Administrative Appointments

  • Director, Training program in Vascular Medicine and Biology (1991 - 2007)
  • Associate Director (Education and Training), Stanford Cardiovascular Institute (2007 - Present)

Honors & Awards

  • President, Society for Vascular Medicine (2005-2007)
  • "Best Doctors in the Bay Area", San Francisco Magazine (2003,2005)
  • Teaching Award, Dept of Medicine (2001)
  • Established Investigator Award, American Heart Association (1995)
  • Master of the Society for Vascular Medicine, Society for Vascular Medicine (2009)

Professional Education

  • PhD, Mayo Graduate School of Medicine, Physiology (1985)
  • MD, WSU School of Medicine, Medicine (1980)

Current Research and Scholarly Interests

I am now Chair of the Department of Cardiovascular Sciences in the Houston Methodist Research Institute (HMRI) in Houston, and the Director of the Center for Cardiovascular Regeneration in the Debakey Heart and Vascular Center. Our laboratory location is in the HMRI, a new (built in 2010) research facility (450,000 sq ft) housing a growing scientific community, with some outstanding investigators such as Neil Copeland and Nancy Jenkins, who are Nobel contenders for their work with the sleeping beauty transposon. I will be recruiting 13 faculty over the next 4 years to build the Department. It is going to be great fun, but I will miss this wonderful Stanford community. I will have some ongoing projects and collaborations here, that will keep me in touch with Stanford, facilitated by my new position as an Emeritus Professor at Stanford.

My Center for Cardiovascular Regeneration in Houston performs fundamental basic and translational work in cardiovascular regeneration from molecule to man. The goal is to transfer basic research insights into clinical trials using a vertically integrated approach with an array of biochemical and molecular tools, cellular and animal models, and clinical research techniques. Our mission is to to generate great ideas and transform cardiovascular care.

My own basic research is focused on induced pluripotent stem cells (iPSCs), as well as direct reprogramming, for vascular regeneration. We have discovered a critical role for activation of innate immune pathways in nuclear reprogramming to pluripotency, or in directed transdifferentiation. We have coined the term "transflammation" to describe the role of innate immune activation in epigenetic plasticity required for reprogramming (see Lee et al, CELL 2012).

We have a long-standing interest in two different pathways regulating endothelial function. Endothelium derived nitric oxide synthase(NOS) plays a critical role in EC survival, proliferation, and angiogenesis. There is an endogenous competitive inhibitor of the NO synthase pathway called ADMA (asymmetric dimethylarginine). We find that this molecule is elevated in disorders associated with endothelial dysfunction, and plays a significant role in causing vascular disease. ADMA becomes elevated in people with hypercholesterolemia, diabetes, and other vascular disorders. We find that oxidative stress impairs the activity of the enzyme (DDAH) that degrades ADMA. ADMA accumulates and blocks NO synthesis. Overexpression of DDAH (in our transgenic mouse or in endothelial cell culture) can reduce ADMA levels and increase NO synthesis, with significant consequences on vascular homeostasis and angiogenesis(Jacobi et al Circulation 2005).

We discovered a pathway modulating angiogenesis (Heeschen et al, Nature Medicine 2001). Nicotinic acetylcholine receptors on endothelial cells are upregulated with hypoxia, and when stimulated (by the endogenous transmitter acetylcholine), these receptors mediate endothelial tube formation in vitro, and angio

Clinical Trials

  • Exercise Therapy to Treat Adults With Abdominal Aortic Aneurysms Not Recruiting

    An abdominal aortic aneurysm (AAA) is a weakened and enlarged area in the abdominal aorta, which is a large blood vessel in the abdomen. If an AAA ruptures, it can be life-threatening. Research has shown that sedentary individuals are at increased risk of developing AAAs. This study will evaluate the effectiveness of an exercise program at limiting the growth of small AAAs in older individuals.

    Stanford is currently not accepting patients for this trial. For more information, please contact Ronald Dalman, (650) 723 - 2169.

    View full details

2023-24 Courses

Graduate and Fellowship Programs

All Publications

  • Therapeutic Transdifferentiation A Novel Approach for Vascular Disease CIRCULATION RESEARCH Cooke, J. P. 2013; 112 (5): 748-750
  • Multifunctional in vivo vascular imaging using near-infrared II fluorescence NATURE MEDICINE Hong, G., Lee, J. C., Robinson, J. T., Raaz, U., Xie, L., Huang, N. F., Cooke, J. P., Dai, H. 2012; 18 (12): 1841-?


    In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (∼30 μm) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1-3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.

    View details for DOI 10.1038/nm.2995

    View details for PubMedID 23160236

  • Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming CELL Lee, J., Sayed, N., Hunter, A., Au, K. F., Wong, W. H., Mocarski, E. S., Pera, R. R., Yakubov, E., Cooke, J. P. 2012; 151 (3): 547-558


    Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripotent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved exceedingly inefficient. We discovered a striking difference in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogramming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain- and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables efficient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory pathways are required for efficient nuclear reprogramming in the induction of pluripotency.

    View details for DOI 10.1016/j.cell.2012.09.034

    View details for PubMedID 23101625

  • Endothelial Cells Derived From Nuclear Reprogramming CIRCULATION RESEARCH Wong, W. T., Huang, N. F., Botham, C. M., Sayed, N., Cooke, J. P. 2012; 111 (10): 1363-1375


    The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health, the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell (EC) culture have been developed and refined. Recently, methods to derive ECs from pluripotent cells have extended the scientific range of human EC studies. Pluripotent stem cells may be generated, expanded, and then differentiated into ECs for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific ECs to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of ECs from stem cells; their characterization by genetic, histological, and functional studies; and their translational applications.

    View details for DOI 10.1161/CIRCRESAHA.111.247213

    View details for Web of Science ID 000310501300017

    View details for PubMedID 23104878

    View details for PubMedCentralID PMC3526979

  • Endothelial Cells Derived From Human iPSCS Increase Capillary Density and Improve Perfusion in a Mouse Model of Peripheral Arterial Disease ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Rufaihah, A. J., Huang, N. F., Jame, S., Lee, J. C., Nguyen, H. N., Byers, B., De, A., Okogbaa, J., Rollins, M., Reijo-Pera, R., Gambhir, S. S., Cooke, J. P. 2011; 31 (11): E72-U44


    Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease.Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×10(5)) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm(2)) (P<0.002).This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

    View details for DOI 10.1161/ATVBAHA.111.230938

    View details for PubMedID 21836062

  • DDAH Says NO to ADMA ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Cooke, J. P., Ghebremariam, Y. T. 2011; 31 (7): 1462-1464

    View details for DOI 10.1161/ATVBAHA.111.228833

    View details for Web of Science ID 000291656300003

    View details for PubMedID 21677286

  • Dietary nitrate, nitric oxide, and restenosis JOURNAL OF CLINICAL INVESTIGATION Cooke, J. P., Ghebremariam, Y. T. 2011; 121 (4): 1258-1260


    Endothelium-derived NO controls the contractility and growth state of the underlying vascular smooth muscle cells and regulates the interaction of the vessel wall with circulating blood elements. Acute injury of the vessel wall denudes the endothelial lining, removing homeostatic regulation and precipitating a wave of events leading to myointimal hyperplasia. In this issue of the JCI, Alef and colleagues provide evidence that in the injured vessel wall, the disruption of the NOS pathway is countered by induction of xanthine oxidoreductase, an enzyme capable of producing NO from nitrite. In addition, they link low dietary nitrite levels to increased severity of myointimal hyperplasia following vessel injury in mice.

    View details for DOI 10.1172/JCI57193

    View details for Web of Science ID 000289174600010

    View details for PubMedID 21436578

  • MicroRNA and Mechanisms of Impaired Angiogenesis in Diabetes Mellitus CIRCULATION Leeper, N. J., Cooke, J. P. 2011; 123 (3): 236-238
  • Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells STEM CELLS AND DEVELOPMENT Huang, N. F., Fleissner, F., Sun, J., Cooke, J. P. 2010; 19 (10): 1617-1625


    Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.

    View details for DOI 10.1089/scd.2009.0417

    View details for Web of Science ID 000282394400014

    View details for PubMedID 20064011

    View details for PubMedCentralID PMC3121801

  • Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. 2010; 30 (5): 984-U224


    We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.

    View details for DOI 10.1161/ATVBAHA.110.202796

    View details for PubMedID 20167654

  • Cholinergic Modulation of Angiogenesis: Role of the 7 Nicotinic Acetylcholine Receptor JOURNAL OF CELLULAR BIOCHEMISTRY Wu, J. C., Chruscinski, A., Perez, V. A., Singh, H., Pitsiouni, M., Rabinovitch, M., Utz, P. J., Cooke, J. P. 2009; 108 (2): 433-446


    Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except alpha(2), alpha(4), gamma, and delta in four different types of ECs. Using siRNA methodology, we found that the alpha(7) nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The alpha(9) and alpha(7) nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the alpha(7) nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.

    View details for DOI 10.1002/jcb.22270

    View details for Web of Science ID 000270438000012

    View details for PubMedID 19623583

    View details for PubMedCentralID PMC3140170

  • Embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia. Journal of visualized experiments : JoVE Huang, N. F., Niiyama, H., De, A., Gambhir, S. S., Cooke, J. P. 2009


    Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD. In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.

    View details for DOI 10.3791/1034

    View details for PubMedID 19229180

    View details for PubMedCentralID PMC2781824

  • Dimethylarginine dimethylaminohydrolase overexpression enhances insulin sensitivity ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Sydow, K., Mondon, C. E., Schrader, J., Konishi, H., Cooke, J. P. 2008; 28 (4): 692-697


    Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity.To test this hypothesis we used a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22 to 70 wk) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistance index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2 mumol/L) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle.These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity.

    View details for DOI 10.1161/ATVBAHA.108.162073

    View details for Web of Science ID 000254179400017

    View details for PubMedID 18239148

    View details for PubMedCentralID PMC3165027

  • A central role for nicotinic cholinergic regulation of growth factor-induced endothelial cell migration ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Ng, M. K., Wu, J., Chang, E., Wang, B., Katzenberg-Clark, R., Ishii-Watabe, A., Cooke, J. P. 2007; 27 (1): 106-112


    An endothelial nicotinic acetylcholine receptor (nAChR) participates in atherogenesis and tumorigenesis by promoting neovascularization. To date, the mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, eg, VEGF and bFGF, are unknown.Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/mL) but less than for VEGF (10 ng/mL). Unexpectedly, nAChR antagonism not only abolished nicotine-induced HMVEC migration but also abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens (nicotine, VEGF, and bFGF), a notable feature of which includes corepression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all 3 angiogens induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogen-induced migration while silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)-mediated induction of thioredoxin activity.Nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor-induced HMVEC migration, a key angiogenesis event, requires nAChR activation--an effect mediated in part by nAChR-dependent regulation of thioredoxin activity.

    View details for DOI 10.1161/01.ATV.0000251517.98396.4a

    View details for Web of Science ID 000243572000018

    View details for PubMedID 17082486

  • Dimethylarginine dimethylaminohydrolase overexpression suppresses graft coronary artery disease CIRCULATION Tanaka, M., Sydow, K., Gunawan, F., Jacobi, J., Tsao, P. S., Robbins, R. C., Cooke, J. P. 2005; 112 (11): 1549-1556


    Graft coronary artery disease (GCAD) is the leading cause of death after the first year of heart transplantation. The reduced bioavailability of endothelium-derived nitric oxide (NO) may play a role in endothelial vasodilator dysfunction and the structural changes that are characteristic of GCAD. A potential contributor to endothelial pathobiology is asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor. We hypothesized that lowering ADMA concentrations by dimethylarginine dimethylaminohydrolase (DDAH) overexpression in the recipient might suppress GCAD and long-term immune responses in murine cardiac allografts.In one series, donor hearts of C-H-2(bm12)KhEg (H-2(bm12)) wild-type (WT) mice were heterotopically transplanted into C57BL/6 (H-2b) transgenic mice overexpressing human DDAH-I or WT littermates and procured after 4 hours of reperfusion (WT and DDAH-I recipients, n=6 each). In a second series, donor hearts were transplanted into DDAH-I-transgenic or WT mice and procured 30 days after transplantation (n=7 each). In DDAH-I recipients, plasma ADMA concentrations were lower, in association with reduced myocardial generation of superoxide anion (WT versus DDAH-I, 465.7+/-79.8 versus 173.4+/-32.3 micromol.L(-1).mg(-1).h(-1); P=0.02), inflammatory cytokines, adhesion molecules, and chemokines. GCAD was markedly reduced in cardiac allografts of DDAH-I-transgenic recipients as assessed by luminal narrowing (WT versus DDAH, 79+/-2% versus 33+/-7%; P<0.01), intima-media ratio (WT versus DDAH, 1.1+/-0.1 versus 0.5+/-0.1; P<0.01), and the percentage of diseased vessels (WT versus DDAH, 100+/-0% versus 62+/-10%; P<0.01).Overexpression of DDAH-I attenuated oxidative stress, inflammatory cytokines, and GCAD in murine cardiac allografts. The effect of DDAH overexpression may be mediated by its reduction of plasma and tissue ADMA concentrations.

    View details for DOI 10.1161/CIRCULATIONAHA.105.537670

    View details for Web of Science ID 000231821200007

    View details for PubMedID 16144995

  • Overexpression of dimethylarginine dimethylaminohydrolase reduces tissue asymmetric dimethylarginine levels and enhances angiogenesis CIRCULATION Jacobi, J., Sydow, K., von Degenfeld, G., Zhang, Y., Dayoub, H., Wang, B. Y., Patterson, A. J., Kimoto, M., Blau, H. M., Cooke, J. P. 2005; 111 (11): 1431-1438


    This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA).In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N(omega)-nitro-L-arginine methyl ester (L-NAME; each 250 micromol x kg(-1) x d(-1)) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity.We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.

    View details for DOI 10.1161/01.CIR.0000158487.80483.09

    View details for Web of Science ID 000227805700012

    View details for PubMedID 15781754

  • Asymmetrical dimethylarginine - The Uber marker? CIRCULATION Cooke, J. P. 2004; 109 (15): 1813-1818
  • Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis - Genetic and physiological evidence CIRCULATION Dayoub, H., Achan, V., Adimoolam, S., Jacobi, J., Stuehlinger, M. C., Wang, B. Y., Tsao, P. S., Kimoto, M., Vallance, P., Patterson, A. J., Cooke, J. P. 2003; 108 (24): 3042-3047


    NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility. Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis.We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed approximately 2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (P<0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production.DDAH I overexpression increases NOS activity in vitro and in vivo. The hDDAH-1 transgenic animal exhibits a reduced systolic blood pressure, systemic vascular resistance, and cardiac stroke volume. This study provides compelling evidence that the elaboration and metabolism of endogenous ADMA plays an important role in regulation of NOS activity.

    View details for DOI 10.1161/01.CIR.0000101924.04515.2E

    View details for Web of Science ID 000187286300018

    View details for PubMedID 14638548

  • Impaired nitric oxide synthase pathway in diabetes mellitus - Role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase CIRCULATION LIN, K. Y., Ito, A., Asagami, T., Tsao, P. S., Adimoolam, S., Kimoto, M., Tsuji, H., Reaven, G. M., Cooke, J. P. 2002; 106 (8): 987-992


    An endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is elevated in patients with type 2 diabetes mellitus (DM). This study explored the mechanisms by which ADMA becomes elevated in DM.Male Sprague-Dawley rats were fed normal chow or high-fat diet (n=5 in each) with moderate streptozotocin injection to induce type 2 DM. Plasma ADMA was elevated in diabetic rats (1.33+/-0.31 versus 0.48+/-0.08 micromol/L; P<0.05). The activity, but not the expression, of dimethylarginine dimethylaminohydrolase (DDAH) was reduced in diabetic rats and negatively correlated with their plasma ADMA levels (P<0.05). DDAH activity was significantly reduced in vascular smooth muscle cells and human endothelial cells (HMEC-1) exposed to high glucose (25.5 mmol/L). The impairment of DDAH activity in vascular cells was associated with an accumulation of ADMA and a reduction in generation of cGMP. In human endothelial cells, coincubation with the antioxidant polyethylene glycol-conjugated superoxide dismutase (22 U/mL) reversed the effects of the high-glucose condition on DDAH activity, ADMA accumulation, and cGMP synthesis.A glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in DM.

    View details for DOI 10.1161/01.CIR.0000027109.14149.67

    View details for Web of Science ID 000177634600019

    View details for PubMedID 12186805

  • A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors JOURNAL OF CLINICAL INVESTIGATION Heeschen, C., Weis, M., Aicher, A., Dimmeler, S., Cooke, J. P. 2002; 110 (4): 527-536


    We have recently reported that nicotine has angiogenic effects, which appear to be mediated through non-neuronal nicotinic acetylcholine receptors (nAChRs). Here, we describe the endogenous cholinergic pathway for angiogenesis. In an in vitro angiogenesis model, increasing concentrations of the nonselective nAChR antagonist mecamylamine completely and reversibly inhibited endothelial network formation. Although several nAChR isoforms are expressed on endothelial cells (ECs), a similar inhibition was only obtained with the selective alpha7-nAChR antagonist alpha-bungarotoxin, whereas other selective antagonists did not result in significant inhibition of network formation. alpha7-nAChR was upregulated during proliferation, by hypoxia in vitro, and by ischemia in vivo. The nAChR-induced network formation was partially dependent on VEGF, was completely dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, and finally resulted in NF-kappaB activation. In vivo, pharmacological inhibition of nAChR as well as genetic disruption of alpha7-nAChR expression significantly inhibited inflammatory angiogenesis and reduced ischemia-induced angiogenesis and tumor growth. Our results suggest that nAChRs may play an important role in physiological and pathological angiogenesis. To our knowledge, this is the first description of a cholinergic angiogenic pathway, and it suggests a novel avenue for therapeutic modulation of angiogenesis.

    View details for DOI 10.1172/JCI200214676

    View details for Web of Science ID 000177557400015

    View details for PubMedID 12189247

    View details for PubMedCentralID PMC150415

  • Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis NATURE MEDICINE Heeschen, C., Jang, J. J., Weis, M., Pathak, A., Kaji, S., Hu, R. S., Tsao, P. S., Johnson, F. L., Cooke, J. P. 2001; 7 (7): 833-839


    We provide anatomic and functional evidence that nicotine induces angiogenesis. We also show that nicotine accelerates the growth of tumor and atheroma in association with increased neovascularization. Nicotine increased endothelial-cell growth and tube formation in vitro, and accelerated fibrovascular growth in vivo. In a mouse model of hind-limb ischemia, nicotine increased capillary and collateral growth, and enhanced tissue perfusion. In mouse models of lung cancer and atherosclerosis, we found that nicotine enhanced lesion growth in association with an increase in lesion vascularity. These effects of nicotine were mediated through nicotinic acetylcholine receptors at nicotine concentrations that are pathophysiologically relevant. The endothelial production of nitric oxide, prostacyclin and vascular endothelial growth factor might have a role in these effects.

    View details for Web of Science ID 000169808600036

    View details for PubMedID 11433349

  • Alternative Ankle-Brachial Index Method Identifies Additional At-Risk Individuals JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Nead, K. T., Cooke, J. P., Olin, J. W., Leeper, N. J. 2013; 62 (6): 553-559


    OBJECTIVES: To determine whether utilization of an alternative ankle-brachial index (ABI) calculation method improves mortality risk prediction compared to traditional methods. BACKGROUND: The ABI is used to diagnose peripheral arterial disease (PAD), and to identify those at risk for cardiovascular events. Traditionally, the ABI is calculated using the higher of the dorsalis pedis and posterior tibial ankle arteries. Studies directly comparing calculation methods are limited. METHODS: The ABI was calculated at baseline in 1,413 study participants undergoing non-emergent coronary angiography subsequently followed for all-cause and cardiovascular mortality. There were 224 individuals assigned to the traditional-PAD group (ABI < 0.90) using the traditional ABI method. Of those remaining, an alternative ABI method utilizing the lower of the two ankle pressures assigned 282 patients to the alternative-PAD group. The 862 individuals not assigned to PAD by either method were the no-PAD group. RESULTS: There were 163 mortalities during a median follow-up of 5.0 years. Adjusted Cox regression models showed that the alternative-PAD group had an increased risk for all-cause (HR=1.49; 95% CI, 1.01-2.19) and cardiovascular mortality (HR=3.21; 95% CI, 1.53-6.37) versus the no-PAD group. Additionally, in the no-PAD group, there was an 11% (HR=1.11; 95% CI, 1.05-1.17) increased risk of all-cause mortality per 1mm Hg increased difference between the left and right brachial systolic pressures. CONCLUSION: The implementation of an alternative ABI method and use of the brachial difference identifies individuals at an increased risk for mortality who are currently missed using traditional ABI methods. Current ABI protocols may need to be evaluated.

    View details for DOI 10.1016/j.jacc.2013.04.061

    View details for Web of Science ID 000322524300011

    View details for PubMedID 23707317

  • The modulation of endothelial cell morphology, function, and survival using anisotropic nanofibrillar collagen scaffolds BIOMATERIALS Huang, N. F., Okogbaa, J., Lee, J. C., Jha, A., Zaitseva, T. S., Paukshto, M. V., Sun, J. S., Punjya, N., Fuller, G. G., Cooke, J. P. 2013; 34 (16): 4038-4047


    Endothelial cells (ECs) are aligned longitudinally under laminar flow, whereas they are polygonal and poorly aligned in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote lesion formation. We demonstrate that the alignment of the ECs may directly influence their biology, independent of fluid flow. We developed aligned nanofibrillar collagen scaffolds that mimic the structure of collagen bundles in blood vessels, and examined the effects of these materials on EC alignment, function, and in vivo survival. ECs cultured on 30-nm diameter aligned fibrils re-organized their F-actin along the nanofibril direction, and were 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. After EC transplantation into both subcutaneous tissue and the ischemic hindlimb, EC viability was enhanced when ECs were cultured and implanted on aligned nanofibrillar scaffolds, in contrast to non-patterned scaffolds. ECs derived from human induced pluripotent stem cells and cultured on aligned scaffolds also persisted for over 28 days, as assessed by bioluminescence imaging, when implanted in ischemic tissue. By contrast, ECs implanted on scaffolds without nanopatterning generated no detectable bioluminescent signal by day 4 in either normal or ischemic tissues. We demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival after implantation in normal and ischemic tissues.

    View details for DOI 10.1016/j.biomaterials.2013.02.036

    View details for PubMedID 23480958

  • Walking impairment questionnaire improves mortality risk prediction models in a high-risk cohort independent of peripheral arterial disease status. Circulation. Cardiovascular quality and outcomes Nead, K. T., Zhou, M., Caceres, R. D., Olin, J. W., Cooke, J. P., Leeper, N. J. 2013; 6 (3): 255-261


    Background- The Walking Impairment Questionnaire (WIQ) is a subjective measure of patient-reported walking performance developed for peripheral arterial disease. The purpose of this study is to examine whether this simple tool can improve the predictive capacity of established risk models and whether the WIQ can be used in patients without peripheral arterial disease. Methods and Results- At baseline we assessed the walking distance, stair-climbing, and walking speed WIQ category scores among individuals who were undergoing coronary angiography. During a median follow-up of 5.0 years, there were 172 mortalities among 1417 study participants. Adjusted Cox proportional hazards models showed that all 3 WIQ categories independently predicted future all-cause and cardiovascular mortality, including among individuals without peripheral arterial disease (P<0.001). Compared with the cardiovascular risk factors model, we observed significantly increased risk discrimination with a C-index of 0.741 (change in C-index, 0.040; 95% confidence interval, 0.011-0.068) and 0.832 (change in C-index, 0.080; 95% confidence interval, 0.034-0.126) for all-cause and cardiovascular mortality, respectively. Examination of risk reclassification using the net reclassification improvement index showed a 48.4% (P<0.001) improvement for all-cause mortality and a 77.4% (P<0.001) improvement for cardiovascular mortality compared with the cardiovascular risk factors model. Conclusions- All 3 WIQ categories independently predicted future all-cause and cardiovascular mortality. Importantly, we found that this subjective measure of walking ability could be extended to patients without peripheral arterial disease. The addition of the WIQ scores to established cardiovascular risk models significantly improved risk discrimination and reclassification, suggesting broad clinical use for this simple, inexpensive test.

    View details for DOI 10.1161/CIRCOUTCOMES.111.000070

    View details for PubMedID 23633217

  • Spatial patterning of endothelium modulates cell morphology, adhesiveness and transcriptional signature BIOMATERIALS Huang, N. F., Lai, E. S., Ribeiro, A. J., Pan, S., Pruitt, B. L., Fuller, G. G., Cooke, J. P. 2013; 34 (12): 2928-2937


    Microscale and nanoscale structures can spatially pattern endothelial cells (ECs) into parallel-aligned organization, mimicking their cellular alignment in blood vessels exposed to laminar shear stress. However, the effects of spatial patterning on the function and global transcriptome of ECs are incompletely characterized. We used both parallel-aligned micropatterned and nanopatterned biomaterials to evaluate the effects of spatial patterning on the phenotype of ECs, based on gene expression profiling, functional characterization of monocyte adhesion, and quantification of cellular morphology. We demonstrate that both micropatterned and aligned nanofibrillar biomaterials could effectively guide EC organization along the direction of the micropatterned channels or nanofibrils, respectively. The ability of ECs to sense spatial patterning cues were abrogated in the presence of cytoskeletal disruption agents. Moreover, both micropatterned and aligned nanofibrillar substrates promoted an athero-resistant EC phenotype by reducing endothelial adhesiveness for monocytes and platelets, as well as by downregulating the expression of adhesion proteins and chemokines. We further found that micropatterned ECs have a transcriptional signature that is unique from non-patterned ECs, as well as from ECs aligned by shear stress. These findings highlight the importance of spatial patterning cues in guiding EC organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

    View details for DOI 10.1016/j.biomaterials.2013.01.017

    View details for Web of Science ID 000316038900008

    View details for PubMedID 23357369

    View details for PubMedCentralID PMC3581686

  • Usefulness of the Addition of Beta-2-Microglobulin, Cystatin C and C-Reactive Protein to an Established Risk Factors Model to Improve Mortality Risk Prediction in Patients Undergoing Coronary Angiography AMERICAN JOURNAL OF CARDIOLOGY Nead, K. T., Zhou, M. J., Caceres, R. D., Sharp, S. J., Wehner, M. R., Olin, J. W., Cooke, J. P., Leeper, N. J. 2013; 111 (6): 851-856


    Evidence-based therapies are available to reduce the risk for death from cardiovascular disease, yet many patients go untreated. Novel methods are needed to identify those at highest risk for cardiovascular death. In this study, the biomarkers β2-microglobulin, cystatin C, and C-reactive protein were measured at baseline in a cohort of participants who underwent coronary angiography. Adjusted Cox proportional-hazards models were used to determine whether the biomarkers predicted all-cause and cardiovascular mortality. Additionally, improvements in risk reclassification and discrimination were evaluated by calculating the net reclassification improvement, C-index, and integrated discrimination improvement with the addition of the biomarkers to a baseline model of risk factors for cardiovascular disease and death. During a median follow-up period of 5.6 years, there were 78 deaths among 470 participants. All biomarkers independently predicted future all-cause and cardiovascular mortality. A significant improvement in risk reclassification was observed for all-cause (net reclassification improvement 35.8%, p = 0.004) and cardiovascular (net reclassification improvement 61.9%, p = 0.008) mortality compared to the baseline risk factors model. Additionally, there was significantly increased risk discrimination with C-indexes of 0.777 (change in C-index 0.057, 95% confidence interval 0.016 to 0.097) and 0.826 (change in C-index 0.071, 95% confidence interval 0.010 to 0.133) for all-cause and cardiovascular mortality, respectively. Improvements in risk discrimination were further supported using the integrated discrimination improvement index. In conclusion, this study provides evidence that β2-microglobulin, cystatin C, and C-reactive protein predict mortality and improve risk reclassification and discrimination for a high-risk cohort of patients who undergo coronary angiography.

    View details for DOI 10.1016/j.amjcard.2012.11.055

    View details for Web of Science ID 000316537700013

    View details for PubMedID 23290308

    View details for PubMedCentralID PMC3594484

  • Exercise capacity is the strongest predictor of mortality in patients with peripheral arterial disease JOURNAL OF VASCULAR SURGERY Leeper, N. J., Myers, J., Zhou, M., Nead, K. T., Syed, A., Kojima, Y., Caceres, R. D., Cooke, J. P. 2013; 57 (3): 728-733


    The objective of this study was to assess the predictive value of clinical and exercise test variables in patients with peripheral arterial disease (PAD).A customized symptom-limited ramp treadmill protocol was used to assess 725 PAD patients referred for exercise testing at the Palo Alto Veterans Hospital between 1997 and 2011. Detailed clinical and exercise test data were collected at baseline, and patients were followed up for a mean of 11.3 ± 6.3 years.During follow-up, there were 364 deaths. Baseline exercise capacity was 7.0 ± 2.6 metabolic equivalents (METs) among survivors and 5.5 ± 2.4 METs in those who died (P < .001). Although several physiologic parameters differed between survivors and nonsurvivors, age-adjusted Cox regression revealed that exercise capacity was the strongest independent predictor of death. Each additional MET achieved was associated with age-adjusted 18% and 20% reductions in all-cause and cardiovascular mortality, respectively (P < .001 for both). This variable surpassed all classical risk factors (including smoking and history of congestive heart failure) and all measured exercise test responses (including symptoms and electrocardiograph abnormalities).Among PAD patients, reduced exercise capacity is the most powerful harbinger of long-term mortality. This factor has predictive power beyond traditional risk factors and confirms the critical importance of fitness in this cohort.

    View details for DOI 10.1016/j.jvs.2012.07.051

    View details for Web of Science ID 000315944400019

    View details for PubMedID 23044259

    View details for PubMedCentralID PMC3543469

  • Limited Gene Expression Variation in Human Embryonic Stem Cell and Induced Pluripotent Stem Cell-Derived Endothelial Cells STEM CELLS White, M. P., Rufaihah, A. J., Liu, L., Ghebremariam, Y. T., Ivey, K. N., Cooke, J. P., Srivastava, D. 2013; 31 (1): 92-103


    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized, and defined growth factors were used to generate KDR(+) EC progenitors. Magnetic purification of a KDR(+) progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders.

    View details for DOI 10.1002/stem.1267

    View details for Web of Science ID 000312561000010

    View details for PubMedID 23079999

    View details for PubMedCentralID PMC3528812

  • FXR agonist INT-747 upregulates DDAH expression and enhances insulin sensitivity in high-salt fed Dahl rats. PloS one Ghebremariam, Y. T., Yamada, K., Lee, J. C., Johnson, C. L., Atzler, D., Anderssohn, M., Agrawal, R., Higgins, J. P., Patterson, A. J., Böger, R. H., Cooke, J. P. 2013; 8 (4)


    Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.

    View details for DOI 10.1371/journal.pone.0060653

    View details for PubMedID 23593273

    View details for PubMedCentralID PMC3617194

  • The role of dimethylarginine dimethylaminohydrolase (DDAH) in pulmonary fibrosis JOURNAL OF PATHOLOGY Janssen, W., Pullamsetti, S. S., Cooke, J., Weissmann, N., Guenther, A., Schermuly, R. T. 2013; 229 (2): 242-249


    Pulmonary fibrosis is a devastating and progressive parenchymal lung disease with an extremely poor prognosis. Patients suffering from idiopathic pulmonary fibrosis (IPF) display a compromised lung function alongside pathophysiological features such as highly increased production of extracellular matrix, alveolar epithelial cell dysfunction, and disordered fibroproliferation - features that are due to a dysregulated response to alveolar injury. Under pathophysiological conditions of IPF, abnormally high concentrations of nitric oxide (NO) are found, likely a result of increased activity of the inducible nitric oxide synthase (NOS2), giving rise to products that contribute to fibrosis development. It is known that pharmacological inhibition or knockdown of NOS2 reduces pulmonary fibrosis, suggesting a role for NOS inhibitors in the treatment of fibrosis. Recent reports identified a critical enzyme, dimethylarginine dimethylaminohydrolase (DDAH), which is exceedingly active in patients suffering from IPF and in mice treated with bleomycin. An up-regulation of DDAH was observed in primary alveolar epithelial type II (ATII) cells from mice and patients with pulmonary fibrosis, where it co-localizes with NOS2. DDAH is a key enzyme that breaks down an endogenous inhibitor of NOS, asymmetric dimethylarginine (ADMA), by metabolizing it to l-citrulline and dimethylamine. DDAH was shown to modulate key fibrotic signalling cascades, and inhibition of this enzyme attenuated many features of the disease in in vivo experiments, suggesting a possible new therapeutic strategy for the treatment of patients suffering from IPF.

