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Protease-Activated Receptors in Cardiovascular Diseases
- Andrew J. Leger,
- Lidija Covic and
- Athan Kuliopulos
Circulation Volume 114, Issue 105 September 2006

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Thrombosis associated with the pathophysiological activation of platelets and vascular cells has brought thrombin and its receptors to the forefront of cardiovascular medicine. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiological responses including platelet activation, intimal hyperplasia, inflammation, and maintenance of vascular tone and barrier function. The thrombin receptors PAR1 and PAR4 can be effectively targeted in animals in which acute or prolonged exposure to thrombin leads to thrombosis and/or restenosis. In the present study, we describe the molecular and pharmacological basis of small-molecule inhibitors that target PAR1. In addition, we discuss a new class of cell-penetrating inhibitors, termed pepducins, that provide insight into previously unidentified roles of PAR1 and PAR4 in protease signaling.
Abstract 15942: Effect of Genetic Deficiency of Matrix Metalloprotease 1a in ApoE^-/-Mouse Models of Atherosclerosis
- Elizabeth Fletcher,
- Rajashree Rana,
- Susan Turner,
- Lidija Covic and
- Athan Kuliopulos
Circulation Volume 138, Issue Suppl_16 November 2018

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Introduction: Emerging evidence suggests that Matrix Metalloprotease-1 (MMP-1) is involved in the progression of atherosclerosis in humans. MMP1 has been recently documented to activate protease-activated Receptor-1 (PAR1) on endothelium and platelets which contributes to arterial thrombosis and endothelial inflammation. However, the mouse homolog of human MMP1, namely MMP1a has not yet been definitively shown to contribute to progression of atherosclerosis.Hypothesis: Here, we test the hypothesis that genetic deficiency of Mmp1a will affect atherosclerotic plaque development and vessel inflammation in ApoE^-/- mouse models.Methods and Results:ApoE^-/-/Mmp1a^-/- and ApoE/Mmp1a^+/+ mice were maintained for 12 weeks on the Western Diet. ApoE-/- mice genetically deficient in Mmp1a had significantly less plaque area in the aortic arch and abdominal aorta as compared to ApoE^-/-/Mmp1a^+/+ mice. FACs analysis of inflammatory cells isolated from the aortic arch plaques of the Mmp1a-deficient mice revealed significant suppressive effects on levels of macrophages and T cells. Circulating MMP1, but not MMP13, was found to correlate with total coronary artery burden in patients undergoing percutaneous coronary interventions from the recently completed TRIP-PCI study.Conclusion: These data suggest that genetic deficiency of Mmp1a results in decreased atherosclerotic burden and inflammatory infiltrates in the aorta of ApoE^-/- mice and that human MMP1 may likewise be involved in the development of human coronary artery disease.
Suppression of Heart Failure With PAR1 Pepducin Technology in a Pressure Overload Model in Mice
- Elizabeth K. Fletcher,
- Njabulo Ngwenyama,
- Nga Nguyen,
- Susan E. Turner,
- Lidija Covic,
- Pilar Alcaide and
- Athan Kuliopulos
Circulation: Heart Failure Volume 16, Issue 101 October 2023

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BACKGROUND:PAR1 (protease-activated receptor-1) contributes to acute thrombosis, but it is not clear whether the receptor is involved in deleterious inflammatory and profibrotic processes in heart failure. Here, we employ the pepducin technology to determine the effects of targeting PAR1 in a mouse heart failure with reduced ejection fraction model.METHODS:After undergoing transverse aortic constriction pressure overload or sham surgery, C57BL/6J mice were randomized to daily sc PZ-128 pepducin or vehicle, and cardiac function, inflammation, fibrosis, and molecular analyses conducted at 7 weeksRESULTS:After 7 weeks of transverse aortic constriction, vehicle mice had marked increases in macrophage/monocyte infiltration and fibrosis of the left ventricle as compared with Sham mice. PZ-128 treatment significantly suppressed the inflammatory cell infiltration and cardiac fibrosis. Despite no effect on myocyte cell hypertrophy, PZ-128 afforded a significant reduction in overall left ventricle weight and completely protected against the transverse aortic constriction-induced impairments in left ventricle ejection fraction. PZ-128 significantly suppressed transverse aortic constriction-induced increases in an array of genes involved in myocardial stress, fibrosis, and inflammation.CONCLUSIONS:The PZ-128 pepducin is highly effective in protecting against cardiac inflammation, fibrosis, and loss of left ventricle function in a mouse model.
Bivalirudin Is a Dual Inhibitor of Thrombin and Collagen-Dependent Platelet Activation in Patients Undergoing Percutaneous Coronary Intervention
- Carey Kimmelstiel,
- Ping Zhang,
- Navin K. Kapur,
- Andrew Weintraub,
- Barath Krishnamurthy,
- Vilma Castaneda,
- Lidija Covic and
- Athan Kuliopulos
Circulation: Cardiovascular Interventions Volume 4, Issue 21 April 2011

