Genotyping, Platelet Activation, and Cardiovascular Outcome in Patients after Percutaneous Coronary Intervention: Two Pieces of the Puzzle of Clopidogrel Resistance.

OBJECTIVES: Individual platelet responses to antiplatelet therapy depend on genetic, cellular, and clinical factors. CYP2C19 and P2Y12 receptor polymorphisms are implicated in platelet responses to antiplatelet treatment. We aimed to evaluate the impact of CYP2C19 and C34T P2Y12 genotyping on platelet reactivity and cardiovascular outcome in patients after percutaneous coronary intervention (PCI) on clopidogrel treatment. METHODS: We enrolled 408 patients with stable coronary artery disease (CAD) receiving aspirin and clopidogrel (75 mg/day) 1 month after PCI. High on-treatment platelet reactivity was evaluated using the VerifyNow Assay in a subset of patients. CYP2C19*2 and C34T P2Y12 genotyping was performed by real-time polymerase chain reaction. The primary end point was the composite of death or hospitalization for cardiovascular causes, and patients were followed for a median time of 13 months. RESULTS: In the total study population, 37% were carriers of at least 1 CYP2C19*2 loss-of-function allele, and 53% were carriers of at least 1 C34T loss-of-function allele. Interestingly, homozygotes of the CYP2C19*2 loss-of-function allele had significantly increased P2Y12 reaction units (PRU) (p = 0.007). However, PRU did not differ between carriers and noncarriers of the C34T loss-of-function allele (p = 0.41). Moreover, carriers of CYP2C19*2 had an increased hazard ratio (HR) for the occurrence of the primary end point (for carriers HR = 1.96, 95% CI 1.05-3.66, p = 0.03), whereas the C34T polymorphism had no impact on the cardiovascular outcome (p = 0.17). Finally, PRU was associated with cardiovascular outcome even after adjustment for the presence of any reduced function allele polymorphism. CONCLUSIONS: We documented a different effect of CYP2C19 and P2Y12 receptor polymorphisms on platelet reactivity and cardiovascular outcome in CAD patients after PCI on clopidogrel treatment. Importantly, increased platelet reactivity adversely affects the cardiovascular outcome independently of the studied polymorphisms.

Effects of CYP2C19 Polymorphism on Endothelial Function, Arterial Stiffness and Inflammation in Coronary Artery Disease Patients Under Clopidogrel Treatment.

BACKGROUND: Clopidogrel's ability to inhibit platelet function determined its clinical usefulness. The role of CYP2C19*2 genotype on antiplatelet treatment is recently under question. Arterial wall properties and inflammation are key players in atherosclerosis development. Hence, we evaluated the impact of CYP2C19*2 genetic polymorphism on endothelial function, arterial stiffness and inflammation in coronary artery disease (CAD) patients receiving clopidogrel treatment. METHODS AND RESULTS: In this study we enrolled 408 consecutive patients with stable CAD under dual antiplatelet therapy (clopidogrel 75mg/day, aspirin 100mg/day), 30 days after percutaneous coronary intervention. Measurement of flow-mediated dilation (FMD) of the brachial artery was used to evaluate endothelial function. Carotid-femoral pulse wave velocity (PWV) and augmentation index (AIx) was measured to estimate arterial stiffness. Real time polymerase chain reaction was used for the genotyping of CYP2C19*2. Levels of tumor necrosis factor alpha (TNF-a) and interleukin 6 (IL-6) were measured with ELISA. We found no difference in basic clinical and demographic characteristics nor in FMD, PWV, AIx and inflammatory status (p=NS for all) between CYP2C19 homozygotes for the wild type; carriers of reduced function allele and homozygotes for the reduced function allele. CONCLUSION: CYP2C19*2 loss of action polymorphism causes no impact on vascular function and inflammatory status in stable CAD patients receiving clopidogrel treatment.

Clopidogrel response variability is associated with endothelial dysfunction in coronary artery disease patients receiving dual antiplatelet therapy.