    View details for DOI 10.1002/path.4127

    View details for Web of Science ID 000312542400011

    View details for PubMedID 23097221

  • Human induced pluripotent stem cell-derived endothelial cells exhibit functional heterogeneity. American journal of translational research Rufaihah, A. J., Huang, N. F., Kim, J., Herold, J., Volz, K. S., Park, T. S., Lee, J. C., Zambidis, E. T., Reijo-Pera, R., Cooke, J. P. 2013; 5 (1): 21-35


    Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are promising for treatment of vascular diseases. However, hiPSC-ECs purified based on CD31 expression are comprised of arterial, venous, and lymphatic subtypes. It is unclear whether hiPSC-ECs are heterogeneous in nature, and whether there may be functional benefits of enriching for specific subtypes. Therefore, we sought to characterize the hiPSC-ECs and enrich for each subtype, and demonstrate whether such enrichment would have functional significance. The hiPSC-ECs were generated from differentiation of hiPSCs using vascular endothelial growth factor (VEGF)-A and bone morphogenetic protein-4. The hiPSC-ECs were purified based on positive expression of CD31. Subsequently, we sought to enrich for each subtype. Arterial hiPSC-ECs were induced using higher concentrations of VEGF-A and 8-bromoadenosine-3':5'-cyclic monophosphate in the media, whereas lower concentrations of VEGF-A favored venous subtype. VEGF-C and angiopoietin-1 promoted the expression of lymphatic phenotype. Upon FACS purification based on CD31+ expression, the hiPSC-EC population was observed to display typical endothelial surface markers and functions. However, the hiPSC-EC population was heterogeneous in that they displayed arterial, venous, and to a lesser degree, lymphatic lineage markers. Upon comparing vascular formation in matrigel plugs in vivo, we observed that arterial enriched hiPSC-ECs formed a more extensive capillary network in this model, by comparison to a heterogeneous population of hiPSC-ECs. This study demonstrates that FACS purification of CD31+ hiPSC-ECs produces a diverse population of ECs. Refining the differentiation methods can enrich for subtype-specific hiPSC-ECs with functional benefits of enhancing neovascularization.

    View details for PubMedID 23390563

  • PPAR delta Activation Protects Endothelial Function in Diabetic Mice DIABETES Tian, X. Y., Wong, W. T., Wang, N., Lu, Y., Cheang, W. S., Liu, J., Liu, L., Liu, Y., Lee, S. S., Chen, Z. Y., Cooke, J. P., Yao, X., Huang, Y. 2012; 61 (12): 3285-3293


    Recent evidence highlights the therapeutic potential of peroxisome proliferator-activated receptor-δ (PPARδ) agonists to increase insulin sensitivity in diabetes. However, the role of PPARδ in regulating vascular function is incompletely characterized. We investigate whether PPARδ activation improves endothelial function in diabetic and obese mice. PPARδ knockout (KO) and wild-type (WT) mice fed with high-fat diet and db/db mice were used as diabetic mouse models, compared with PPARδ KO and WT mice on normal diet and db/m(+) mice. Endothelium-dependent relaxation (EDR) was measured by wire myograph. Flow-mediated vasodilatation (FMD) was measured by pressure myograph. Nitric oxide (NO) production was examined in primary endothelial cells from mouse aortae. PPARδ agonist GW1516 restored EDRs in mouse aortae under high-glucose conditions or in db/db mouse aortae ex vivo. After oral treatment with GW1516, EDRs in aortae and FMDs in mesenteric resistance arteries were improved in obese mice in a PPARδ-specific manner. The effects of GW1516 on endothelial function were mediated through phosphatidylinositol 3-kinase (PI3K) and Akt with a subsequent increase of endothelial nitric oxide synthase (eNOS) activity and NO production. The current study demonstrates an endothelial-protective effect of PPARδ agonists in diabetic mice through PI3K/Akt/eNOS signaling, suggesting the therapeutic potential of PPARδ agonists for diabetic vasculopathy.

    View details for DOI 10.2337/db12-0117

    View details for Web of Science ID 000312041700036

    View details for PubMedID 22933110

    View details for PubMedCentralID PMC3501853

  • A validated biomarker panel to identify peripheral artery disease VASCULAR MEDICINE Hiatt, W. R., Zakharyan, A., Fung, E. T., Crutcher, G., Smith, A., Stanford, C., Cooke, J. 2012; 17 (6): 386-393


    Current guidelines recommend obtaining an ankle-brachial index (ABI) to screen for peripheral artery disease (PAD) in subjects at risk. Previous work demonstrated that a combination of β(2)-microglobulin, cystatin C, high-sensitivity C-reactive protein and glucose was associated with PAD. This study evaluated the ability of these biomarkers combined with clinical parameters to predict PAD in at-risk subjects. This study enrolled 1025 subjects from 99 primary care clinics who were smokers and/or diabetics ≥ 50 years or any individual ≥ 70 years. Consented subjects underwent a clinical assessment, fasting blood draw, and an ABI measurement with PAD defined as an ABI < 0.90 in either leg. The biomarkers and their interactions were evaluated using logistic regression and performance was evaluated at a cut point of the biomarker panel selected to maximize sensitivity while minimizing the false positive rate of the test. Of the 1025 subjects enrolled, 46 did not meet the ABI or other criteria for inclusion in the analysis. Among the evaluable subjects (n = 979), PAD was detected in 83 (8.5%). The model had a C-statistic of 0.73 (95% CI 0.67-0.79). There were 20 patients with PAD who were judged to be at low to moderate risk for cardiovascular events by clinical assessment; the model correctly identified 17 of these 20 patients. The model also performed well in subjects with no prior history of PAD. Thus, a biomarker panel may have a role for identifying PAD.

    View details for DOI 10.1177/1358863X12463491

    View details for Web of Science ID 000311516300003

    View details for PubMedID 23086582

  • Nicotine and pathological angiogenesis 3rd International Symposium on Non-Neuronal Acetylcholine Lee, J., Cooke, J. P. PERGAMON-ELSEVIER SCIENCE LTD. 2012: 1058–64


    This paper describes the role of endothelial nicotinic acetylcholine receptors (nAChR) in diseases where pathological angiogenesis plays a role. An extensive review of the literature was performed, focusing on studies that investigated the effect of nicotine upon angiogenesis. Nicotine induces pathological angiogenesis at clinically relevant concentrations (i.e. at tissue and plasma concentrations similar to those of a light to moderate smoker). Nicotine promotes endothelial cell migration, proliferation, survival, tube formation and nitric oxide (NO) production in vitro, mimicking the effect of other angiogenic growth factors. These in vitro findings indicate that there may be an angiogenic component to the pathophysiology of major tobacco related diseases such as carcinoma, atherosclerosis, and age-related macular degeneration. Indeed, nicotine stimulates pathological angiogenesis in pre-clinical models of these disorders. Subsequently, it has been demonstrated that nicotine stimulates nAChRs on the endothelium to induce angiogenic processes, that these nAChRs are largely of the α7 homomeric type, and that there are synergistic interactions between the nAChRs and angiogenic growth factor receptors at the phosphoproteomic and genomic levels. These findings are of potential clinical relevance, and provide mechanistic insights into tobacco-related disease. Furthermore, these findings may lead to novel therapies for diseases characterized by insufficient or inappropriate angiogenesis.

    View details for DOI 10.1016/j.lfs.2012.06.032

    View details for PubMedID 22796717

  • Aligned nanofibrillar collagen regulates endothelial organization and migration REGENERATIVE MEDICINE Lai, E. S., Huang, N. F., Cooke, J. P., Fuller, G. G. 2012; 7 (5): 649-661


    Modulating endothelial cell (EC) morphology and motility, with the aim to influence their biology, might be beneficial for the treatment of vascular disease. We examined the effect of nanoscale matrix anisotropy on EC organization and migration for vascular tissue engineering applications.We developed a flow processing technique to generate anisotropic nanofibrillar collagen. Human ECs were cultured on aligned or on randomly oriented collagen, and their cellular alignment and cytoskeletal organization were characterized by immunofluorescence staining and time-lapse microscopy.ECs were elongated along the direction of aligned collagen nanofibrils and had organized focal adhesions. Cellular protrusion migrated with greater directionality and higher velocity along the anisotropic nanofibrils compared with cells on random nanofibrils. The flow technique can be adapted to fabricate vascular grafts that support the endothelial phenotype.Aligned nanofibrillar collagen regulates EC organization and migration, which can significantly contribute to the development of vascular grafts.

    View details for DOI 10.2217/RME.12.48

    View details for Web of Science ID 000308387900011

    View details for PubMedID 22954436

    View details for PubMedCentralID PMC3589994

  • Genetics of Peripheral Artery Disease CIRCULATION Leeper, N. J., Kullo, I. J., Cooke, J. P. 2012; 125 (25): 3220-3228
  • Development of a Dimethylarginine Dimethylaminohydrolase (DDAH) Assay for High-Throughput Chemical Screening JOURNAL OF BIOMOLECULAR SCREENING Ghebremariam, Y. T., Erlanson, D. A., Yamada, K., Cooke, J. P. 2012; 17 (5): 651-661


    Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.

    View details for DOI 10.1177/1087057112441521

    View details for Web of Science ID 000304715800009

    View details for PubMedID 22460174

  • Endovascular correction of cerebrovenous anomalies in multiple sclerosis: A retrospective review of an uncontrolled case series VASCULAR MEDICINE Dake, M. D., Dantzker, N., Bennett, W. L., Cooke, J. P. 2012; 17 (3): 131-137


    Endovascular intervention for obstruction to venous drainage of the head and neck is an established treatment for disorders such as superior vena cava syndrome. Some patients with multiple sclerosis have been observed to have anomalies of the veins draining the head and neck. It is possible that some symptoms associated with multiple sclerosis may be secondary to disturbed venous flow. In an uncontrolled clinical series of 40 patients who had been previously diagnosed with multiple sclerosis, anomalies of the venous drainage of the head and neck were observed, including venous stenoses of the internal jugular veins. In 38 of 40 patients, venous stents were placed with restoration of luminal dimensions and abrogation of the venous pressure gradient. The angiographic and hemodynamic improvement was associated with improvement in symptomatology, most particularly in cognitive and constitutional symptoms that may be related to cerebrovenous flow. Serious complications included death in one subject and stent embolization requiring open heart surgery in another. In conclusion, in this series, endovascular intervention to correct venous stenosis associated with multiple sclerosis was associated with improvement in symptoms possibly related to disturbed venous hemodynamics. However, given the serious adverse events in this small series, a randomized clinical trial is required to confirm these findings, and to determine if the procedure has any effect on the progression of multiple sclerosis, or untoward long-term adverse effects.

    View details for DOI 10.1177/1358863X12440125

    View details for Web of Science ID 000304719900001

    View details for PubMedID 22496109

  • Genetic determinants of the ankle-brachial index: A meta-analysis of a cardiovascular candidate gene 50K SNP panel in the candidate gene association resource (CARe) consortium ATHEROSCLEROSIS Wassel, C. L., Lamina, C., Nambi, V., Coassin, S., Mukamal, K. J., Ganesh, S. K., Jacobs, D. R., Franceschini, N., Papanicolaou, G. J., Gibson, Q., Yanek, L. R., van der Harst, P., Ferguson, J. F., Crawford, D. C., Waite, L. L., Allison, M. A., Criqui, M. H., McDermott, M. M., Mehra, R., Cupples, L. A., Hwang, S., Redline, S., Kaplan, R. C., Heiss, G., Rotter, J. I., Boerwinkle, E., Taylor, H. A., Eraso, L. H., Haun, M., Li, M., Meisinger, C., O'Connell, J. R., Shuldineri, A. R., Tybjaerg-Hansen, A., Frikke-Schmidt, R., Kollerits, B., Rantner, B., Dieplinger, B., Stadler, M., Mueller, T., Haltmayer, M., Klein-Weigel, P., Summerer, M., Wichmann, H., Asselbergs, F. W., Navis, G., Leach, I. M., Brown-Gentry, K., Goodloe, R., Assimes, T. L., Becker, D. M., Cooke, J. P., Absher, D. M., Olin, J. W., Mitchell, B. D., Reilly, M. P., Mohler, E. R., North, K. E., Reiner, A. P., Kronenberg, F., Murabito, J. M. 2012; 222 (1): 138-147


    Candidate gene association studies for peripheral artery disease (PAD), including subclinical disease assessed with the ankle-brachial index (ABI), have been limited by the modest number of genes examined. We conducted a two stage meta-analysis of ∼50,000 SNPs across ∼2100 candidate genes to identify genetic variants for ABI.We studied subjects of European ancestry from 8 studies (n=21,547, 55% women, mean age 44-73 years) and African American ancestry from 5 studies (n=7267, 60% women, mean age 41-73 years) involved in the candidate gene association resource (CARe) consortium. In each ethnic group, additive genetic models were used (with each additional copy of the minor allele corresponding to the given beta) to test each SNP for association with continuous ABI (excluding ABI>1.40) and PAD (defined as ABI<0.90) using linear or logistic regression with adjustment for known PAD risk factors and population stratification. We then conducted a fixed-effects inverse-variance weighted meta-analyses considering a p<2×10(-6) to denote statistical significance.In the European ancestry discovery meta-analyses, rs2171209 in SYTL3 (β=-0.007, p=6.02×10(-7)) and rs290481 in TCF7L2 (β=-0.008, p=7.01×10(-7)) were significantly associated with ABI. None of the SNP associations for PAD were significant, though a SNP in CYP2B6 (p=4.99×10(-5)) was among the strongest associations. These 3 genes are linked to key PAD risk factors (lipoprotein(a), type 2 diabetes, and smoking behavior, respectively). We sought replication in 6 population-based and 3 clinical samples (n=15,440) for rs290481 and rs2171209. However, in the replication stage (rs2171209, p=0.75; rs290481, p=0.19) and in the combined discovery and replication analysis the SNP-ABI associations were no longer significant (rs2171209, p=1.14×10(-3); rs290481, p=8.88×10(-5)). In African Americans, none of the SNP associations for ABI or PAD achieved an experiment-wide level of significance.Genetic determinants of ABI and PAD remain elusive. Follow-up of these preliminary findings may uncover important biology given the known gene-risk factor associations. New and more powerful approaches to PAD gene discovery are warranted.

    View details for DOI 10.1016/j.atherosclerosis.2012.01.039

    View details for Web of Science ID 000302960600022

    View details for PubMedID 22361517

    View details for PubMedCentralID PMC3596171

  • Imaging Vascular Nicotine Receptors A New Window Onto Vascular Disease JACC-CARDIOVASCULAR IMAGING Cooke, J. P. 2012; 5 (5): 537-539

    View details for DOI 10.1016/j.jcmg.2012.03.004

    View details for Web of Science ID 000304675600009

    View details for PubMedID 22595162

  • Development of pluripotent stem cells for vascular therapy VASCULAR PHARMACOLOGY Volz, K. S., Miljan, E., Khoo, A., Cooke, J. P. 2012; 56 (5-6): 288-296


    Peripheral arterial disease (PAD) is characterized by reduced limb blood flow due to arterial obstruction. Current treatment includes surgical or endovascular procedures, the failure of which may result in amputation of the affected limb. An emerging therapeutic approach is cell therapy to enhance angiogenesis and tissue survival. Small clinical trials of adult progenitor cell therapies have generated promising results, although large randomized clinical trials using well-defined cells have not been performed. Intriguing pre-clinical studies have been performed using vascular cells derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSCs). In particular, hiPSC-derived vascular cells may be a superior approach for vascular regeneration. The regulatory roadmap to the clinic will be arduous, but achievable with further understanding of the reprogramming and differentiation processes; with meticulous attention to quality control; and perseverance.

    View details for DOI 10.1016/j.vph.2012.02.010

    View details for Web of Science ID 000306889400014

    View details for PubMedID 22387745

    View details for PubMedCentralID PMC3595194

  • Lymphangiogenesis A Potential New Therapy for Lymphedema? CIRCULATION Cooke, J. P. 2012; 125 (7): 853-855
  • Oxidative Stress-Dependent Cyclooxygenase-2-Derived Prostaglandin F-2 alpha Impairs Endothelial Function in Renovascular Hypertensive Rats ANTIOXIDANTS & REDOX SIGNALING Tian, X. Y., Wong, W. T., Leung, F. P., Zhang, Y., Wang, Y., Lee, H. K., Ng, C. F., Chen, Z. Y., Yao, X., Au, C. L., Lau, C. W., Vanhoutte, P. M., Cooke, J. P., Huang, Y. 2012; 16 (4): 363-373


    Abstract Aims: The role of endothelium-derived contracting factors (EDCFs) in regulating renovascular function is yet to be elucidated in renovascular hypertension (RH). The current study investigated whether oxidative stress-dependent cyclooxygenase (COX)-2-derived prostaglandin F(2α) (PGF(2α)) impairs endothelial function in renal arteries of renovascular hypertensive rats (RHR). Results: Renal hypertension was induced in rats by renal artery stenosis of both kidneys using the 2-kidney 2-clip model. Acute treatment with reactive oxygen species (ROS) scavengers, COX-2 inhibitors, and thromboxane-prostanoid receptor antagonists, but not COX-1 inhibitors, improved endothelium-dependent relaxations and eliminated endothelium-dependent contractions in RHR renal arteries. Five weeks of treatment with celecoxib or tempol reduced blood pressure, increased renal blood flow, and restored endothelial function in RHRs. Increased ROS production in RHR arteries was inhibited by ROS scavengers, but unaffected by COX-2 inhibitors; whereas increased PGF(2α) release was reduced by both ROS scavengers and COX-2 inhibitors. ROS also induced COX-2-dependent contraction in RHR renal arteries, which was accompanied by the release of COX-2-derived PGF(2α). Further, chronic tempol treatment reduced COX-2 and BMP4 upregulation, p38MAPK phosphorylation, and the nitrotyrosine level in RHR renal arteries. Conclusion: These findings demonstrate the functional importance of oxidative stress, which serves as an initiator of increased COX-2 activity, and that COX-2-derived PGF(2α) plays an important role in mediating endothelial dysfunction in RH. Innovation: The current study, thus, suggests that drugs targeting oxidative stress-dependent COX-2-derived PGF(2α) may be useful in the prevention and management of RH. Antioxid. Redox Signal. 16, 363-373.

    View details for DOI 10.1089/ars.2010.3874

    View details for Web of Science ID 000298814600007

    View details for PubMedID 21951274

    View details for PubMedCentralID PMC3584508

  • In Memoriam of John T. Shepherd, MD, DSc CIRCULATION Vanhoutte, P. M., Cooke, J. P., Cohen, R. a. 2012; 125 (2): 393-394
  • Bioluminescence Imaging of Stem Cell-Based Therapeutics for Vascular Regeneration THERANOSTICS Huang, N. F., Okogbaa, J., Babakhanyan, A., Cooke, J. P. 2012; 2 (4): 346-354


    Stem cell-based therapeutics show promise for treatment of vascular diseases. However, the survival of the cells after in vivo injection into diseased tissues remains a concern. In the advent of non-invasive optical imaging techniques such as bioluminescence imaging (BLI), cell localization and survival can be easily monitored over time. This approach has recently been applied towards monitoring stem cell treatments for vascular regeneration of the coronary or peripheral arteries. In this review, we will describe the application of BLI for tracking transplanted stem cells and associating their viability with therapeutic efficacy, in preclinical disease models of vascular disease.

    View details for DOI 10.7150/thno.3694

    View details for Web of Science ID 000304031200003

    View details for PubMedID 22509198

    View details for PubMedCentralID PMC3326722

  • Solubility partner IF2 Domain I enables high yield synthesis of transducible transcription factors in Escherichia coli PROTEIN EXPRESSION AND PURIFICATION Yang, W. C., Welsh, J. P., Lee, J., Cooke, J. P., Swartz, J. R. 2011; 80 (1): 145-151


    Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a certain set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. However, the search for an efficient reprogramming approach utilizing the transcription factors in protein form requires a significant amount of protein material. Milligram quantities of transcription factors are challenging to obtain due to low yields and poor solubility. In this work, we describe enhanced production of the pluripotency transcription factors Oct4, Sox2, Klf4, Nanog, and Lin28 after fusing them to a solubility partner, IF2 Domain I (IF2D1). We expressed and purified milligram quantities of the fusion proteins. Though the transcription factor passenger proteins became insoluble after removal of the IF2D1, the un-cleaved Oct4, Sox2, Klf4, and Nanog fusion proteins exhibited specific binding to their consensus DNA sequences. However, when we administered the un-cleaved IF2D1-Oct4-R9 and IF2D1-Sox2-R9 to fibroblasts and measured their ability to influence transcriptional activity, we found that they were not fully bioactive; IF2D1-Oct4-R9 and IF2D1-Sox2-R9 influenced only a subset of their downstream gene targets. Thus, while the IF2D1 solubility partner enabled soluble production of the fusion protein at high levels, it did not yield fully bioactive transcription factors.

    View details for DOI 10.1016/j.pep.2011.06.017

    View details for PubMedID 21757009

  • Two Decades of Progress in Vascular Medicine AMERICAN JOURNAL OF MEDICINE Leeper, N. J., Lee, J. T., Cooke, J. P. 2011; 124 (9): 791-792

    View details for DOI 10.1016/j.amjmed.2011.03.017

    View details for PubMedID 21683936

  • Meal-induced increases in C-reactive protein, interleukin-6 and tumour necrosis factor alpha are attenuated by prandial plus basal insulin in patients with Type 2 diabetes DIABETIC MEDICINE Beisswenger, P. J., Brown, W. V., Ceriello, A., Le, N. A., Goldberg, R. B., Cooke, J. P., Robbins, D. C., Sarwat, S., Yuan, H., Jones, C. A., Tan, M. H. 2011; 28 (9): 1088-1095


    To determine if a regimen with prandial + basal insulin compared with basal insulin attenuates post-meal inflammatory and glycative biomarkers in patients with Type 2 diabetes.This test-meal sub-study in the USA is from a previously reported clinical trial comparing the effect on glycaemic control of 24 weeks of thrice-daily pre-meal insulin lispro mix 50 (50% insulin lispro, 50% insulin lispro protamine suspension) or bedtime insulin glargine, both plus metformin. In the sub-study, glucose, insulin, triglycerides, high-sensitivity C-reactive protein, tumour necrosis factor α, interleukin-6, methylglyoxal and 3-deoxyglucosone were measured during the post-meal period of a mixed-meal breakfast at the final visit. Prandial + basal (n = 25) and basal (n = 21) insulin were administered at the same times as during the previous 24 weeks.Post-meal, the prandial + basal insulin group had significantly higher insulin, lower glucose and triglycerides, as well as lower high-sensitivity C-reactive protein, tumour necrosis factor α and interleukin-6, than the basal insulin group. Glucose incremental area under the concentration curve significantly correlated with high-sensitivity C-reactive protein, tumour necrosis factor α, interleukin-6, methylglyoxal and 3-deoxyglucosone incremental area under the concentration curve. Insulin incremental area under the concentration curve correlated inversely with high-sensitivity C-reactive protein and tumour necrosis factor α incremental area under the concentration curve. However, after adjusting for glucose incremental area under the concentration curve, these inverse correlations were no longer significant. Triglyceride incremental area under the concentration curve was not correlated with any biomarker incremental area under the concentration curve.Controlling post-meal hyperglycaemia with prandial + basal insulin in patients with Type 2 diabetes attenuates meal-induced increases in high-sensitivity C-reactive protein, interleukin-6 and tumour necrosis factor α compared with basal insulin. The rise in post-meal glucose, but not triglycerides, significantly correlated with the rise in post-meal inflammatory and glycative biomarkers.

    View details for DOI 10.1111/j.1464-5491.2011.03324.x

    View details for Web of Science ID 000293905000014

    View details for PubMedID 21517955

  • Low lifetime recreational activity is a risk factor for peripheral arterial disease JOURNAL OF VASCULAR SURGERY Wilson, A. M., Sadrzadeh-Rafie, A. H., Myers, J., Assimes, T., Nead, K. T., Higgins, M., Gabriel, A., Olin, J., Cooke, J. P. 2011; 54 (2): 427-432


    The relationship between lifetime physical activity and the risk of developing peripheral arterial disease (PAD) is not known.We studied 1381 patients referred for elective coronary angiography in a point prevalence analysis. PAD was defined as ankle-brachial index (ABI) <0.9 at the time or a history of revascularization of the lower extremities regardless of ABI measure. We used a validated physical activity questionnaire to retrospectively measure each patient's lifetime recreational activity (LRA). Multivariate and logistic regression analyses were used to assess the independent association of LRA to ABI and the presence of PAD.PAD was present in 19% (n = 258) of all subjects. Subjects reporting no regular LRA had greater diastolic blood pressure and were more likely to be female. They had lower average ABI, and a higher proportion had PAD (25.6%). In a regression model, including traditional risk factors and LRA, multivariate analysis showed that age (P < .001), female gender (P < .001), systolic blood pressure (P = .014), fasting glucose (P < .001), serum triglycerides (P = .02), and cumulative pack years (P < .001) were independent negative predictors of ABI, and LRA was a positive predictor of ABI (P < .001). History of sedentary lifestyle independently increased the odds ratio for PAD (odds ratio, 0.46; 95% confidence interval, 1.01-2.10) when assessed by logistic regression. Intriguingly, there is a correlation between physical activity and gender, such that women with low LRA are at greatest risk.Recalled LRA is positively correlated to ABI and associated with PAD. Whereas the mechanism for this effect is not clear, LRA may be a useful clinical screening tool for PAD risk, and strategies to increase adult recreational activity may reduce the burden of PAD later in life.

    View details for DOI 10.1016/j.jvs.2011.02.052

    View details for Web of Science ID 000293814400025

    View details for PubMedID 21664093

    View details for PubMedCentralID PMC3152670

  • Increased nitric oxide availability attenuates high fat diet metabolic alterations and gene expression associated with insulin resistance CARDIOVASCULAR DIABETOLOGY Razny, U., Kiec-Wilk, B., Wator, L., Polus, A., Dyduch, G., Solnica, B., Malecki, M., Tomaszewska, R., Cooke, J. P., Dembinska-Kiec, A. 2011; 10


    High fat diet impairs nitric oxide (NO) bioavailability, and induces insulin resistance. The link between NO availability and the metabolic adaptation to a high fat diet is not well characterized. The purpose of this study was to investigate the effect of high fat diet on metabolism in mice with decreased (eNOS-/-) and increased (DDAH overexpressed) NO bioavailability.eNOS-/- (n = 16), DDAH (n = 24), and WT (n = 19) mice were fed a high fat diet (HFD) for 13 weeks. Body weight, biochemical parameters, adipokines and insulin were monitored. The matrigel in vivo model with CD31 immunostaining was used to assess angiogenesis. Gene expression in adipose tissues was analyzed by microarray and Real Time PCR. Comparisons of the mean values were made using the unpaired Student t test and p < 0.05 were considered statistically significant.eNOS-/- mice gained less weight than control WT and DDAH mice. In DDAH mice, a greater increase in serum adiponectin and a lesser increment in glucose level was observed. Fasting insulin and cholesterol levels remained unchanged. The angiogenic response was increased in DDAH mice. In adipose tissue of DDAH mice, genes characteristic of differentiated adipocytes were down-regulated, whereas in eNOS-/- mice, genes associated with adipogenesis, fatty acid and triglyceride synthesis were upregulated.Our results indicate that increased NO availability attenuates some HFD induced alterations in metabolism and gene expression associated with insulin resistance.

    View details for DOI 10.1186/1475-2840-10-68

    View details for Web of Science ID 000293516900001

    View details for PubMedID 21781316

  • Identification and Classification of Acute Cardiac Rejection by Intragraft Transcriptional Profiling CIRCULATION Holweg, C. T., Potena, L., Luikart, H., Yu, T., Berry, G. J., Cooke, J. P., Valantine, H. A., Mocarski, E. S. 2011; 123 (20): 2236-U154


    Treatment of acute rejection (AR) in heart transplantation relies on histopathological grading of endomyocardial biopsies according to International Society for Heart and Lung Transplantation guidelines. Intragraft gene expression profiling may be a way to complement histological evaluation.Transcriptional profiling was performed on 26 endomyocardial biopsies, and expression patterns were compared with the 1990 International Society for Heart and Lung Transplantation AR grades. Importantly, transcriptional profiles from settings with an equivalent AR grade appeared the same. In addition, grade 0 profiles could not be distinguished from 1A profiles, and grade 3A profiles could not be distinguished from 3B profiles. Comparing the AR groupings (0+1A, 1B, and 3A+3B), 0+1A showed more striking differences from 1B than from 3A+3B. When these findings were extrapolated to the 2005 revised guidelines, the combination of 1A and 1B into a single category (1R) appears to have brought together endomyocardial biopsies with different underlying processes that are not evident from histological evaluation. Grade 1B was associated with upregulated immune response genes, as 1 categorical distinction from grade 1A. Although grade 1B was distinct from the clinically relevant AR grades 3A and 3B, all of these grades shared a small number of overlapping pathways consistent with common physiological underpinnings.The gene expression similarities and differences identified here in different AR settings have the potential to revise the clinical perspective on acute graft rejection, pending the results of larger studies.

    View details for DOI 10.1161/CIRCULATIONAHA.109.913921

    View details for Web of Science ID 000290852200018

    View details for PubMedID 21555702

    View details for PubMedCentralID PMC3115694

  • The role of nicotine in the pathogenesis of atherosclerosis ATHEROSCLEROSIS Lee, J., Cooke, J. P. 2011; 215 (2): 281-283
  • A matrix micropatterning platform for cell localization and stem cell fate determination ACTA BIOMATERIALIA Huang, N. F., Patlolla, B., Abilez, O., Sharma, H., Rajadas, J., Beygui, R. E., Zarins, C. K., Cooke, J. P. 2010; 6 (12): 4614-4621


    To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.

    View details for DOI 10.1016/j.actbio.2010.06.033

    View details for Web of Science ID 000284385300018

    View details for PubMedID 20601236

    View details for PubMedCentralID PMC2957527

  • Sex differences in the prevalence of peripheral artery disease in patients undergoing coronary catheterization VASCULAR MEDICINE Rafie, A. H., Stefanick, M. L., Sims, S. T., Phan, T., Higgins, M., Gabriel, A., Assimes, T., Narasimhan, B., Nead, K. T., Myers, J., Olin, J., Cooke, J. P. 2010; 15 (6): 443-450


    To determine whether there are sex differences in the prevalence of peripheral artery disease, we performed an observational study of 1014 men and 547 women, aged ≥ 40 years, referred for elective coronary angiography. Women were slightly older, more obese, had higher low-density lipoprotein cholesterol (LDL-C) levels and systolic blood pressure (BP), and were more likely to be African American. Women had higher high-density lipoprotein cholesterol (HDL-C) levels, lower diastolic BP, and were less likely to smoke or to have a history of cardiovascular disease. Women had less prevalent (62% vs 81%) and less severe coronary artery disease (CAD) (p < 0.001 for both) by coronary angiography, but more prevalent peripheral artery disease (PAD) as determined by the ankle-brachial index (ABI) than men (23.6% versus 17.2%). Independent predictors of lower ABI were female sex, black race, older age, tobacco use, CAD, diabetes, and triglyceride level. In a full multivariable logistic regression model, women had a risk-adjusted odds ratio for PAD of 1.78 (95% CI 1.25-2.54) relative to men. Among patients referred for coronary angiography, women have less prevalent and less severe CAD, but more prevalent PAD, a sex difference that is not explained by traditional cardiovascular disease risk factors or CAD severity. Clinical Trial Registration-URL: Unique identifier: NCT00380185.

    View details for DOI 10.1177/1358863X10388345

    View details for Web of Science ID 000285574400002

    View details for PubMedID 21183651

  • The Emerging Role of the Thioredoxin System in Angiogenesis ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Dunn, L. L., Buckle, A. M., Cooke, J. P., Ng, M. K. 2010; 30 (11): 2089-2098


    Although there have been a multitude of studies, the mechanisms of angiogenesis remain incompletely understood. Increasing evidence suggests that cellular redox homeostasis is an important regulator of angiogenesis. The thioredoxin (TRX) system functions as an endogenous antioxidant that can exert influence over endothelial cell function via modulation of cellular redox status. It has become apparent that the cytosolic TRX1 isoform participates in both canonical and novel angiogenic signaling pathways and may represent an avenue for therapeutic exploitation. Recent studies have further identified a role for the mitochondrial isoform TRX2 in ischemia-induced angiogenesis. TRX-interacting protein (TXNIP) is the endogenous inhibitor of TRX redox activity that has been implicated in growth factor-mediated angiogenesis. As TXNIP is strongly induced by glucose, this molecule could be of consequence to disordered angiogenesis manifest in diabetes mellitus. This review will focus on data implicating the TRX system in endothelial cell homeostasis and angiogenesis.

    View details for DOI 10.1161/ATVBAHA.110.209643

    View details for Web of Science ID 000283234800630

    View details for PubMedID 20798378

  • Cholinergic activation of hematopoietic stem cells: role in tobacco-related disease? VASCULAR MEDICINE Chang, E., Forsberg, E. C., Wu, J., Wang, B., Prohaska, S. S., Allsopp, R., Weissman, I. L., Cooke, J. P. 2010; 15 (5): 375-385


    Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

    View details for DOI 10.1177/1358863X10378377

    View details for Web of Science ID 000282582300004

    View details for PubMedID 20926497

    View details for PubMedCentralID PMC3110740

  • Stem Cell Therapy for Vascular Regeneration Adult, Embryonic, and Induced Pluripotent Stem Cells CIRCULATION Leeper, N. J., Hunter, A. L., Cooke, J. P. 2010; 122 (5): 517-526
  • Asymmetric dimethylarginine correlates with measures of disease severity, major adverse cardiovascular events and all-cause mortality in patients with peripheral arterial disease VASCULAR MEDICINE Wilson, A. M., Shin, D. S., Weatherby, C., Harada, R. K., Ng, M. K., Nair, N., Kielstein, J., Cooke, J. P. 2010; 15 (4): 267-274


    Peripheral arterial disease (PAD) is associated with major cardiovascular morbidity and mortality. Abnormalities in nitric oxide metabolism due to excess of the NO synthase inhibitor asymmetric dimethylarginine (ADMA) may be pathogenic in PAD. We explored the association between ADMA levels and markers of atherosclerosis, function, and prognosis. A total of 133 patients with symptomatic PAD were enrolled. Ankle-brachial index (ABI), walking time, vascular function measures (arterial compliance and flow-mediated vasodilatation) and plasma ADMA level were assessed for each patient at baseline. ADMA correlated inversely with ABI (r = -0.238, p = 0.003) and walking time (r = -0.255, p = 0.001), independent of other vascular risk factors. We followed up 125 (94%) of our 133 initial subjects with baseline measurements (mean 35 months). Subjects with ADMA levels in the highest quartile (> 0.84 mumol/l) showed a significantly greater occurrence of a major adverse cardiovascular event (MACE) compared to those with ADMA levels in the lower three quartiles (p = 0.001). Cox proportional-hazards regression analysis revealed that ADMA was a significant predictor of MACE, independent of other risk factors including age, sex, blood pressure, smoking history, diabetes and ABI (hazard ratio = 5.1, p < 0.001). Measures of vascular function, such as compliance, flow-mediated vasodilatation (FMVD) and blood pressure, as well as markers of PAD severity, including ABI and walking time, were not predictive. In conclusion, circulating levels of ADMA correlate independently with measures of disease severity and major adverse cardiovascular events. Agents that target this pathway may be useful for this patient population. Clinical Trial Registration - URL: http:// Unique identifier: NCT00284076.