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Background—Bivalirudin, a direct thrombin inhibitor, is a widely used adjunctive therapy in patients undergoing percutaneous intervention (PCI). Thrombin is a highly potent agonist of platelets and activates the protease-activated receptors, PAR1 and PAR4, but it is not known whether bivalirudin exerts antiplatelet effects in PCI patients. We tested the hypothesis that bivalirudin acts as an antiplatelet agent in PCI patients by preventing activation of PARs on the platelet surface.Methods and Results—The effect of bivalirudin on platelet function and systemic thrombin levels was assessed in patients undergoing elective PCI. Mean plasma levels of bivalirudin were 2.7±0.5 μmol/L during PCI, which correlated with marked inhibition of thrombin-induced platelet aggregation and significantly inhibited cleavage of PAR1. Unexpectedly, bivalirudin also significantly inhibited collagen-platelet aggregation during PCI. Collagen induced a conversion of the platelet surface to a procoagulant state in a thrombin-dependent manner that was blocked by bivalirudin. Consistent with this result, bivalirudin reduced systemic thrombin levels by >50% during PCI. Termination of the bivalirudin infusion resulted in rapid clearance of the drug with a half-life of 29.3 minutes.Conclusions—Bivalirudin effectively suppresses thrombin-dependent platelet activation via inhibition of PAR1 cleavage and inhibits collagen-induced platelet procoagulant activity as well as systemic thrombin levels in patients undergoing PCI.
Protease-Activated Receptor-2 Modulates Protease-Activated Receptor-1–Driven Neointimal Hyperplasia
- Leila M. Sevigny,
- Karyn M. Austin,
- Ping Zhang,
- Shogo Kasuda,
- Georgios Koukos,
- Sheida Sharifi,
- Lidija Covic and
- Athan Kuliopulos
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 31, Issue 121 December 2011

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Objective—Emerging evidence suggests that protease-activated receptors-1 and -2 (PAR1 and PAR2) can signal together in response to proteases found in the rapidly changing microenvironment of damaged blood vessels. However, it is unknown whether PAR1 and PAR2 promote or mitigate the hyperplastic response to arterial injury. Using cell-penetrating PAR1 pepducins and mice deficient in PAR1 or PAR2, we set out to determine the respective contributions of the receptors to hyperplasia and phenotypic modulation of smooth muscle cells (SMCs) in response to arterial injury.Methods and Results—SMCs were strongly activated by PAR1 stimulation, as evidenced by increased mitogenesis, mitochondrial activity, and calcium mobilization. The effects of chronic PAR1 stimulation following vascular injury were studied by performing carotid artery ligations in mice treated with the PAR1 agonist pepducin, P1pal-13. Histological analysis revealed that PAR1 stimulation caused striking hyperplasia, which was ablated in PAR1^−/− and, surprisingly, PAR2^−/− mice. P1pal-13 treatment yielded an expression pattern consistent with a dedifferentiated phenotype in carotid artery SMCs. Detection of PAR1-PAR2 complexes provided an explanation for the hyperplastic effects of the PAR1 agonist requiring the presence of both receptors.Conclusion—We conclude that PAR2 regulates the PAR1 hyperplastic response to arterial injury leading to stenosis.
Lipid Receptor GPR31 (G-Protein–Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis
- Layla Van Doren,
- Nga Nguyen,
- Christopher Garzia,
- Elizabeth K. Fletcher,
- Ryan Stevenson,
- David Jaramillo,
- Athan Kuliopulos and
- Lidija Covic
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 41, Issue 11 January 2021

