Publications by authors named "Weifei Zhu"

25 Publications

  • Page 1 of 1

A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Cell 2020 03;180(5):862-877.e22

Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44106, USA; Center for Microbiome & Human Health, Cleveland Clinic, Cleveland, OH 44106, USA; Heart and Vascular Institute, Cleveland Clinic, Cleveland, OH 44106, USA. Electronic address:

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and β2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.
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http://dx.doi.org/10.1016/j.cell.2020.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402401PMC
March 2020

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Arterioscler Thromb Vasc Biol 2019 06;39(6):1045-1054

From the Department of Medicine/Division of Cardiology (D.M.S., Y.M., A.M., A.J.L.), University of California, Los Angeles.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.
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http://dx.doi.org/10.1161/ATVBAHA.119.312592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531332PMC
June 2019

Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential.

Circ Res 2018 10;123(10):1164-1176

Center for Microbiome and Human Health, Lerner Research Institute (S.M.S., W.Z., K.A.R., Z.W., J.K., J.A.D., W.H.W.T., S.L.H.), Cleveland Clinic, OH.

Rationale: Gut microbes influence cardiovascular disease and thrombosis risks through the production of trimethylamine N-oxide (TMAO). Microbiota-dependent generation of trimethylamine (TMA)-the precursor to TMAO-is rate limiting in the metaorganismal TMAO pathway in most humans and is catalyzed by several distinct microbial choline TMA-lyases, including the proteins encoded by the cutC/D (choline utilization C/D) genes in multiple human commensals.

Objective: Direct demonstration that the gut microbial cutC gene is sufficient to transmit enhanced platelet reactivity and thrombosis potential in a host via TMA/TMAO generation has not yet been reported.

Methods And Results: Herein, we use gnotobiotic mice and a series of microbial colonization studies to show that microbial cutC-dependent TMA/TMAO production is sufficient to transmit heightened platelet reactivity and thrombosis potential in a host. Specifically, we examine in vivo thrombosis potential employing germ-free mice colonized with either high TMA-producing stable human fecal polymcrobial communities or a defined CutC-deficient background microbial community coupled with a CutC-expressing human commensal±genetic disruption of its cutC gene (ie, Clostridium sporogenes Δ cutC).

Conclusions: Collectively, these studies point to the microbial choline TMA-lyase pathway as a rational molecular target for the treatment of atherothrombotic heart disease.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223262PMC
October 2018

Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.

Nat Med 2018 09 6;24(9):1407-1417. Epub 2018 Aug 6.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite that enhances both platelet responsiveness and in vivo thrombosis potential in animal models, and TMAO plasma levels predict incident atherothrombotic event risks in human clinical studies. TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA) moiety-containing nutrients, which are abundant in a Western diet. Here, using a mechanism-based inhibitor approach targeting a major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D), we developed inhibitors that are potent, time-dependent, and irreversible and that do not affect commensal viability. In animal models, a single oral dose of a CutC/D inhibitor significantly reduced plasma TMAO levels for up to 3 d and rescued diet-induced enhanced platelet responsiveness and thrombus formation, without observable toxicity or increased bleeding risk. The inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over 1-million-fold higher than needed for a therapeutic effect. These studies reveal that mechanism-based inhibition of gut microbial TMA and TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective nonlethal targeting of gut microbial enzymes linked to host disease limiting systemic exposure of the inhibitor in the host.
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http://dx.doi.org/10.1038/s41591-018-0128-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129214PMC
September 2018

Gut Microbiota-Dependent Trimethylamine N-Oxide Predicts Risk of Cardiovascular Events in Patients With Stroke and Is Related to Proinflammatory Monocytes.

Arterioscler Thromb Vasc Biol 2018 09;38(9):2225-2235

From the Department of Cardiology (A.H., D.S., F.Z., N.K., U.L.).