    View details for DOI 10.1177/1358863X10364552

    View details for Web of Science ID 000281080700003

    View details for PubMedID 20484311

    View details for PubMedCentralID PMC3131178

  • Relationship of Asymmetric Dimethylarginine and Homocysteine to Vascular Aging in Systemic Lupus Erythematosus Patients ARTHRITIS AND RHEUMATISM Perna, M., Roman, M. J., Alpert, D. R., Crow, M. K., Lockshin, M. D., Sammaritano, L., Devereux, R. B., Cooke, J. P., Salmon, J. E. 2010; 62 (6): 1718-1722


    Systemic lupus erythematosus (SLE) is independently associated with accelerated atherosclerosis and premature arterial stiffening. Asymmetric dimethylarginine (ADMA) and homocysteine are mechanistically interrelated mediators of endothelial dysfunction and correlates of atherosclerosis in the general population. The aim of this study was to assess the relationship of ADMA and homocysteine to subclinical vascular disease in patients with SLE.One hundred twenty-five patients with SLE who were participating in a study of cardiovascular disease underwent clinical and laboratory assessment, carotid artery ultrasonography to detect atherosclerosis, and radial artery applanation tonometry to measure arterial stiffness.Neither ADMA nor homocysteine correlated with the presence or extent of carotid atherosclerosis. In contrast, ADMA was significantly related to the arterial stiffness index. Independent correlates of arterial stiffening included the ADMA concentration, the presence of diabetes mellitus, older age at the time of diagnosis, longer disease duration, and the absence of anti-Sm or anti-RNP antibodies. A secondary multivariable analysis substituting homocysteine for ADMA demonstrated comparable relationships with arterial stiffness (r(2) = 0.616 for homocysteine and r(2) = 0.595 for ADMA).ADMA and homocysteine are biomarkers for and may be mediators of premature arterial stiffening in patients with SLE. Because arterial stiffness has independent prognostic value for cardiovascular morbidity and mortality, its predictors may identify patients who are at increased risk of cardiovascular disease.

    View details for DOI 10.1002/art.27392

    View details for Web of Science ID 000279432500022

    View details for PubMedID 20155836

  • DDAH: A target for vascular therapy? VASCULAR MEDICINE Cooke, J. P. 2010; 15 (3): 235-238

    View details for DOI 10.1177/1358863X10362605

    View details for Web of Science ID 000277807800010

    View details for PubMedID 20385710

    View details for PubMedCentralID PMC3133479

  • Biomarkers of Peripheral Arterial Disease JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Cooke, J. P., Wilson, A. M. 2010; 55 (19): 2017-2023


    Atherosclerotic arterial occlusive disease affecting the lower extremities is also known as peripheral artery disease (PAD). This disorder affects 8 to 12 million individuals in the U.S. and is increasingly prevalent in Europe and Asia. Unfortunately, most patients are not diagnosed and are not optimally treated. A blood test for PAD, if sufficiently sensitive and specific, would be expected to improve recognition and treatment of these individuals. Even a biomarker panel of moderate sensitivity and specificity for PAD could refine risk stratification to select individuals for diagnostic vascular examination. Alternatively, biomarkers for PAD may be useful in determining prognosis, the risk for progression, or the response to therapy. Finally, the discovery of biomarkers associated with PAD may provide novel insights into the pathophysiology of PAD and new therapeutic avenues to pursue. Biomarkers may be derived from studies of the genome, transcriptome, proteome, or metabolome. The focus of this review is on proteomic biomarkers associated with PAD.

    View details for DOI 10.1016/j.jacc.2009.08.090

    View details for Web of Science ID 000277303100002

    View details for PubMedID 20447524

    View details for PubMedCentralID PMC3157958

  • Topical Mecamylamine for Diabetic Macular Edema AMERICAN JOURNAL OF OPHTHALMOLOGY Campochiaro, P. A., Shah, S. M., Hafiz, G., Heier, J. S., Lit, E. S., Zimmer-Galler, I., Channa, R., Nguyen, Q. D., Syed, B., Do, D. V., Lu, L., Monk, J., Cooke, J. P., Kengatharan, M. K., Hsu, H. H. 2010; 149 (5): 839-851


    Stimulation of nicotinic acetylcholine (nACh) receptors on vascular endothelial cells promotes angiogenesis and vascular permeability in animal models. The safety and bioactivity of topical mecamylamine, an antagonist of nACh receptors, was tested in patients with diabetic macular edema.A multicenter phase I/II clinical trial.Twenty-three patients with chronic diabetic macular edema received 1% mecamylamine topically twice daily for 12 weeks, the primary end point. Patients underwent safety assessments, measurement of best-corrected visual acuity (BCVA), and measurement of foveal thickness using optical coherence tomography at baseline, 1, 4, 8, 12, and 16 weeks.Mecamylamine drops were well tolerated and there were no drug-related safety problems. Mean improvement in BCVA at 1, 4, 8, 12, and 16 weeks was 2.8, 1.9, 2.4, 0.8, and 3.1 letters, respectively. There was little change in mean excess foveal thickness. There was substantial heterogeneity in response, because 8 patients showed convincing improvement in BCVA, foveal thickness, or both, 9 patients showed equivocal or no substantial changes, and 4 patients showed worsening. Five patients showed a substantial improvement in BCVA, foveal thickness, or both between their last visit while receiving mecamylamine and 1 month after stopping mecamylamine.This study suggested that administration of topical mecamylamine, a nonspecific nACh receptor blocker, may have heterogeneous effects in patients with diabetic macular edema. Variable expression of nACh receptor subtypes on endothelial cells that have different effects on permeability would provide an explanation for these results and should be investigated, because more specific nACh receptor blockers may dissociate antipermeability and propermeability effects.

    View details for DOI 10.1016/j.ajo.2009.12.005

    View details for Web of Science ID 000277493800021

    View details for PubMedID 20189159

    View details for PubMedCentralID PMC3143041

  • Overexpression of Dimethylarginine Dimethylaminohydrolase Protects Against Cerebral Vascular Effects of Hyperhomocysteinemia CIRCULATION RESEARCH Rodionov, R. N., Dayoub, H., Lynch, C. M., Wilson, K. M., Stevens, J. W., Murry, D. J., Kimoto, M., Arning, E., Bottiglieri, T., Cooke, J. P., Baumbach, G. L., Faraci, F. M., Lentz, S. R. 2010; 106 (3): 551-U65


    Hyperhomocysteinemia is a cardiovascular risk factor that is associated with elevation of the nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA).Using mice transgenic for overexpression of the ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1), we tested the hypothesis that overexpression of DDAH1 protects from adverse structural and functional changes in cerebral arterioles in hyperhomocysteinemia.Hyperhomocysteinemia was induced in DDAH1 transgenic (DDAH1 Tg) mice and wild-type littermates using a high methionine/low folate (HM/LF) diet. Plasma total homocysteine was elevated approximately 3-fold in both wild-type and DDAH1 Tg mice fed the HM/LF diet compared with the control diet (P<0.001). Plasma ADMA was approximately 40% lower in DDAH1 Tg mice compared with wild-type mice (P<0.001) irrespective of diet. Compared with the control diet, the HM/LF diet diminished endothelium-dependent dilation to 10 micromol/L acetylcholine in cerebral arterioles of both wild-type (12 + or - 2 versus 29 + or - 3%; P<0.001) and DDAH1 Tg (14 + or - 3 versus 28 + or - 2%; P<0.001) mice. Responses to 10 micromol/L papaverine, a direct smooth muscle dilator, were impaired with the HM/LF diet in wild-type mice (30 + or - 3 versus 45 + or - 5%; P<0.05) but not DDAH1 Tg mice (45 + or - 7 versus 48 + or - 6%). DDAH1 Tg mice also were protected from hypertrophy of cerebral arterioles (P<0.05) but not from accelerated carotid artery thrombosis induced by the HM/LF diet.Overexpression of DDAH1 protects from hyperhomocysteinemia-induced alterations in cerebral arteriolar structure and vascular muscle function.

    View details for DOI 10.1161/CIRCRESAHA.109.200360

    View details for Web of Science ID 000274651400018

    View details for PubMedID 20019334

    View details for PubMedCentralID PMC2831416

  • Chronic exposure to nicotine impairs cholinergic angiogenesis VASCULAR MEDICINE Konishi, H., Wu, J., Cooke, J. P. 2010; 15 (1): 47-54


    Cholinergic angiogenesis is mediated by an endothelial nicotinic acetylcholine receptor (EC nAChR). Short-term administration of nicotine stimulates angiogenesis via EC nAChRs.The long-term effects of nicotine upon cholinergic angiogenesis are unknown. The objective of this study was to determine whether chronic nicotine exposure blunts angiogenesis. We exposed C57/Bl6 male mice (n = 42) to nicotine (200 microg/ml drinking water) or vehicle for 8 or 16 weeks. Subsequently, hindlimb ischemia was induced by ligation of the left femoral artery. After surgery, animals in the vehicle-treated group were re-randomized to vehicle (vehicle group) or nicotine (acute exposure group) for 2 weeks; whereas animals that had been previously treated (for 8 or 16 weeks with nicotine) continued to receive nicotine (8 WK or 16 WK groups). After 2 weeks, animals were sacrificed for immunohistochemical, gene expression, and angiogenesis studies. Capillary density of the ischemic hindlimb was increased by nicotine in naïve animals (vehicle vs acute exposure: 2.40 +/- 0.09 vs 2.82 +/- 0.10 capillaries/myocyte, p < 0.05). However, prior exposure to nicotine for 16 weeks (16 WK) abolished the effects of nicotine to increase capillary density in the ischemic hindlimb (acute vs 16 WK: 2.82 +/- 0.10 vs 2.47 +/- 0.03 capillaries/ myocyte; p < 0.05). The impairment of cholinergic angiogenesis was associated with a reduction in nAChR expression and plasma VEGF levels. Chronic exposure to nicotine impaired capillary sprouting of aortic segments ex vivo (vehicle vs 16 WK: 0.303 +/- 0.029 vs 0.204 +/- 0.017 mm(2), p < 0.05, n = 3 in each group). In conclusion, the current study shows for the first time that chronic exposure to nicotine impairs cholinergic angiogenesis, an effect mediated by downregulation of the vascular nAChR, and attenuation of nicotine-induced VEGF release. These studies may explain the impairment in angiogenic processes observed in long-term smokers.

    View details for DOI 10.1177/1358863X09106326

    View details for Web of Science ID 000273768800008

    View details for PubMedID 19778953

    View details for PubMedCentralID PMC3142172

  • Assessing endothelial vasodilator function with the Endo-PAT 2000. Journal of visualized experiments : JoVE Axtell, A. L., Gomari, F. A., Cooke, J. P. 2010


    The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction. In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT (peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response. In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

    View details for DOI 10.3791/2167

    View details for PubMedID 20972417

    View details for PubMedCentralID PMC3143035

  • Cell-Free Production of Transducible Transcription Factors for Nuclear Reprogramming BIOTECHNOLOGY AND BIOENGINEERING Yang, W. C., Patel, K. G., Lee, J., Ghebremariam, Y. T., Wong, H. E., Cooke, J. P., Swartz, J. R. 2009; 104 (6): 1047-1058


    Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target.

    View details for DOI 10.1002/bit.22517

    View details for PubMedID 19718703

  • Does improving mood in depressed patients alter factors that may affect cardiovascular disease risk? JOURNAL OF PSYCHIATRIC RESEARCH Taylor, C. B., Conrad, A., Wilhelm, F. H., Strachowski, D., Khaylis, A., Neri, E., Giese-Davis, J., Roth, W. T., Cooke, J. P., Kraemer, H., Spiegel, D. 2009; 43 (16): 1246-1252


    To determine if improvement in mood would ameliorate autonomic dysregulation, HPA dysfunction, typical risk factors and C-reactive protein in depressed patients with elevated cardiovascular disease risk (CVD), 48 depressed participants with elevated cardiovascular risk factors were randomized to a cognitive behavioral intervention (CBT) or a waiting list control (WLC) condition. Twenty non-depressed age and risk-matched controls were also recruited. Traditional risk factors (e.g., lipids, blood pressure) and C-reactive protein were assessed pre- and post-treatment six months later. Subjects also underwent a psychophysiological stress test while cardiovascular physiology was measured. Salivary cortisol was measured during the day and during the psychological stress test. At post-treatment, the CBT subjects were significantly less depressed than WLC subjects. There was no significant difference in change scores on any of the traditional risk factors or C-reactive protein, cortisol measures, or cardiovascular physiology, except for triglyceride levels and heart rate, which were significantly lower in treatment compared to control subjects. The normal controls exhibited no change in the variables measured during the same time. A significant improvement in mood may have little impact on most traditional or atypical risk factors, cortisol or cardiophysiology.

    View details for DOI 10.1016/j.jpsychires.2009.05.006

    View details for Web of Science ID 000272860300002

    View details for PubMedID 19577757

    View details for PubMedCentralID PMC2783820

  • Extensive characterization of the human DDAH1 transgenic mice PHARMACOLOGICAL RESEARCH Schwedhelm, E., von Leitner, E., Atzler, D., Schmitz, C., Jacobi, J., Meinertz, T., Muenzel, T., Baldus, S., Cooke, J. P., Boeger, R. H., Maas, R., Sydow, K. 2009; 60 (6): 494-502


    Overexpression of the human dimethylarginine dimethylaminohydrolase type 1 (hDDAH1) gene was reported to have beneficial cardiovascular effects in mice. To date, it is unclear whether these effects are related to enhanced metabolic clearance of asymmetric dimethylarginine (ADMA) and l-N(G)-mono-methyl-l-arginine (l-NMMA) or increased DDAH1 expression and activity in cardiovascular tissues of hDDAH1 transgenic mice.DDAH activity (DDAH1+DDAH2) was found to be markedly increased in aortic and heart tissues but unaltered in liver and kidney tissues of hDDAH1 transgenic as compared to wild-type (WT) mice. In WT mice, DDAH activity was more abundant in liver and kidney as compared to aorta and heart, suggesting a possible ceiling effect of activity which was unsurpassed by hDDAH1 overexpression.Overexpression of hDDAH1 in healthy mice does not result in an improved DDAH-metabolic capacity of kidney and liver under normal, i.e. unchallenged conditions. The most likely explanation for low ADMA and l-NMMA concentrations in hDDAH1 transgenic mice is a decreased release of ADMA from aorta, heart, and possibly other organs. The protective cardiovascular effects seen in these animals may therefore be related to an improved activity of the DDAH enzyme in the cardiovascular system and not be related to improved renal and hepatic clearance of ADMA and l-NMMA.

    View details for DOI 10.1016/j.phrs.2009.08.001

    View details for Web of Science ID 000271797500008

    View details for PubMedID 19666120

  • nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation, Apoptosis, and Angiogenesis PLOS ONE Yu, J., Huang, N. F., Wilson, K. D., Velotta, J. B., Huang, M., Li, Z., Lee, A., Robbins, R. C., Cooke, J. P., Wu, J. C. 2009; 4 (9)


    Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%), CD31 (97.2+/-2.5%), and VE-cadherin (93.7+/-2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways.

    View details for DOI 10.1371/journal.pone.0007040

    View details for Web of Science ID 000269796500020

    View details for PubMedID 19753305

    View details for PubMedCentralID PMC2737633

  • Hypercholesterolemia impairs exercise capacity in mice VASCULAR MEDICINE Maxwell, A. J., Niebauer, J., Lin, P. S., Tsao, P. S., Bernstein, D., Cooke, J. P. 2009; 14 (3): 249-257


    We previously reported an attenuation of both exercise hyperemia and measures of aerobic capacity in hypercholesterolemic mice. In this study, we expanded upon the previous findings by examining the temporal and quantitative relationship of hypercholesterolemia to aerobic and anaerobic capacity and by exploring several potential mechanisms of dysfunction. Eight-week-old wild type (n = 123) and apoE knockout (n = 79) C57BL/6J mice were divided into groups with distinct cholesterol levels by feeding with regular or high-fat diets. At various ages, the mice underwent treadmill ergospirometry. To explore mechanisms, aortic ring vasodilator function and nitrate (NO(x)) activity, urinary excretion of NO(x), running muscle microvascular density and citrate synthase activity, as well as myocardial mass and histologic evidence of ischemia were measured. At 8 weeks of age, all mice had similar measures of exercise capacity. All indices of aerobic exercise capacity progressively declined at 12 and 20 weeks of age in the hypercholesterolemic mice as cholesterol levels increased while indices of anaerobic capacity remained unaffected. Across the four cholesterol groups, the degree of aerobic dysfunction was related to serum cholesterol levels; a relationship that was maintained after correcting for confounding factors. Associated with the deterioration in exercise capacity was a decline in measures of nitric oxide-mediated vascular function while there was no evidence of aberrations in functional or oxidative capacities or in other components of transport capacity. In conclusion, aerobic exercise dysfunction is observed in murine models of genetic and diet-induced hypercholesterolemia and is associated with a reduction in vascular nitric oxide production.

    View details for DOI 10.1177/1358863X08100040

    View details for Web of Science ID 000268568300008

    View details for PubMedID 19651675

    View details for PubMedCentralID PMC3140166

  • Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-beta-catenin and Wnt-RhoA-Rac1 pathways JOURNAL OF CELL BIOLOGY Perez, V. A., Alastalo, T., Wu, J. C., Axelrod, J. D., Cooke, J. P., Amieva, M., Rabinovitch, M. 2009; 184 (1): 83-99


    Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-beta, induces beta-catenin (beta-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA-Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both beta-C- and Dvl-mediated RhoA-Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2-mediated angiogenesis.

    View details for DOI 10.1083/jcb.200806049

    View details for Web of Science ID 000262867000010

    View details for PubMedID 19139264

    View details for PubMedCentralID PMC2615088

  • Murine model of hindlimb ischemia. Journal of visualized experiments : JoVE Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. 2009


    In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.

    View details for DOI 10.3791/1035

    View details for PubMedID 19229179

    View details for PubMedCentralID PMC2763292

  • Endothelial Nicotinic Acetylcholine Receptors and Angiogenesis TRENDS IN CARDIOVASCULAR MEDICINE Cooke, J. P., Ghebremariam, Y. T. 2008; 18 (7): 247-253


    Nicotinic acetylcholine receptors (nAChRs) were first described in non-excitable cells just more than a decade ago. The nAChRs on endothelial cells modulate key angiogenic processes, including endothelial cell survival, proliferation, and migration. The receptors may be stimulated by endogenous agonists such as acetylcholine, or exogenous chemicals such as nicotine, to activate physiologic angiogenesis (such as in wound healing) or pathologic angiogenesis (such as retinal neovascularization or tumor angiogenesis). The endothelial nAChRs may represent a target for therapeutic modulation of disorders characterized by insufficient or pathologic angiogenesis.

    View details for Web of Science ID 000264060100003

    View details for PubMedID 19232953

  • Asymmetric dimethylarginine and cardiac allograft vasculopathy progression: Modulation by sirolimus TRANSPLANTATION Potena, L., Fearon, W. F., Sydow, K., Holweg, C., Luikart, H., Chin, C., Weisshaar, D., Mocarski, E. S., Lewis, D. B., Valantine, H. A., Cooke, J. P. 2008; 85 (6): 827-833


    Cardiac allograft vasculopathy (CAV) is a major cause of death after heart transplantation (HT). The reduced bioavailability of endothelium-derived nitric oxide may play a role in endothelial vasodilator dysfunction and thus in the structural changes characterizing CAV. A potential contributor to endothelial pathobiology is asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor. It was hypothesized that ADMA concentrations may influence CAV progression during the first postoperative year.Thirty-two consecutive HT recipients underwent intravascular ultrasound evaluation at month 1 and year 1 after HT. Immunosuppression included mycophenolate mofetil (MMF, n=16) and sirolimus (n=16). Change in intimal volume greater than the median and vascular remodeling were major outcome measures.Plasma ADMA levels were associated with subsequent development of intimal hyperplasia (risk ratio [95% confidence interval] =2.72 [1.06-6.94]; P=0.038), and plasma ADMA levels greater than 0.70 micromol/L most accurately identified patients who would have developed intimal hyperplasia. However, ADMA levels did not correlate with negative coronary remodeling. Treatment with sirolimus, as compared with MMF, was associated with significantly lower ADMA levels (0.65+/-0.12 vs. 0.77+/-0.10 micromol/L; P<0.01) and less intimal hyperplasia (risk ratio [95% confidence interval] = 0.08 [0.01-0.56]; P=0.01).Elevated plasma ADMA is associated with coronary intimal hyperplasia, supporting the importance of nitric oxide synthase inhibition in CAV pathogenesis. Treatment with sirolimus (rather than MMF) is associated with lower ADMA levels and reduced risk of accelerated CAV.

    View details for DOI 10.1097/TP.0b013e318166a3a4

    View details for PubMedID 18360263

  • Overexpression of dimethylarginine dimethylaminohydrolase inhibits asymmetric dimethylarginine-induced endothelial dysfunction in the cerebral circulation STROKE Dayoub, H., Rodionov, R., Lynch, C., Cooke, J. P., Arning, E., Bottiglieri, T., Lentz, S. R., Faraci, F. M. 2008; 39 (1): 180-184


    Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). An elevation of plasma ADMA levels is associated with cardiovascular disease. ADMA is hydrolyzed by dimethylarginine dimethylaminohydrolases (DDAHs). The goal of this study was to determine whether overexpression of human DDAH-1 in transgenic (DDAH-1-Tg) mice inhibits the vascular effects of ADMA.Using nontransgenic (non-Tg) and DDAH-1-Tg mice, we compared responses of the carotid artery and aorta (in vitro) and of the cerebral arterioles (in vivo) in the absence or presence of ADMA. DDAH-1 expression and plasma levels of ADMA were also measured.Western blotting indicated that vascular expression of DDAH-1 was increased markedly in DDAH-1-Tg mice. Plasma levels of ADMA were reduced by approximately 50% in DDAH-1-Tg mice compared with non-Tg mice (0.19+/-0.02 vs 0.37+/-0.04 micromol/L, P<0.05). Contraction of the aorta to nitro-l-arginine methyl ester (an inhibitor of NOS), an index of basal production of NO, was increased in DDAH-1-Tg mice compared with controls (50+/-4% vs 34+/-4%, P<0.05). Relaxation of the carotid artery to acetylcholine (an endothelium-dependent agonist) was enhanced in DDAH-1-Tg animals compared with control mice (relaxation of 74+/-6% vs 59+/-5%, respectively, in response to 10 micromol/L acetylcholine, P<0.05). ADMA (100 micromol/L) impaired the vascular response to acetylcholine in both non-Tg and DDAH-1-Tg mice, but the relative difference between the 2 strains remained. Responses to the endothelium-independent NO donor nitroprusside were similar in all groups. In vivo, ADMA (10 micromol/L) reduced responses of the cerebral arterioles to acetylcholine by approximately 70% in non-Tg mice (P<0.05), and this inhibitory effect was largely absent in DDAH-1-Tg mice.These findings provide the first evidence that overexpression of DDAH-1 increases basal levels of vascular NO and protects against ADMA-induced endothelial dysfunction in the cerebral circulation.

    View details for DOI 10.1161/STROKEAHA.107.490631

    View details for Web of Science ID 000251924600030

    View details for PubMedID 18063827

  • Two cardiovascular risk factors in one? Homocysteine and its relation to glomerular filtration rate KIDNEY & BLOOD PRESSURE RESEARCH Kielstein, J. T., Salpeter, S. R., Buckley, N. S., Cooke, J. P., Fliser, D. 2008; 31 (4): 259-267


    Hyperhomocysteinemia is thought to be an independent risk factor for cardiovascular disease, but the association between renal dysfunction and homocysteine may not have been fully taken into account. We performed a meta-analysis of studies that report correlations between glomerular filtration rate (GFR) and homocysteine plasma levels.Using a prespecified research strategy, we identified 41 studies involving 26,617 participants that reported Pearson or Spearman correlation coefficients for the association between 1/GFR and homocysteine. The summary correlation coefficients with 95% CI were obtained by pooling the logarithmic Z values derived from the individual trial correlation coefficients. Subgroup analysis was performed to compare results for measured GFR using clearance methods and various estimates of GFR.The pooled correlation coefficient between homocysteine and 1/GFR was 0.37 (CI 0.32-0.40, p < 0.0001). The correlation coefficient based on various estimates of GFR was 0.33 (CI 0.29-0.38, p < 0.0001), and for measured GFR it was 0.45 (CI 0.39-0.51, p < 0.0001). The correlation coefficient was higher when GFR was measured using clearance methods compared with various estimates GFR (1.36 [CI 1.13-1.65], p = 0.0014).Homocysteine plasma levels significantly depend on renal function. This correlation is even more robust when GFR is measured using clearance methods. Therefore, in order to assess whether homocysteine is an independent cardiovascular risk factor, accurate adjustments for renal dysfunction are essential.

    View details for DOI 10.1159/000142725

    View details for Web of Science ID 000259265900006

    View details for PubMedID 18689992

  • Genetic susceptibility to peripheral arterial disease: A dark corner in vascular biology ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Knowles, J. W., Assimes, T. L., Li, J., Quertermous, T., Cooke, J. P. 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

  • beta 2-microglobulin as a biomarker in peripheral arterial disease - Proteomic profiling and clinical studies CIRCULATION Wilson, A. M., Kimura, E., Harada, R. K., Nair, N., Narasimhan, B., Meng, X., Zhang, F., Beck, K. R., Olin, J. W., Fung, E. T., Cooke, J. P. 2007; 116 (12): 1396-1403


    Peripheral arterial disease (PAD) is common but commonly unrecognized. Improved recognition of PAD is needed. We used high-throughput proteomic profiling to find PAD-associated biomarkers.Plasma was collected from PAD patients (ankle brachial index of <0.90; n=45) and subjects with risk factors but without PAD (n=43). Plasma was analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to quantify 1619 protein peaks. The peak intensity of a 12-kDa protein was higher in PAD patients. Western blot analyses and immunoaffinity studies confirmed that this protein was beta2-microglobulin (B2M). In a validation study, B2M was measured by ELISA in plasma in age- and gender-matched PAD (n=20) and non-PAD (n=20) subjects. Finally, we studied a larger cohort of subjects (n=237) referred for coronary angiography but without known PAD. Plasma B2M levels were higher in PAD patients than in non-PAD patients with coronary artery disease. Plasma B2M correlated with ankle brachial index and functional capacity. Independent predictors of PAD were diabetes mellitus, age, and the combination of B2M and C-reactive protein level.In PAD patients, circulating B2M is elevated and correlates with the severity of disease independent of other risk factors. These findings might provide a needed biomarker for PAD and new insight into its pathophysiology. Further studies in other populations are needed to confirm the utility of measuring B2M in cardiovascular disease risk assessment.

    View details for DOI 10.1161/CIRCULATIONAHA.106.683722

    View details for Web of Science ID 000249536300011

    View details for PubMedID 17724262

  • Differentiation, survival, and function of embryonic stem cell-derived endothelial cells for ischemic heart disease 79th Annual Scientific Session of the American-Heart-Association Li, Z., Wu, J. C., Sheikh, A. Y., Kraft, D., Cao, F., Xie, X., Patel, M., Gambhir, S. S., Robbins, R. C., Cooke, J. P., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2007: I46–I54


    Embryonic stem (ES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of ES cell-derived endothelial cells (ESC-ECs), use molecular imaging techniques to examine their survival in vivo, and determine the therapeutic efficacy of ESC-ECs for restoration of cardiac function after ischemic injury.Murine ES cells were transfected with a construct composed of a vascular endothelial cadherin promoter driving enhanced green fluorescence protein (pVE-cadherin-eGFP). Differentiation of ES cells to ECs was detected by FACS analysis using Flk-1 (early EC marker at day 4) and VE-cadherin (late EC marker at day 8). After isolation, these ESC-ECs express endothelial cell markers similar to adult mouse lung endothelial cells, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). For in vivo imaging, ES cells were transduced with an ubiquitin promoter driving firefly luciferase and monomeric red fluorescence protein (pUb-Fluc-mRFP). A robust correlation exists between Fluc signals and cell numbers by ex vivo imaging analysis (R2=0.98) and by in vitro enzyme assay (R2=0.94). Afterward, 5x10(5) ESC-ECs or PBS (as control) was injected into the hearts of mice undergoing LAD ligation (n=15 per group). Bioluminescence imaging showed longitudinal survival of transplanted ESC-ECs for approximately 8 weeks. Echocardiogram demonstrated significant functional improvement in the ESC-EC group compared with control (P=0.04). Finally, postmortem analysis confirmed increased presence of small capillaries and venules in the infarcted zones by CD31 staining.This is the first study to track the fate and function of transplanted ESC-ECs in the heart. With further validation, these ESC-ECs could become a valuable source of cell therapy for induction of angiogenesis in the treatment of myocardial ischemia.

    View details for DOI 10.1161/CIRCULATIONAHA.106.680561

    View details for PubMedID 17846325

  • L-arginine supplementation in peripheral arterial disease - No benefit and possible harm CIRCULATION Wilson, A. M., Harada, R., Nair, N., Balasubramanian, N., Cooke, J. P. 2007; 116 (2): 188-195


    L-arginine is the precursor of endothelium-derived nitric oxide, an endogenous vasodilator. L-arginine supplementation improves vascular reactivity and functional capacity in peripheral arterial disease (PAD) in small, short-term studies. We aimed to determine the effects of long-term administration of L-arginine on vascular reactivity and functional capacity in patients with PAD.The Nitric Oxide in Peripheral Arterial Insufficiency (NO-PAIN) study was a randomized clinical trial of oral L-arginine (3 g/d) versus placebo for 6 months in 133 subjects with intermittent claudication due to PAD in a single-center setting. The primary end point was the change at 6 months in the absolute claudication distance as assessed by the Skinner-Gardner treadmill protocol. L-arginine supplementation significantly increased plasma L-arginine levels. However, measures of nitric oxide availability (including flow-mediated vasodilation, vascular compliance, plasma and urinary nitrogen oxides, and plasma citrulline formation) were reduced or not improved compared with placebo. Although absolute claudication distance improved in both L-arginine- and placebo-treated patients, the improvement in the L-arginine-treated group was significantly less than that in the placebo group (28.3% versus 11.5%; P=0.024).In patients with PAD, long-term administration of L-arginine does not increase nitric oxide synthesis or improve vascular reactivity. Furthermore, the expected placebo effect observed in studies of functional capacity was attenuated in the L-arginine-treated group. As opposed to its short-term administration, long-term administration of L-arginine is not useful in patients with intermittent claudication and PAD.

    View details for DOI 10.1161/CIRCULATIONAHA.106.683656

    View details for Web of Science ID 000247902600010

    View details for PubMedID 17592080

  • Frequent occult infection with cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis JOURNAL OF CLINICAL MICROBIOLOGY Potena, L., Holweg, C. T., Vana, M. L., Bashyam, L., Rajamani, J., McCormick, A. L., Cooke, J. P., Valantine, H. A., Mocarski, E. S. 2007; 45 (6): 1804-1810


    Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

    View details for DOI 10.1128/JCM.01362-06

    View details for Web of Science ID 000247286500022

    View details for PubMedID 17409205

    View details for PubMedCentralID PMC1933112

  • A novel bioluminescent tumor model of human renal cancer cell lines: An in vitro and in vivo characterization JOURNAL OF UROLOGY Peter, C., Kielstein, J. T., Clarke-Katzenberg, R., Adams, M. C., Pitsiouni, M., Kambham, N., Karimi, M. A., Kengatharan, K. M., Cooke, J. P. 2007; 177 (6): 2342-2346


    Bioluminescent imaging permits sensitive in vivo detection and quantification of cells engineered to emit light. We developed a bioluminescent human renal cancer cell line for in vitro and in vivo studies.The 2 human renal cell carcinoma cell lines SN12-C and SN12-L1 were stably transfected to constitutively express luciferase using a retroviral shuttle. The bioluminescent signal was correlated with tumor cell numbers in vitro. Parental and transfected cells were compared by growth kinetics and histology. Tumor burden after heterotopic injection in immune deficient mice was monitored up to 39 days. The kinetics of the bioluminescent signal was evaluated for 1 to 60 minutes following luciferin injection.Bioengineered renal cancer cell lines stably expressed luciferase. The growth kinetics of the cells in vitro and the histology of tumors resulting from implantation of these cells were unaffected by retroviral transfection with the luciferase gene. As few as 1,000 cells could be reliably detected. The intensity of the bioluminescent signal correlated with the number of tumor cells in vitro. Photon emission in vivo and ex vivo correlated significantly with tumor weight at sacrifice. After intraperitoneal injection of luciferin there was a time dependent change in the intensity of the bioluminescent signal with maximum photon emission at 20 minutes (optimal 17 to 25).Luciferase transfected human renal cancer lines allow reliable, rapid, noninvasive and longitudinal monitoring of tumor growth in vivo. The ability to assess tumor development in vivo with time is economical and effective compared to end point data experiments.