OBJECTIVES: Dual antiplatelet therapy with aspirin and a platelet P2Y12 ADP receptor antagonist is the cornerstone of treatment following percutaneous coronary intervention (PCI). Several clinical and genetic factors can cause suboptimal clopidogrel response. We examined the impact of endothelial dysfunction on clopidogrel response variability in subjects with stable coronary artery disease (CAD) after PCI. METHODS: We consecutively enrolled 198 patients with stable CAD one month after successful PCI. All patients were receiving dual antiplatelet therapy (clopidogrel 75 mg and aspirin 100 mg/day). Platelet reactivity was measured by VerifyNow P2Y12 assay (Accumetrics, San Diego, CA). VerifyNow reports its results in P2Y12 reaction units (PRU) and the diagnostic cut-off value is 230. Endothelial function was evaluated by flow mediated dilation (FMD). RESULTS: Patients with high on treatment platelet reactivity (32% of the study population), compared to subjects with low on treatment platelet reactivity, presented decreased FMD values (4.35 ± 2.22% vs. 5.74 ± 3.29%, p = 0.01). Moreover, an inverse association between endothelial function measurement and platelet reactivity (r = -0.24, p = 0.001) was found. Importantly, multivariate analysis after adjustment for age, gender and confounders revealed by the univariate analysis (left ventricle ejection fraction, body mass index, diabetes, dyslipidemia, coronary lesion number) showed that for every decrease in FMD by 1% there is an anticipated increased in the odds of patients to have HPR by 1.66 (95% CI 1.03-2.57, p = 0.037). CONCLUSIONS: Endothelial dysfunction is associated with clopidogrel response variability in patients after PCI receiving dual antiplatelet therapy. These findings shed some light on the mechanisms affecting individual platelet response to antiplatelet therapy and may explain the non-straight forward association between clopidogrel dose, platelet inhibition and cardiovascular outcome.

High platelet reactivity is associated with vascular function in patients after percutaneous coronary intervention receiving clopidogrel.

BACKGROUND: In the present study, we evaluated the association of platelet reactivity with vascular function in patients after percutaneous coronary intervention receiving clopidogrel treatment. METHODS: We enrolled 150 patients with stable CAD receiving clopidogrel regimen (75 mg/d), 1 month after percutaneous coronary intervention. Carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness and augmentation index (AIx) as an index of arterial wave reflections. High on treatment platelet reactivity (HPR) was evaluated using VerifyNow Assay. VerifyNow reports its results in P2Y12 reaction units (PRU), and the diagnostic cutoff value is 230 PRU. Patients were evaluated prospectively up to 24 months. The primary end point was a composite of death from cardiovascular causes, nonfatal major cardiovascular events and hospitalization for cardiovascular causes. RESULTS: There was no difference in the basic clinical and demographic characteristics between subjects with HPR and non-HPR. Subjects with high on treatment platelet reactivity and PRU>230 had significantly increased PWV (8.81 ± 2.25 m/s vs. 7.69 ± 1.95 m/s, p = 0.001) and AIx (25.27 ± 8.67% vs. 20.87 ± 10.57%, p = 0.04) compared to subjects with PRU≤230. PWV was also associated with PRU (r = 0.23, p = 0.02). HPR was associated with significantly increased risk of primary end point [HR = 5.38, 95%CI:(1.15, 26.04), p = 0.03]. CONCLUSIONS: Increased platelet reactivity is associated with impaired arterial stiffness in patients after percutaneous coronary intervention receiving clopidogrel treatment, highlighting another clinical factor implicated in individual platelet response to antiplatelet therapy. Moreover, increased platelet reactivity is associated with adverse outcome in these patients.

Vitamin D3, D2 and arterial wall properties in coronary artery disease.

OBJECTIVES: There are two major forms of vitamin D, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). We studied the effect of the different vitamin D fractions (D3/D2) on arterial wall properties in coronary artery disease (CAD) patients. METHODS: We included 252 subjects with CAD. Endothelial function was evaluated by flow mediated dilation (FMD). Carotid femoral pulse wave velocity (PWV) was measured as an index of arterial stiffness and augmentation index (AI) as a measure of reflected waves. Measures for 25(OH)D2 and 25(OH)D3 were performed using Liquid Chromatography Mass Spectrometry technology. RESULTS: From the study population, 155(62%), 66(26%) and 31(12%) were categorized as having vitamin D deficiency, insufficiency and sufficiency respectively. There was no difference between subjects with vitamin D deficiency, insufficiency and sufficiency in FMD, AI and PWV (p=NS for all). Subjects with vitamin D insufficiency/deficiency had significantly higher D2 to D ratio compared to subjects with vitamin D sufficiency. Interestingly, FMD was positively associated with D2 to D ratio (rho=0.13, p=0.02) and subjects with D2 levels<0.3ng/ml had impaired FMD compared to those with increased D2 levels (p=0.048). CONCLUSION: Vitamin D insufficiency/deficiency is highly prevalent in CAD subjects. Vitamin D2 concentrations are positively associated with endothelial function. These findings may suggest a beneficial role of vitamin D2 levels in vascular health.

Serum osteoprotegerin and osteopontin levels are associated with arterial stiffness and the presence and severity of coronary artery disease.