Objective- Gut microbiota-dependent metabolites, in particular trimethylamine N-oxide (TMAO), have recently been reported to promote atherosclerosis and thrombosis. Here, we examined for the first time the relation of TMAO and the risk of incident cardiovascular events in patients with recent first-ever ischemic stroke in 2 independent prospective cohorts. Moreover, the link between TMAO and proinflammatory monocytes as a potential contributing factor for cardiovascular risk in stroke patients was studied. Approach and Results- In a first study (n=78), higher TMAO plasma levels were linked with an increased risk of incident cardiovascular events including myocardial infarction, recurrent stroke, and cardiovascular death (fourth quartile versus first quartile; hazard ratio, 2.31; 95% CI, 1.25-4.23; P<0.01). In the second independent validation cohort (n=593), high TMAO levels again heralded marked increased risk of adverse cardiovascular events (fourth quartile versus first quartile; hazard ratio, 5.0; 95% CI, 1.7-14.8; P<0.01), and also after adjustments for cardiovascular risk factors including hypertension, diabetes mellitus, LDL (low-density lipoprotein) cholesterol, and estimated glomerular filtration rate (hazard ratio, 3.3; 95% CI, 1.2-10.9; P=0.04). A significant correlation was also found between TMAO levels and percentage of proinflammatory intermediate CD14CD16 monocytes ( r=0.70; P<0.01). Moreover, in mice fed a diet enriched with choline to increase TMAO synthesis, levels of proinflammatory murine Ly6C monocytes were higher than in the chow-fed control group (choline: 9.2±0.5×10 per mL versus control: 6.5±0.5×10 per mL; P<0.01). This increase was abolished in mice with depleted gut microbiota (choline+antibiotics: 5.4±0.7×10 per mL; P<0.001 versus choline). Conclusions- The present study demonstrates for the first time a graded relation between TMAO levels and the risk of subsequent cardiovascular events in patients with recent prior ischemic stroke. Our data support the notion that TMAO-related increase of proinflammatory monocytes may add to elevated cardiovascular risk of patients with increased TMAO levels.
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http://dx.doi.org/10.1161/ATVBAHA.118.311023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202215PMC
September 2018

Gut Microbe-Generated Trimethylamine -Oxide From Dietary Choline Is Prothrombotic in Subjects.

Circulation 2017 04;135(17):1671-1673

From Department of Cellular and Molecular Medicine, Lerner Research Institute (W.Z., Z.W., W.H.W.T., S.L.H.), Center for Microbiome & Human Health (W.Z., Z.W., W.H.W.T., S.L.H.), and Heart and Vascular Institute (W.H.W.T., S.L.H.), Cleveland Clinic, OH.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.025338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460631PMC
April 2017

Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

Cell 2016 Mar 10;165(1):111-124. Epub 2016 Mar 10.

Department of Cellular & Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA. Electronic address:

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.
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http://dx.doi.org/10.1016/j.cell.2016.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862743PMC
March 2016

Trimethylamine N-Oxide Promotes Vascular Inflammation Through Signaling of Mitogen-Activated Protein Kinase and Nuclear Factor-κB.

J Am Heart Assoc 2016 Feb 22;5(2). Epub 2016 Feb 22.

Department of Medicine, Cardiology Division at the University of California, Los Angeles, CA

Background: The choline-derived metabolite trimethylamine N-oxide (TMAO) has been demonstrated to contribute to atherosclerosis and is associated with coronary artery disease risk.

Methods And Results: We explored the impact of TMAO on endothelial and smooth muscle cell function in vivo, focusing on disease-relevant outcomes for atherogenesis. Initially, we observed that aortas of LDLR(-/-) mice fed a choline diet showed elevated inflammatory gene expression compared with controls. Acute TMAO injection at physiological levels was sufficient to induce the same inflammatory markers and activate the well-known mitogen-activated protein kinase, extracellular signal-related kinase, and nuclear factor-κB signaling cascade. These observations were recapitulated in primary human aortic endothelial cells and vascular smooth muscle cells. We also found that TMAO promotes recruitment of activated leukocytes to endothelial cells. Through pharmacological inhibition, we further showed that activation of nuclear factor-κB signaling was necessary for TMAO to induce inflammatory gene expression in both of these relevant cell types as well as endothelial cell adhesion of leukocytes.

Conclusions: Our results suggest a likely contributory mechanism for TMAO-dependent enhancement in atherosclerosis and cardiovascular risks.
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http://dx.doi.org/10.1161/JAHA.115.002767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802459PMC
February 2016

Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis.