    View details for PubMedID 17509355

  • Angiogenesis and the role of the endothelial nicotinic acetylcholine receptor 2nd International Symposium on Non-Neuronal Acetylcholine Cooke, J. P. PERGAMON-ELSEVIER SCIENCE LTD. 2007: 2347–51


    An endothelial nicotinic acetycholine receptor (nAChR) mediates endothelial proliferation, survival, migration and tube formation in vitro, and angiogenesis in vivo. Exogenous nicotine stimulates this angiogenic pathway. This action of nicotine may contribute to tumor angiogenesis and tumor growth; atherosclerotic plaque neovascularization and progression; and other tobacco-related diseases. The endothelial nAChR mediates an angiogenic pathway that is interdependent with growth factor mediated pathways, as shown by pharmacological and molecular studies. The characterization of this new angiogenic pathway may provide a new therapeutic avenue for disorders of insufficient or pathological angiogenesis.

    View details for DOI 10.1016/j.lfs.2007.01.061

    View details for Web of Science ID 000247520900035

    View details for PubMedID 17383685

    View details for PubMedCentralID PMC1941778

  • NOx and ADMA changes with focal ischemia, amelioration with the chaperonin GroEL NEUROSCIENCE LETTERS Xu, L., Wang, B., Kaur, K., Kho, M. F., Cooke, J. P., Giffard, R. G. 2007; 418 (2): 201-204


    Both nitric oxide and asymmetric dimethylarginine (ADMA) play a critical role in the regulation of cerebral blood flow, though their neuroprotective and cytotoxic effects are still under investigation. In this study, we found that nitrate/nitrite (NOx) levels in plasma, ischemic brain tissue, and cerebrospinal fluid (CSF) increased significantly 24h after 2h transient middle cerebral artery occlusion (MCAO) in rats. ADMA levels were unchanged in plasma, but decreased significantly in CSF 24h following MCAO. The CSF ADMA/NOx ratio decreased markedly following ischemia. Rats protected by expression of the chaperonin GroEL or its folding deficient mutant D87K had lower plasma NOx levels at 24h reperfusion. ADMA, NO, and their ratio in CSF merit further study as biomarkers for ischemic brain injury.

    View details for DOI 10.1016/j.neulet.2007.03.021

    View details for Web of Science ID 000246871600017

    View details for PubMedID 17398004

    View details for PubMedCentralID PMC2430977

  • Changes in coronary arterial dimensions early after cardiac transplantation TRANSPLANTATION Fearon, W. F., Potena, L., Hirohata, A., Sakurai, R., Yamasaki, M., Luikart, H., Lee, J., Vana, M. L., Cooke, J. P., Mocarski, E. S., Yeung, A. C., Valantine, H. A. 2007; 83 (6): 700-705


    Significant changes in coronary artery structure, including intimal thickening and vessel remodeling, occur early after cardiac transplantation. The degree to which these changes compromise coronary lumen dimensions, and the clinical factors that affect these changes, remain controversial.Thirty-eight adult cardiac transplant recipients underwent coronary angiography and volumetric intravascular ultrasound (IVUS) evaluation of the left anterior descending artery within 8 weeks of transplantation and at 1 year. Clinical parameters including donor and recipient characteristics, rejection episodes, and serology were prospectively recorded. Two-dimensional IVUS measurements and vessel, lumen and plaque volume were calculated at both time points and compared. Multivariate regression analysis was performed to reveal clinical predictors of change in coronary dimensions.During the first year after transplantation, significant decreases in vessel size (negative remodeling) and lumen size were observed with significant increases in plaque burden based on IVUS analyses. Loss of lumen volume correlated significantly with the degree of negative remodeling (R=0.82, P<0.0001), but not with changes in plaque burden (R=0.08, P=0.64). Patients with the greatest increase in plaque volume had significantly less negative remodeling (R=0.53, P=0.0006). Transplant recipient cytomegalovirus (CMV) antibody seropositivity and lack of aggressive prophylaxis against CMV infection/reactivation were significant independent predictors of greater negative remodeling (P<0.01 and P=0.03, respectively) and greater lumen loss (P=0.02 and P=0.03, respectively).Negative remodeling is primarily responsible for coronary artery lumen loss during the first year after cardiac transplantation. CMV seropositivity and lack of aggressive CMV prophylaxis correlate with increased negative remodeling, resulting in greater lumen loss.

    View details for DOI 10.1097/

    View details for PubMedID 17414701

  • Dimethylarginine dimethylaminohydrolase promotes endothelial repair after vascular injury JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Konishi, H., Sydow, K., Cooke, J. P. 2007; 49 (10): 1099-1105


    We sought to determine if a reduction in asymmetric dimethylarginine (ADMA) enhances endothelial regeneration.Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthase (NOS). Increased plasma levels of ADMA are associated with endothelial vasodilator dysfunction in patients with vascular disease or risk factors. Asymmetric dimethylarginine is eliminated largely by the action of dimethylarginine dimethylaminohydrolase (DDAH), which exists in 2 isoforms. Dimethylarginine dimethylaminohydrolase-1 transgenic (TG) mice manifest increased DDAH activity, reduced plasma and tissue ADMA levels, increased nitric oxide synthesis, and reduced systemic vascular resistance.The left femoral arteries of DDAH1 TG mice and wild-type (WT) mice were injured by a straight spring wire, and regeneration of the endothelial cell (EC) monolayer was assessed. Endothelial sprouting was assayed with growth factor-reduced Matrigel.Regeneration of the EC monolayer was more complete 1 week after injury in TG mice (WT vs. TG: 40.0 +/- 6.5% vs. 61.2 +/- 6.4%, p < 0.05). The number of CD45 positive cells at the injured sites was reduced by 62% in DDAH TG mice (p < 0.05). Four weeks after injury, the neointima area and intima/media ratio were attenuated in DDAH TG mice (WT vs. TG: 0.049 +/- 0.050 mm2 vs. 0.031 +/- 0.060 mm2, 3.1 +/- 0.5 vs. 1.7 +/- 0.2, respectively, p < 0.05). Endothelial cell sprouting from vascular segments increased in TG mice (WT vs. TG: 24.3 +/- 3.9 vs. 39.0 +/- 2.2, p < 0.05).We find for the first time an important role for DDAH in EC regeneration and in neointima formation. Strategies to enhance DDAH expression or activity might be useful in restoring the endothelial monolayer and in treating vascular disease.

    View details for DOI 10.1016/j.jacc.2006.10.068

    View details for Web of Science ID 000244808700012

    View details for PubMedID 17349891

  • Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia CIRCULATION RESEARCH Aicher, A., Rentsch, M., Sasaki, K., Ellwart, J. W., Faendrich, F., Siebert, R., Cooke, J. P., Dimmeler, S., Heeschen, C. 2007; 100 (4): 581-589


    Circulating progenitor cells home to sites of postnatal neovascularization and differentiate into endothelial cells but questions remain regarding the source of these cells. Indeed, a recent study suggests that nonbone marrow-derived cells may be even more important than bone marrow-derived cells in the setting of transplant arteriosclerosis. Thus, we aimed to thoroughly investigate the contribution of nonbone marrow-derived progenitor cells for neovascularization. We exclusively identified nonbone marrow-derived progenitor cells by combining a parabiosis model with reverse bone marrow transplantation followed by hindlimb ischemia. In this model, nonbone marrow-derived circulating progenitor cells attributed for 74+/-13% of the circulating progenitor cells that incorporated into the ischemic hindlimb. Increasing evidence suggests that organs such as small intestine and liver contain a considerable number of tissue resident progenitor cells and, thus, represent putative sources for nonbone marrow-derived progenitors. To track organ-derived progenitors, we transplanted sex-mismatched small intestine or liver, respectively, into rats followed by induction of hindlimb ischemia. These experiments show that organ-derived progenitor cells are contributing to postnatal vasculogenesis (intestine: 4.7+/-3.7%; liver: 6.3+/-2.2%). Based on the subsequent observation that liver-derived nonhematopoietic c-kit(+)CD45(-) progenitors are mobilized on induction of hindlimb ischemia, we prospectively isolated and intravenously infused these progenitors from murine livers. The isolated cells demonstrated a marked capacity for enhancing neovascularization and restoring blood flow to the ischemic hindlimb (no cells: 26.4+/-4.8% of normal blood flow; c-kit(+)CD45(-) cells: 67.0+/-8.0% of normal flow; P<0.01). In conclusion, we find that nonbone marrow-derived c-kit(+)CD45(-) progenitors contribute to postnatal neovascularization to an extent that is similar to that of bone marrow-derived progenitor cells. Intestine and liver represent a rich source for mobilized tissue-residing progenitor cells.

    View details for DOI 10.1161/01.RES.0000259562.63718.35

    View details for Web of Science ID 000244571600018

    View details for PubMedID 17272807

  • Should we measure asymmetric dimethylarginine in patients with coronary artery disease? CLINICAL CHEMISTRY Kielstein, J. T., Cooke, J. P. 2007; 53 (2): 161-163

    View details for DOI 10.1373/clinchem.2006.078881

    View details for Web of Science ID 000243965500002

    View details for PubMedID 17259245

  • Endothelial progenitor cells participate in nicotine-mediated angiogenesis JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Heeschen, C., Chang, E., Aicher, A., Cooke, J. P. 2006; 48 (12): 2553-2560


    We aimed to determine the role of endothelial progenitor cells (EPCs) in cholinergic angiogenesis.Recently, we provided evidence for a new angiogenic pathway mediated by endothelial nicotinic acetylcholine receptors (nAChR). Increasing evidence suggests that circulating EPCs also contribute to postnatal neovascularization by homing to sites of neovascularization, a process termed postnatal vasculogenesis. Therefore, we investigated whether nAChR activation increases mobilization and/or recruitment of EPCs to a site of angiogenesis.To identify EPCs from reservoirs both inside and outside of the bone marrow and to avoid the adverse effects of total body irradiation, we employed a murine parabiosis model with tie-2-LacZ FvB/N mice connected to wild-type FvB/N mice and induced unilateral hind limb ischemia in the wild-type animal.Administration of nicotine increased capillary density in the ischemic hind limb, and increased soluble Kit ligand plasma levels. The effect of systemic administration was greater than that of local delivery of nicotine (45% vs. 76% increase in capillary density by comparison to vehicle control, intramuscular vs. oral administration of nicotine; p < 0.05). Ischemia-induced incorporation of EPC in the control group was rare, but was increased 5-fold by systemic administration of nicotine. Exposure to nicotine in vitro increased EPC count and EPC transmigration. Finally, systemic administration of nicotine increased EPC number in the bone marrow and spleen during hind limb ischemia.Nicotine treatment increased the number of EPCs in the bone marrow and spleen, and increased their incorporation into the vasculature of ischemic tissue. Administration of nicotine increased markers of EPC mobilization. This study indicates that the known angiogenic effect of nicotine may be mediated in part by mobilization of precursor cells.

    View details for DOI 10.1016/j.jacc.2006.07.066

    View details for Web of Science ID 000242916100022

    View details for PubMedID 17174197

  • Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia FASEB JOURNAL von Degenfeld, G., Banfi, A., Springer, M. L., Wagner, R. A., Jacobi, J., Ozawa, C. R., Merchant, M. J., Cooke, J. P., Blau, H. M. 2006; 20 (14): 2657-?


    The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.

    View details for DOI 10.1096/fj.06-6568fje

    View details for Web of Science ID 000242490700056

    View details for PubMedID 17095533

  • A pilot study of ranolazine in patients with intermittent claudication INTERNATIONAL ANGIOLOGY Ma, A., Garland, W. T., Smith, W. B., Skettino, S., Navarro, M. T., Chan, A. Q., Anderson, B. E., Cooke, J. P. 2006; 25 (4): 361-369


    This pilot study provides preliminary information regarding safety and changes in exercise performance during treatment with ranolazine extended-release in patients with reproducible claudication during exercise treadmill testing (ETT).We enrolled 45 patients with documented peripheral arterial disease, reproducible claudication on ETT, and ankle-brachial indices <0.85 at rest that decreased by at least 0.15% or 20% immediately postexercise. Randomized patients received double-blind treatment with either ranolazine 1 000 mg b.i.d. (n=22) or placebo (n=23) for 4 weeks.Compared with baseline, peak walking time (PWT) increased (mean+/-SEM) by 53+/-34 s with ranolazine (P=0.13) and by 41+/-33 s with placebo (P=0.22). Pain-free walking time during ETT increased by 62+/-18 s with ranolazine (P=0.002) and 36+/-18 s with placebo (P=0.045). Supplemental analyses, excluding patients with baseline exercise duration (16 min and (12 min, showed additional improvement with ranolazine on PWT.Ranolazine was well tolerated and these data provide a rationale for proceeding with a definitive trial.

    View details for Web of Science ID 000244592700005

    View details for PubMedID 17164742

  • T-cell immunity to subclinical cytomegalovirus infection reduces cardiac allograft disease CIRCULATION Tu, W., Potena, L., Stepick-Biek, P., Liu, L., Dionis, K. Y., Luikart, H., Fearon, W. F., Holmes, T. H., Chin, C., Cooke, J. P., Valantine, H. A., Mocarski, E. S., Lewis, D. B. 2006; 114 (15): 1608-1615


    Asymptomatic cytomegalovirus (CMV) replication is frequent after cardiac transplantation in recipients with pretransplantation CMV infection. How subclinical viral replication influences cardiac allograft disease remains poorly understood, as does the importance of T-cell immunity in controlling such replication.Thirty-nine cardiac recipients who were pretransplantation CMV antibody positive were longitudinally studied for circulating CMV-specific CD4 and CD8 T-cell responses, CMV viral load in blood neutrophils, and allograft rejection during the first posttransplantation year. Nineteen of these recipients were also analyzed for changes of coronary artery intimal, lumen, and whole-vessel area. All recipients received early prophylactic therapy with ganciclovir. No recipients developed overt CMV disease. Those with detectable levels of CMV-specific CD4 T cells in the first month after transplantation were significantly protected from high mean and peak posttransplantation viral load (P<0.05), acute rejection (P<0.005), and loss of allograft coronary artery lumen (P<0.05) and of whole-vessel area (P<0.05) compared with those who lacked this immune response. The losses of lumen and vessel area were both significantly correlated with the time after transplantation at which a CD4 T-cell response was first detected (P<0.05) and with the cumulative graft rejection score (P<0.05).The early control of subclinical CMV replication after transplantation by T-cell immunity may limit cardiac allograft rejection and vascular disease. Interventions to increase T-cell immunity might be clinically useful in limiting these adverse viral effects.

    View details for DOI 10.1161/CIRCULATIONAHA.105.607549

    View details for PubMedID 17015794

  • Symmetric dimethylarginine (SDMA) as endogenous marker of renal function - a meta-analysis NEPHROLOGY DIALYSIS TRANSPLANTATION Kielstein, J. T., Salpeter, S. R., Bode-Boeger, S. M., Cooke, J. P., Fliser, D. 2006; 21 (9): 2446-2451


    Dosing of most drugs must be adapted in renal insufficiency, making accurate assessment of renal function essential in clinical medicine. Furthermore, even modest impairment of renal function has been recognized as a cardiovascular risk factor. The purpose of this analysis was to identify the role of symmetric dimethylarginine (SDMA), the structural isomer of the cardiovascular risk marker asymmetric dimethylarginine, as an endogenous marker of renal function.Comprehensive searches of Medline and the Cochrane Library from 1970 to February 2006 were performed to identify studies that evaluated the correlation between SDMA and renal function. The search was augmented by scanning references of identified articles and reviews. The correlation coefficients (R) were recorded from each study for the values of 1/SDMA and clearance estimates and for SDMA and creatinine levels. The summary correlation coefficients with 95% confidence intervals (CIs) were pooled using the random-effects method.In 18 studies involving 2136 patients systemic SDMA concentrations correlated highly with inulin clearance [R = 0.85 (CI 0.76-0.91, P < 0.0001)], as well as with various clearance estimates combined [R = 0.77 (CI 0.65-0.85, P < 0.0001)] and serum creatinine [R = 0.75 (CI 0.46-089, P < 0.0001)].SDMA exhibits some properties of a reliable marker of renal function. Future studies have to clarify whether SDMA is indeed suited to improve diagnosis and eventually optimize care of patients.

    View details for DOI 10.1093/ndt/gfl292

    View details for Web of Science ID 000240694200019

    View details for PubMedID 16766542

  • Acute rejection and cardiac allograft vascular disease is reduced by suppression of subclinical cytomegalovirus infection TRANSPLANTATION Potena, L., Holweg, C. T., Chin, C., Luikart, H., Weisshaar, D., Narasimhan, B., Fearon, W. F., Lewis, D. B., Cooke, J. P., Mocarski, E. S., Valantine, H. A. 2006; 82 (3): 398-405


    Anticytomegalovirus (CMV) prophylaxis prevents the acute disease but its impact on subclinical infection and allograft outcome is unknown. We sought to determine whether CMV prophylaxis administered for three months after heart transplant would improve patient outcomes.This prospective cohort study of 66 heart transplant recipients compared aggressive CMV prophylaxis (n = 21, CMV hyperimmune globulin [CMVIG] plus four weeks of intravenous ganciclovir followed by two months of valganciclovir); with standard prophylaxis (n = 45, intravenous ganciclovir for four weeks). Prophylaxis was based on pretransplant donor (D) and recipient (R) CMV serology: R-/D+ received aggressive prophylaxis; R+ received standard prophylaxis. Outcome measures were: CMV infection assessed by DNA-polymerase chain reaction on peripheral blood polymorphonuclear leukocytes, acute rejection, and cardiac allograft vascular disease (CAV) assessed by intravascular ultrasound. All patients completed one year of follow-up. RESULTS.: CMV infection was subclinical in all but four patients (two in each group). Aggressively treated patients had a lower incidence of CMV infection (73 +/- 10% vs. 94 +/- 4%; P = 0.038), and an independent reduced relative risk for acute rejection graded > or =3A (relative risk [95% CI] = 0.55 [0.26-0.96]; P = 0.03), as compared with the standard prophylaxis group. Aggressively prophylaxed patients also showed a slower progression of CAV, in terms of coronary artery lumen loss (lumen volume change=-21 +/- 13% vs. -10+/-14%; P = 0.05); and vessel shrinkage (vessel volume change = -15 +/- 11% vs. -3 +/- 18%; P = 0.03).Prolonged (val)ganciclovir plus CMVIG reduces viral levels, acute rejection, and allograft vascular disease, suggesting a role for chronic subclinical infection in the pathophysiology of the most common diseases affecting heart transplant recipients.

    View details for DOI 10.1097/

    View details for PubMedID 16906040

  • ADMA increases arterial stiffness and decreases cerebral blood flow in humans STROKE Kielstein, J. T., Donnerstag, F., Gasper, S., Menne, J., Kielstein, A., Martens-Lobenhoffer, J., Scalera, F., Cooke, J. P., Fliser, D., Bode-Boeger, S. M. 2006; 37 (8): 2024-2029


    Preclinical studies have revealed that the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), increases vascular tone in cerebral blood vessels. Marked elevations of ADMA blood levels were found in patients with diseases characterized by decreased cerebral perfusion, such as ischemic stroke. Arterial stiffness is an independent predictor of stroke and other adverse cardiovascular events. The aim of this study was to investigate the influence of a systemic subpressor dose of ADMA on arterial stiffness and cerebral perfusion in humans.Using a double-blind, vehicle-controlled study design, we allocated 20 healthy men in random order to infusion of either ADMA (0.10 mg ADMA/kg per min) or vehicle over a period of 40 minutes. Arterial stiffness was assessed noninvasively by pulse wave analysis. All volunteers underwent measurement of cerebral perfusion by dynamic contrast-enhanced perfusion magnetic resonance imaging of the brain.Infusion of ADMA significantly decreased total cerebral perfusion by 15.1+/-4.5% (P=0.007), whereas blood flow in the vehicle group increased by 7.7+/-2.8% (P=0.02). ADMA also increased arterial stiffness as assessed by measurement of the augmentation index (-12.6+/-1.9 to -9.6+/-1.5, P=0.007).Our results document for the first time that subpressor doses of ADMA increase vascular stiffness and decrease cerebral perfusion in healthy subjects. Thus, ADMA is an important endogenous modulator of cerebral vascular tone and may be involved in the pathogenesis of cerebrovascular disease.

    View details for DOI 10.1161/01.STR.0000231640.32543.11

    View details for Web of Science ID 000239459000020

    View details for PubMedID 16809568

  • Psychophysiological and cortisol responses to psychological stress in depressed and nondepressed older men and women with elevated cardiovascular disease risk PSYCHOSOMATIC MEDICINE Taylor, C. B., Conrad, A., Wilhelm, F. H., Neri, E., DeLorenzo, A., Kramer, M. A., Giese-Davis, J., Roth, W. T., Oka, R., Cooke, J. P., Kraemer, H., Spiegel, D. 2006; 68 (4): 538-546


    The objective of this study was to compare psychophysiological and cortisol reactions to psychological stress in older depressed and nondepressed patients at risk for cardiovascular disease (CVD).Forty-eight depressed participants and 20 controls with elevated cardiovascular risk factors underwent a psychological stress test during which cardiovascular variables were measured. Salivary cortisol was collected after each test segment. Traditional (e.g., lipids) and atypical (e.g., C-reactive protein) CVD risk factors were also obtained.At baseline, the groups did not differ on lipid levels, flow-mediated vasodilation, body mass index, or asymmetric dimethylarginine. However, the depressed patients had significantly higher C-reactive protein levels. Contrary to our hypothesis, there were no differences in baseline cortisol levels or diurnal cortisol slopes, but depressed patients showed significantly lower cortisol levels during the stress test (p = .03) and less cortisol response to stress. Compared with nondepressed subjects, depressed subjects also showed lower levels of respiratory sinus arrhythmia (RSA(TF)) during the stress test (p = .02).In this sample, older depressed subjects with elevated risk for CVD exhibited a hypocortisol response to acute stress. This impaired cortisol response might contribute to chronic inflammation (as reflected in the elevated C-reactive proteins in depressed patients) and in other ways increase CVD risk. The reduced RSA(TF) activity may also increase CVD risk in depressed patients through impaired autonomic nervous system response to cardiophysiological demands.

    View details for DOI 10.1097/01.psy.0000222372.16274.92

    View details for Web of Science ID 000239330600005

    View details for PubMedID 16868262

  • Limb hemodynamics are not predictive of functional capacity in patients with PAD VASCULAR MEDICINE Szuba, A., Oka, R. K., Harada, R., Cooke, J. P. 2006; 11 (3): 155-163


    To the practicing clinician, it seems obvious that limb hemodynamics would be the primary determinant of walking distance. However, other determinants, such as skeletal muscle metabolism, may play a role. Accordingly, in the current study, we examined the relationship between measures of limb hemodynamics and walking capacity in patients with peripheral arterial disease (PAD). We measured toe and ankle pressures for calculation of toe- (TBI) and ankle (ABI)-brachial indices; basal and hyperemic calf blood flow (CBF; by plethysmography); and initial (ICT) and absolute (ACT) claudication time using the Skinner-Gardner protocol. As expected, PAD patients had impaired limb hemodynamics with reduced TBI, ABI and a reduction in ABI post-exercise. However, there was no relationship between any of the hemodynamic variables (including ABI, ABI reduction post-exercise, TBI, baseline or maximal CBF) and walking distance as assessed by ICT or ACT. A subset of PAD patients with an ACT >750s (n = 16; 'long claudicators') were compared with a subset of PAD patients with an ACT <260s (n = 16; 'short claudicators'). The average ACT in the long claudicants was over fivefold greater than the short claudicators. Surprisingly, there were no differences between the two groups in any of the hemodynamic variables. There was also no relationship between the initial ABI, TBI, toe pressure, baseline or hyperemic CBF, and the improvement in ACT over the 3-month course of the study. This study found little relationship between hemodynamic variables and functional capacity in PAD. Accordingly, to assess the response to therapeutic interventions, exercise performance and functional status need to be directly measured, and cannot be predicted from hemodynamic measurements.

    View details for DOI 10.1177/1358863x06074828

    View details for Web of Science ID 000243489600003

    View details for PubMedID 17288121

  • The time has come for vascular medicine VASCULAR MEDICINE Cooke, J. P. 2006; 11 (3): 177-180
  • Vascular compliance versus flow-mediated vasodilation: correlation with cardiovascular risk factors VASCULAR MEDICINE Nair, N., Oka, R. K., Waring, L. D., Umoh, E. M., Taylor, C. B., Cooke, J. P. 2005; 10 (4): 275-283


    Cardiovascular risk factors are associated with impaired endothelium dependent vasodilation and reduced vascular compliance. In this study, the correlation with cardiovascular risk factor score of two common techniques for assessing vascular function was compared. Risk factors and vascular function were evaluated in a study population of 122 people with peripheral arterial disease (PAD) or with risk factors for PAD (73 men and 49 women; mean age 69 years). A risk factor score was determined using Framingham criteria. Vascular compliance was assessed by pulse waveform analysis and simultaneous blood pressure measurement. Flow-mediated vasodilation of the brachial artery was measured using duplex ultrasonography. Participants with a high risk factor score had significantly reduced vascular compliance of large and small vessels. By contrast, the difference in flow-mediated vasodilation between those with a high or low risk factor score did not reach statistical significance. There was a significant negative correlation between vascular compliance and risk factor score. There was a similar trend between flow-mediated vasodilation and risk factor score, but this did not reach statistical significance. A measure of vascular compliance was more significantly correlated with cardiovascular risk factor score than was a measure of flow-mediated vasodilation in the study population. Neither technique provided values that were highly correlated with risk factor burden. Although flow-mediated vasodilation is a preferred research tool for assessing vascular function, technical limitations and biological variability may reduce its clinical application in assessing individual cardiovascular risk.

    View details for DOI 10.1191/1358863x05vm633oa

    View details for Web of Science ID 000234803000004

    View details for PubMedID 16444856

  • Insulin resistance: potential role of the endogenous nitric oxide synthase inhibitor ADMA 2nd International Symposium on AMDA - An Emerging Cardiovascular Risk Factor Sydow, K., Mondon, C. E., Cooke, J. P. SAGE PUBLICATIONS LTD. 2005: S35–S43


    The insulin resistance syndrome (IRS) is considered to be a new target of risk-reduction therapy. The IRS is a cluster of closely associated and interdependent abnormalities and clinical outcomes that occur more commonly in insulin-resistant/hyperinsulinemic individuals. This syndrome predisposes individuals to type 2 diabetes, cardiovascular diseases, essential hypertension, certain forms of cancer, polycystic ovary syndrome, nonalcoholic fatty liver disease, and sleep apnea. In patients at high risk for cardiovascular diseases, endothelial dysfunction is observed in morphologically intact vessels even before the onset of clinically manifest vascular disease. Indeed, there are several lines of evidence that indicate that endothelial function is compromised in situations where there is reduced sensitivity to endogenous insulin. It is well established that a decreased bioavailability of nitric oxide (NO) contributes to endothelial dysfunction. Furthermore, NO may modulate insulin sensitivity. Activation of NO synthase (NOS) augments blood flow to insulin-sensitive tissues (i.e. skeletal muscle, liver, adipose tissue), and its activity is impaired in insulin resistance. Inhibition of NOS reduces the microvascular delivery of nutrients and blunts insulin-stimulated glucose uptake in skeletal muscle. Furthermore, induction of hypertension by administration of the NOS inhibitor NG-monomethyl-L-arginine is also associated with insulin resistance in rats. Increased levels of asymmetric dimethylarginine (ADMA) are associated with endothelial vasodilator dysfunction and increased risk of cardiovascular diseases. An intriguing relationship exists between insulin resistance and ADMA. Plasma levels of ADMA are positively correlated with insulin resistance in nondiabetic, normotensive people. New basic research insights that provide possible mechanisms underlying the development of insulin resistance in the setting of impaired NO bioavailability will be discussed.

    View details for Web of Science ID 000234895900006

    View details for PubMedID 16444867

  • ADMA: its role in vascular disease 2nd International Symposium on AMDA - An Emerging Cardiovascular Risk Factor Cooke, J. P. SAGE PUBLICATIONS LTD. 2005: S11–S17


    Endothelium-derived nitric oxide (NO) is the most potent endogenous vasodilator and, by virtue of its anti-inflammatory and anti-thrombotic effects, it is an endogenous anti-atherogenic agent. Accordingly, impairment of NO synthesis or bioactivity may increase the risk of vascular disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the NO synthase pathway. Plasma levels of ADMA are increased in patients with vascular disease, or with risk factors for vascular disease. Preclinical and clinical studies indicate that ADMA may mediate the adverse effects of traditional risk factors on endothelial vasodilator function. By impairing endothelial function, ADMA may contribute to pulmonary or systemic hypertension, as well as to vascular disease. Several drugs known to treat cardiovascular disease also reduce plasma ADMA levels, such as angiotensin receptor antagonists, converting enzyme inhibitors, and insulin sensitizing agents. Plasma ADMA may be a common mediator of endothelial dysfunction induced by vascular risk factors. Insights into the mechanisms by which plasma ADMA is regulated may lead to new therapeutic knowledge.

    View details for Web of Science ID 000234895900003

    View details for PubMedID 16444864

  • Mechanisms of Raynaud's disease VASCULAR MEDICINE Cooke, J. P., Marshall, J. M. 2005; 10 (4): 293-307


    Raynaud's phenomenon is due to transient cessation of blood flow to the digits of the hands or feet. An attack of Raynaud's phenomenon is classically manifested as triphasic color changes. The white phase is due to excessive vasoconstriction and cessation of regional blood flow. This phase is followed by a cyanotic phase, as the residual blood in the finger desaturates. The red phase is due to hyperemia as the attack subsides and blood flow is restored. An attack is frequently associated with pain and/or paresthesia due to sensory nerve ischemia. Variants of Raynaud's phenomenon include acrocyanosis and primary livedo reticularis, each of which is associated with reduced skin blood flow, exacerbated by cold or emotional upset. Raynaud's phenomenon in the absence of other disorders is primary Raynaud's phenomenon, or Raynaud's disease. The mechanisms of Raynaud's disease include increased activation of the sympathetic nerves, in response to cold or emotion; an impaired habituation of the cardiovascular response to stress may contribute. In addition, there appears to be a local fault, which is likely multifactorial. This local fault is due to an alteration in vascular function rather than vascular structure. The alteration in vascular function may be related to increased sensitivity to cold of the adrenergic receptors on the digital artery vascular smooth muscle. In some cases, locally released or systemically circulating vasoconstrictors may participate, including endothelin, 5-hydroxytryptamine and thromboxane. A deficiency or increased degradation of nitric oxide, possibly due to increased oxidative stress, may be involved in some cases. These recent pathophysiological insights may lead to new therapeutic options.

    View details for DOI 10.1191/1358863x05vm639ra

    View details for Web of Science ID 000234803000006

    View details for PubMedID 16444858

  • ADMA regulates angiogenesis: genetic and metabolic evidence VASCULAR MEDICINE Achan, V., Ho, H. K., Heeschen, C., Stuehlinger, M., Jang, J. J., Kimoto, M., Vallance, P., Cooke, J. P. 2005; 10 (1): 7-14


    Endothelium-derived nitric oxide (NO) plays an important role in transducing the effects of angiogenic factors. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We used a murine model of hindlimb ischemia to investigate whether genetic or metabolic changes in ADMA levels could impair angiogenic response in vivo. Hindlimb ischemia was surgically induced in C57BL/6J mice, apo E-deficient mice, or transgenic mice overexpressing dimethylarginine dimethylaminohydrolase (DDAH). Some animals were also treated with the NOS antagonist L-nitro-arginine, or the NO precursor L-arginine. Angiogenesis was quantified in the hindlimb skeletal muscle by capillary/myocyte ratio. Plasma or tissue ADMA levels were measured by HPLC. In normal mice, hindlimb ischemia increased tissue ADMA twofold, and reduced DDAH and NOS expression. This was associated with a reduced NOS activity (by over 80%) three days following surgery. On day seven, a threefold increase in DDAH expression and a fall in tissue ADMA levels were associated with a sevenfold increase in NOS activity, whereas NOS expression did not increase above baseline. In DDAH transgenic mice, the elevation of ADMA and decrement in NOS activity was blunted during hindlimb ischemia. Plasma ADMA levels were increased in apo E-mice (1.79 +/- 0.45 versus 1.07 +/- 0.08 pmol/l; p = 0.008). Capillary index was significantly reduced in apo E-mice up to seven weeks after surgery (0.25 +/- 0.05 versus 0.62 +/- 0.08; p < 0.001). The effect of hypercholesterolemia on capillary index was reversed by L-arginine, and (in wild-type mice) mimicked by administration of the NOS antagonist L-nitro-arginine. In conclusion, metabolic or genetic changes in plasma and tissue ADMA levels affect tissue NO production and angiogenic response to ischemia.