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Objective:12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein–coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity.Approach and Results:Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4–mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop–derived pepducin, GPR310 (G-protein–coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride–induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE–mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y₁₂ receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems.Conclusions:The 12-LOX product 12(S)-HETE stimulates GPR31-Gi–signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.
Abstract 19606: PZ-128: First in Human Pepducin Inhibitor of PAR-1 Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability
- Paul A Gurbel,
- Kevin P Bliden,
- Udaya S Tantry,
- Susan E Turner,
- Martin Gesheff,
- Lidija Covic and
- Athan Kuliopulos
Circulation Volume 132, Issue suppl_310 November 2015

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Background: PZ-128 is a first-in-class cell penetrating lipopeptide pepducin that inhibits PAR-1-G protein signaling by forming a structure highly similar to the corresponding off-state juxtamembrane region of the GPCR. PZ-128 inhibited PAR-1 and thrombosis in non-human primates. Its effects in humans are unknown.Methods and Results: PZ-128 was administered by continuous intravenous infusion (0.01 mg/kg to 2 mg/kg) to patients with multiple cardiovascular risk factors (n=31). Safety, tolerability, and pharmacokinetic (PK) effects were assessed. Platelet receptor function was assessed by conventional aggregation at baseline and 0.5, 1, 2, 6, 24 h and 8-11 d post-dosing. There were no effects on ECG, hematologic or clinical chemistry parameters. At 0.5 mg/kg given over 2 h there was 40 % and 50 % inhibition (8 μM SFLLRN) at 0.5 and 2 h, respectively. At 1-2 mg/kg, there was 25% and 75% inhibition at 0.5 h and 95-100% inhibition at 1-2 h (p<0.05 for 1 mg dose). Maximal platelet aggregation (8 μM SFLLRN) was 66-100% recovered by 24 h at 0.5-2 mg/kg. There was no significant inhibition of PAR-4, ADP, or collagen receptors at any time point. PZ-128 plasma levels were linear with dose (R=0.99) and reached Cmax levels of 2 μM, 2.5 μM, 5 μM, and 12 μM at 0.3, 0.5, 1 and 2 mg/kg doses, respectively. The terminal elimination plasma half-life was 49 ± 6 min.Conclusion: PZ-128 is a promising parenteral antiplatelet agent that provided rapid, specific, dose-dependent, and reversible inhibition of platelet PAR-1 through a novel mechanism.
Blocking the Protease-Activated Receptor 1-4 Heterodimer in Platelet-Mediated Thrombosis
- Andrew J. Leger,
- Suzanne L. Jacques,
- Jehangir Badar,
- Nicole C. Kaneider,
- Claudia K. Derian,
- Patricia Andrade-Gordon,
- Lidija Covic and
- Athan Kuliopulos
Circulation Volume 113, Issue 97 March 2006

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Background— Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors.Methods and Results— Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin–PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1.Conclusions— We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.
Cell-Penetrating Pepducin Therapy Targeting PAR1 in Subjects With Coronary Artery Disease
- Paul A. Gurbel,
- Kevin P. Bliden,
- Susan E. Turner,
- Udaya S. Tantry,
- Martin G. Gesheff,
- Travis P. Barr,
- Lidija Covic and
- Athan Kuliopulos
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 36, Issue 11 January 2016

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Objective—Pepducins are membrane-tethered, cell-penetrating lipopeptides that target the cytoplasmic surface of their cognate receptor. Here, we report the first human use of a protease-activated receptor-1–based pepducin, which is intended as an antiplatelet agent to prevent ischemic complications of percutaneous coronary interventions.Approach and Results—PZ-128 was administered by 1 to 2 hours continuous intravenous infusion (0.01–2 mg/kg) to 31 subjects with coronary artery disease or multiple coronary artery disease risk factors. Safety, antiplatelet efficacy, and pharmacokinetics were assessed at baseline and 0.5, 1, 2, 6, 24 hours, and 7 to 10 days postdosing. The inhibitory effects of PZ-128 on platelet aggregation stimulated by the protease-activated receptor-1 agonist SFLLRN (8 μmol/L) at 30 minutes to 6 hours were dose dependent with 20% to 40% inhibition at 0.3 mg/kg, 40% to 60% at 0.5 mg/kg, and ≥80% to 100% at 1 to 2 mg/kg. The subgroup receiving aspirin in the 0.5 and 1-mg/kg dose cohorts had 65% to 100% inhibition of final aggregation to SFLLRN at 30 minutes to 2 hours and 95% to 100% inhibition by 6 hours. The inhibitory effects of 0.5 mg/kg PZ-128 were reversible with 50% recovery of aggregation to SFLLRN by 24 hours. There were no significant effects of PZ-128 on aggregation induced by AYPGKF, ADP, or collagen, indicating that the observed effects were specific to protease-activated receptor-1. The plasma half-life was 1.3 to 1.8 hours, and PZ-128 was nondetectable in urine. There were no effects on bleeding, coagulation, clinical chemistry, or ECG parameters.Conclusions—PZ-128 is a promising antiplatelet agent that provides rapid, specific, dose dependent, and reversible inhibition of platelet protease-activated receptor-1 through a novel intracellular mechanism.Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01806077.
Suppression of Arterial Thrombosis Without Affecting Hemostatic Parameters With a Cell-Penetrating PAR1 Pepducin
- Ping Zhang,
- András Gruber,
- Shogo Kasuda,
- Carey Kimmelstiel,
- Katie O'Callaghan,
- Daniel H. Cox,
- Andrew Bohm,
- James D. Baleja,
- Lidija Covic and
- Athan Kuliopulos
Circulation Volume 126, Issue 13 July 2012