Cell 2015 Dec;163(7):1585-95

Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA. Electronic address:

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.
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http://dx.doi.org/10.1016/j.cell.2015.11.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871610PMC
December 2015

PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

PLoS One 2015 17;10(4):e0124494. Epub 2015 Apr 17.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401672PMC
April 2016

Transmission of atherosclerosis susceptibility with gut microbial transplantation.

J Biol Chem 2015 Feb 30;290(9):5647-60. Epub 2014 Dec 30.

From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195,

Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.
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http://dx.doi.org/10.1074/jbc.M114.618249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342477PMC
February 2015

Techniques to harvest diseased human peripheral arteries and measure endothelial function in an ex vivo model.

J Vasc Surg 2013 Aug 1;58(2):470-7. Epub 2013 Feb 1.

Harrington Heart and Vascular Institute, University Hospitals Case Medical Center, Cleveland Clinic, Cleveland, Ohio 44106, USA.

Objective: Endothelial dysfunction has been studied in animal models. However, direct evidence of endothelial function from human vessels is limited. Our objectives were to optimize methods in harvesting human arteries from amputation specimens, determine endothelial function, and measure responsiveness to l-arginine, a nitric oxide precursor.

Methods: Fresh amputation specimens were transferred expeditiously from the operating room to the bench laboratory for dissection and arterial harvest in an Investigational Review Board-approved protocol. Popliteal and tibial vessels were examined in pilot experiments leading to the use of the anterior tibial artery in consecutive experiments. Human lower extremity anterior tibial artery segments were harvested from 14 amputation specimens. Specimens were rapidly collected and divided for endothelial-dependent relaxation (EDR) studies in a tissue bath apparatus, immunohistochemistry, and intravascular ultrasound-derived virtual histology. A total of 47 ring segments were studied. The data were compared with two-way analysis of variance.

Results: Human lower extremity arteries exhibited low responsiveness to acetylcholine (EDR, 24.9%; acetylcholine, 10(-4)). L-arginine supplementation enhanced EDR by 38.5% (P < .0001). N-nitro-L-arginine methyl ester abrogated EDR (P < .0001) in vessels exposed to L-arginine. Arterial responsiveness was intact in all vessels (endothelial independent relaxation to sodium nitroprusside, 113.2% ± 28.1%). Histology and immunohistochemistry confirmed intact endothelium by morphometric analysis, cluster of differentiation 31, endothelial nitric oxide synthase, and arginase II staining. Intravascular ultrasound-derived virtual histology indicated atheroma burden was 11.9 ± 4.7 mm(3)/cm, and plaque stratification indicated fibrous morphology was predominant (59.9%; necrotic core, 16.9%; calcium, 11.2%). Variations in plaque morphology did not correlate with endothelial function or responsiveness to L-arginine.

Conclusions: Human lower extremity arteries demonstrate low baseline endothelial function in patients requiring amputation. Endothelial dysfunction is improved by L-arginine supplementation in an ex vivo model. These results support strategies to increase local levels of nitric oxide in human vessels.
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http://dx.doi.org/10.1016/j.jvs.2012.10.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644533PMC
August 2013

Advanced glycation end products induce a prothrombotic phenotype in mice via interaction with platelet CD36.

Blood 2012 Jun 19;119(25):6136-44. Epub 2012 Mar 19.

Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA.

Diabetes mellitus has been associated with platelet hyperreactivity, which plays a central role in the hyperglycemia-related prothrombotic phenotype. The mechanisms responsible for this phenomenon are not established. In the present study, we investigated the role of CD36, a class-B scavenger receptor, in this process. Using both in vitro and in vivo mouse models, we demonstrated direct and specific interactions of platelet CD36 with advanced glycation end products (AGEs) generated under hyperglycemic conditions. AGEs bound to platelet CD36 in a specific and dose-dependent manner, and binding was inhibited by the high-affinity CD36 ligand NO(2)LDL. Cd36-null platelets did not bind AGE. Using diet- and drug-induced mouse models of diabetes, we have shown that cd36-null mice had a delayed time to the formation of occlusive thrombi compared with wild-type (WT) in a FeCl(3)-induced carotid artery injury model. Cd36-null mice had a similar level of hyperglycemia and a similar level of plasma AGEs compared with WT mice under this condition, but WT mice had more AGEs incorporated into thrombi. Mechanistic studies revealed that CD36-dependent JNK2 activation is involved in this prothrombotic pathway. Therefore, the results of the present study couple vascular complications in diabetes mellitus with AGE-CD36-mediated platelet signaling and hyperreactivity.
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http://dx.doi.org/10.1182/blood-2011-10-387506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383021PMC
June 2012

Coupling of Fcγ receptor I to Fcγ receptor IIb by SRC kinase mediates C-reactive protein impairment of endothelial function.