    View details for DOI 10.1191/1358863x05vm580oa

    View details for Web of Science ID 000228828200002

    View details for PubMedID 15920994

  • Capillary electrophoretic and micellar electrokinetic separations of asymmetric dimethyl-L-arginine and structurally related amino acids: Quantitation in human plasma JOURNAL OF SEPARATION SCIENCE Trapp, G., Sydow, K., Dulay, M. T., Chou, T., Cooke, J. P., Zare, R. N. 2004; 27 (17-18): 1483-1490


    We report the development of efficient electrophoretic methods for the separation and quantification of L-arginine and six naturally occurring derivatives that are structurally and functionally related. Capillary electrophoresis (CE) employing a concentrated borate buffer at pH 9.4 achieves the separation of mixtures containing dimethyl-L-arginine, NG-monomethyl-L-arginine, L-arginine, L-homoarginine, L-ornithine, and L-citrulline as 4-fluoro-7-nitrobenzofurazan derivatives. In addition, the separation of the isomeric dimethyl-L-arginine derivatives (symmetric and asymmetric) is attained with baseline resolution by micellar electrokinetic chromatography (MEKC) when a high concentration of deoxycholic acid is added as a surfactant to the same running buffer. The influence of buffer type, concentration, and pH on the separation was studied to optimize separation conditions. The limit of quantitation (LOQ) for asymmetric dimethyl-L-arginine in aqueous solution was determined to be 20 microM using UV absorption in a CE separation and 0.1 microM using laser induced fluorescence (LIF) detection in an MEKC separation. This newly developed method was successfully applied for the quantitation of asymmetric dimethyl-L-arginine and L-arginine in human plasma samples at levels that might be used as a clinical diagnostic for cardiovascular disease (0.125 microM LOQ).

    View details for DOI 10.1002/jssc.200401918

    View details for Web of Science ID 000225938100011

    View details for PubMedID 15638156

  • Adenoviral gene transfer with soluble vascular endothelial growth factor receptors impairs angiogenesis and perfusion in a murine model of hindlimb ischemia CIRCULATION Jacobi, J., Tam, B. Y., Wu, G., Hoffman, J., Cooke, J. P., Kuo, C. J. 2004; 110 (16): 2424-2429


    The purpose of the current study was to examine the contribution of endogenous vascular endothelial growth factor (VEGF) to ischemia-induced angiogenesis and perfusion.C57BL/6J mice (n=28) were subjected to unilateral hindlimb ischemia after intravenous injection of recombinant adenoviruses (10(9) plaque-forming units) encoding the ligand-binding ectodomain of VEGF receptor 1 (VEGFR1/Ad Flt1), VEGF receptor 2 (VEGFR2/Ad Flk1-Fc), a control murine IgG2alpha Fc fragment (Ad Fc), or vehicle (phosphate-buffered saline). Hindlimb perfusion was assessed by both laser Doppler and fluorescent microsphere injection 10 days after surgery. The role of endogenous VEGF in ischemia-induced angiogenesis and arteriogenesis was measured by capillary density and microangiography, respectively. Adenoviral gene transfer with soluble VEGFRs significantly attenuated hindlimb perfusion as assessed by laser Doppler and microsphere analysis (P<0.05). Furthermore, soluble VEGFRs significantly reduced ischemia-induced angiogenesis and collateral growth and inhibited histological recovery of muscle tissue. Adverse events consistent with ongoing vascular insufficiency such as limb necrosis or gangrene were observed only in animals expressing soluble VEGFRs and not in control animals.Systemic, soluble receptor-mediated VEGF inhibition indicates an essential role for endogenous VEGF during postischemic angiogenesis and hindlimb perfusion.

    View details for DOI 10.1161/01.CIR.0000145142.85645.EA

    View details for Web of Science ID 000224553000025

    View details for PubMedID 15477417

  • The pivotal role of nitric oxide for vascular health Advances in Vascular Biology Symposium Cooke, J. P. PULSUS GROUP INC. 2004: 7B–15B


    The endothelium is a single cell layer that lines all blood vessels. This tissue produces an impressive array of paracrine factors that maintain vascular homeostasis. One of these factors is the potent vasodilator endothelium-derived nitric oxide (NO). Impairment in the synthesis or bioactivity of NO, as manifested by reduced endothelium-dependent vasodilation, has been shown to be an independent risk factor for major adverse cardiovascular events. The linkage between endothelium-derived NO and cardiovascular health is likely due to the pleiotropic effects of NO on the vessel wall. NO inhibits the proliferation of vascular smooth muscle cells, the aggregation of platelets, and the adherence and infiltration of inflammatory cells. Thus, an impairment of NO bioactivity or synthesis will reduce its braking effect on processes involved in atherogenesis. New insights have been made regarding the mechanisms by which NO bioactivity becomes impaired. Cardiovascular risk factors induce vascular oxidative stress, which accelerates the degradation of NO. In addition, oxidative stress causes the accumulation of asymmetric dimethylarginine (ADMA). ADMA is an endogenous inhibitor of NO synthesis. Elevation of ADMA appears to be a common mediator by which cardiovascular risk factors impair NO synthesis. Elevation of plasma ADMA has been linked to impaired endothelium-dependent vasodilation, carotid intimal thickening and adverse cardiovascular events. A number of modern therapies directed against atherosclerosis also improve the function of the NO synthase pathway. New agents that specifically target the NO synthase pathway have been developed, and represent a new front on the war against heart disease.

    View details for Web of Science ID 000223420600002

    View details for PubMedID 15309199

  • Developmental endothelial locus-1 (Del-1), a novel angiogenic protein - Its role in ischemia CIRCULATION Ho, H. K., Jang, J. J., Kaji, S., Spektor, G., Fong, A., Yang, P., Hu, B. S., Schatzman, R., Quertermous, T., Cooke, J. P. 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 PubMedID 14981004

  • Cytomegalovirus infection impairs the nitric oxide synthase pathway - Role of asymmetric dimethylarginine in transplant arteriosclerosis CIRCULATION Weis, M., Kledal, T. N., LIN, K. Y., Panchal, S. N., Gao, S. Z., Valantine, H. A., Mocarski, E. S., Cooke, J. P. 2004; 109 (4): 500-505


    We hypothesized that cytomegalovirus (CMV) may contribute to the vasculopathy observed in cardiac allograft recipients by impairing the endothelial nitric oxide synthase pathway. We focused on asymmetric dimethylarginine (ADMA, the endogenous inhibitor of nitric oxide synthase) as a potential mediator of the adverse vascular effect of CMV.Heart transplant recipients manifested elevated plasma ADMA levels compared with healthy control subjects. Transplant patients with CMV DNA-positive leukocytes had higher plasma ADMA concentrations and more extensive transplant arteriopathy (TA). Human microvascular endothelial cells infected with the CMV isolates elaborated more ADMA. The increase in ADMA was temporally associated with a reduction in the activity of dimethylarginine dimethylaminohydrolase (DDAH, the enzyme that metabolizes ADMA). Infected cultures showed high levels of oxidative stress with enhanced endothelial production of superoxide anion.CMV infection in human heart transplant recipients is associated with higher ADMA elevation and more severe TA. CMV infection in endothelial cells increases oxidative stress, impairs DDAH activity, and increases ADMA elaboration. CMV infection may contribute to endothelial dysfunction and TA by dysregulation of the endothelial nitric oxide synthase pathway.

    View details for DOI 10.1161/01.CIR.0000109692.16004.AF

    View details for Web of Science ID 000188669400011

    View details for PubMedID 14732750

  • Discordant effects of a soluble VEGF receptor on wound healing and angiogenesis GENE THERAPY Jacobi, J., Tam, B. Y., Sundram, U., von Degenfeld, G., Blau, H. M., Kuo, C. J., Cooke, J. P. 2004; 11 (3): 302-309


    Soluble receptors to vascular endothelial growth factor (VEGF) can inhibit its angiogenic effect. Since angiogenesis is involved in wound repair, we hypothesized that adenovirus-mediated gene transfer of a soluble form of VEGF receptor 2 (Flk-1) would attenuate wound healing in mice. C57Bl/6J and genetically diabetic (db/db) mice (each n=20) received intravenous (i.v.) injections of recombinant adenoviruses (10(9) PFU) encoding the ligand-binding ectodomain of VEGF receptor 2 (Flk-1) or cDNA encoding the murine IgG2alpha Fc fragment (each n=10). At 4 days after gene transfer, two full-thickness skin wounds (0.8 cm) were created on the dorsum of each animal. Wound closure was measured over 9-14 days after which wounds were resected for histological analysis. Prior to killing, fluorescent microspheres were systemically injected for quantitation of wound vascularity. Single i.v. injections of adenoviruses encoding soluble Flk-1 significantly decreased wound angiogenesis in both wild-type and diabetic mice. Fluorescence microscopy revealed a 2.0-fold (wild type) and 2.9-fold (diabetic) reduction in wound vascularity in Flk-1-treated animals (p<0.05). Impairment of angiogenesis was confirmed by CD31 immunohistochemistry. Interestingly, despite significant reductions in wound vascularity, wound closure was not grossly delayed. Our data indicates that while VEGF function is essential for optimal wound angiogenesis, it is not required for wound closure.

    View details for DOI 10.1038/

    View details for Web of Science ID 000188329600011

    View details for PubMedID 14737090

  • Nicotine and angiogenesis: a new paradigm for tobacco-related diseases ANNALS OF MEDICINE Cooke, J. P., Bitterman, H. 2004; 36 (1): 33-40


    The pathophysiology of tobacco-related diseases is complex and multifactorial. Among the approximately 4,000 compounds in tobacco smoke are carcinogens such as nitrosamines, irritants such as a variety of phenolic compounds, volatiles such as carbon monoxide, and of course nicotine. Nicotine itself has quite complex actions, mediated in part by nicotinic cholinergic receptors that may have extraneuronal, as well as neuronal distribution. This review discusses the mechanisms by which nicotine contributes to tobacco-related disease, with a focus on the surprising new finding that nicotine is a potent angiogenic agent. Nicotine hijacks an endogenous nicotinic cholinergic pathway present in endothelial cells that is involved in physiological, as well as pathological angiogenesis.

    View details for DOI 10.1080/07853890310017576

    View details for Web of Science ID 000189109500004

    View details for PubMedID 15000345

  • Isoflavones improve vascular reactivity in post-menopausal women with hypercholesterolemia VASCULAR MEDICINE Lissin, L. W., Oka, R., Lakshmi, S., Cooke, J. P. 2004; 9 (1): 26-30


    This randomized clinical trial was designed to assess the effects of dietary isoflavones on vascular reactivity, lipid levels, and markers of inflammation in post-menopausal women. Epidemiological studies have revealed that populations consuming large amounts of soy protein have lower cardiovascular morbidity and mortality. The benefits of soy protein may be due to its hypolipidemic effects; its anti-oxidant properties; its high content of L-arginine; and/or or its phytoestrogen content. Two putative mediators of the effects of soy protein are the isoflavones genistein and daidzein. Forty post-menopausal, hypercholesterolemic women who did not take estrogen replacement therapy were recruited for this study of isoflavone supplementation. Baseline flow-mediated vasodilation and response to nitroglycerin were measured, along with urinary isoflavone and nitrite/nitrate levels and serum lipids. After 6 weeks of 90 mg of isoflavones daily versus placebo, women receiving isoflavones demonstrated improved responsiveness to nitroglycerin, an assessment of endothelium-independent vasodilation, with an effect size (percentage points change from baseline) of 7.2 +/- 1.9 versus 1.2 +/- 1.3; p = 0.01. There was a trend towards improvement of flow-mediated vasodilation, which is an endothelium-dependent response (effect size: 3.4 +/- 2.0% versus -0.6 +/- 1.7%; p = 0.12). Lipid levels were unchanged after isoflavone treatment. In conclusion, dietary isoflavones may have cardiovascular benefit in the form of improved vascular reactivity, but not by lowering cholesterol, for women who do not take estrogen replacement therapy.

    View details for Web of Science ID 000222186300005

    View details for PubMedID 15230485

  • NO and angiogenesis 1st International Symposium on ADMA as A Risk Factor Cooke, J. P. ELSEVIER IRELAND LTD. 2003: 53–60


    Angiogenesis requires the elaboration of endothelium-derived nitric oxide (NO). Angiogenic factors induce the release of NO from endothelial cells, which mediates a multiplicity of processes involved in angiogenesis. These NO-modulated processes include endothelial cell survival, proliferation, migration, and interaction with the extracellular matrix. Derangements of the NO synthase pathway impair angiogenesis. Accordingly, the competitive inhibitor of the NOS pathway asymmetric dimethylarginine (ADMA) acts as an endogenous inhibitor of angiogenesis. By contrast, agents which increase NO synthesis, such as low dose statins, enhance angiogenesis. Modulation of the NO synthase pathway could become a new therapeutic avenue for angiogenesis-related disorders.

    View details for DOI 10.1016/S1567-5688(03)00034-5

    View details for Web of Science ID 000187946900008

    View details for PubMedID 14664903

  • A novel mechanism for pulmonary arterial hypertension? CIRCULATION Cooke, J. P. 2003; 108 (12): 1420-1421
  • Endothelial dysfunction induced by hyperhomocyst(e)inemia - Role of asymmetric dimethylarginine CIRCULATION Stuhlinger, M. C., Oka, R. K., Graf, E. E., Schmolzer, I., Upson, B. M., Kapoor, O., Szuba, A., Malinow, M. R., Wascher, T. C., Pachinger, O., Cooke, J. P. 2003; 108 (8): 933-938


    Endothelial function is impaired by hyperhomocyst(e)inemia. We have previously shown that homocyst(e)ine (Hcy) inhibits NO production by cultured endothelial cells by causing the accumulation of asymmetric dimethylarginine (ADMA). The present study was designed to determine if the same mechanism is operative in humans.We studied 9 patients with documented peripheral arterial disease (6 men; 3 women; age, 64+/-3 years), 9 age-matched individuals at risk for atherosclerosis (older adults; 9 men; age, 65+/-1 years), and 5 young control subjects (younger adults; 5 men; age, 31+/-1 years) without evidence of or risk factors for atherosclerosis. Endothelial function was measured by flow-mediated vasodilatation of the brachial artery before and 4 hours after a methionine-loading test (100 mg/kg body weight, administered orally). In addition, blood was drawn at both time points for measurements of Hcy and ADMA concentrations. Plasma Hcy increased after the methionine-loading test in each group (all, P<0.001). Plasma ADMA levels rose in all subjects, from 0.9+/-0.2 to 1.6+/-0.2 micromol/L in younger adults, from 1.5+/-0.2 to 3.0+/-0.4 micromol/L in older adults, and from 1.8+/-0.1 to 3.9+/-0.3 micromol/L in peripheral arterial disease patients (all, P<0.001). Flow-mediated vasodilatation was reduced from 13+/-2% to 10+/-1% in younger adults, from 6+/-1% to 5+/-1% in older adults, and from 7+/-1% to 3+/-1% in peripheral arterial disease patients (all, P<0.001). Furthermore, we found positive correlations between plasma Hcy and ADMA concentrations (P=0.03, r=0.450), as well as ADMA and flow-mediated vasodilatation (P=0.002, r=0.623).Our results suggest that experimental hyperhomocyst(e)inemia leads to accumulation of the endogenous NO synthase inhibitor ADMA, accompanied by varying degrees of endothelial dysfunction according to the preexisting state of cardiovascular health.

    View details for DOI 10.1161/01.CIR.0000085067.55901.89

    View details for Web of Science ID 000184954900008

    View details for PubMedID 12912818

  • NOS inhibition accelerates atherogenesis: reversal by exercise AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Niebauer, J., Maxwell, A. J., Lin, P. S., Wang, D., Tsao, P. S., Cooke, J. P. 2003; 285 (2): H535-H540


    In this study, we assessed the effects of chronic exercise training (12 wk) on atherosclerotic lesion formation in hypercholesterolemic apolipoprotein E-deficient mice (n = 31). At the age of 9 wk, mice were assigned to the following groups: sedentary (Sed; n = 9); exercise (Ex; n = 12); sedentary and oral NG-nitro-L-arginine (L-NNA, Sed-NA; n = 4), or exercise and oral L-NNA (Ex-NA; n = 6). Chronic exercise training was performed on a treadmill for 12 wk (6 times/wk and twice for 1 h/day) at a final speed of 22 m/min, and an 8 degrees grade. L-NNA was discontinued 5 days before final treadmill testing. The farthest distance run to exhaustion was observed in Ex-NA mice (Sed: 306 +/- 32 m; Ex: 640 +/- 87; Sed-NA: 451 +/- 109 m; Ex-NA: 820 +/- 49 m; all P < 0.05). Lesion formation was assessed in the proximal ascending aorta by dissection microscopy after oil red O staining. The aortas of Sed-NA mice manifested a threefold increase in lesion formation compared with the other groups. This L-NNA-induced lesion formation was reduced by chronic exercise training (Sed, 786 +/- 144; Ex, 780 +/- 206; Sed-NA, 2,147 +/- 522; Ex-NA, 851 +/- 253; Sed-NA vs. all other groups: P < 0.001). In conclusion, treatment with oral L-NNA (an nitric oxide synthase antagonist) leads to accelerated atherogenesis in genetically determined hypercholesterolemic mice. This adverse effect can be overcome by chronic exercise training.

    View details for DOI 10.1152/ajpheart.00360.2001

    View details for PubMedID 12598230

  • Flow-responsive remodeling after angioplasty is enhanced by high cholesterol diet. Prevention with pyrrolidine dithiocarbamate ATHEROSCLEROSIS Ward, M. R., Tsao, P. S., Herity, N. A., Cooke, J. P., Yeung, A. C. 2003; 168 (2): 333-341


    We examined the effects of high cholesterol diet and pyrrolidine dithiocarbamate (PDTC) on flow-dependent remodeling after angioplasty. After right common carotid balloon-injury, the right external carotid (low flow) or left common carotid artery were ligated (high flow) in rabbits fed normal diet, 1% cholesterol diet without or with the antioxidant PDTC for 7 days pre- and 7-28 days post-injury. Angiographic lumen diameter was significantly greater at 28 days in high flow than low flow normal diet animals, attributable on perfusion-fixed vessel morphometry to altered remodeling (area within the external elastic lamina: high flow 1.85+/-0.24 vs. low flow 1.31+/-0.04 mm(2), P<0.05) rather than differences in neointima formation or vessel tone. In animals on 1% cholesterol diet high flow remodeling was significantly enhanced (area within the external elastic lamina 3.13+/-0.17 mm(2), P<0.05 vs. high flow normal diet) but low flow inward remodeling was similar (area within the external elastic lamina 1.29+/-0.07 mm(2)). Mean Doppler flow velocities (initial/post-ligation/28 day follow-up, cm/s) had almost normalized in normal diet animals (high flow 30/49/35, low flow 32/9/26) but showed overcompensation in 1% cholesterol diet animals (high flow 32/49/22, low flow 30/11/25). PDTC therapy markedly attenuated remodeling (area within the external elastic lamina: high flow 2.20+/-0.18, and low flow 2.00+/-0.11 both P<0.05 vs. 1% cholesterol diet alone) and flow velocities only partially normalized (high flow 26/42/34, low flow 27/7/16). We conclude that hypercholesterolemia enhances and PDTC attenuates flow-dependent remodeling after angioplasty.

    View details for DOI 10.1016/S0021-9150(03)00103-5

    View details for Web of Science ID 000183784900016

    View details for PubMedID 12801617

  • Cardiac allograft vasculopathy and dysregulation of the NO synthase pathway ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Weis, M., Cooke, J. P. 2003; 23 (4): 567-575


    Cardiac allograft vasculopathy is the most aggressive form of atherosclerosis in humans and is the leading cause of death after the first year of heart transplantation. Endothelial dysfunction is a major contributing factor to the acceleration of coronary vascular disease in these individuals. A reflection of this endothelial dysfunction is the severe impairment in endothelium-dependent vasodilation that occurs early after transplantation. The etiology of this allograft endothelial alteration is multifactorial and may include preexisting atherosclerosis of the graft vessels, reperfusion injury during transplantation, denervation, disruption of the lymphatic system, and acute and chronic immune injury, as well as traditional risk factors for coronary artery disease (hyperlipidemia, diabetes, hypertension, or hyperhomocysteinemia) and pathogens, such as cytomegalovirus. The alteration in endothelial function affects vasomotor tone of the coronary arteries. Evidence indicates that there may be an impairment of endothelial production and/or activity of NO. Because NO is a potent vasodilator, its deficiency would explain the abnormal vasomotor tone in these individuals. In addition, because NO inhibits key processes in vascular inflammation and atherosclerosis, its absence may contribute to the acceleration of transplant vascular disease. Recent studies from our group and others have shed light on the mechanisms of endothelial dysfunction and its importance in cardiac allograft vasculopathy. In addition, the alteration in endothelial function contributes to vascular inflammation and progression of the disease.

    View details for DOI 10.1161/01.ATV.0000067060.31369.F9

    View details for Web of Science ID 000182165100007

    View details for PubMedID 12649081

  • Nicotine promotes arteriogenesis JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Heeschen, C., Weis, M., Cooke, J. P. 2003; 41 (3): 489-496


    In the current study, we used a model of limb ischemia to determine whether nicotine could enhance arteriogenesis, to compare the magnitude of this effect to the angiogenic factor basic fibroblast growth factor (bFGF), and to investigate the mechanisms of the effect.We have shown previously that nicotine stimulates angiogenesis via stimulation of endothelial nicotinic cholinergic receptors. Stimulation of endothelial nicotinic cholinergic receptors causes endothelial cell proliferation, migration, and formation of capillary networks in vitro and angiogenesis in vivo in conditions of ischemia and inflammation.New Zealand White rabbits (n = 85) underwent unilateral femoral artery occlusion and were randomized to nicotine (0.05 to 5.0 microg/kg/day), bFGF (10 microg/kg/day), or vehicle delivered intra-arterially via osmotic minipumps. At day 21, morphologic changes were assessed by immunohistochemistry and angiography. Blood flow in the ischemic limb was determined by intra-arterial Doppler flow measurements and microsphere distribution.Nicotine enhanced capillary density in the ischemic hind-limb to a similar extent as bFGF. Nicotine also increased angiographic score, calf blood pressure ratio, intra-arterial Doppler flow, and microsphere distribution. In vitro, nicotine stimulated monocyte adhesion and transmigration. Nicotine increased by two- to three-fold the expression of monocyte adhesion molecules CD11b and CD11a; the expression of the endothelial adhesion molecule intercellular adhesion molecule-1; and the endothelial release of monocyte chemoattractant protein-1.In the short term, nicotine promotes angiogenesis and arteriogenesis in the setting of ischemia. The effect of nicotine may be mediated in part by activation of endothelial-monocyte interactions involved in arteriogenesis.

    View details for DOI 10.1016/S0735-1097(02)02818-8

    View details for Web of Science ID 000180759800022

    View details for PubMedID 12575981

  • Flow, NO, and atherogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cooke, J. P. 2003; 100 (3): 768-770

    View details for DOI 10.1073/pnas.0430082100

    View details for Web of Science ID 000180838100002

    View details for PubMedID 12552094

    View details for PubMedCentralID PMC298674

  • Short polymers of arginine rapidly translocate into vascular cells - Effects on nitric oxide synthesis CIRCULATION JOURNAL Uemura, S., Rothbard, J. B., Matsushita, H., Tsao, P. S., Fathman, C. G., Cooke, J. P. 2002; 66 (12): 1155-1160


    The present study was designed to determine the efficiency of translocation of short polymers of arginine into vascular smooth muscle cells (VSMC) and to determine their effect on nitric oxide (NO) synthesis. Immunostaining revealed that heptamers of L-arginine (R7) rapidly translocated into the VSMC. This rapid transport was not observed with shorter polymers of L-arginine (R5) nor heptamers of lysine (K7). Translocation of R7 was not inhibited by the addition of free L-arginine into the media. When cells were transiently pretreated with R7 or a nonamer of arginine (R9), NO(2) production from cytokine stimulated VSMC was significantly increased, whereas incubation with R5 and K7 had no effect. Short polymers of arginine not only have a unique ability of rapid VSMC translocation but once internalized enhance NO production. Heptamers (or larger polypeptides) of arginine may be useful in therapy to enhance NO production in the vascular system.

    View details for Web of Science ID 000179496000015

    View details for PubMedID 12499624

  • Endothelial determinants of dendritic cell adhesion and migration - New implications for vascular diseases ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Weis, M., Schlichting, C. L., Engleman, E. G., Cooke, J. P. 2002; 22 (11): 1817-1823


    Atherosclerosis is a chronic disease triggered by endothelial injury and sustained by inflammation. Dendritic cells (DCs) are critical for the cell-mediated arm of an immune response and are known to influence inflammatory immunity. A fundamental aspect of DC function is their capacity to adhere and migrate through vascular endothelial cells (ECs). We investigated the role of endothelial activation and dysregulation of the NO pathway on DC adhesion and migration.We discovered that DC adhesion and migration are modulated by changes in endothelial function. DC adhesion and transmigration were markedly increased after exposing ECs to hypoxia, oxidized low density lipoprotein, or tumor necrosis factor-alpha. Specifically, inhibition of endothelial NO synthase increased DC binding and transmigration. L-Arginine or 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition partially decreased DC-EC interaction.The results of this study suggest that the adhesion and migration of DCs are increased by stimuli known to accelerate atherogenesis. Vice versa, augmentation of endothelial NO synthase activity prevents DC adhesion. These findings may provide insight into the inflammatory processes occurring in atherosclerosis. Because DCs control immunity, regulating DC-EC interaction may be relevant to inflammation and atherogenesis.

    View details for DOI 10.1161/01.ATV.0000036418.04998.D5

    View details for Web of Science ID 000179258000015

    View details for PubMedID 12426210

  • Nicotine accelerates angiogenesis and wound healing in genetically diabetic mice AMERICAN JOURNAL OF PATHOLOGY Jacobi, J., Jang, J. J., Sundram, U., Dayoub, H., Fajardo, L. F., Cooke, J. P. 2002; 161 (1): 97-104


    Recently, we have discovered an endogenous cholinergic pathway for angiogenesis mediated by endothelial nicotinic acetylcholine receptors (nAChRs). Since angiogenesis plays a major role in wound repair, we hypothesized that activation of nAChRs with nicotine would accelerate wound healing in a murine excisional wound model. In genetically diabetic and control mice full-thickness skin wounds (0.8 cm) were created on the dorsum and topically treated over 7 days with either vehicle (phosphate-buffered saline, PBS) or nicotine (10(-8) mol/L, 10(-9) mol/L; each, n = 5). Wound size was measured over 14 days followed by resection, histological analysis, and quantitation of vascularity. In diabetic animals an agonist (epibatidine, 10(-10) mol/L) or antagonist (hexamethonium, 10(-4) mol/L) of nAChRs as well as the positive control basic fibroblast growth factor (bFGF, 25 microg/kg) were also tested. To further study the role of endothelial nAChRs in angiogenesis, we used an ex vivo vascular explant model. In diabetic mice wound healing was markedly impaired. Nicotine significantly accelerated wound healing as assessed by closure rate and histological score. The effects of nicotine were equal to bFGF and were mimicked by epibatidine and blocked by hexamethonium. Histomorphometry revealed increased neovascularization in animals treated with nicotine. Furthermore, capillary-like sprouting from vascular explants was significantly enhanced by nicotine. In conclusion, agonist-induced stimulation of nAChRs accelerates wound healing in diabetic mice by promoting angiogenesis. We have discovered a cholinergic pathway for angiogenesis that is involved in wound healing, and which is a potential target for therapeutic angiogenesis.

    View details for Web of Science ID 000176718300013

    View details for PubMedID 12107094

  • Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes METABOLISM-CLINICAL AND EXPERIMENTAL Asagami, T., Abbasi, F., Stuelinger, M., Lamendola, C., McLaughlin, T., Cooke, J. P., Reaven, G. M., Tsao, P. S. 2002; 51 (7): 843-846


    This study was initiated to see if plasma asymmetric dimethylarginine (ADMA) concentrations decreased in hyperglycemic patients with type 2 diabetes following metformin treatment, either as monotherapy or following its addition to sulfonylurea-treated patients. Fasting plasma glucose, dimethylarginine, and L-arginine concentrations were measured before and 3 months after the administration of a maximally effective dose of metformin to 31 patients with type 2 diabetes in poor glycemic control (fasting plasma concentrations > 9.7 mmol/L), while being treated with either diet (n = 16) or a maximal amount of a sulfonylurea compound (n = 15). Fasting plasma glucose concentration (mean +/- SEM) decreased to a similar degree (P <.01) in patients treated with either metformin alone (12.4 +/- 0.5 to 9.5 +/- 0.5 mmol/L) or when it was added to a sulfonylurea compound (14.1 +/- 0.5 to 10.6 +/- 0.9 mmol/L). The improvement in glycemic control was associated with similar decreases (P <.01) in ADMA concentrations in metformin (1.65 +/- 0.21 to 1.18 +/- 0.13 micromol/L) and sulfonylurea + metformin-treated patients (1.75 +/- 0.13 to 1.19 +/- 0.08 micromol/L). Plasma L-arginine concentrations were similar in the 2 groups at baseline and did not change in response to metformin. Thus, metformin treatment was associated with a favorable increase in the plasma L-arginine/ADMA ratio. These results provide the first evidence that plasma ADMA concentrations decrease in association with improved glycemic control in patients with type 2 diabetes and demonstrate that the magnitude of the change in metformin-treated patients was similar, irrespective of whether it was used as monotherapy or in combination with sulfonylurea treatment.

    View details for DOI 10.1053/meta.2002.33349

    View details for Web of Science ID 000176578200007

    View details for PubMedID 12077728

  • Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Stuhlinger, M. C., Abbasi, F., Chu, J. W., Lamendola, C., McLaughlin, T. L., Cooke, J. P., Reaven, G. M., Tsao, P. S. 2002; 287 (11): 1420-1426


    Increased levels of asymmetric dimethylarginine (ADMA) are associated with endothelial dysfunction and increased risk of cardiovascular disease. Several cardiovascular risk factors are associated with reduced sensitivity to insulin, but elevated ADMA concentrations have not been fully linked to the metabolic syndrome.To evaluate the relationship between insulin sensitivity and plasma ADMA concentrations, and to determine whether a pharmacological treatment that increases insulin sensitivity would also modulate ADMA concentrations.Cross-sectional study, containing a nonrandomized controlled trial component, of 64 healthy volunteers without diabetes (42 women, 22 men; 48 with normal blood pressure and 16 with hypertension), which was conducted at a university medical center between October 2000 and July 2001.Rosiglitazone (4 mg/d for 4 weeks and then 4 mg twice daily for 8 weeks), an insulin-sensitizing agent, was given to 7 insulin-resistant subjects with hypertension. These subjects were studied before and after 12-week treatment.Insulin sensitivity measured by the insulin suppression test, and fasting plasma levels of low-density lipoprotein cholesterol, triglycerides, high-density lipoprotein cholesterol, glucose, insulin, and ADMA concentrations.Plasma ADMA concentrations were positively correlated with impairment of insulin-mediated glucose disposal in nondiabetic, normotensive subjects (r = 0.73; P<.001). Consistent with the metabolic syndrome, ADMA levels were also positively correlated with fasting triglyceride levels (r = 0.52; P<.001) but not with low-density lipoprotein cholesterol levels (r = 0.19; P =.20). Plasma ADMA concentrations increased in insulin-resistant subjects independent of hypertension. Pharmacological treatment improved insulin sensitivity and reduced mean (SD) plasma ADMA concentrations from 1.50 (0.30) to 1.05 (0.33) micromol/L (P =.001).A significant relationship exists between insulin resistance and plasma concentrations of ADMA. Pharmacological intervention with rosiglitazone enhanced insulin sensitivity and reduced ADMA levels. Increases in plasma ADMA concentrations may contribute to the endothelial dysfunction observed in insulin-resistant patients.

    View details for Web of Science ID 000174465000024

    View details for PubMedID 11903029

  • Effect of local delivery of L-arginine on in-stent restenosis in humans AMERICAN JOURNAL OF CARDIOLOGY Suzuki, T., Hayase, M., Hibi, K., Hosokawa, H., Yokoya, K., Fitzgerald, P. J., Yock, P. G., Cooke, J. P., Suzuki, T., Yeung, A. C. 2002; 89 (4): 363-367


    To determine whether intramural administration of L-arginine reduces intimal thickening after optimal Palmaz-Schatz stent deployment in humans, 50 patients with native coronary artery disease who received a single Palmaz-Schatz stent were enrolled in this pilot study. Patients were randomized into 2 treatment groups: an L-arginine group (n = 25) and a saline group (n = 25). After stent deployment, L-arginine (600 mg/6 ml) or saline (6 ml) was locally delivered via the Dispatch catheter (Scimed) over 15 minutes. Serial angiography and intravascular ultrasound examinations (motorized pull-back at 0.5 mm/s) were performed before and after the procedure, and at 6-month follow-up. Measurements of stent area, lumen area, and neointimal area were computed within the stents at 1-mm intervals, by technicians who were blinded to the treatment assignment. Using Simpson's rule, stent, plaque, and lumen volumes, neointimal volume within the stent, and percent neointimal volume were measured before and after the procedure, and at 6-month follow-up. The 6-month volume data in quantitative coronary ultrasound showed that neointimal volume in the L-arginine group was significantly less than in the saline group (25 vs 39 mm(3); p = 0.049). Similarly, percent neointimal volume was significantly less in the L-arginine group at 6-month follow-up (17 +/- 13% vs 27 +/- 21%; p = 0.048). Thus, these results showed that local delivery of L-arginine reduces in-stent neointimal hyperplasia in humans, indicating that this approach may be a novel strategy to prevent in-stent restenosis.