BACKGROUND: Osteopontin (OPN) and osteoprotegerin (OPG) have recently emerged as key factors in both vascular remodeling and development of atherosclerosis. Arterial stiffness has an independent predictive value for cardiovascular events. We evaluate the relationship between OPG, OPN serum levels and vascular function in coronary artery disease (CAD) patients. METHODS: The study population was consisted of 409 subjects (280 with CAD and 129 without CAD). Carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness. OPG and OPN levels were measured, as markers of vascular remodeling and calcification, by ELISA. Gensini score was used to evaluate the extent of CAD. RESULTS: CAD patients, compared to those without CAD, had higher OPG (3.91 ± 1.87 pmol/l vs. 2.88 ± 1.32 pmol/l, p<0.001) and logOPN levels (1.81 ± 0.18 ng/ml vs. 1.71 ± 0.24 ng/ml, p<0.001) and impaired PWV (8.94 ± 2.21 m/s vs. 8.28 ± 1.91 m/s, p=0.006). Furthermore, PWV was associated with serum OPG levels (r=0.19, p<0.001) and with serum logOPN levels (r=0.10, p=0.049). Multivariate linear regression analysis revealed that increased OPG (p=0.013) and logOPN (p=0.006) levels are associated with 3-vessel CAD and Gensini score (p=0.04 for OPG and p=0.09 for OPN), independently of other known cardiovascular risk factors. CONCLUSION: The present study revealed that serum OPG and OPN levels are positively associated with arterial stiffness, and with the extent of CAD. These preliminary results suggest that OPG and OPN levels are significantly correlated with vascular function contributing to the pathogenesis of atherosclerosis in CAD. Further studies are needed to explore the mechanisms of action of OPG and OPN in CAD.

Effects of Ω-3 fatty acids on endothelial function, arterial wall properties, inflammatory and fibrinolytic status in smokers: a cross over study.

BACKGROUND: Smoking is associated with endothelial dysfunction and arterial stiffness. Supplementation of Ω-3 PUFAs is associated with better prognosis. Aim of this study was to evaluate the effects of Ω-3 polyunsaturated fatty acids (PUFAs) supplementation on smoking-induced impairment of arterial function. METHODS: We studied the effect of a 12 weeks oral treatment with 2gr/day of Ω-3 PUFAs in 20 healthy smokers on three occasions (day 0:baseline, day 28 and day 84). The study was carried out on two separate arms (Ω-3 fatty acids and placebo), according to a randomized, placebo-controlled, double-blind, cross-over design. Measurements were carried out before (pSm), immediately and 20min after cigarette smoking. Endothelial function was evaluated by flow-mediated dilation (FMD) of the brachial artery. Carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness and augmentation index (AIx) as a measure of arterial wave reflections. Circulating levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) were measured. RESULTS: Compared with placebo, Ω-3 PUFAs treatment resulted in a significant improvement in pSm values of FMD (p<0.05), AIx (p<0.001) and PWV (p<0.01). Although, acute cigarette smoking decreased FMD and caused an increase in AIx and PWV, Ω-3 PUFAs treatment blunted the acute smoking-induced impairment of FMD (p<0.001), AIx (p<0.05) and PWV (p<0.05) and significantly decreased levels of TNFα (p<0.05) and IL-6 (p=0.01) and increased levels of PAI-1 (p=0.05). CONCLUSIONS: Ω-3 PUFAs improved endothelial function and the elastic properties of the arterial tree in healthy smokers, with a parallel anti-inflammatory effect.

Inflammatory mechanisms in atherosclerosis: the impact of matrix metalloproteinases.

Inflammatory process is essential for the initiation and progression of vascular remodeling, entailing degradation and reorganization of the extra-cellular matrix (ECM) scaffold of the vessel wall, leading to the development of atherosclerotic lesions. Matrix metalloproteinases (MMPs) are zing dependent endo-peptidases found in most living organisms and act mainly by degrading ECM components. Most MMPs are formed as inactive proenzymes and are activated by proteolysis. This process depends and is regulated by other proteases and endogenous MMP inhibitors (TIMPs). MMPs and TIMPs play a major role not only in ECM degradation but also in mediating cell migration, proliferation, tissue remodeling; acting as a signal for the production and secretion of growth factors and cytokines. More importantly MMPs through proteolysis and degradation of ECM contribute in many physiological and pathological processes including organ development, wound healing, tissue support, vascular remodeling and restenosis, atherosclerosis progression, acute coronary syndromes, myocardial infarction, cardiomyopathy, aneurysms remodeling, cancer, arthritis, and chronic inflammatory diseases. A substantial body of evidence support the notion that imbalance between the activity of MMPs and their tissue inhibitors (TIMPs) contribute to the pathogenesis of cardiovascular diseases such as atherosclerosis, vascular remodeling and progression of heart failure. In this review, we will discuss the relationship between MMPs, inflammation and atherosclerosis under the topic of cardiovascular disease.