Circ Res 2011 Oct 22;109(10):1132-40. Epub 2011 Sep 22.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Rationale: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fcγ receptor IIB (FcγRIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5'-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization.

Objective: How CRP activates FcγRIIB in endothelium is not known. We determined the role of Fcγ receptor I (FcγRI) and the basis for coupling of FcγRI to FcγRIIB in endothelium.

Methods And Results: In cultured endothelial cells, FcγRI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via FcγRI. CRP-induced increases in FcγRIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5'-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the γ subunit of FcγRI (FcRγ(-/-)) or FcγRIIB(-/-) mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcRγ(-/-); TG-CRP and FcγRIIB(-/-); TG-CRP mice.

Conclusions: CRP antagonism of eNOS is mediated by the coupling of FcγRI to FcγRIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5'-phosphatase 1, and consistent with this mechanism, both FcγRI and FcγRIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. FcγRI and FcγRIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.
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http://dx.doi.org/10.1161/CIRCRESAHA.111.254573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215097PMC
October 2011

Thrombin induces endothelial arginase through AP-1 activation.

Am J Physiol Cell Physiol 2010 Apr 23;298(4):C952-60. Epub 2009 Dec 23.

Department of Cell Biology, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA.

Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction.
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http://dx.doi.org/10.1152/ajpcell.00466.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853217PMC
April 2010

Arginase blockade lessens endothelial dysfunction after thrombosis.

J Vasc Surg 2008 Aug 16;48(2):441-6. Epub 2008 May 16.

Department of Cell Biology, the Lerner Research Institute, Cleveland, Ohio; Department of Vascular Surgery, Cleveland Clinic, Cleveland, Ohio, USA.

Introduction: Acute arterial thrombosis causes endothelial dysfunction due to decreased nitric oxide bioactivity. Increased arginase activity may modulate intracellular L-arginine levels, the substrate for nitric oxide. The purpose of this study was to identify the role of arginase in endothelial dysfunction in cell culture and in the vasomotor response of arteries exposed to thrombus.

Methods: Rat aortic endothelial cells were exposed to thrombin at different time points. The cell extract was analyzed by immunoblotting and real-time polymerase chain reaction. Adult male rats underwent infrarenal aortic thrombosis by clip ligature for 1 hour. Infrarenal aortic ring segments were harvested and placed in physiologic buffer baths, and a force transducer was used to measure endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR). Arginase blockade was performed by incubating infrarenal aortic ring segments with arginase inhibitors for 1 hour before measuring EDR. Whole tissue extracts also underwent immunoblot analysis. The EDR and EIR curves were compared with analyses of variance.

Results: A 6.76 +/- 1.4-fold induction in arginase I message levels (P = .001) was found in rat aortic endothelial cells exposed to thrombin (30 U/mL), and arginase I protein levels increased 2.1 times. The eight infrarenal aortic ring segments exposed to thrombosis for 1 hour had diminished EDR curves compared with 14 nonthrombosed normal segments (controls). The maximum (+/- SEM) EDR (acetylcholine 10(-5)M dose) in control infrarenal aortic ring segments was 108% +/- 4.3% compared with 63% +/- 6.2% for thrombosed infrarenal aortic ring segments (P < .001). Exposure to arterial thrombosis resulted in a 3.8-times increase in arginase I protein levels in infrarenal aortic ring segments. Preincubation of nine infrarenal aortic ring segments with the nonspecific (difluoromethylornithine) and six with specific ([S]-[2-boronoethyl]-L-Cysteine-HCl [BEC]) arginase inhibitor for 1 hour significantly increased the maximum EDR compared with untreated thrombosed segments (104 +/- 5.2, 108 +/- 7.6 vs 63% +/- 6.2, P < .001). EDR curves for difluoromethylornithine- and BEC-treated infrarenal aortic ring segments were superimposed on control EDR curves. The EIR and the vasoconstriction with norepinephrine for all groups were similar.