    View details for Web of Science ID 000173816400001

    View details for PubMedID 11835911

  • Statins have biphasic effects on angiogenesis CIRCULATION Weis, M., Heeschen, C., Glassford, A. J., Cooke, J. P. 2002; 105 (6): 739-745


    Statins inhibit HMG-CoA reductase to reduce the synthesis of cholesterol and isoprenoids that modulate diverse cell functions. We investigated the effect of the statins cerivastatin and atorvastatin on angiogenesis in vitro and in vivo.Endothelial cell proliferation, migration, and differentiation were enhanced at low concentrations (0.005 to 0.01 micromol/L) but significantly inhibited at high statin concentrations (0.05 to 1 micromol/L). Antiangiogenic effects at high concentrations were associated with decreased endothelial release of vascular endothelial growth factor and increased endothelial apoptosis and were reversed by geranylgeranyl pyrophosphate. In murine models, inflammation-induced angiogenesis was enhanced with low-dose statin therapy (0.5 mg x kg(-1) x d(-1)) but significantly inhibited with high concentrations of cerivastatin or atorvastatin (2.5 mg x kg(-1) x d(-1)). Despite the fact that high-dose statin treatment was effective at reducing lipid levels in hyperlipidemic apolipoprotein E-deficient mice, it impaired rather than enhanced angiogenesis. Finally, high-dose cerivastatin decreased tumor growth and tumor vascularization in a murine Lewis lung cancer model.HMG-CoA reductase inhibition has a biphasic dose-dependent effect on angiogenesis that is lipid independent and associated with alterations in endothelial apoptosis and vascular endothelial growth factor signaling. Statins have proangiogenic effects at low therapeutic concentrations but angiostatic effects at high concentrations that are reversed by geranylgeranyl pyrophosphate. At clinically relevant doses, statins may modulate angiogenesis in humans via effects on geranylated proteins.

    View details for DOI 10.1161/hc0602.103393

    View details for Web of Science ID 000173924600025

    View details for PubMedID 11839631

  • Homocysteine impairs the nitric oxide synthase pathway - Role of asymmetric dimethylarginine CIRCULATION Stuhlinger, M. C., Tsao, P. S., Her, J. H., Kimoto, M., Balint, R. F., Cooke, J. P. 2001; 104 (21): 2569-2575


    Hyperhomocysteinemia is a putative risk factor for cardiovascular disease, which also impairs endothelium-dependent vasodilatation. A number of other risk factors for cardiovascular disease may exert their adverse vascular effects in part by elevating plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Accordingly, we determined if homocysteine could increase ADMA levels.When endothelial or nonvascular cells were exposed to DL-homocysteine or to its precursor L-methionine, ADMA concentration in the cell culture medium increased in a dose- and time-dependent fashion. This effect was associated with the reduced activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Furthermore, homocysteine-induced accumulation of ADMA was associated with reduced nitric oxide synthesis by endothelial cells and segments of pig aorta. The antioxidant pyrrollidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation. Moreover, homocysteine dose-dependently reduced the activity of recombinant human DDAH in a cell free system, an effect that was due to a direct interaction between homocysteine and DDAH.Homocysteine post-translationally inhibits DDAH enzyme activity, causing ADMA to accumulate and inhibit nitric oxide synthesis. This may explain the known effect of homocysteine to impair endothelium-mediated nitric oxide-dependent vasodilatation.

    View details for Web of Science ID 000172307200013

    View details for PubMedID 11714652

  • Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus AMERICAN JOURNAL OF CARDIOLOGY Abbasi, F., Asagmi, T., Cooke, J. P., Lamendola, C., McLaughlin, T., Reaven, G. M., Stuehlinger, M., Tsao, P. S. 2001; 88 (10): 1201-?

    View details for Web of Science ID 000172412300025

    View details for PubMedID 11703973

  • eNOS activity is reduced in senescent human endothelial cells - Preservation by hTERT immortalization CIRCULATION RESEARCH Matsushita, H., Chang, E., Glassford, A. J., Cooke, J. P., Chiu, C. P., Tsao, P. S. 2001; 89 (9): 793-798


    Advanced age is associated with endothelial dysfunction and increased risk for atherosclerosis. However, the mechanisms for these observed effects are not clear. To clarify the association between aging and loss of endothelial function, young human aortic endothelial cells (HAECs), senescent HAECs transfected with control vector, and immortalized HAECs containing human telomerase reverse transcriptase (hTERT) were compared for expression of endothelial nitric oxide synthase (eNOS) and production of NO. To investigate a specific function modulated by endothelial NO, adhesion of monocytes under basal conditions as well as after exposure to TNF-alpha was assessed. A decrease in eNOS mRNA, protein, and activity was observed in endothelial cells at senescence as compared with young HAEC; this effect was blunted in hTERT cells. In all cells, shear stress induced a greater increase in the expression of eNOS protein with the final result being higher levels in hTERT compared with senescent cells. Basal monocyte binding was significantly elevated on aged endothelial cells compared with parental and hTERT cells. Exposure of TNF-alpha resulted in a 2-fold increase in monocyte adhesion in senescent cells, whereas this effect was reduced in cells transfected with hTERT. Prior exposure to fluid flow significantly reduced subsequent monocyte adhesion in all groups. These studies demonstrate that replicative aging results in decreased endothelial expression of eNOS accompanied by enhanced monocyte binding. Stable expression of hTERT results in endothelial cells with a younger phenotype with greater amount of eNOS and NO activity. Thus, telomerase transfection may have important functional consequences on endothelial cells.

    View details for Web of Science ID 000171974800010

    View details for PubMedID 11679409

  • Cholesterol-induced upregulation of angiotensin II and its effects on monocyte-endothelial interaction and superoxide production VASCULAR MEDICINE Niebauer, J., Tsao, P. S., Lin, P. S., Pratt, R. E., Cooke, J. P. 2001; 6 (3): 133-138


    Atherogenesis involves an early endothelial dysfunction hallmarked by elevated free radical production and increased adhesiveness for monocytes. It was hypothesized that activation of the tissue renin angiotensin system may contribute to the endothelial alteration. To test this hypothesis, thoracic aortae were isolated from normocholesterolemic (NC; n = 6) and hypercholesterolemic (HC; n = 6; diet: 0.5% cholesterol; 6 weeks) New Zealand white rabbits, and incubated for 2 h with the angiotensin II (Ang II) receptor antagonist Sar-1,Ile-8-Ang II, the antioxidant pyrolidine dithiocarbamate (PDTC) and the protein kinase C (PKC) antagonist staurosporin. Superoxide production from aortic segments was measured by lucigenin-enhanced chemiluminescence. In comparison to the normocholesterolemic state, hypercholesterolemia led to a significant increase in superoxide production (221 +/- 44%, p < 0.02); this was reduced by ex vivo treatment of the vessel segment with Ang II-antagonist (to 130 +/- 29%; p < 0.04 vs HC), or PKC-antagonist (to 86 +/- 26%; p < 0.001 vs HC), or PDTC (to 103 +/- 27%; p < 0.02 vs HC). Monocyte-endothelial interaction was assessed by functional binding assay. When compared to normocholesterolemic rabbits, hypercholesterolemia led to a twofold increase in monocyte binding (74 +/- 13 vs 37 +/- 4 monocytoid cells per high power field (m/hpf); p < 0.03). The Ang II-antagonist and the PKC-antagonist led to a normalization of monocyte-endothelial binding (Ang II-antagonist: 37 +/- 9 m/hpf; PKC-antagonist: 41 +/- 17 m/hpf; p < 0.05). In conclusion, these results indicate that hypercholesterolemia activates the tissue renin angiotensin system, which results in an increased endothelial production of superoxide and monocyte adhesiveness. Ang II-antagonist inhibits free radical production and monocyte adhesion through a mechanism which may include PKC.

    View details for Web of Science ID 000172694700002

    View details for PubMedID 11789966

  • Atherogenesis and the arginine hypothesis. Current atherosclerosis reports Cooke, J. P., Oka, R. K. 2001; 3 (3): 252-259


    In patients who have elevated levels of plasma ADMA, a relative deficiency of L-arginine has been found to contribute to the pathophysiology of athersclerosis, causing vasoconstriction, and accelerating atherogenesis. This finding--that there is a relative deficiency of L-arginine in atherosclerotic disease--is a breakthrough that will open new avenues of therapy.

    View details for PubMedID 11286647

  • L-Arginine enhances aerobic exercise capacity in association with augmented nitric oxide production JOURNAL OF APPLIED PHYSIOLOGY Maxwell, A. J., Ho, H. V., Le, C. Q., Lin, P. S., Bernstein, D., Cooke, J. P. 2001; 90 (3): 933-938


    We tested whether supplementation with L-arginine can augment aerobic capacity, particularly in conditions where endothelium-derived nitric oxide (EDNO) activity is reduced. Eight-week-old wild-type (E(+)) and apolipoprotein E-deficient mice (E(-)) were divided into six groups; two groups (LE(+) and LE(-)) were given L-arginine (6% in drinking water), two were given D-arginine (DE(+) and DE(-)), and two control groups (NE(+) and NE(-)) received no arginine supplementation. At 12-16 wk of age, the mice were treadmill tested, and urine was collected after exercise for determination of EDNO production. NE(-) mice demonstrated a reduced aerobic capacity compared with NE(+) controls [maximal oxygen uptake (VO(2 max)) of NE(-) = 110 +/- 2 (SE) vs. NE(+) = 122 +/- 3 ml O(2). min(-1). kg(-1), P < 0.001]. This decline in aerobic capacity was associated with a diminished postexercise urinary nitrate excretion. Mice given L-arginine demonstrated an increase in postexercise urinary nitrate excretion and aerobic capacity in both groups (VO(2 max) of LE(-) = 120 +/- 1 ml O(2). min(-1). kg(-1), P < 0.05 vs. NE(-); VO(2 max) of LE(+) = 133 +/- 4 ml O(2). min(-1). kg(-1), P < 0.01 vs. NE(+)). Mice administered D-arginine demonstrated an intermediate increase in aerobic capacity in both groups. We conclude that administration of L-arginine restores exercise-induced EDNO synthesis and normalizes aerobic capacity in hypercholesterolemic mice. In normal mice, L-arginine enhances exercise-induced EDNO synthesis and aerobic capacity.

    View details for Web of Science ID 000167051400024

    View details for PubMedID 11181603

  • Maintaining the endothelium: preventive strategies for vessel integrity. Preventive cardiology Lissin, L. W., Cooke, J. P. 2001; 4 (1): 28-37


    The endothelium is a diaphanous membrane, only one cell layer thick, that lines all of our blood vessels. Despite its apparent fragility, it exerts profound control over vascular tone, structure, and intersection with circulating blood elements. One of the factors that the endothelium synthesizes is nitric oxide, which is the most potent endogenous vasodilator known. In addition to its blood flow regulating effects, nitric oxide also inhibits key processes in atherosclerosis, including monocyte adherence, platelet aggregation, and proliferation of vascular smooth muscle cells. Nitric oxide synthesis is impaired, and its degradation is accelerated, in many of the conditions associated with atherosclerosis, including hypercholesterolemia. Restoration of nitric oxide synthesis and activity in these disorders can improve blood flow, relieve symptoms, and perhaps reduce the progression of atherosclerosis.(c) 2001 by CHF, Inc.

    View details for PubMedID 11828196

  • Nicotine is an agent of angiogenesis. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med Heeschen C, et al 2001; 7 (7): 833-9
  • An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Boger, R. H., Bode-Boger, S. M., Tsao, P. S., Lin, P. S., Chan, J. R., Cooke, J. P. 2000; 36 (7): 2287-2295


    We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes.Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase.Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2-) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-KB by electromobility gel shift assay.Incubation of endothelial cells with ADMA (0.1 microM to 100 microM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 microM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2- to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-KB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody.We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

    View details for Web of Science ID 000165600400041

    View details for PubMedID 11127475

  • Rapid and efficient vascular transport of arginine polymers inhibits myointimal hyperplasia CIRCULATION Uemura, S., Fathman, C. G., Rothbard, J. B., Cooke, J. P. 2000; 102 (21): 2629-2635


    We recently discovered that short polymers of arginine efficiently translocate across the cytoplasmic membrane independent of the basic amino acid transporter. We evaluated the kinetics and biological effects of heptamers of L-arginine and D-arginine (L-R7 and D-R7, respectively) in vascular cells. We assessed the effects of these peptides on the NO synthesis pathway and vascular cell proliferation.Human umbilical vein endothelial cell and rabbit vascular segments were incubated in medium containing biotin-labeled L-R7 or D-R7. Both polymers rapidly translocated through the vessel wall and into the vascular cells in a dose- and time-dependent fashion. At a dose of 10 micromol/L for 30 minutes, 100% of the endothelial cells showed evidence of cytoplasmic and nuclear localization of the peptides. To evaluate the biological effects of the polymer translocation on myointimal formation, rabbit jugular vein segments were incubated with polymers (10 micromol/L, 30 minutes) or vehicle before arterial interposition grafting. Planimetric measurement 28 days after surgery revealed that L-R7 and D-R7 substantially reduced myointimal formation compared with the control condition (intima/media ratio: control 1. 50.5, L-R7 0.40.2, and D-R7 0.80.2; P:<0.05). Furthermore, basal nitrate and nitrite production from L-R7-treated grafts was significantly higher than that from both control and D-R7-treated veins. Studies in vitro of cultured vascular smooth muscle cells revealed that both polymers also exhibit an NO-independent inhibition of vascular smooth muscle cell proliferation.Short polymers of arginine have the unique ability of vascular cell translocation, and they also have direct biological effects. These attributes are potentially useful in treating myointimal hyperplasia.

    View details for Web of Science ID 000165405800013

    View details for PubMedID 11085967

  • Photoangioplasty for human peripheral atherosclerosis - Results of a phase I trial of photodynamic therapy with motexafin lutetium (Antrin) 72nd Annual Scientific Session of the American-Heart-Association Rockson, S. G., Kramer, P., Razavi, M., Szuba, A., Filardo, S., Fitzgerald, P., Cooke, J. P., Yousuf, S., DeVault, A. R., Renschler, M. F., Adelman, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2000: 2322–24


    In photoangioplasty, light activation of a photosensitive drug offers the potential for treatment of long segments of vascular disease. This is a brief description of a study designed to evaluate the safety and tolerability of a new photosensitizer, Antrin (motexafin lutetium), in the endovascular treatment of atherosclerosis.An open-label, single-dose, escalating drug- and light-dose study was performed in patients with atherosclerotic peripheral arterial insufficiency. Clinical evaluation, serial quantitative angiography, and intravascular ultrasonography were performed. Therapy was well tolerated, and only minor side effects were observed. Treatment produced no deleterious vascular effects. Although this study was not designed to examine clinical efficacy, several secondary end points suggested a favorable therapeutic effect.This phase I study demonstrates that photoangioplasty with motexafin lutetium is well tolerated and safe. Preliminary efficacy data suggest a future role for the treatment of flow-limiting atherosclerosis.

    View details for Web of Science ID 000165169200001

    View details for PubMedID 11067782

  • Maintaining the endothelium: preventive strategies for vessel integrity. Preventive cardiology Wroblewski Lissin, L., Cooke, J. P. 2000; 3 (4): 172-177


    The endothelium is a diaphanous membrane, only one cell layer thick, that lines all of our blood vessels. Despite its apparent fragility, it exerts profound control over vascular tone, structure, and interaction with circulating blood elements. One of the factors that the endothelium synthesizes is nitric oxide, which is the most potent endogenous vasodilator known. In addition to its blood flow regulating effects, nitric oxide also inhibits key processes in atherosclerosis, including monocyte adherence, platelet aggregation, and proliferation of vascular smooth muscle cells. Nitric oxide synthesis is impaired, and its degradation is accelerated, in many of the conditions associated with atherosclerosis, including hypercholesterolemia. Restoration of nitric oxide synthesis and activity in these disorders can improve blood flow, relieve symptoms, and perhaps reduce the progression of atherosclerosis (part 1 of 2 parts). (c) 2000 by CHF, Inc.

    View details for PubMedID 11834938

  • Angiogenesis is impaired by hypercholesterolemia - Role of asymmetric dimethylarginine CIRCULATION Jang, J. J., Ho, H. K., Kwan, H. H., Fajardo, L. F., Cooke, J. P. 2000; 102 (12): 1414-1419


    Many angiogenic factors require endothelium-derived nitric oxide (NO) to exert their effects. Recently, an endogenous competitive antagonist of NO synthase has been characterized: asymmetric dimethylarginine (ADMA). Elevated plasma levels of ADMA reduce NO synthesis in hypercholesterolemia. Accordingly, we hypothesized that hypercholesterolemia impairs angiogenesis by an ADMA-dependent mechanism.Angiogenesis was assessed with the use of a disk angiogenesis system implanted subcutaneously in normal (E(+)) mice or apolipoprotein (apo)E-deficient hypercholesterolemic (E(-)) mice. After 2 weeks, the disks were removed, and the fibrovascular growth area was used as an index of angiogenesis. Basal and fibroblast growth factor-stimulated angiogenesis was impaired in E(-) mice, associated with an elevation in plasma ADMA. Oral administration of L-arginine reversed the impairment of angiogenesis in E(-) mice. By contrast, oral administration of L-nitroarginine (an exogenous antagonist of NO synthase) reduced angiogenesis. When added directly to the disk, ADMA dose-dependently inhibited basal and fibroblast growth factor-induced angiogenesis, an effect that was reversed by oral administration of L-arginine.The derangement of the NO synthase pathway that occurs in hypercholesterolemia is associated with an impairment of angiogenesis. The lipid-induced impairment of angiogenesis can be reversed by oral administration of L-arginine and can be mimicked in normocholesterolemic animals by administration of an NO synthase antagonist. The data are consistent with the hypothesis that ADMA is an endogenous inhibitor of angiogenesis.

    View details for Web of Science ID 000089335900014

    View details for PubMedID 10993861

  • Decongestive lymphatic therapy for patients with cancer-related or primary lymphedema AMERICAN JOURNAL OF MEDICINE Szuba, A., Cooke, J. P., Yousuf, S., Rockson, S. G. 2000; 109 (4): 296-300


    A prospective evaluation was undertaken to assess the efficacy of intensive, short-term decongestive lymphatic therapy coupled with focused patient instruction in long-term self-care for the management of lymphedema.The therapeutic responses of 79 patients with lymphedema were analyzed prospectively. Each patient received intensive, short-term decongestive lymphatic therapy, with quantification of the extent and durability of the clinical response. Decongestive lymphatic therapy was performed by therapists trained in these techniques. The mean (+/-SD) duration of therapy was 8+/-3 days. Instruction in self-management techniques was incorporated into the therapeutic regimen by day 3 of the patient's treatment. The mean period of follow-up was 38+/-52 days. Changes in the volume of the affected limb were assessed with a geometric approximation derived from serial measurements of circumference along the axis of the limb.The mean short-term reduction in limb volume was 44%+/-62% of the excess volume in the upper extremities and 42%+/-40% in the lower extremities. At follow-up, these results were adequately sustained: mean long-term excess volume reductions of 38%+/-56% (upper extremities) and 41%+/-27% (lower extremities) were observed.Decongestive lymphatic therapy, combined with long-term self-management, is efficacious in treating patients with lymphedema of the extremity.

    View details for Web of Science ID 000089356600005

    View details for PubMedID 10996580

  • Does ADMA cause endothelial dysfunction? ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Cooke, J. P. 2000; 20 (9): 2032-2037


    Asymmetric dimethylarginine (ADMA) is an endogenous and competitive inhibitor of nitric oxide synthase. Plasma levels of this inhibitor are elevated in patients with atherosclerosis and in those with risk factors for atherosclerosis. In these patients, plasma ADMA levels are correlated with the severity of endothelial dysfunction and atherosclerosis. By inhibiting the production of nitric oxide, ADMA may impair blood flow, accelerate atherogenesis, and interfere with angiogenesis. ADMA may be a novel risk factor for vascular disease.

    View details for Web of Science ID 000089333600002

    View details for PubMedID 10978245

  • Differential expression of nitric oxide by dermal microvascular endothelial cells from patients with scleroderma VASCULAR MEDICINE Romero, L. I., Zhang, D. N., Cooke, J. P., Ho, H. K., Avalos, E., Herrera, R., Herron, G. S. 2000; 5 (3): 147-158


    Vascular abnormalities in scleroderma are fundamental to the pathogenesis of this disease. The objective of this study was to characterize dermal microvascular endothelial cells (DMEC) isolated from scleroderma patients with respect to growth and expression of the constitutive form of endothelial nitric oxide synthase (eNOS). DMEC from patients with both systemic sclerosis (SSc) and localized scleroderma (Loc Scl) contained small intact microvascular structures in contrast to single cell isolations obtained from control skin. Immunoaffinity selection on anti-PECAM-1 beads yielded pure populations of DMEC expressing normal markers. While the morphology and initial growth of SSc DMEC closely paralleled control cells, the growth of SSc DMEC decreased with time in culture (doubling time of 3 days vs. 5 days). Expression of ecNOS mRNA was reduced in both Loc Scl and SSc as shown by semi-quantitative RT-PCR (p < 0.001). Western blots showed variable but generally lower ecNOS protein levels and decreased levels of nitrogen oxides in media were found from both SSc and Loc Scl relative to control cells. The results indicate an intrinsic defect in the mechanism of nitric oxide production in DMEC isolated from scleroderma patients and suggest its possible involvement in the pathophysiology of scleroderma.

    View details for Web of Science ID 000165361400004

    View details for PubMedID 11104297

  • Phytoestrogens and cardiovascular health JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Lissin, L. W., Cooke, J. P. 2000; 35 (6): 1403-1410


    Coronary artery disease is the leading overall cause of mortality for women and increases dramatically after menopause. Estrogen has many beneficial cardiovascular actions although concerns have been raised about its effects on the progression of breast and uterine neoplasms and its tendency to increase coagulability. Selective estrogen agonists may be superior to conventional estrogens. A dietary source of a partial estrogen agonist is the plant-based group of phytoestrogens, which include isoflavones, lignans and coumestans. Phytoestrogens have a similar structure to estradiol and have weak affinity for the estrogen receptor. Epidemiologic data indicate that women ingesting high amounts of phytoestrogens, particularly as isoflavones in soy products, have less cardiovascular disease, breast and uterine cancer and menopausal symptoms than those eating Western diets. Preclinical and clinical studies have found that isoflavones have lipid-lowering effects as well as the ability to inhibit low-density lipoprotein oxidation. They have been shown to normalize vascular reactivity in estrogen-deprived primates. Furthermore, phytoestrogens have antineoplastic effects with inhibition of cellular proliferation as well as angiogenesis, properties that could be protective against cancer development. Finally, menopausal symptoms and bone density may be favorably influenced by phytoestrogens. In summary, phytoestrogens, in the form of dietary isoflavones, represent a new area to explore in pursuit of nutritional approaches to cardiovascular protection.

    View details for Web of Science ID 000086828700001

    View details for PubMedID 10807439

  • Asymmetric dimethylarginine increases mononuclear cell adhesiveness in hypercholesterolemic humans ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Chan, J. R., Boger, R. H., Bode-Boger, S. M., Tangphao, O., Tsao, P. S., Blaschke, T. F., Cooke, J. P. 2000; 20 (4): 1040-1046


    Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is elevated in hypercholesterolemia. This study was designed to determine the role of ADMA in the increased mononuclear cell adhesiveness observed in human hypercholesterolemia. In patient studies, plasma ADMA levels were determined by high-performance liquid chromatography. Functional mononuclear leukocyte adhesion assays were performed in parallel, and flow cytometry was used to characterize bound monocytes and T lymphocytes. Hypercholesterolemic patients were then placed on an oral L-arginine regimen of 14 or 21 g/d and studied over 12 weeks. In cell culture studies, bovine aortic endothelial cells were incubated with varied concentrations of ADMA. Monocytoid cells were cocultured with these bovine aortic endothelial cells, and their adhesiveness was assessed by use of a binding assay. Flow cytometry was used to quantify adhesion molecule expression. Plasma ADMA levels and adhesiveness of mononuclear cells (specifically, monocytes and T lymphocytes) were elevated in hypercholesterolemic patients. Adhesiveness was inversely correlated with the plasma L-arginine/ADMA ratio. Oral administration of L-arginine normalized plasma L-arginine/ADMA ratios and attenuated monocyte and T-lymphocyte adhesiveness. ADMA had no direct effect on the adhesiveness of mononuclear cells. However, monocytes became hyperadhesive when cocultured with ADMA-exposed endothelial cells. In human hypercholesterolemia, the plasma L-arginine/ADMA ratio is inversely correlated with mononuclear cell adhesiveness. Restoration of the L-arginine/ADMA ratio to control levels normalizes mononuclear cell adhesiveness. Our studies suggest that the elaboration of endothelium-derived nitric oxide affects the behavior of circulating T lymphocytes and monocytes.

    View details for Web of Science ID 000086468300020

    View details for PubMedID 10764670

  • The endothelium: a new target for therapy VASCULAR MEDICINE Cooke, J. P. 2000; 5 (1): 49-53


    At one time considered merely a monolayer of cells lining the vascular conduit, the endothelium has emerged recently as an organ with functions as complex as any in the body. A highly active regulatory organ, the endothelium senses and assesses the hemodynamic, humoral, and inflammatory signals to which it is constantly exposed by the blood and responds by secreting factors that affect vessel tone and structure. These interactions are not merely of academic interest. It has been increasingly recognized that endothelial dysfunction plays a pivotal role in the development and progression of atherosclerosis and coronary artery disease.

    View details for Web of Science ID 000085754700008

    View details for PubMedID 10737156

  • Local L-arginine delivery after balloon angioplasty reduces monocyte binding and induces apoptosis CIRCULATION Niebauer, J., Schwarzacher, S. P., Hayase, M., Wang, B. Y., Kernoff, R. S., Cooke, J. P., Yeung, A. C. 1999; 100 (17): 1830-1835


    Local administration of L-arginine after balloon angioplasty has been shown to enhance NO generation and inhibit lesion formation. In this study, we assessed the mechanisms by which local delivery of L-arginine inhibits lesion formation.New Zealand White rabbits (n=56) were fed a 1% cholesterol diet. After 1 week, both iliac arteries were balloon-denuded, and a local drug delivery catheter was introduced into both iliac arteries to deliver either L-arginine (800 mg/5 mL with and without 100 microCi L-[2,3-(3)H]-arginine) or saline. Monocyte-endothelial interaction was assessed by functional binding assay; NO activity was measured by chemiluminescence. Intramural administration of radioactively labeled L-arginine led to significantly higher counts in comparison to the contralateral segment for up to 1 week after delivery (676+/-223 versus 453+/-93 cpm/mg; P<0.02); this was associated with significantly higher NO levels in the L-arginine-treated segments (394.4+/-141.6 versus 86.3+/-34.3 nmol/mg; P<0.01). Even after 2 to 3 weeks, monocyte binding was significantly decreased by treatment with L-arginine as compared with saline infusion (P<0.01). After 4 weeks, there was a 9-fold greater number of apoptotic cells in the vessel wall of L-arginine as compared with the saline-treated segments (P<0.05).Intramural delivery of L-arginine immediately after angioplasty causes a sustained increase in tissue L-arginine levels associated with enhancement of local NO synthesis. The local increase in NO synthesis is associated with an attenuation of monocyte binding and increased apoptosis of resident macrophages. This treatment strategy could be valuable for the prevention and management of restenosis.

    View details for Web of Science ID 000083348100022

    View details for PubMedID 10534472

  • Gene transfer of nitric oxide synthase - Effects on endothelial biology JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Niebauer, J., Dulak, J., Chan, J. R., Tsao, P. S., Cooke, J. P. 1999; 34 (4): 1201-1207


    The purpose of the study was to investigate the role of nitric oxide (NO) in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA.Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC.Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence.The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by +60% (n = 6), which increased further when EC were stimulated by shear stress (24 h) by +137% (n = 5) as compared with untransfected, unstimulated EC (both p < 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r = -0.5669, p < 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha.Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation.

    View details for Web of Science ID 000082936900039

    View details for PubMedID 10520813

  • Mononuclear cell adherence to cultured endothelium is enhanced by hypertension and insulin resistance in healthy nondiabetic volunteers CIRCULATION Chen, N. G., Abbasi, F., Lamendola, C., McLaughlin, T., Cooke, J. P., Tsao, P. S., Reaven, G. M. 1999; 100 (9): 940-943


    This study was initiated to compare the adherence to cultured endothelial cells of mononuclear cells isolated from normotensive and hypertensive individuals.Mononuclear cell binding to endothelium was greater in patients with hypertension (32+/-1 versus 25+/-2; P<0.001) than in normal volunteers. There was a significant relationship (r=0.42, P<0. 01) between mononuclear cell binding and mean arterial pressure, independent of differences in age, sex, and body mass index. A significant relationship also existed between insulin resistance (estimated by the steady-state plasma glucose concentration during the insulin suppression test) and mononuclear cell binding in both the normotensive (r=0.86, P<0.001) and hypertensive (r=0.74, P<0. 001) groups. Furthermore, multiple regression analysis demonstrated an independent relationship (P<0.001) between mononuclear cell binding and both steady-state plasma glucose and hypertensive status.These results indicate that both hypertension and insulin resistance lead to changes in mononuclear cells that increase their adherence to cultured endothelial cells.

    View details for Web of Science ID 000082353000009

    View details for PubMedID 10468524

  • Novel mechanism for endothelial dysfunction - Dysregulation of dimethylarginine dimethylaminohydrolase CIRCULATION Ito, A., Tsao, P. S., Adimoolam, S., Kimoto, M., Ogawa, T., Cooke, J. P. 1999; 99 (24): 3092-3095


    Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA.ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity.These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.

    View details for Web of Science ID 000080943900003

    View details for PubMedID 10377069

  • Fibroblast growth factor as therapy for critical limb ischemia: a case report VASCULAR MEDICINE Cooke, J. P., Bhatnagar, R., Szuba, A., Rockson, S. G. 1999; 4 (2): 89-91


    In an attempt to avert impending, primary amputation, an 85-year-old woman with chronic critical leg ischemia was enrolled in an experimental protocol to induce therapeutic angiogenesis. Treatment consisted of six consecutive, weekly intravenous infusions of recombinant basic fibroblast growth factor (bFGF). Angiographic evaluation was performed before and after therapy. The patient's clinical response was monitored through serial measurements of the ankle/brachial index and by repetitive assessment of limb flow by mercury strain-gauge plethysmography. A beneficial clinical response was detectable by week 4 of therapy, which was characterized by an improved walking distance, relief of ischemic pain, a marked reduction in analgesic consumption, and healing of persistent, unresponsive, painful inflammation of the hallux. The clinical improvement was sustained throughout the remaining weeks of therapy and follow-up evaluation. Plethysmography documented improved blood flow; specifically, the augmentation of digital flow was sustained and correlated with the marked improvement in the patient's clinical status.

    View details for Web of Science ID 000081421600006

    View details for PubMedID 10406455

  • Impaired aerobic capacity in hypercholesterolemic mice: partial reversal by exercise training AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Niebauer, J., Maxwell, A. J., Lin, P. S., Tsao, P. S., Kosek, J., Bernstein, D., Cooke, J. P. 1999; 276 (4): H1346-H1354


    The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised (E+ex, n = 13; E-ex, n = 7) and high-fat chow, sedentary (E+chol, n = 6; E-chol, n = 8) or exercised (E+chol-ex, n = 6; E-chol-ex, n = 7). Mice were trained on a treadmill 2 x 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake (r = -0.35; P < 0. 02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match E+ex (all P < 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold (r = 0.36; P < 0.025) and maximal distance run (r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.

    View details for Web of Science ID 000079554200030

    View details for PubMedID 10199861

  • Regression of atherosclerosis - Role of nitric oxide and apoptosis CIRCULATION Wang, B. Y., Ho, H. K., Lin, P. S., Schwarzacher, S. P., Pollman, M. J., Gibbons, G. H., Tsao, P. S., Cooke, J. P. 1999; 99 (9): 1236-1241


    We have recently found that administration of L-arginine to hypercholesterolemic rabbits induces regression of preexisting lesions. Others have previously shown that activation of the L-arginine/nitric oxide (NO) synthase pathway can induce apoptosis of vascular cells in vitro. Accordingly, the current study was designed to determine if dietary supplementation of L-arginine induces apoptosis of intimal lesions and if this effect is mediated through the NO synthase pathway.Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water, and the cholesterol diet was continued for 2 weeks, at which time the aortas were harvested for histological studies. L-Arginine treatment increased the number of apoptotic cells (largely macrophages) in the intimal lesions by 3-fold (11.9+/-3.9 vs 3.9+/-1. 4 apoptotic cells/mm2, P<0.01). In subsequent studies, aortas were harvested for ex vivo studies. Aortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of the NO synthase pathway. The tissues were then collected for histological studies and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside (10(-5) mol/L) to the medium caused a time-dependent increase in apoptosis of vascular cells (largely macrophages) in the intimal lesion. L-Arginine (10(-3) mol/L) had an identical effect on apoptosis, which was associated with an increase in nitrogen oxides released into the medium. These effects were not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor L-nitro-arginine (10(-4) mol/L). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-BR cGMP.These results indicate that supplemental L-arginine induces apoptosis of macrophages in intimal lesions by its metabolism to NO, which acts through a cGMP-independent pathway. These studies are consistent with our previous observation that supplementation of dietary arginine induces regression of atheroma in this animal model. These studies provide a rationale for further investigation of the therapeutic potential of manipulating the NO synthase pathway in atherosclerosis.