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Background—Thrombin-dependent platelet activation is heightened in the setting of percutaneous coronary intervention and may cause arterial thrombosis with consequent myocardial necrosis. Given the high incidence of adverse effects in patients with acute coronary syndromes, there remains an unmet need for the development of new therapeutics that target platelet activation without unduly affecting hemostasis. The thrombin receptor, PAR1, has recently emerged as a promising new target for therapeutic intervention in patients with acute coronary syndromes.Methods and Results—We report the development of a first-in-class intracellular PAR1 inhibitor with optimized pharmacokinetic properties for use during percutaneous coronary intervention in patients with acute coronary syndromes. PZ-128 is a cell-penetrating pepducin inhibitor of PAR1 that targets the receptor–G-protein interface on the inside surface of platelets. The structure of PZ-128 closely resembles the predicted off-state of the corresponding juxtamembrane region of the third intracellular loop of PAR1. The onset of action of PZ-128 was rapid and suppressed PAR1 aggregation and arterial thrombosis in guinea pigs and baboons and strongly synergized with oral clopidogrel. There was full recovery of platelet function by 24 hours. Importantly, PZ-128 had no effect on bleeding or coagulation parameters in primates or in blood from patients undergoing percutaneous coronary intervention.Conclusions—Based on the efficacy data in nonhuman primates with no noted adverse effects on hemostasis, we anticipate that the rapid onset of platelet inhibition and reversible properties of PZ-128 are well suited to the acute interventional setting of percutaneous coronary intervention and may provide an alternative to long-acting small-molecule inhibitors of PAR1.
Abstract 449: Pepducin Inhibition of Protease-activated Receptor 2 Attenuates a Mouse Model of Abdominal Aortic Aneurysm
- Hannah M Russell,
- Kelsey A Conrad,
- Joshua A Benoit,
- Anders Wanhainen,
- Doran S Mix,
- Scott J Cameron,
- Lidija Covic,
- Athan Kuliopulos and
- A. Phillip Owens
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 39, Issue Suppl_11 May 2019