Conclusion: Endothelial cells exposed to thrombin have increased arginase I messenger RNA and protein levels. Arterial thrombosis causes endothelial dysfunction without affecting smooth muscle responsiveness. Arginase blockade can lead to normalization of arterial vasomotor function.
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http://dx.doi.org/10.1016/j.jvs.2008.02.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517129PMC
August 2008

The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity.

Circ Res 2008 Feb 3;102(4):480-7. Epub 2008 Jan 3.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas, USA.

Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.
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http://dx.doi.org/10.1161/CIRCRESAHA.107.159079DOI Listing
February 2008

C-reactive protein downregulates endothelial NO synthase and attenuates reendothelialization in vivo in mice.

Circ Res 2007 May 19;100(10):1452-9. Epub 2007 Apr 19.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

C-reactive protein (CRP) is an acute-phase reactant that is positively associated with cardiovascular disease risk and endothelial dysfunction. In cell culture, CRP decreases the expression of endothelial NO synthase (eNOS), which regulates diverse endothelial cell (EC) functions including migration. To determine whether CRP alters EC gene expression and phenotype in vivo, we studied CF1 transgenic mice expressing rabbit CRP (CF1-CRP) regulated by the phosphoenolpyruvate carboxykinase promoter such that levels could be altered by changing carbohydrate intake. Compared with CF1 controls with CRP of <1 microg/mL, carotid artery reendothelialization after perivascular electric injury was blunted in CF1-CRP mice, with CRP levels as low as 9 microg/mL. eNOS mRNA and enzyme abundance in carotid arteries was also blunted by CRP at 9 microg/mL in vivo, and ex vivo studies of isolated arteries showed that this occurs via direct action on the endothelium. The impaired reendothelialization with CRP was mimicked by NOS antagonism in CF1 mice; conversely, in cultured ECs CRP attenuation of migration was prevented by exogenous NO. Studies of EC transfected with human eNOS 5' flanking sequence fused to luciferase indicated that CRP decreases eNOS gene transcription. Both mutagenesis and electrophoretic mobility shift assays further revealed that CRP-responsive elements reside within the first 79 bp of the eNOS promoter. Thus, CRP downregulates eNOS and attenuates reendothelialization in vivo in mice, and this action of CRP on eNOS is mediated at the level of gene transcription.
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http://dx.doi.org/10.1161/01.RES.0000267745.03488.47DOI Listing
May 2007

Increased expression of glutathione reductase in macrophages decreases atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice.

Arterioscler Thromb Vasc Biol 2007 Jun 15;27(6):1375-82. Epub 2007 Mar 15.

Division of Nephrology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.

Objective: Thiol oxidative stress leads to macrophage dysfunction and cell injury, and has been implicated in the development of atherosclerotic lesions. We investigated if strengthening the glutathione-dependent antioxidant system in macrophages by overexpressing glutathione reductase (GR) decreases the severity of atherosclerosis.

Methods And Results: Bone marrow cells infected with retroviral vectors expressing either enhanced green fluorescent protein (EGFP) or an EGFP-fusion protein of cytosolic GR (GR(cyto)-EGFP) or mitochondrial GR (GR(mito)-EGFP) were transplanted into low-density lipoprotein receptor-deficient mice. Five weeks after bone marrow transplantation, animals were challenged with a Western diet for 10 weeks. No differences in either plasma cholesterol and triglyceride levels or peritoneal macrophage content were observed. However, mice reconstituted with either GR(cyto)-EGFP or GR(mito)-EGFP-expressing bone marrow had lesion areas (P<0.009) that were 32% smaller than recipients of EGFP-expressing bone marrow. In cultured macrophages, adenovirus-mediated overexpression of GR(cyto)-EGFP or GR(mito)-EGFP protected cells from mitochondrial hyperpolarization induced by oxidized low-density lipoprotein.

Conclusion: This study provides direct evidence that the glutathione-dependent antioxidant system in macrophages plays a critical role in atherogenesis, and suggests that thiol oxidative stress-induced mitochondrial dysfunction contributes to macrophage injury in atherosclerotic lesions.
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http://dx.doi.org/10.1161/ATVBAHA.107.142109DOI Listing
June 2007

Myristic acid stimulates endothelial nitric-oxide synthase in a CD36- and an AMP kinase-dependent manner.