    View details for Web of Science ID 000078978500016

    View details for PubMedID 10069793

  • [Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis]. Journal of cardiology Miyazaki, H., Matsuoka, H., Cooke, J. P., Usui, M., Ueda, S., Okuda, S., Imaizumi, T. 1999; 33 (2): 105-106

    View details for PubMedID 10087482

  • L-arginine and nitric oxide-related compounds in plasma: comparison of normal and arginine-free diets in a 24-h crossover study VASCULAR MEDICINE Tangphao, O., Chalon, S., Coulston, A. M., Moreno, H., Chan, J. R., Cooke, J. P., Hoffman, B. B., Blaschke, T. F. 1999; 4 (1): 27-32


    The amino acid L-arginine is the precursor of nitric oxide (NO), a powerful vasodilator with antiplatelet properties. The availability of L-arginine has been suggested to be a rate-limiting factor in the production of NO in conditions such as hypercholesterolemia. It was speculated that fluctuations in plasma concentrations of L-arginine during the day may be dependent upon dietary intake of the amino acid, or other variables, and might modify the elaboration of endogenous NO. Over a 24-h period, the plasma concentrations of L-arginine and NO-related compounds (NOx) were measured during an L-arginine and nitrate/nitrite-free diet (diet A) or a nitrate/nitrite-free diet with a fixed amount of L-arginine intake (3.8 g/d) (diet B) in eight healthy volunteers during a 2-day crossover study. Subjects were randomly selected to begin with diet A or diet B and consumed the other diet on the second day. During diet A, plasma L-arginine decreased significantly from 09.00 to 16.00 (21.4+/-2.0 to 11.9+/-1.1 microg/ml), rose slightly in the evening (to 16.6+/-1.7 microg/ml) and gradually increased during the night. During diet B, plasma L-arginine showed a peak after each meal (approximately 23 microg/ml). Plasma NOx concentrations measured by chemiluminescence did not show any circadian variation on either diet. Plasma L-arginine concentrations change during the day and are influenced by dietary intake. Importantly, plasma NOx do not seem to vary with this pattern in healthy individuals.

    View details for Web of Science ID 000081421400005

    View details for PubMedID 10355867

  • Nutriceuticals for cardiovascular health Symposium on Endothelial Function and Cardiovascular Disease - Potential Mechanisms and Interventions, at the Vascular-Biology-Working-Group Meeting Cooke, J. P. EXCERPTA MEDICA INC-ELSEVIER SCIENCE INC. 1998: 43S–45S

    View details for Web of Science ID 000077693900025

    View details for PubMedID 9860358

  • Asymmetric dimethylarginine (ADMA): A novel risk factor for endothelial dysfunction - Its role in hypercholesterolemia CIRCULATION Boger, R. H., Bode-Boger, S. M., Szuba, A., Tsao, P. S., Chan, J. R., Tangphao, O., Blaschke, T. F., Cooke, J. P. 1998; 98 (18): 1842-1847


    Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase. Because endothelial NO elaboration is impaired in hypercholesterolemia, we investigated whether plasma concentrations of ADMA are elevated in young, clinically asymptomatic hypercholesterolemic adults. We further studied whether such elevation of ADMA levels was correlated with impaired endothelium-dependent, NO-mediated vasodilation and urinary nitrate excretion. In a randomized, double-blind, placebo-controlled study, we investigated whether these changes could be reversed with exogenous L-arginine.We measured plasma levels of L-arginine, ADMA, and symmetrical dimethylarginine (SDMA) by high-performance liquid chromatography in 49 hypercholesterolemic (HC) and 31 normocholesterolemic (NC) humans. In 8 HC subjects, endothelium-dependent forearm vasodilation was assessed before and after an intravenous infusion of L-arginine or placebo and compared with 8 NC control subjects. ADMA levels were significantly elevated by >100% (2.17+/-0.15 versus 1.03+/-0.09 micromol/L; P<0.05) in HC subjects compared with NC adults. L-Arginine levels were similar, resulting in a significantly decreased L-arginine/ADMA ratio in HC subjects (27.7+/-2.4 versus 55. 7+/-5.4; P<0.05). In 8 HC subjects, intravenous infusion of L-arginine significantly increased the L-arginine/ADMA ratio and normalized endothelium-dependent vasodilation and urinary nitrate excretion. ADMA levels were inversely correlated with endothelium-mediated vasodilation (R=0.762, P<0.01) and urinary nitrate excretion rates (R=0.534, P<0.01).We find that ADMA is elevated in young HC individuals. Elevation of ADMA is associated with impaired endothelium-dependent vasodilation and reduced urinary nitrate excretion. This abnormality is reversed by administration of L-arginine. ADMA may be a novel risk factor for endothelial dysfunction in humans.

    View details for Web of Science ID 000076718900005

    View details for PubMedID 9799202

  • Is atherosclerosis an arginine deficiency disease? JOURNAL OF INVESTIGATIVE MEDICINE Cooke, J. P. 1998; 46 (8): 377-380


    To conclude, an impairment of the NO synthase pathway may be one of the earliest events in atherogenesis. A reduction in NO synthesis and/or activity may contribute to the initiation and progressive of atherosclerosis. Derangement of the NO synthase pathway may occur by several mechanisms, including lipoproptein-induced alterations in signal transduction; increases in superoxide anion elaboration (and degradation of NO); reduced affinity of NOS for L-arginine; and/or elevated levels of circulating antagonists. NO is a potent vasodilator, a regulator of vascular structure, and an inhibitor of endothelial interactions and circulating blood elements. A loss of endothelial NO activity may contribute to the abnormal vasomotion observed in coronary artery disease, as well as the progression of atherosclerosis. Strategies to enhance NO synthesis and/or activity may be useful in maintaining cardiovascular health.

    View details for Web of Science ID 000076725100013

    View details for PubMedID 9805422

  • Limb blood flow during exercise is dependent on nitric oxide CIRCULATION Maxwell, A. J., Schauble, E., Bernstein, D., Cooke, J. P. 1998; 98 (4): 369-374


    We have recently reported that hypercholesterolemia reduces aerobic exercise capacity in mice and that this is associated with a reduced endothelium-dependent vasodilator function, endothelium-derived nitric oxide (EDNO) production, and urinary nitrate excretion. These findings led us to test the hypothesis that EDNO production contributes significantly to limb blood flow during exercise and to determine whether loss of EDNO production is responsible for the decline in exercise capacity observed in hypercholesterolemia.Twelve-week-old wild-type (E+; n=9) and apoE-deficient (E-; n=9) C57BL/6J mice were treadmill-tested to measure indices defining exercise capacity on a metabolic chamber-enclosed treadmill capable of measuring oxygen uptake and carbon dioxide excretion. Urine was collected before and after treadmill exercise for determination of vascular NO production assessed by urinary nitrate excretion. The wild-type mice were then given nitro-L-arginine (E+LNA) in the drinking water (6 mg/dL) for 4 days before undergoing a second treadmill testing and urinary nitrate measurement. An additional set of 12-week-old wild-type mice was divided into 2 groups: 1 receiving regular water (E+; n=8) and 1 administered LNA for 4 days (E+LNA; n=8). These mice, along with an additional set of E mice (n=8), underwent treadmill testing to determine maximal oxygen uptake (VO2max). The mice were then cannulated such that the tip of the tubing was positioned in the ascending aorta. Fluorescent microspheres (20000) were infused into the carotid cannula while the mice were sedentary and again while approaching VO2max. When the mice were euthanized, the running muscles were collected and fluorescence intensity was measured to determine the peak-exercise redistribution of blood flow to the running muscles (expressed as percentage of total cardiac output, %COrm) during both states. Both E+LNA and E- mice demonstrated a markedly reduced postexercise urinary nitrate excretion, aerobic capacity, and %COrm at VO2max compared with E+.EDNO contributes significantly to limb blood flow during exercise. Conditions that reduce EDNO production disturb the hyperemic response to exercise, resulting in a reduced exercise capacity.

    View details for Web of Science ID 000074984800014

    View details for PubMedID 9711943

  • Interaction of diabetes and hypertension on determinants of endothelial adhesiveness ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Tsao, P. S., Niebauer, J., Buitrago, R., Lin, P. S., Wang, B. Y., Cooke, J. P., CHEN, Y. D., Reaven, G. M. 1998; 18 (6): 947-953


    Epidemiological studies have established that diabetes mellitus and hypertension are independent risk factors for atherosclerosis. One of the earliest abnormalities seen in atherogenesis is enhanced monocyte adherence to the endothelium. The mechanisms by which diabetes mellitus or hypertension enhances monocyte-endothelial cell interactions are incompletely characterized. It is not known whether there are additive interactions between these risk factors on endothelial adhesiveness for monocytes. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were fed a normal or fructose-enriched diet. In some cases, animals were injected with streptozotocin (35 mg/kg body weight) to induce diabetes. After 2 weeks, plasma was drawn for biochemical measurements, and thoracic aortas were harvested, opened longitudinally, and exposed to fluorescently labeled mouse monocytoid cells (WEHI 78/24, 2 x 10(6)/mL) for 30 minutes on a rocking platform. Adherent cells were counted by epifluorescence microscopy. WEHI 78/24 binding to aortic segments from SHR animals was elevated compared with segments from WKYs. Fructose feeding alone had no effect on endothelial adhesiveness. When WKYs were made hyperglycemic by STZ injection, monocyte binding was 160% of the control value. Elevated monocyte binding was also observed in aortas derived from SHR animals injected with STZ, indicating an additive effect of hypertension and hyperglycemia. To determine whether alterations in oxidative state played a role in the endothelial adhesiveness, aortic segments were exposed to lucigenin (250 micromol/L) for measurement of superoxide anion. Aortic segments from SHR elaborated 120% more superoxide anion than did controls. Elevated free-radical production was also observed in aortas from diabetic WKYs. Furthermore, thoracic aortas derived from diabetic SHR animals elaborated more superoxide anion than did any of the other groups (374%, P<0.05). Immunohistochemical staining for monocyte chemotactic protein-1 demonstrated increased expression in aortas isolated from diabetic WKY and SHR compared with control vessels. These studies demonstrate that both diabetes and hypertension lead to increased monocyte adherence to the endothelium. This abnormality is associated with increased vascular superoxide production and monocyte chemotactic protein-1 expression. Furthermore, there appears to be an additive interaction between hyperglycemia and hypertension in their effects on endothelial adhesiveness and its determinants.

    View details for Web of Science ID 000074175500014

    View details for PubMedID 9633936

  • Thromboangiitis obliterans an update on Buerger's disease WESTERN JOURNAL OF MEDICINE Szuba, A., Cooke, J. P. 1998; 168 (4): 255-260


    Buerger's disease (thromboangiitis obliterans) is a nonnecrotizing vasculitis affecting small and medium-sized arteries, typically in young male smokers. The diagnosis can often be made on the basis of a careful history and physical examination, together with ancillary laboratory studies. Occasionally arteriography is warranted to confirm the diagnosis. The pathological findings are distinctive and distinguish this disorder from other arterial occlusive diseases. Successful therapy is possible only with absolute abstinence from tobacco.

    View details for Web of Science ID 000073259300004

    View details for PubMedID 9584663

    View details for PubMedCentralID PMC1304949

  • Peripheral arterial insufficiency: mechanisms, natural history, and therapeutic options. Advances in internal medicine Rockson, S. G., Cooke, J. P. 1998; 43: 253-277

    View details for PubMedID 9506185

  • Endothelial alterations in hypercholesterolemia: More than simply vasodilator dysfunction 1st Meeting of the International Scientific Faculty on Endothelial Function Tsao, P. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 1998: S48–S53


    Occlusive vascular disease begins with an alteration of the endothelium, which is characterized by a decrease in nitric oxide (NO) activity. Endogenous NO inhibits many key processes in atherogenesis, including monocyte adherence, platelet activation, and smooth muscle proliferation. The mechanism by which NO activity is reduced in hypercholesterolemia and in other metabolic disorders associated with atherogenesis appears to be multifactorial. It includes increased production of oxygen-derived free radicals, alterations in NO synthase, and the accumulation of endogenous inhibitors (ADMA) of NO synthase. Plasma concentrations of ADMA are elevated in hypercholesterolemic humans. Elevated ADMA concentrations are associated with impaired endothelium-dependent, NO-mediated vasodilatation and reduced urinary nitrate exertion. These effects of ADMA are counteracted by administration of the NO precursor L-arginine. It is likely that basic insights regarding the mechanisms of endothelial dysfunction will lead to new therapeutic strategies for atherosclerosis.

    View details for Web of Science ID 000077892100009

    View details for PubMedID 9883748

  • Cardiovascular effects of L-arginine CURRENT OPINION IN NEPHROLOGY AND HYPERTENSION Maxwell, A. J., Cooke, J. P. 1998; 7 (1): 63-70


    Most of the known cardiovascular effects of L-arginine are exerted via its conversion to nitric oxide by nitric oxide synthase. Accumulating evidence indicates that supplemental administration of L-arginine is sufficient to restore endothelium-derived nitric oxide production in many disorders in which endothelium-derived nitric oxide production is altered. L-arginine may enhance nitric oxide production by competing as a substrate with an endogenous antagonist for nitric oxide synthase. In other cases, L-arginine may act by competing with molecular oxygen as a substrate so as to reduce the production of superoxide anion. It is likely that other mechanisms exist by which the nitric oxide synthase pathway can be perturbed. Regardless of the mechanism, a wide array of cardiovascular disorders characterized by endothelial dysfunction are reversible by L-arginine.

    View details for Web of Science ID 000071425600011

    View details for PubMedID 9442365

  • Images in vascular medicine. Phlegmasia coerulea dolens--venous gangrene. Vascular medicine Szuba, A., Cooke, J. P., Rockson, S. G. 1998; 3 (1): 29-31

    View details for PubMedID 9666529

  • Adhesiveness of mononuclear cells in hypercholesterolemic humans is normalized by dietary L-arginine ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Theilmeier, G., Chan, J. R., Zalpour, C., Anderson, B., Wang, B. Y., Wolf, A., Tsao, P. S., Cooke, J. P. 1997; 17 (12): 3557-3564


    Hypercholesterolemia reduces vascular nitric oxide (NO) activity. This dysfunction may promote endothelial monocyte interaction, as NO is a potent inhibitor of cell adhesion. We have previously shown that in hypercholesterolemic (HC) rabbits, chronic oral supplementation of L-arginine (Arg) restores NO activity and inhibits monocyte-endothelial cell interaction, in association with a reduction in atherogenesis. We hypothesized that enhancement of endothelial NO activity in HC humans would reduce monocyte adhesiveness. We used a functional binding assay to assess the adhesiveness of human mononuclear cells (MNCs) ex vivo to determine the effects of hypercholesterolemia and L-arginine administration. MNCs from HC subjects adhered in greater numbers (50% more cells per high-power field; P < .0001) than cells derived from normocholesterolemic (NC) subjects. To determine whether enhancement of endogenous NO activity could inhibit mononuclear cell adhesiveness, in a double-blinded placebo-controlled study, oral arginine HCl (8.4 g/d) was administered to HC subjects. Over a course of 2 weeks, this treatment abolished the increased adhesiveness of HC MNCs (160 +/- 11% versus 104 +/- 5%; before and after 2 weeks of Arg treatment; results expressed as a percentage of the binding values obtained using cells derived from paired NC individuals). By contrast, MNC adhesion remained significantly elevated in placebo-treated HC subjects. To examine whether endothelium-derived NO could act as a paracrine modulator of monocyte behavior, monocytes were exposed to NO donors or cocultered in the presence of endothelial cells exposed to antagonists of NO synthase in the presence or absence of L-arginine. NO donors inhibited monocyte adhesiveness. Furthermore, the adhesiveness of monocytes cocultured with endothelial cells was increased by antagonists of NO synthase; this effect was reversed by L-arginine. This study shows that the adhesiveness of human MNCs is increased by hypercholesterolemia. The increase in adhesiveness was reversed in vivo by administration of the NO precursor L-arginine. NO donors or endothelium-derived NO inhibits the adhesiveness of monocytes in vitro, supporting the hypothesis that the effects of L-arginine are mediated by NO.

    View details for Web of Science ID 000072212600025

    View details for PubMedID 9437205

  • Anti-CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis BLOOD McEvoy, L. M., JUTILA, M. A., Tsao, P. S., Cooke, J. P., BUTCHER, E. C. 1997; 90 (9): 3587-3594


    Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti-L-selectin and anti-alpha4 and beta2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

    View details for Web of Science ID A1997YD10800034

    View details for PubMedID 9345042

  • Therapeutic interventions in endothelial dysfunction: endothelium as a target organ. Clinical cardiology Cooke, J. P. 1997; 20 (11): II-45 51


    Endothelial dysfunction is recognized as the initial step in the atherosclerotic process. To date, most interventions attempting to improve endothelial dysfunction have targeted one or more of the numerous risk factors that can cause endothelial damage: hypertension (angiotensin-converting enzyme inhibitors and calcium antagonists), hypercholesterolemia (lipid-lowering agents), cigarette smoking (cessation), sedentary lifestyle (increased physical activity), menopause (estrogen replacement therapy), and diabetes mellitus (control of associated metabolic abnormalities). Interventions targeted specifically to the endothelium remain speculative, as the precise mechanisms of endothelial dysfunction are still being elucidated. Several pharmacologic agents have been suggested to achieve vascular protection through mechanisms that go beyond their primary therapeutic (e.g., hypotensive or hypocholesterolemic) actions; examples of these are angiotensin-converting enzyme inhibitors or HMG-CoA reductase inhibitors. Beneficial changes to the endothelium might result from promotion of vasorelaxation, inhibition of vasoconstriction, reduction in the production of free radicals, or other mechanisms that protect the endothelium from injury.

    View details for PubMedID 9422852

  • Adherence of mononuclear cells to endothelium in vitro is increased in patients with NIDDM DIABETES CARE Carantoni, M., Abbasi, F., Chu, L., CHEN, Y. D., Reaven, G. M., Tsao, P. S., Varasteh, B., Cooke, J. P. 1997; 20 (9): 1462-1465


    To compare the binding to cultured endothelial cells of mononuclear cells isolated from healthy volunteers and patients with NIDDM.Mononuclear cells were isolated from healthy volunteers (n = 11) and patients with NIDDM (n = 14) and incubated with ECV 304 cells, a human umbilical endothelial cell-derived transformed cell line. Following a period of incubation, the adherence of mononuclear cells to endothelial cells was determined.Adherence of mononuclear cells from patients with NIDDM was significantly greater (P < 0.05) than that of cells isolated from the healthy volunteers, and this difference persisted when adjusted for age, sex, and degree of obesity. Mononuclear cell binding to ECV 304 cells correlated significantly with fasting plasma glucose (r = 0.52, P < 0.01), insulin (r = 0.51, P < 0.01), triglyceride (r = 0.54, P < 0.01), and VLDL (r = 0.54, P < 0.01) and HDL cholesterol (r = -0.45, P < 0.05) levels, but not with either total or LDL cholesterol levels or blood pressure.Since the adherence of mononuclear cells to the endothelium represents the earliest step in atherogenesis, the observation that mononuclear cells from patients with NIDDM bind more avidly to cultured endothelial cells may help explain why accelerated atherosclerosis occurs in patients with NIDDM. The metabolic abnormality, or abnormalities, present in patients with NIDDM that is responsible for the enhanced adhesiveness of mononuclear cells requires further examination.

    View details for PubMedID 9283798

  • Nitric oxide regulates monocyte chemotactic protein-1 CIRCULATION Tsao, P. S., Wang, B. Y., Buitrago, R., Shyy, J. Y., Cooke, J. P. 1997; 96 (3): 934-940


    Monocyte chemotactic protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. We and others have demonstrated that NO inhibits monocyte-endothelial cell interactions and atherogenesis. We hypothesize that the antiatherogenic effect of NO may be due in part to its inhibition of MCP-1 expression.Smooth muscle cells (SMCs) were isolated from normal rabbit aortas by the explant method. Cells were then exposed to LPS (10 microg/mL), native LDL, or oxidized LDL (30 microg/mL) for 6 hours. The expression of MCP-1 in SMCs and chemotactic activity in the conditioned medium were induced by lipopolysaccharide (LPS) or by oxidized LDL but not native LDL. The induction of MCP-1 by cytokines or oxidized lipoproteins was associated with an increased generation of superoxide anion by the SMCs and increased activity of the transcriptional protein nuclear factor-kappaB (NFkappaB). The induced expression of MCP-1 and activation of NFkappaB were reduced by previous exposure of the SMCs to the NO donor DETA-NONOate (100 micromol/L) (P<.05). To determine whether NO exerted its effect at a transcriptional level, SMCs and COS cells were transfected with a 400-bp fragment of the MCP-1 promoter. Promoter activity was enhanced by oxidized LDL, and LPS was inhibited by DETA-NO. Nuclear run-on assays confirmed that the effect of NO occurred at a transcriptional level. To investigate the role of endogenous NO in the regulation of MCP-1 in vivo, New Zealand White rabbits were fed normal chow, normal chow plus nitro-L-arginine (LNA), high-cholesterol diet (Chol), or high-cholesterol diet supplemented with L-arginine (Arg). After 2 weeks, thoracic aortas were harvested and total RNA was isolated. Northern analysis using full-length MCP-1 cDNA demonstrated increased expression in Chol and LNA aortas; this expression was decreased in aortas from Arg animals.These studies indicate that the antiatherogenic effect of NO may be mediated in part by its inhibition of MCP-1 expression.

    View details for Web of Science ID A1997XP13000035

    View details for PubMedID 9264504

  • Novel vascular molecule involved in monocyte adhesion to aortic endothelium in models of atherogenesis JOURNAL OF EXPERIMENTAL MEDICINE McEvoy, L. M., Sun, H. L., Tsao, P. S., Cooke, J. P., Berliner, J. A., BUTCHER, E. C. 1997; 185 (12): 2069-2077


    Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.

    View details for Web of Science ID A1997XF79100005

    View details for PubMedID 9182678

  • Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty CIRCULATION Schwarzacher, S. P., Lim, T. T., Wang, B. Y., Kernoff, R. S., Niebauer, J., Cooke, J. P., Yeung, A. C. 1997; 95 (7): 1863-1869


    Long-term oral administration of L-arginine has been shown to enhance production of nitric oxide (NO) and to reduce lesion formation. The goal of this study was to determine whether local intramural administration of L-arginine could enhance NO generation and reduce intimal thickening.New Zealand White rabbits (n = 27) received a 1% cholesterol diet. For the short-term study, after 1 week of diet, both iliac arteries were balloon injured. Four weeks later, vasoreactivity was assessed angiographically during infusion of acetylcholine (Ach) before and after delivery of L-arginine or saline into the right or left iliac artery (800 mg/5 mL; 0.2 mL/min, 15 minutes) by use of a local drug-delivery balloon. Vessels were then harvested for measurements of NO. For the long-term study, after balloon injury, drugs were delivered as above into the iliac arteries. Two and 4 weeks after L-arginine delivery, vasoreactivity was determined. Subsequently, the iliac arteries were harvested for histomorphometric analysis and measurements of NO. In the short-term study, local delivery of L-arginine restored endothelium-dependent vasodilatation (Ach 10(-5) mol/L; L-arginine +35 +/- 10%; saline -14 +/- 5%; P < .001) and enhanced local production of nitrogen oxides (L-arginine 152 +/- 28; saline 78 +/- 12 nmol/L per milligram of tissue per hour; P < .04). In the long-term study, local administration of L-arginine enhanced vascular NO production as long as 1 week after the injury (L-arginine 394.4 +/- 141.6; saline 86.3 +/- 34.3 nmol/L per milligram of tissue per hour; P < .01) and reduced intimal thickening 4 weeks later (intima/ media ratio: L-arginine 0.56 +/- 0.1; saline 1.40 +/- 0.2; P < .001), largely due to suppression of macrophage accumulation.A single intramural administration of L-arginine enhances vascular NO generation and inhibits lesion formation. Local augmentation of NO production at the site of balloon angioplasty may be a novel strategy to prevent restenosis.

    View details for Web of Science ID A1997WR51900020

    View details for PubMedID 9107174

  • Dietary L-arginine supplementation normalizes platelet aggregation in hypercholesterolemic humans JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Wolf, A., Zalpour, C., Theilmeier, G., Wang, B. Y., Ma, A., Anderson, B., Tsao, P. S., Cooke, J. P. 1997; 29 (3): 479-485


    The present study was designed to test the hypothesis that long-term dietary supplementation with the nitric oxide precursor L-arginine would enhance vascular or platelet-derived nitric oxide activity, or both, and thereby inhibit platelet reactivity in hypercholesterolemic humans.We have shown that reduced vascular activity of nitric oxide in hypercholesterolemic rabbits can be restored by L-arginine supplementation. The improvement in nitric oxide activity is associated with an inhibition of platelet aggregation ex vivo. This effect is most likely due to increased elaboration of endothelium- or platelet-derived nitric oxide, or both, because the inhibition of platelet reactivity was associated with elevation of intraplatelet cyclic guanosine monophosphate and was reversed by the nitric oxide synthase antagonist N-methyl-arginine.In a double-blinded, randomized, placebo-controlled trial, hypercholesterolemic patients were assigned to L-arginine hydrochloride, 8.4 g/day orally, or placebo for 2 weeks. Platelet-rich plasma was obtained for aggregometry induced by collagen (1 to 10 micrograms/ml) at four points: baseline, after 2 weeks of treatment, after a 2-week washout and after a long-term washout of 16 weeks on average. Aggregation was quantified by light transmittance and expressed as a percent transmittance observed with platelet-poor plasma.Compared with normocholesterolemic control subjects, platelets from hypercholesterolemic subjects stimulated with 5 micrograms/ml of collagen showed increased aggregability (68.6% in hypercholesterolemic patients vs. 54.5% in normocholesterolemic control subjects, p < or = 0.02). After 2 weeks of treatment with L-arginine (but not placebo), platelet reactivity was modestly reduced; this effect persisted for 2 weeks after discontinuation of arginine (52.6% in arginine-treated patients vs. 65.1% in normocholesterolemic control subjects, p = 0.07). After 18 weeks (i.e., 16 weeks after discontinuing arginine treatment), the platelets of hypercholesterolemic patients once again became hyperaggregable, and the extent of platelet aggregation was significantly increased compared with the 4-week point (73.6% after vs. 52.6% during arginine treatment, p < 0.01). No significant change in platelet reactivity was seen in placebo-treated hypercholesterolemic patients throughout the study. L-Arginine treatment was well tolerated without side effects.This double-blinded, placebo-controlled study demonstrates that dietary supplementation with L-arginine can modestly attenuate the increased platelet reactivity seen in hypercholesterolemic patients. The data are consistent with our previous studies in hypercholesterolemic animals, demonstrating that L-arginine restores endogenous nitric oxide activity and inhibits platelet aggregation. Enhancement of endogenous nitric oxide activity is a potential novel therapeutic strategy worthy of further study.

    View details for Web of Science ID A1997WL49000002

    View details for PubMedID 9060881

  • Nitric oxide synthase: Role in the genesis of vascular disease ANNUAL REVIEW OF MEDICINE Cooke, J. P., Dzau, V. J. 1997; 48: 489-509


    The product of nitric oxide (NO) synthase is the most potent endogenous vasodilator known. No not only is a potent vasodilator, it also inhibits platelet adherence and aggregation, reduces adherence of leukocytes to the endothelium, and suppresses proliferation of vascular smooth muscle cells. A number of disorders are associated with reduced synthesis and/or increased degradation of vascular NO. These include hypercholesterolemia, diabetes mellitus, hypertension, and tobacco use. The endothelial dysfunction caused by these disorders contributes to the alterations in vascular function and structure observed in these conditions. A reduction in the activity of vascular NO likely plays a significant role in the development of atherosclerosis. Insights into the mechanisms by which NO production or activity is altered in these states will lead to new therapeutic strategies in the treatment of a number of vascular disorders, including hypertension, atherosclerosis, restenosis, and thrombosis.

    View details for Web of Science ID A1997WH48600040

    View details for PubMedID 9046979

  • The pathophysiology of peripheral arterial disease: rational targets for drug intervention. Vascular medicine Cooke, J. P. 1997; 2 (3): 227-230


    The most common cause of peripheral arterial disease (PAD) is atherosclerosis, which begins with an alteration in endothelial biology due to hypercholesterolemia, diabetes mellitus, hypertension, tobacco use, elevated levels of lipoprotein(a) or homocystinemia. With chronic and severe arterial disease, changes begin to occur in the microcirculation, including obstruction at the microvascular level and tissue injury. Based on insights into the vascular biology of PAD, new therapies have been developed and are at various stages of clinical trials. Future pharmacotherapy for PAD will include agents that have one or more of the following attributes; (1) reduce, or even reverse, the progression of atherosclerosis; (2) inhibit plaque rupture; (3) inhibit thrombosis by a novel mechanism; (4) induce angiogenesis; (5) reverse microvascular derangements; (6) affect blood rheology; and (7) enhance skeletal muscle's ability to use available nutrients.

    View details for PubMedID 9546972

  • Role of digital artery adrenoceptors in Raynaud's disease. Vascular medicine Cooke, J. P., Creager, S. J., SCALES, K. M., Ren, C., Tsapatsaris, N. P., BEETHAM, W. P., Creager, M. A. 1997; 2 (1): 1-7


    Raynaud's disease is characterized by excessive cutaneous vasoconstriction in response to ambient cold. A functional disturbance in the local regulation of digital vasomotion has been proposed. The purpose of this study was to determine whether there is an alteration in the postjunctional adrenergic receptors in the digital circulation of patients with Raynaud's disease. Furthermore, we sought to determine whether this abnormality was responsible for the excessive cold-induced vasoconstriction in these patients. Finger blood flow was measured by strain-gauge venous occlusion plethysmography in 10 patients with Raynaud's disease and in 10 normal volunteers in a 22 degrees C room. Measurements of finger blood flow and mean systemic arterial pressure were made during intra-arterial infusions of the alpha 1-adrenergic antagonist, prazosin, or the alpha 2-adrenergic antagonist, yohimbine, at room temperature and during local cooling of the hand. Basal finger blood flow in normal subjects was significantly greater than that of patients (8.6 +/- 2.7 vs 1.7 +/- 0.5 ml/100 ml per min; normal vs Raynaud's subjects; p < 0.05). In normal subjects, either prazosin or yohimbine induced dose-dependent increases in finger blood flow. The maximal increase in finger blood flow induced by prazosin was significantly greater than that in response to yohimbine (29.2 +/- 10.1 vs 2.8 +/- 2.1 ml/100 ml per min; prazosin vs yohimbine; p < 0.05). By contrast, in the Raynaud's patients, prazosin or yohimbine induced maximal increases in finger blood flow that were not significant (7.1 +/- 1.8 vs 5.0 +/- 2.2 ml/100 ml per min; prazosin vs yohimbine; p = NS). The response to prazosin in Raynaud's patients was significantly less than that of the normal volunteers (p < 0.05). In normal subjects, during intra-arterial infusion of vehicle alone, cooling induced a 52.6 +/- 5.8% reduction in finger blood flow. This cold-induced vasoconstriction was blunted, but not qualitatively altered, by either adrenergic antagonist. In the Raynaud's patients, during the intra-arterial infusion of the vehicle, cooling induced a 68.2 +/- 7.8% reduction in finger blood flow. Infusion of either adrenergic antagonist blunted, but did not qualitatively alter, the response to cold. Finger blood flow is less in patients with Raynaud's disease than in normal subjects when studied in a 22 degrees C room. In normal subjects, postjunctional alpha 1-adrenergic receptors appear to predominate in the control of digital vasoconstriction. Postjunctional alpha 1- and alpha 2-adrenoceptors play an equal role in adrenergic regulation of finger blood flow in patients with Raynaud's disease. In both normal and Raynaud's subjects, selective antagonism of alpha 1- or alpha 2-adrenergic receptors does not abolish local cold-induced vasoconstriction. Therefore, it is likely that a nonadrenergic mechanism contributes to local cold-induced vasoconstriction.

    View details for PubMedID 9546943

  • Cardiovascular effects of exercise: Role of endothelial shear stress JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Niebauer, J., Cooke, J. P. 1996; 28 (7): 1652-1660


    Experimental, epidemiologic and clinical studies have provided strong evidence that physical exercise has beneficial effects on multiple physiological variables affecting cardiovascular health (lipoprotein levels, rest blood pressure and heart rate, carbohydrate tolerance, neurohormonal activity). Regular exercise has been shown to slow the progression of cardiovascular disease and to reduce cardiovascular morbidity and mortality. More recently, exercise-induced increases in blood flow and shear stress have been observed to enhance vascular function and structure. By increasing the release of nitric oxide and prostacyclin, shear stress augments endothelium-dependent vasodilation and inhibits multiple processes involved in atherogenesis and restenosis. In this review we discuss the underlying mechanisms by which exercise-induced blood flow and shear stress exert their salutary effects on cardiovascular remodeling.

