac progenitor cells: from embryonic to the aging heart. Aging Health 2010; 6 (6): 679-686
Myocardial Injury Induces the Expansion and Cardiomyogenic Differentiation of Postnatal Nkx2.5 Progenitor Cells via Inflammatory Signals
2009; 120 (18): S756-S756
View details for Web of Science ID 000271831502235
- VISIONS: the art of science. Molecular reproduction and development 2009; 76 (6): 525-?
Committed Ventricular Progenitors in the Islet-1 Lineage Expand and Assemble Into Functional Ventricular Heart Muscle
JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
2009; 53 (10): A468-A468
View details for Web of Science ID 000263864201940
- Platypnea-orthodeoxia syndrome in two previously healthy adults: a case-based review Clinical Medicine Insights: Cardiology 2009; 3: 37-43
Derivation and Functional Characterization of Nkx2.5+Cardiac Progenitor Cells from Mouse Induced Pluripotent Stem Cells
2008; 118 (18): S428-S428
View details for Web of Science ID 000262104500714
Mesp1 at the heart of mesoderm lineage specification
CELL STEM CELL
2008; 3 (1): 1-2
Stem cell-based cardiac regeneration requires a detailed understanding of the factors that induce cardiac lineage commitment. In this issue of Cell Stem Cell, Lindsley et al. (2008) and Bondue et al. (2008) use embryonic stem cells to identify a key role for Mesp1 in this process.
View details for DOI 10.1016/j.stem.2008.06.017
View details for Web of Science ID 000257622300001
View details for PubMedID 18593549
Multipotent stem cells in cardiac regenerative therapy
2008; 3 (2): 189-198
The potential for stem cells to ameliorate or cure heart diseases has galvanized a cadre of cardiovascular translational and clinical scientists to take a 'first-in-man' approach using autologous stem cells from a variety of tissues. However, recent clinical trial data show that when these cells are given by intracoronary infusion or direct myocardial injection, limited improvement in heart function occurs with no evidence of cardiomyogenesis. These studies illustrate the great need to understand the logic of cell-lineage commitment and the principles of cardiac differentiation. Recent identification of stem/progenitor cells of embryological origin with intrinsic competence to differentiate into multiple lineages within the heart offers new possibilities for cardiac regeneration. When combined with developments in nuclear reprogramming and provided that tumor risks and other challenges of embryonic cell transplantation can be overcome, the prospect of achieving autologous, cardiomyogenic, stem cell-based therapy may be within reach.
View details for DOI 10.2217/174607184.108.40.206
View details for Web of Science ID 000257995000013
View details for PubMedID 18307403
Cardiovascular Stem Cells in Regenerative Medicine: Ready for Prime Time?
Drug discovery today. Therapeutic strategies
2008; 5 (4): 201-207
Restoration of cardiovascular function is the ultimate goal of stem cell-based therapy. In principle, cardiovascular stem cells can improve cardiac function via de novo cardiomyogenesis, enhanced myocardial neovascularization, and prevention of post-infarct remodeling. Stem cell transplantation to improve cardiac function has received mixed results in human clinical trials. These early data suggest that a critical reassessment of the scientific basis to stem cell-based therapy is needed in order to bring this highly promising treatment modality to mainstream clinical care.
View details for PubMedID 20054428
alpha(2)-Macraglobulin from rheumatoid arthritis synovial fluid: Functional analysis defines a role for oxidation in inflammation
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
2001; 391 (1): 119-126
A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.
View details for Web of Science ID 000169700200015
View details for PubMedID 11414692
Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis
2001; 97 (11): 3450-3457
The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated alpha(2)M (alpha(2)M*) to bind to members of the FGF family. Both alpha(2)M and alpha(2)M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to alpha(2)M* is of significantly higher affinity. Neither alpha(2)M nor alpha(2)M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-beta binding site. The alpha(2)M* inhibits FGF-2-dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, alpha(2)M* does not affect FGF-2-induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase alpha(2)M* and solid-phase Matrigel or collagen. These studies suggest that the ability of alpha(2)M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components. (Blood. 2001;97:3450-3457)
View details for Web of Science ID 000168927900019
View details for PubMedID 11369636
The conformation-dependent interaction of alpha(2)-macroglobulin with vascular endothelial growth factor - A novel mechanism of alpha(2)-macroglobulin/growth factor binding
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (35): 26806-26811
alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).
View details for Web of Science ID 000089144800021
View details for PubMedID 10862607
- a-Macroglobulins/Kunins In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 4th Ed. 2000: 367-379
Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha(2)-Macroglobulin
1999; 38 (42): 13983-13990
The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.
View details for Web of Science ID 000083288400025
View details for PubMedID 10529245
Oxidized alpha(2)-macroglobulin (alpha(2)M) differentially regulates receptor binding by cytokines growth factors: Implications for tissue injury and repair mechanisms in inflammation
JOURNAL OF IMMUNOLOGY
1998; 161 (8): 4356-4365
Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.
View details for Web of Science ID 000076343300073
View details for PubMedID 9780213
The binding of receptor-recognized alpha(2)-macroglobulin to the low density lipoprotein receptor-related protein and the alpha(2)M signaling receptor is decoupled by oxidation
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (33): 20627-20635
Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.
View details for Web of Science ID A1997XR22100048
View details for PubMedID 9252378
- Crashing the Boards: A User Friendly Study Guide for the USMLE Step 1 Lippincott-Raven 1997; 1
Low-density lipoprotein receptor-related protein alpha(2)-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
1996; 326 (1): 39-47
Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.
View details for Web of Science ID A1996TT88500006
View details for PubMedID 8579370