J Biol Chem 2005 Aug 21;280(33):29543-50. Epub 2005 Jun 21.

Department of Pediatrics, University of Kentucky, Lexington, 40536, USA.

Dietary free fatty acids have been reported to have various effects on the endothelium including the generation of nitric oxide. The goal of the current study was to determine the mechanism whereby free fatty acid causes an increase in nitric oxide synthesis. The specific hypothesis tested was that free fatty acid association with CD36, a class B scavenger receptor, induces the activation of endothelial nitric-oxide synthase (eNOS). A human microvascular endothelial cell line and a transfected Chinese hamster ovary cell system were used to determine which free fatty acids stimulate eNOS. Surprisingly, only myristic acid, and to a lesser extent palmitic acid, stimulated eNOS. The stimulation of eNOS was dose- and time-dependent. Competition experiments with other free fatty acids and with a CD36-blocking antibody demonstrated that the effects of myristic acid on eNOS required association with CD36. Further mechanistic studies demonstrated that the effects of myristic acid on eNOS function were not dependent on PI 3-kinase, Akt kinase, or calcium. Pharmacological studies and dominant negative constructs were used to demonstrate that myristic acid/CD36 stimulation of eNOS activity was dependent on the activation of AMP kinase. These data demonstrate an unexpected link among myristic acid, CD36, AMP kinase, and eNOS activity.
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http://dx.doi.org/10.1074/jbc.M501238200DOI Listing
August 2005

Estrogen in cardiovascular disease.

Curr Opin Lipidol 2004 Oct;15(5):589-93

Department of Pediatrics, University of Kentucky Medical School, Lexington, Kentucky 40536-0230, USA.

Purpose Of Review: The controversy surrounding hormone replacement therapy has induced fear in patients and left many researchers with the impression that estrogen produces negative effects on cardiovascular function. The aim of this review is to summarize recent findings illustrating that estrogen also has positive effects even if estrogen replacement therapy is not a cure-all.

Recent Findings: Studies have unveiled new aspects of estrogen action in the cardiovascular system; however, clinical trials have not demonstrated a protective effect of the most widely used modalities of hormone replacement therapy against cardiovascular disease. New information has emerged showing that estrogen has both beneficial and detrimental effects. Further mechanistic studies and use of well defined forms of estrogens and selective estrogen receptor modulators will continue to provide novel mechanistic information that will likely lead to the development of new avenues for therapeutic interventions.

Summary: Estrogens, like other steroid hormones, are potent actors in the cardiovascular system. Since half the population have high levels of estrogen most of their lives it is plain that estrogen has a variety of beneficial physiologic functions. Clinical studies, however, have demonstrated that a specific formulation of a combination of potent estrogens and metabolites is not a magic bullet, but induces both positive and negative impacts on different organ systems. More research into the mechanistic actions of estrogens in specific pathways in individual cell types is necessary to determine appropriate therapeutic interventions to replace the loss of positive effects of estrogens while minimizing the negative effects in postmenopausal women.
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http://dx.doi.org/10.1097/00041433-200410000-00014DOI Listing
October 2004

Lipids: potential regulators of nitric oxide generation.

Am J Physiol Endocrinol Metab 2004 Sep;287(3):E386-9

Department of Pediatrics, University of Kentucky Medical School, Lexington, Kentucky 40536-0230, USA.

The endothelium is a dynamic organ that secretes several biologically active substances and plays a major functional role in the health of an organism in both physiological and pathological conditions. For instance, the endothelium is involved in control of the exchange of plasma and tissue biomolecules, regulation of vessel tone, inflammation, lipid metabolism, vessel growth and remodeling, and modulation of coagulation and fibrinolysis. The endothelium generates nitric oxide, which is a key regulator of vasodilation and plays important roles in preventing, or in some cases promoting, numerous cardiovascular diseases. Several recent studies have examined the interplay between lipids and nitric oxide generation, especially in relation to atherosclerosis. The endothelium is continuously exposed to circulating lipids in the form of lipoproteins and protein carriers that may have a direct impact on nitric oxide synthesis and function. The purpose of this review is to illustrate some of the recent findings that link lipids (plasma and cellular) to nitric oxide generation (see Fig. 1).
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http://dx.doi.org/10.1152/ajpendo.00106.2004DOI Listing
September 2004

HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner.