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Background: Vascular smooth muscle cells are an abundant source of protease-activated receptor 2 (PAR2) in the aortic media. Accumulating evidence suggests PAR2 signaling promotes the release of proinflammatory cytokines which contribute to the pathogenesis of several cardiovascular diseases. Our laboratory has previously shown that PAR2 deficiency in low-density lipoprotein receptor deficient (Ldlr^-/-) mice attenuates the incidence of abdominal aortic aneurysm (AAA) in a mouse model. The purpose of this study was to determine whether pharmacologic inhibition of PAR2 with a cell-penetrating pepducin could inhibit both the initiation and progression of AAA.Methods and Results: Male Ldlr^-/- mice received daily subcutaneous (S.C.) injections with placebo (10% DMSO; n = 11) or PAR2 pepducin (PZ-235; 10mg/kg/day dissolved in 10% DMSO; n = 11) and were placed on a fat and cholesterol-enriched diet (Western: 42% kcal/fat; 0.2% cholesterol) for 1 week prior to and throughout AngII administration for 28 days. PAR2 pepducin attenuated abdominal aortic diameters (Placebo: 1.59 ± 0.14 mm; PZ-235: 1.01 ± 0.06 mm; P = 0.001) and the incidence of AAA (91% versus 9.1%; P < 0.001) when compared to placebo controls. To determine if delayed PAR2 pepducin treatment could blunt the progression of aneurysm, male Ldlr^-/- mice (n = 40) were fed a Western diet with concomitant AngII (1,000 ng/kg/min) for 28 days. After 28 days, mice were divided into two groups (with equal abdominal diameters, as determined by ultrasound) and then administered either placebo (10% DMSO; n = 8) or PAR2 pepducin (PZ-235; n = 12) S.C. daily for an additional 14 days. PZ-235 intervention significantly attenuated the progression of abdominal diameter after 14 days (Placebo: 1.83 ± 0.05 mm; PZ-235: 1.62 ± 0.03 mm; P < 0.001). PZ-235 reduced plasma anti-inflammatory cytokines (IL-1β and MCP-1), macrophage accumulation and matrix metalloproteinase 2 expression in the abdominal aorta. None of the treatments affected plasma cholesterol, lipoprotein distributions, or increases in systolic blood pressure in either study.Conclusion: These results suggest that pepducin-based inhibition of PAR2 can attenuate the initiation and progression of AAA in a mouse model via an anti-inflammatory mechanism.
PAR1 (Protease-Activated Receptor 1) Pepducin Therapy Targeting Myocardial Necrosis in Coronary Artery Disease and Acute Coronary Syndrome Patients Undergoing Cardiac Catheterization: A Randomized, Placebo-Controlled, Phase 2 Study
- Athan Kuliopulos,
- Paul A. Gurbel,
- Jeffrey J. Rade,
- Carey D. Kimmelstiel,
- Susan E. Turner,
- Kevin P. Bliden,
- Elizabeth K. Fletcher,
- Daniel H. Cox,
- Lidija Covic and
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 40, Issue 121 December 2020

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Objective:Arterial thrombosis leading to ischemic injury worsens the prognosis of many patients with cardiovascular disease. PZ-128 is a first-in-class pepducin that reversibly inhibits PAR1 (protease-activated receptor 1) on platelets and other vascular cells by targeting the intracellular surface of the receptor. The TRIP-PCI (Thrombin Receptor Inhibitory Pepducin in Percutaneous Coronary Intervention) trial was conducted to assess the safety and efficacy of PZ-128 in patients undergoing cardiac catheterization with intent to perform percutaneous coronary intervention.Approach and Results:In this randomized, double-blind, placebo-controlled, phase 2 trial, 100 patients were randomly assigned (2:1) to receive PZ-128 (0.3 or 0.5 mg/kg), or placebo in a 2-hour infusion initiated just before the start of cardiac catheterization, on top of standard oral antiplatelet therapy. Rates of the primary end point of bleeding were not different between the combined PZ-128 doses (1.6%, 1/62) and placebo group (0%, 0/35). The secondary end points of major adverse coronary events at 30 and 90 days did not significantly differ but were numerically lower in the PZ-128 groups (0% and 2% in the PZ-128 groups, 6% and 6% with placebo, p=0.13, p=0.29, respectively). In the subgroup of patients with elevated baseline cardiac troponin I, the exploratory end point of 30-day major adverse coronary events + myocardial injury showed 83% events in the placebo group versus 31% events in the combined PZ-128 drug groups, an adjusted relative risk of 0.14 (95% CI, 0.02–0.75); P=0.02.Conclusions:In this first-in-patient experience, PZ-128 added to standard antiplatelet therapy appeared to be safe, well tolerated, and potentially reduced periprocedural myonecrosis, thus providing the basis for further clinical trials.Registration:URL: https://www.clinicaltrials.gov. Unique identifier: NCT02561000.
Deficiency of MMP1a (Matrix Metalloprotease 1a) Collagenase Suppresses Development of Atherosclerosis in Mice: Translational Implications for Human Coronary Artery Disease
- Elizabeth K. Fletcher,
- Yanling Wang,
- Laura K. Flynn,
- Susan E. Turner,
- Jeffrey J. Rade,
- Carey D. Kimmelstiel,
- Paul A. Gurbel,
- Kevin P. Bliden,
- Lidija Covic and
- Athan Kuliopulos
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 41, Issue 55 May 2021