    View details for Web of Science ID A1996VY32500002

    View details for PubMedID 8962548

  • Arginine restores nitric oxide activity and inhibits monocyte accumulation after vascular injury in hypercholesterolemic rabbits JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Wang, B. Y., Candipan, R. C., Arjomandi, M., HSIUN, P. T., Tsao, P. S., Cooke, J. P. 1996; 28 (6): 1573-1579


    This study sought to determine whether the alterations in vascular function and structure after balloon injury in hypercholesterolemic rabbits could be inhibited by dietary arginine.Administration of arginine (the nitric oxide [NO] precursor) restores vascular NO activity in hypercholesterolemic animals. We and other investigators have shown that enhancement of vascular NO activity can inhibit myointimal hyperplasia after vascular injury in normocholesterolemic animals.Twenty-eight New Zealand White rabbits received either normal rabbit chow, 0.5% cholesterol diet or 0.5% cholesterol diet plus L-arginine hydrochloride (2.25% wt/vol) in the drinking water. After 6 weeks of dietary intervention, the left iliac artery of each animal was subjected to a balloon injury. Four weeks later, the iliac arteries were harvested for vascular reactivity studies and immunohistochemical analysis.Vascular injury induced intimal thickening that was largely composed of vascular smooth muscle cells and extracellular matrix. In the setting of hypercholesterolemia, vascular injury induced an exuberant myointimal lesion that was augmented by the accumulation of lipid-laden macrophages. Dietary arginine reduced intimal thickening in the injured vessels of hypercholes-terolemic animals and substantially inhibited the accumulation of macrophages in the lesion (from 28% to 5% of the lesion area, p < 0.001).We report that lesions induced by vascular injury in hypercholesterolemic animals are markedly reduced by oral administration of arginine. Moreover, we find that the nature of the lesion is altered, with a striking reduction in the percentage of macrophages comprising the lesion.

    View details for Web of Science ID A1996VT31000020

    View details for PubMedID 8917274

  • Fluid flow inhibits endothelial adhesiveness - Nitric oxide and transcriptional regulation of VCAM-1 CIRCULATION Tsao, P. S., Buitrago, R., Chan, J. R., Cooke, J. P. 1996; 94 (7): 1682-1689


    In the arterial tree, regions exposed to reduced shear stress (low and/or disturbed flow) are predisposed to atherogenesis. Fluid flow is a potent stimulus for the release of endothelium-derived nitric oxide (NO). Because NO inhibits monocyte-endothelial cell interaction, we speculated that the effects of flow in inhibiting atherogenesis might be mediated in part by NO.Confluent monolayers of human aortic endothelial cells were exposed to static or fluid flow conditions for 4 hours. The medium was replaced, and cells were then incubated with native LDL (50 micrograms/mL), oxidized LDL (30 micrograms/mL), or lipopolysaccharide (LPS) (10 ng/mL)+tumor necrosis factor-alpha (TNF-alpha) (10 U/mL) for an additional 4 hours. Functional binding assays using THP-1 monocytes were then performed. Superoxide production by human aortic endothelial cells was monitored by lucigenin chemiluminescence, and expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were quantified by flow cytometry. Whereas native LDL had little effect, incubation with either oxidized LDL or LPS/TNF-alpha significantly increased superoxide production, nuclear factor-kappa B activity, VCAM-1 expression, and endothelial adhesiveness for monocytes. Previous exposure to fluid flow inhibited these sequelae of exposure to cytokines or oxidized lipoprotein. The effect of fluid flow appears to be due in part to shear-induced release of NO, because coincubation with nitro-L-arginine completely abolished these effects of flow. Furthermore, the NO donor PAPA-NONO-ate and 8-Br-cGMP (but not 8-Br-cAMP) mimicked the effects of flow.Previous exposure to fluid flow decreased cytokine- or lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 expression, and monocyte binding; the effects of flow appear to be due to NO. Flow-mediated NO-dependent regulation of oxidant-responsive transcription may influence the site of a lesion.

    View details for Web of Science ID A1996VJ97900030

    View details for PubMedID 8840861

  • Role of nitric oxide in progression and regression of atherosclerosis WESTERN JOURNAL OF MEDICINE Cooke, J. P. 1996; 164 (5): 419-424


    Endothelium-derived nitric oxide is a potent endogenous vasodilator that is derived from the metabolism of L-arginine. This endothelial factor inhibits circulating blood elements from interacting with the vessel wall. Platelet adherence and aggregation as well as monocyte adherence and infiltration are opposed by this paracrine substance. By virtue of these characteristics, endothelium-derived nitric oxide inhibits atherogenesis in animal models and may even induce regression.

    View details for Web of Science ID A1996UR78800005

    View details for PubMedID 8686299

    View details for PubMedCentralID PMC1303540

  • Hypertension-enhanced monocyte adhesion in experimental atherosclerosis JOURNAL OF VASCULAR SURGERY Tropea, B. I., Huie, P., Cooke, J. P., Tsao, P. S., Sibley, R. K., Zarins, C. K. 1996; 23 (4): 596-605


    Hypertension is a known clinical risk factor for atherosclerosis. In experimental atherosclerosis, monocyte adhesion to the endothelial surface is enhanced and is considered to be an important early stage in plaque formation. We tested the hypothesis that hypertension enhances monocyte adhesion in experimental atherosclerosis.Twenty-two New Zealand White rabbits were fed an atherogenic diet for 3 weeks to induce plaque formation. Aortic coarctation was created in eight rabbits by wrapping a Dacron band around the midportion of the descending thoracic aorta (stenosis group), whereas six rabbits underwent banding without aortic constriction (no stenosis group). Eight rabbits served as nonoperated controls. Monocyte binding to the aortic endothelial surface was counted with epifluorescent microscopy on standard aortic segments proximal and distal to the band. Immunohistochemistry was performed for the following antibodies: VCAM-1, RAM11, CD11b, and factor VIII.Mean blood pressure was 89 +/- 3 mm Hg in the aorta proximal to the stenosis, compared with 64 +/- 4 mm Hg in the no stenosis group and 74 +/- 3 mm Hg in the control group (p < 0.01). The mean aortic blood pressure gradient across the stenosis was 16 +/- 2 mm Hg in the stenosis group, whereas the aortic blood pressure gradient was 0.2 +/- 0.6 mm Hg in the no stenosis group and -0.3 +/- 0.4 mm Hg in the control group (p < 0.001). Monocyte adhesion to the aortic endothelial surface proximal to the stenosis was increased twofold compared with adhesion to the aorta distal to the stenosis and compared with the proximal aorta in the control group (p < 0.02). The proximal-to-distal aortic ratio of monocyte binding was enhanced in the stenosis group (2.2) compared with the no stenosis (0.76) and control (0.83) groups (p < 0.01). The intima area of the aorta proximal to the stenosis was significantly increased compared with the proximal aortas in the no stenosis and control groups (p < 0.01). RAM11, CD11b, and endothelial VCAM-1 expression were enhanced in the hypertensive region proximal to the stenosis.In the hypertensive region in the aorta proximal to the stenosis, monocyte adhesion and endothelial VCAM-1 expression were increased, with intimal thickening and accumulation of macrophages. These findings suggest that hypertension may promote atherosclerotic plaque formation by enhancing monocyte adhesion.

    View details for Web of Science ID A1996UJ12600010

    View details for PubMedID 8627894

  • Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle CIRCULATION Lewis, N. P., Tsao, P. S., Rickenbacher, P. R., Xue, C., Johns, R. A., Haywood, G. A., VONDERLEYEN, H., Trindade, P. T., Cooke, J. P., Hunt, S. A., Billingham, M. E., Valantine, H. A., Fowler, M. B. 1996; 93 (4): 720-729


    The mechanisms underlying cardiac contractile dysfunction after transplantation remain poorly defined. Previous work has revealed that inducible nitric oxide synthase (iNOS) is expressed in the rat heterotopic cardiac allograft during rejection; resultant overproduction of nitric oxide (NO) might cause cardiac contractile dysfunction via the negative inotropic and cytotoxic actions of NO. In this investigation, we tested the hypothesis that induction of iNOS may occur and be associated with cardiac allograft contractile dysfunction in humans.We prospectively studied 16 patients in the first year after cardiac transplantation at the time of serial surveillance endomyocardial biopsy. Clinical data, the results of biopsy histology, and echocardiographic and Doppler evaluation of left ventricular systolic and diastolic function were recorded. Total RNA was extracted from biopsy specimens, and mRNA for beta-actin, detected by reverse transcription-polymerase chain reaction (RT-PCR) using human specific primers, was used as a constitutive gene control; iNOS mRNA was similarly detected by RT-PCR using human specific primers. iNOS protein was detected in biopsy frozen sections by immunofluorescence. Myocardial cGMP was measured by radioimmunoassay, and serum nitrogen oxide levels (NOx = NO2 + NO3) were measured by chemiluminescence. iNOS mRNA was detected in allograft myocardium at some point in each patient and in 59 of 123 biopsies (48%) overall. In individual patients, iNOS mRNA expression was episodic and time dependent; the frequency of expression was highest during the first 180 days after transplant (P = .0006). iNOS protein associated with iNOS mRNA was detected by immunofluorescence in cardiac myocytes. iNOS mRNA expression was not related to the ISHLT histological grade of rejection or to serum levels of NOx but was associated with increased levels of myocardial cGMP (P = .01) and with both systolic (P = .024) and diastolic (P = .006) left ventricular contractile dysfunction measured by echocardiography and Doppler.These data support a relation between iNOS mRNA expression and contractile dysfunction in the human cardiac allograft.

    View details for Web of Science ID A1996TV05300015

    View details for PubMedID 8641001

  • Regression or progression - Dependency on vascular nitric oxide ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Candipan, R. C., Wang, B. Y., Buitrago, R., Tsao, P. S., Cooke, J. P. 1996; 16 (1): 44-50


    We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine inhibits atherogenesis in the hypercholesterolemic rabbit. However, the effect of supplemental arginine on preexisting lesions is not known and was the focus of the present study. New Zealand White rabbits received normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently, L-arginine (2.25% in drinking water; ARG group) or vehicle (CHOL group) was administered for an additional 13 weeks, while the high-cholesterol diet was continued. Thoracic aortae were harvested at weeks 10, 14, 18, or 23. Rings of aorta were used to assess NO-dependent vasodilation to acetylcholine. Maximal relaxation to acetylcholine in the CHOL rabbits became progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). Planimetry of the luminal surface of the aortae from CHOL animals revealed a progressive increase in lesion surface area from 30.3% (at week 10) to 56.5% (by week 23). By contrast, animals in the ARG groups manifested improved endothelium-dependent relaxation associated with a reduction of lesion surface area at 14 and 18 weeks. The arginine-induced improvement in endothelium-dependent relaxation was associated with an increased generation of vascular NO and a reduced generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had persistent improvement in NO-dependent vasodilation and exhibited a further reduction of lesion surface area tc 5.4%. We conclude that hypercholesterolemia induces a progressive loss of NO-dependent vasodilation associated with progressive intimal lesion formation. Administration of L-arginine to animals with preexisting intimal lesions augments vascular NO elaboration, reduces superoxide anion generation, and is associated with a reduction in lesion surface area. This is the first demonstration that restoration of NO activity can induce regression of preexisting intimal lesions and provides evidence that L-arginine therapy may be of potential clinical benefit.

    View details for Web of Science ID A1996TP12400006

    View details for PubMedID 8548425

  • Acute myocardial infarction in a young woman with systemic lupus erythematosus. Vascular medicine Fearon, W. F., Cooke, J. P. 1996; 1 (1): 19-23


    A young woman was diagnosed with systemic lupus erythematosus at the age of 7 years and incurred an acute myocardial infarction at the age of 17 years. Her risk factors for coronary artery disease include hypertension, hypercholesterolemia, a relatively long disease duration, a fairly active disease as evidenced by the history of nephrotic syndrome and other organ system involvement, and a long history of prednisone use. It is difficult to determine the etiology of this patient's acute myocardial infarction without coronary artery histopathology, but aspects of her presentation (a history of virulent systemic lupus erythematosus, and the angiographic findings of ectasia and aneurysm) suggest that coronary arteritis was the etiology of her accelerated coronary artery disease and subsequent myocardial infarction. Acute myocardial infarction is an uncommon occurrence in premenopausal women less than 30 years old.35 These patients are typically found to have an associated systemic disease such as diabetes mellitus or familial hypercholesterolemia. Systemic lupus erythematosus is a less common systemic disease associated with premature coronary artery disease. Mechanisms of acute coronary syndromes in these patients include accelerated atherosclerosis, active coronary vasculitis, and/or vasospasm with superimposed thrombosis.

    View details for PubMedID 9546909

  • A novel therapy for lymphedema complicated by lymphorrhea. Vascular medicine Szuba, A., Cooke, J. P., Rockson, S. G. 1996; 1 (4): 247-250


    Lymphorrhea is a rarely described complication of chronic lymphedema, in which the disrupted flow through diseased lymphatic channels gives rise to the external drainage of lymph, often heralded by the presence of an enlarging lymphocele. This report documents the applicability of the Reid sleeve, a novel, conservative form of therapy, in an unusually severe and protracted example of lymphorrhea.

    View details for PubMedID 9552579

  • Felodipine inhibits intimal lesion formation in the hypercholesterolemic rabbit: differential effects on endothelial and monocyte determinants of atherogenesis. Vascular medicine Wang, B. Y., Niebauer, J., Singer, A. H., Tsao, P. S., Cooke, J. P. 1996; 1 (3): 173-179


    The purpose of this study was to determine if the calcium entry antagonist felodipine inhibited intimal lesion formation in hypercholesterolemic rabbits, and to determine if this was due to an effect upon monocyte and/or endothelial determinants of this interaction. Twenty-three male New Zealand White rabbits received the following treatment regimen for 10 weeks: normal chow (NP, n = 3); normal chow with felodipine infusion (NF, n = 6); 0.5% cholesterol chow (CP, n = 7); or 0.5% cholesterol chow and felodipine infusion (CF, n = 7). After 10 weeks blood was collected for biochemical measurements and mononuclear cell binding assays, and thoracic aortae were harvested for vascular reactivity studies and histomorphometry. In the animals receiving normal chow, felodipine did not significantly affect blood pressure, plasma cholesterol levels, binding studies, vascular reactivity, or structure; therefore these animals were analyzed as one group (N). Plasma cholesterol levels were significantly elevated in groups receiving the 0.5% cholesterol diet (N, 29 +/- 3 mg/dl; CP, 1221 +/- 73 mg/dl; CF, 979 +/- 108 mg/dl). High-density lipoprotein cholesterol was not different between the groups (25 +/- 4 vs 23 +/- 4 vs 27 +/- 4 mg/dl; N vs CF vs CP respectively; p = NS). Cholesterol feeding markedly augmented the adhesiveness of mononuclear cells, as demonstrated by a 250% increase in cell binding. Felodipine did not alter the adhesiveness of mononuclear cells in hypercholesterolemic animals. Cholesterol feeding significantly impaired endothelium-dependent relaxations. Endothelium-dependent relaxations were restored by felodipine treatment as reflected by the maximal responses to acetylcholine (40 +/- 7% vs 58 +/- 4% vs 67 +/- 5%; CP vs CF vs N respectively). The improvement in endothelium-dependent relaxation in the felodipine-treated animals was associated with a 2.2-fold reduction in lesion surface area of the thoracic aorta (8.2 +/- 6.3% vs 18.2 +/- 9.5%; CF vs CP; p < 0.01). Moreover, the intima/media ratio reflecting lesion thickness was substantially reduced by felodipine treatment (0.05 +/- 0.02 vs 0.20 +/- 0.07; CF vs CP; p = 0.006). Ex vivo studies revealed that felodipine inhibited the adhesiveness of vascular endothelium, but not mononuclear cells, derived from hypercholesterolemic animals. Low-dose felodipine appears to inhibit monocyte-endothelial interaction, as indicated by a reduction in the formation of lesions in hypercholesterolemic animals. This effect is not due to an alteration in adhesiveness of mononuclear cells. The salutary effect of felodipine is associated with an increase in vascular nitric oxide activity which may reduce endothelial adhesiveness.

    View details for PubMedID 9546935



    Shear stress increases the release of nitric oxide (NO) by endothelial cells (ECs). We and others have provided evidence that endothelium-derived NO inhibits monocyte adhesion to the vessel wall. We therefore hypothesized that previous exposure to shear stress would inhibit endothelial adhesiveness for monocytes by virtue of its effect to increase NO release.Confluent monolayers of bovine aortic endothelial cells, human aortic endothelial cells, or human venous endothelial cells were exposed to laminar fluid flow. Culture media were collected for measurement of NO (by chemiluminescence) and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha. NOx and 6-keto-prostaglandin F1 alpha accumulated in the conditioned medium during laminar fluid flow from 30 minutes to 24 hours in a time-dependent fashion. In another set of studies, ECs previously exposed to flow or to static conditions were washed with Hanks' buffer and exposed to THP-1 cells for 30 minutes. Adherent cells were counted by microscopy. Previous exposure to flow reduced endothelial adhesiveness for monocytes by 50% (P < .05). The effect of flow on endothelial adhesiveness occurred within 30 minutes. This effect was abrogated by nitro-L-arginine (an antagonist of NO synthesis), as well as by tetraethylammonium ion (an antagonist of the flow-activated potassium channel); the effects of these inhibitors were reversed by the NO donor SPM-5185. Although the cyclo-oxygenase inhibitor indomethacin totally inhibited the flow-induced production of prostacyclin by ECs, it minimally affected adherence of THP-1 cells. The early effect of flow on endothelial adhesiveness was not mediated by alterations in the expression of the endothelial adhesion molecules VCAM-1 or ICAM-1 as assessed by fluorescent activated cell sorting.Shear stress alters endothelial adhesiveness for monocytes; at early time points, this effect is largely due to flow-stimulated release of NO and, to a lesser extent, prostacyclin. This effect of flow occurs within 30 minutes and is probably due to alterations in the signal transduction or activation state (rather than the expression) of endothelial adhesion molecules.

    View details for Web of Science ID A1995TJ65500026

    View details for PubMedID 8521574



    Lowering the overall morbidity rate of peripheral vascular disease requires a systematic approach to the patient with atherosclerosis. This includes comprehensive assessments of risk factors, appropriate modifications of diet and lifestyle as well as an effective and sustaining exercise regimen. The role of new pharmacologic agents is discussed.

    View details for Web of Science ID A1995RN07700004

    View details for PubMedID 7638705



    To determine whether preoperative coronary angiography and revascularization improve short-term outcomes in patients undergoing noncardiac vascular surgery.Decision analysis.Patients undergoing elective vascular surgery who had either no angina or mild angina and a positive dipyridamole-thallium scan result.Three strategies were compared. The first strategy was to proceed directly to vascular surgery. The second was to perform coronary angiography, followed by selective coronary revascularization, before proceeding to vascular surgery and to cancel vascular surgery in patients with severe inoperable coronary artery disease (CAD). The third was to perform coronary angiography, followed by selective coronary revascularization, before proceeding to vascular surgery and to perform vascular surgery in patients with inoperable CAD.Mortality, nonfatal myocardial infarction, stroke, uncorrected vascular disease, and cost. All outcomes were assessed within 3 months.Proceeding directly to vascular surgery led to lower morbidity and cost in the base case analysis. The coronary angiography strategy led to higher mortality if vascular surgery would proceed in patients with inoperable CAD, but led to slightly lower mortality if vascular surgery were canceled in patients with inoperable CAD. The coronary angiography strategy also led to lower mortality when vascular surgery was particularly risky.Decision analysis indicates vascular surgery without preoperative coronary angiography generally leads to better outcomes. Preoperative coronary angiography should be reserved for patients whose estimated mortality from vascular surgery is substantially higher than average.

    View details for PubMedID 7783301



    Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

    View details for Web of Science ID A1995QX87600085

    View details for PubMedID 7753833

    View details for PubMedCentralID PMC41972



    We investigated the effect of dietary supplementation of L-arginine (L-Arg), the precursor of endothelial nitric oxide (NO), on endothelium-dependent and endothelium-independent vascular responses, as well as vascular structure, in the abdominal aorta of hypercholesterolemic rabbits. Rabbits were fed (a) normal rabbit chow, (b) 1% cholesterol diet, or (c) 1% cholesterol diet supplemented with 2.25% L-Arg HCl in drinking water. After 10 weeks, the abdominal aorta was harvested for study of vascular reactivity and histomorphometry. L-Arg did not affect serum cholesterol levels. Histomorphometric analysis demonstrated an eightfold reduction in intimal thickening in the abdominal aorta of the arginine-supplemented hypercholesterolemic rabbits. By contrast, the effects on vascular reactivity were subtle. Contraction to norepinephrine (NE) was not altered by hypercholesterolemia or L-Arg. Contraction to acetylcholine (ACh) was increased in hypercholesterolemic animals; this was normalized by dietary arginine supplementation. Relaxation to nitroglycerin (NTG) was not altered by hypercholesterolemia but was attenuated in the arginine-supplemented rabbits. Endothelium-dependent relaxation to ACh was impaired in both hypercholesterolemic groups. Dietary L-Arg has a dramatic antiatherogenic effect in hypercholesterolemic rabbits. This effect is associated with rather slight changes in vascular reactivity that are suggestive of a slight increase in NO elaboration by the endothelium. The discordance between the effects of dietary arginine on vascular structure and reactivity suggests that the antiatherogenic effects of the NO precursor may not be mediated entirely by its effect on the endothelium.

    View details for Web of Science ID A1995QV17000005

    View details for PubMedID 7630149



    The endothelium has the capacity to modulate vascular structure in response to hemodynamic stimuli. We tested the hypothesis that exposure of the endothelium to increased laminar shear stress induces the expression of TGF beta 1 via a signal transduction pathway modulated by K+ channel currents. Although TGF beta 1 is normally secreted in a latent, inactive form, exposure of cultured endothelial cells to steady laminar shear stress (20 dynes/cm2) induced increased generation of biologically active TGF beta 1. This increase in active TGF beta 1 was associated with a sustained increase in TGF beta 1 mRNA expression within 2 h of stimulation. TGF beta 1 mRNA levels increased in direct proportion to the intensity of the shear stress within the physiologic range. The effect of shear stress on TGF beta 1 mRNA expression was regulated at the transcriptional level as defined by nuclear run-off studies and transient transfection of a TGF beta 1 promoter-reporter gene construct. Blockade of endothelial K+ channels with tetraethylammonium significantly inhibited: activation of TGF beta 1 gene transcription; increase in steady state mRNA levels; and generation of active TGF beta 1 in response to shear stress. These data suggest that endothelial K+ channels and autocrine-paracrine TGF beta 1 may be involved in the mechanotransduction mechanisms mediating flow-induced vascular remodeling.

    View details for Web of Science ID A1995QM01200057

    View details for PubMedID 7883983



    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as beta-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessels. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

    View details for Web of Science ID A1995QG77000043

    View details for PubMedID 7532305

    View details for PubMedCentralID PMC42653



    Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.

    View details for Web of Science ID A1994PL39400002

    View details for PubMedID 7918301

  • THE VASCULOPATHY OF AGING JOURNALS OF GERONTOLOGY Cooper, L. T., Cooke, J. P., Dzau, V. J. 1994; 49 (5): B191-B196

    View details for Web of Science ID A1994PE10600007

    View details for PubMedID 8056931



    We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine normalizes NO-dependent vasodilation and markedly inhibits atherogenesis in a hypercholesterolemic rabbit model. We hypothesized that this antiatherogenic effect is due to modulation of endothelial adhesiveness by endothelium-derived NO.New Zealand White rabbits were fed normal chow (Cont), a high-cholesterol diet (Chol), a high-cholesterol diet supplemented with L-arginine (Arg), or a normal diet supplemented with the NO synthase antagonist L-nitroarginine (L-NA) for 2 weeks. In additional studies, some animals receiving L-NA were also treated with hydralazine to normalize blood pressure. After 2 weeks, thoracic aortas were harvested, opened longitudinally, and placed in a culture dish with the endothelial surface exposed to medium containing WEHI 78/24 cells, a monocytoid cell line. After incubation with the monocytoid cells for 30 minutes on a rocking platform, the aortic segments were washed repeatedly to remove nonadherent cells and adherent cells counted by epifluorescent microscopy. Monocytoid cell binding to aortic endothelium was significantly increased in Chol (P < .001 versus Cont); binding was markedly reduced in arginine-fed hypercholesterolemic animals (P < .05, Arg versus Chol). Monocytoid cell binding to aortic endothelium was also significantly increased in L-NA (P < .05); hydralazine normalized blood pressure but did not reduce monocytoid cell binding. To confirm that alterations in NO activity modulate endothelial cell-monocyte interaction, the release of nitrogen oxides (NOx) by thoracic aortas was assessed by a chemiluminescent technique. The concentration of NOx in the conditioned medium from segments of Arg thoracic aortas was significantly greater than that from Cont aortas, whereas that from L-NA aortas was significantly less.Hypercholesterolemia enhances the adhesiveness of aortic endothelium for monocytes; this effect is attenuated by dietary L-arginine. Conversely, inhibition of NO synthesis enhances monocyte binding. The results suggest that endothelium-derived NO plays an important role in regulating the endothelial adhesiveness for monocytes. Alterations in NO activity may play a critical role in atherogenesis.

    View details for Web of Science ID A1994NL67500034

    View details for PubMedID 8181143



    Coronary endothelial vasodilator dysfunction is a common finding in cardiac transplant recipients and may represent an early marker for the development of intimal thickening and graft atherosclerosis. The present study tested the hypothesis that endothelial dysfunction precedes intimal thickening and that administration of L-arginine, the precursor of endothelium-derived relaxing factor, improves endothelial vasodilator function of coronary conduit and resistance vessels if given at an early stage of graft atherosclerosis.Acetylcholine (10(-6), 10(-5), 10(-4) mol/L) was infused into the left anterior descending or circumflex artery and repeated after intravenous infusion of L-arginine (10 over 20 minutes) in 18 cardiac transplant recipients. Epicardial responses were evaluated by quantitative angiography, and the microcirculation was studied by determination of coronary blood flow with a Doppler flow velocity wire. Intimal thickening was assessed by intravascular ultrasound (n = 14). In epicardial coronary arteries, acetylcholine tended to elicit vasoconstriction. Epicardial coronary vasoconstriction elicited by acetylcholine was attenuated by infusion of L-arginine (10(-4) mol/L, -6.8% versus -2.8%; P < .01); this beneficial effect was observed predominantly in patients with normal intravascular ultrasound characteristics. In coronary resistance vessels, acetylcholine induced vasodilation, reflected by increases in coronary blood flow. The acetylcholine-induced increase in blood flow was significantly enhanced with L-arginine (at a dose of 10(-4) mol/L, + 121% versus 176%; before versus after L-arginine, P < .002).The coronary vasculature of cardiac transplant recipients exhibits a generalized endothelial dysfunction of conduit and resistance vessels. L-Arginine improves endothelial dysfunction of both coronary microvasculature and epicardial coronary arteries. The reversibility of epicardial endothelial dysfunction by L-arginine is more likely in vessels with normal wall morphology.

    View details for Web of Science ID A1994NG46900019

    View details for PubMedID 8149529



    This study was designed to test the hypothesis that long-term oral supplementation of dietary L-arginine (to provide a sustained elevation of nitric oxide activity) would inhibit atherogenesis in hypercholesterolemic rabbits, as assessed by histomorphometric measurements.Endothelium-derived nitric oxide inhibits a number of processes that are critical in atherogenesis. Hypercholesterolemia reduces endothelial nitric oxide activity, and we postulate that this may promote atherogenesis. This reduction in nitric oxide activity can be reversed acutely by intravenous infusion of L-arginine, the precursor of nitric oxide. We show that dietary supplementation of L-arginine abrogates the development of coronary atheroma in hypercholesterolemic rabbits.Male New Zealand White rabbits were fed normal rabbit chow, 1% cholesterol chow or 1% cholesterol chow with dietary arginine or methionine supplementation to increase their intake of these amino acids sixfold. After 1 or 10 weeks of dietary intervention, the left main and left anterior descending coronary arteries were harvested for histologic study. Plasma cholesterol measurements were elevated to the same degree in all groups of rabbits receiving the 1% cholesterol diet, whereas plasma arginine levels were doubled in the arginine-treated group. High density lipoprotein (HDL) cholesterol values were not affected by arginine treatment.In rabbits receiving the 1% cholesterol diet, with or without methionine supplementation, light and electron microscopy revealed a marked increase from 1 to 10 weeks in the intimal accumulation of macrophages, associated with an increase in the intimal area of the left main coronary artery. By contrast, in arginine-treated hypercholesterolemic rabbits, there was a near absence of adherent monocytes and tissue macrophages and no progression of intimal thickness from 1 to 10 weeks.Dietary supplements of L-arginine prevent intimal thickening in the coronary arteries of hypercholesterolemic rabbits. This antiatherogenic effect is not due to an alteration in plasma total cholesterol, HDL cholesterol or caloric or nitrogen balance. The data are consistent with the hypothesis that nitric oxide has antiatherogenic properties.

    View details for Web of Science ID A1994NR75200026

    View details for PubMedID 8294700



    We have observed persistent desensitization to exogenous norepinephrine after balloon injury. We postulated that this desensitization may be due to a local increase in the release of neuronal norepinephrine.New Zealand White rabbits underwent left iliac artery angioplasty; 4 weeks later, both iliac arteries were harvested. Maximal response to exogenous norepinephrine was reduced in injured compared with noninjured vessels (12.3 +/- 1.0 g versus 10.3 +/- 1.5 g; n = 7, P = .056). By contrast, response to electrical stimulation (to induce neuronal norepinephrine release) was significantly greater in injured tissues (36 +/- 7% versus 14 +/- 3%; values expressed as percent of maximal contraction to exogenous norepinephrine; P = .025). Direct measurement of tissue norepinephrine revealed a threefold increase 4 weeks after injury (1236 +/- 410 versus 466 +/- 97 pg/mg; injured versus noninjured). To determine if desensitization to exogenous norepinephrine was due to a persistent increase in neuronal norepinephrine release, the experiments were repeated after chemical sympatholysis using 6-hydroxydopamine (6-OHDA) (65 mg/kg). To determine if activation of vascular angiotensin II contributed to facilitation of adrenergic neurotransmission, other animals received ramipril (RAM; 1 mg/kg per day). Both treatments were initiated 7 days before angioplasty. In the 6-OHDA group there was no evidence of desensitization, judged by maximal response to exogenous norepinephrine (7.5 +/- 0.6 versus 7.5 +/- 0.8, noninjured versus injured). Similar results were obtained in RAM animals (9.9 +/- 0.8 versus 9.6 +/- 1.2, noninjured versus injured).This is the first study to demonstrate enhanced adrenergic neurotransmission after balloon injury. The facilitation of adrenergic neurotransmission may be due to increased local concentrations of angiotensin II and is associated with desensitization to exogenous norepinephrine.

    View details for Web of Science ID A1994MW36200031

    View details for PubMedID 8313566



    The endothelium acts as the sensor of shear stress and as the mediator of flow-induced changes in vessel tone and structure. The purpose of this study was to delineate the signal transduction pathway of flow-induced release of endothelium-derived relaxing factor (EDRF).We used a shear stress apparatus (a modified cone-plate viscometer) to expose cultured endothelial cells to a well-defined laminar fluid flow. Confluent bovine aortic endothelial cells (BAECs) were subjected to varying levels of shear stress, and intracellular cyclic GMP (cGMP) in the BAECs was measured by radioimmunoassay. After 60 seconds of laminar fluid flow, BAEC cGMP increased by 300% from basal levels (from 0.54 to 1.70 pmol/mg protein, P < 0.05). The elevation in intracellular cGMP was proportional to the intensity of shear stress within a physiological range up to 40 dynes/cm2. This increase in cGMP was abrogated by L-N-methyl-arginine (the competitive antagonist of nitric oxide [NO] synthase), indicating that the flow-induced activation of soluble guanylate cyclase was mediated by autocrine NO production. Furthermore, a potassium channel antagonist, tetraethylammonium ion (TEA [3 mmol/L]) and a G(i) or G(o) protein inhibitor, pertussis toxin (100 ng/mL) also blocked the flow-induced increase in cGMP. By contrast, calcium ionophore or atrial natriuretic peptide caused elevations of cGMP that were not affected by TEA or pertussis toxin.These findings indicate that shear stress elevates endothelial cGMP via an NO-dependent mechanism. The effect of shear stress is mediated by a unique signal transduction pathway that is coupled to a pertussis toxin-sensitive G protein and that requires the activity of an endothelial potassium channel.

    View details for Web of Science ID A1993LM12700026

    View details for PubMedID 8391400


    View details for Web of Science ID A1993KQ92300001

    View details for PubMedID 8443891

  • The role of endothelial dysfunction in restenosis. Revista portuguesa de cardiologia Cooke, J. P., Tsao, P. S. 1992; 11 (10): 889-892

    View details for PubMedID 1285965



    The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.

    View details for Web of Science ID A1992JN95900065

    View details for PubMedID 1522225



    Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide.

    View details for Web of Science ID A1991GN72700031

    View details for PubMedID 1719029



    Hypercholesterolemia attenuates endothelium-dependent vasorelaxation and augments the responses to vasoconstrictor agents. Both effects are largely due to a reduction in the release of endothelium-derived relaxing factor. Since endothelium-derived relaxing factor is now known to be nitric oxide derived from the metabolism of L-arginine, we hypothesized that the abnormal vascular response in hypercholesterolemia could be corrected by supplying the precursor to EDRF, L-arginine. In a series of studies, we have found that conduit and resistance vessels of hypercholesterolemic animals demonstrate endothelial dysfunction which is reversed after exposure to high concentrations of exogenous L-arginine. The experiments suggest that hypercholesterolemia induces a reversible dysfunction of arginine availability or metabolism.

    View details for Web of Science ID A1991GA89600017

    View details for PubMedID 1953609