J Clin Invest 2003 May;111(10):1579-87

Department of Pediatrics, University of Kentucky Medical School, Lexington, Kentucky, USA.

Cardiovascular diseases remain the leading cause of death in the United States. Two factors associated with a decreased risk of developing cardiovascular disease are elevated HDL levels and sex - specifically, a decreased risk is found in premenopausal women. HDL and estrogen stimulate eNOS and the production of nitric oxide, which has numerous protective effects in the vascular system including vasodilation, antiadhesion, and anti-inflammatory effects. We tested the hypothesis that HDL binds to its receptor, scavenger receptor class B type I (SR-BI), and delivers estrogen to eNOS, thereby stimulating the enzyme. HDL isolated from women stimulated eNOS, whereas HDL isolated from men had minimal activity. Studies with ovariectomized and ovariectomized/estrogen replacement mouse models demonstrated that HDL-associated estradiol stimulation of eNOS is SR-BI dependent. Furthermore, female HDL, but not male HDL, promoted the relaxation of muscle strips isolated from C57BL/6 mice but not SR-BI null mice. Finally, HDL isolated from premenopausal women or postmenopausal women receiving estradiol replacement therapy stimulated eNOS, whereas HDL isolated from postmenopausal women did not stimulate eNOS. We conclude that HDL-associated estrodial is capable of the stimulating eNOS. These studies establish a new paradigm for examining the cardiovascular effects of HDL and estrogen.
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http://dx.doi.org/10.1172/JCI16777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155043PMC
May 2003

Caveolae, estrogen and nitric oxide.

Trends Endocrinol Metab 2003 Apr;14(3):114-7

University of Kentucky Medical School, Dept of Physiology, Lexington 40536-0230, USA.

Estradiol has numerous physiological and pathophysiological effects on the cardiovascular system, and the ongoing controversy surrounding estrogen replacement therapy clearly illustrates the importance of understanding the molecular mechanisms of estrogen action. Many recent mechanistic studies have focused on the ability of estradiol to stimulate endothelial nitric oxide synthase (eNOS) and the subsequent generation of nitric oxide (NO). NO is centrally involved in many processes such as mitogenesis, cell adhesion, thrombosis, atherosclerosis and hypertension. Consequently, elucidating the mechanisms whereby estradiol influences NO production will directly impact on our understanding of the advantages and disadvantages of estrogen replacement therapy. An exciting aspect of this emerging area of study is that the estrogen, NO and caveolae research fields have merged to identify a novel and clinically relevant molecular process. The goal of this review is to highlight the recent findings in this area and to point out areas of controversy and areas where more studies are needed.
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http://dx.doi.org/10.1016/s1043-2760(03)00027-4DOI Listing
April 2003

HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages.

J Clin Invest 2003 Feb;111(3):389-97

Department of Physiology, University of Kentucky Medical School, Lexington, Kentucky 40536, USA.

Protease inhibitors decrease the viral load in HIV patients, however the patients develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. It has been assumed that protease inhibitor-dependent increases in atherosclerosis are secondary to the dyslipidemia. Incubation of THP-1 cells or human PBMCs with protease inhibitors caused upregulation of CD36 and the accumulation of cholesteryl esters. The use of CD36-blocking antibodies, a CD36 morpholino, and monocytes isolated from CD36 null mice demonstrated that protease inhibitor-induced increases in cholesteryl esters were dependent on CD36 upregulation. These data led to the hypothesis that protease inhibitors induce foam cell formation and consequently atherosclerosis by upregulating CD36 and cholesteryl ester accumulation independent of dyslipidemia. Studies with LDL receptor null mice demonstrated that low doses of protease inhibitors induce an increase in the level of CD36 and cholesteryl ester in peritoneal macrophages and the development of atherosclerosis without altering plasma lipids. Furthermore, the lack of CD36 protected the animals from protease inhibitor-induced atherosclerosis. Finally, ritonavir increased PPAR-gamma and CD36 mRNA levels in a PKC- and PPAR-gamma-dependent manner. We conclude that protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages.
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http://dx.doi.org/10.1172/JCI16261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC151854PMC
February 2003