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Graphical AbstractObjective:Destruction of arterial collagen allows monocyte and macrophage infiltration leading to atherosclerotic plaque formation, but it is not clear what role the MMP1 (matrix metalloprotease 1) collagenase plays in this process in vivo. To define the specific contribution of MMP1 to atherosclerotic plaque burden and pathogenesis, we generated ApoE^−/− mice deficient in the human MMP1 ortholog, MMP1a.Approach and Results:After 12 to 16 weeks of Western diet, genetic loss of MMP1a resulted in a significant 50% reduction in total aortic plaque burden compared with control ApoE^−/− mice. MMP1a deficiency led to significant reductions in plaque monocytes/macrophages, SMCs (smooth muscle cells), and necrosis, with increases in collagen content. Collagen invasion of oxidized-LDL (low-density lipoprotein) activated peripheral blood mononuclear cells from MMP1a-deficient mice was markedly attenuated and was similar to suppressive effects with pharmacological inhibitors of MMP1 and its receptor, PAR1 (protease-activated receptor 1). Patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization in the TRIP-PCI trial (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention) were evaluated for circulating levels of all 3 major secreted collagenases, MMP1, MMP8, and MMP13 and total number of coronary lesions with ≥50% stenosis (coronary artery disease burden). MMP1 was significantly (P<0.001) higher by 19-fold and 5.7-fold relative to MMP13 and MMP8, respectively. MMP1 correlated with stenotic coronary artery disease burden, TNFα (tumor necrosis factor alpha) levels, and was co-expressed with PAR1 on monocytes. Treatment of patients with the PAR1 inhibitor, PZ-128, prevented a drop in monocytes following coronary catheterization, an acute protective effect that was reproduced in mice undergoing cardiac ischemia reperfusion.Conclusions:These data provide evidence for an important role for the MMP1a collagenase in atherosclerotic lesion development and leukocyte behavior and validate MMP1 as a compelling target in patients with coronary artery disease/acute coronary syndrome.Registration:URL: https://www.clinicaltrials.gov; Unique identifier: NCT02561000.
Noncanonical Matrix Metalloprotease 1–Protease-Activated Receptor 1 Signaling Drives Progression of Atherosclerosis
- Rajashree Rana,
- Tianfang Huang,
- Georgios Koukos,
- Elizabeth K. Fletcher,
- Susan E. Turner,
- Andrew Shearer,
- Paul A. Gurbel,
- Jeffrey J. Rade,
- Carey D. Kimmelstiel,
- Kevin P. Bliden,
- Lidija Covic and
- Athan Kuliopulos
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 38, Issue 61 June 2018

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Objective—Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist.Approach and Results—We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E–deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1–PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells.Conclusions—These data suggest that targeting the MMP1–PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.
Vascular Damage in Children With Chronic Kidney Disease: The Fog Is Dispersing
- Luminita Voroneanu and
- Adrian Covic
Hypertension Volume 69, Issue 51 May 2017

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Pulse Wave Velocity Ratio: The New “Gold Standard” for Measuring Arterial Stiffness
- Adrian Covic and
- Dimitrie Siriopol
Hypertension Volume 65, Issue 21 February 2015

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Meta-Analysis of the Effects of Treating Blood Pressure on Cardiovascular Outcomes of Dialysis Patients
- David J.A. Goldsmith
- Adrian C. Covic
Hypertension Volume 54, Issue 11 July 2009

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Role of Klotho in the Development of Essential Hypertension
- Mehmet Kanbay,
- Atalay Demiray,
- Baris Afsar,
- Adrian Covic,
- Laura Tapoi,
- Carina Ureche and
- Alberto Ortiz
Hypertension Volume 77, Issue 33 March 2021

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Klotho has antiaging properties, and serum levels decrease with physiological aging and aging-related diseases, such as hypertension, cardiovascular, and chronic kidney disease. Klotho deficiency in mice results in accelerated aging and cardiovascular injury, whereas Klotho supplementation slows down the progression of aging-related diseases. The pleiotropic functions of Klotho include, but are not limited to, inhibition of insulin/IGF-1 (insulin-like growth factor 1) and WNT (wingless-related integration site) signaling pathways, suppression of oxidative stress and aldosterone secretion, regulation of calcium-phosphate homeostasis, and modulation of autophagy with inhibition of apoptosis, fibrosis, and cell senescence. Accumulating evidence shows an interconnection between Klotho deficiency and hypertension, and Klotho gene polymorphisms are associated with hypertension in humans. In this review, we critically review the current understanding of the role of Klotho in the development of essential hypertension and the most important underlying pathways involved, such as the FGF23 (fibroblast growth factor 23)/Klotho axis, aldosterone, Wnt5a/RhoA, and SIRT1 (Sirtuin1). Based on this critical review, we suggest avenues for further research.
Inferior and Lateral Electrocardiographic Repolarization Abnormalities in Brugada Syndrome
- Andrea Sarkozy,
- Gian-Battista Chierchia,
- Gaetano Paparella,
- Tim Boussy,
- Carlo De Asmundis,
- Marcus Roos,
- Stefan Henkens,
- Leonard Kaufman,
- Ronald Buyl,
- Ramon Brugada,
- Josep Brugada and
- Pedro Brugada
Circulation: Arrhythmia and Electrophysiology Volume 2, Issue 21 April 2009

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Background— Repolarization abnormalities in the inferior-lateral leads in Brugada syndrome (BS) have not been systematically investigated.Methods and Results— 280 patients (age, 41�18 years; 168 males) with BS were screened for inferior-lateral repolarization abnormalities. The repolarization abnormalities were classified either as early repolarization pattern or coved ≥2-mm Brugada pattern and as spontaneous or class I antiarrhythmic drug (AAD) induced. Thirty-two patients (11%) had inferior-lateral spontaneous early repolarization pattern. These patients were less likely to be asymptomatic at first presentation (13 of 32 versus 156 of 248 patients, P=0.02), and spontaneous type I ECG was more frequent among them (38% versus 21%, P=0.05). The spontaneous early repolarization pattern occurred more frequently among patients with BS than in 283 family members not having BS (11% versus 6%, P=0.03). Class I AAD administration provoked inferior-lateral coved Brugada pattern in 13 patients with BS. These patients had longer baseline PR intervals (206�48 versus 172�31 ms, P<0.001) and class I AAD–induced QRS interval prolongation (108 to 178 versus 102 ms to 131 ms, P<0.001). In 3 patients, the class I AAD–provoked coved Brugada pattern was only present in the inferior leads.Conclusions— Inferior-lateral early repolarization pattern occurs spontaneously relatively frequently in BS. These patients have a more severe phenotype. Class I AAD administration provokes inferior-lateral coved Brugada pattern in 4.6% of patients. We report for the first time 3 patients in whom the class I AAD–provoked coved Brugada pattern was only observed in the inferior leads.
Atherosclerotic Renal Artery Stenosis Prevalence and Correlations in Acute Myocardial Infarction Patients Undergoing Primary Percutaneous Coronary Interventions: Data From Nonrandomized Single‐Center Study (REN‐ACS)—A Single Center, Prospective, Observational Study
- Alexandru Burlacu,
- Dimitrie Siriopol,
- Luminita Voroneanu,
- Ionut Nistor,
- Simona Hogas,
- Ana Nicolae,
- Igor Nedelciuc,
- Grigore Tinica and
- Adrian Covic
Journal of the American Heart Association Volume 4, Issue 10

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BackgroundWe are the first to evaluate the prevalence of renal artery stenosis (RAS) in consecutive patients with acute myocardial infarction (AMI) referred for primary percutaneous coronary intervention from a single tertiary center. As a novelty, we assessed hydration and metabolic status and measured arterial stiffness. We elaborated a predicting model for RAS in AMI.Methods and ResultsOne hundred and eighty‐one patients with AMI underwent concomitantly primary percutaneous coronary intervention and renal angiography. We obtained data on demographics, medical history, cardiovascular risk factors, echocardiography, Killip class, and blood tests. In the first 24 hours post–primary percutaneous coronary intervention, we assessed bioimpedance through Body Composition Monitoring^® and arterial stiffness through pulsed‐wave velocity, SphygmoCor^®. Significant RAS (>50% lumen narrowing, RAS+) was present in 16.6% patients. In the RAS+ group we recorded significantly higher stiffness, CRUSADE score and dehydration, and more women with higher prevalence of multivascular coronary artery disease and heart failure. In our multivariate models, variables independently associated with RAS+ were previous percutaneous coronary intervention, low estimated glomerular filtration rate, multivascular coronary artery disease, and total/extracellular body water. These models had good specificity and low sensitivity.ConclusionsWe observed that RAS+ AMI patients have a particular hydration, metabolic, and endothelial profile that could generate more future major adverse cardiac events. Hence, renal angiography in AMI should be considered in specific subsets of patients.Clinical Trial RegistrationURL: https://www.clinicaltrials.gov/. Unique identifier: NCT02388139.

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