Vinod K. Shah

Polymorphisms of MDR1, CYP2C19 and P2Y12 genes in Indian population: effects on clopidogrel response.

AIMS/OBJECTIVE Influence of genetic variations on the response of clopidogrel, an antiplatelet drug is implicated. In the present study, the prevalence of single nucleotide polymorphisms of MDR1 (C3435T), CYP2C19 [CYP2C19*2 CYP2C19*3, CYP2C19*17] and P2Y12 (i-T744C) in Indian population and their effects on clopidogrel response was analyzed. METHODS AND RESULTS To analyze the prevalence of polymorphisms, 102 healthy individuals were recruited. Clopidogrel response was assessed by ADP induced platelet aggregation in clopidogrel naïve acute myocardial infarction (AMI) patients (n = 26) screened from 100 AMI cases, before loading dose of 300 mg, at 24 h before next dose and 6 days after on 75 mg per day and platelet aggregation inhibition (PAI) was calculated between these time intervals. Genotyping was carried out by PCR-based restriction enzyme digestion method for C3435T of MDR1 and i-T744C of P2Y12, by multiplex PCR for CYP2C19*2 (G681A) and CYP2C19*3 (G636A) and by nested PCR for CYP2C19*17 (C806T). The effect of the above mentioned genetic variations on PAI was analyzed. Variant allele of CYP2C19*3 was not observed while the prevalence of 3435T of MDR1 (0.524), CYP2C19*2 (681A, 0.352); i-744C of P2Y12 (0.088), as well as wild type allele CYP2C19*17 (C806, 0.897) associated with decrease clopidogrel response were observed. Trend toward poor response to clopidogrel was observed at 24 h with the variant genotypes of CYP2C19*2 and i-T744C of P2Y12 as compared to wild type. CONCLUSION The present study did show a trend toward impaired response of clopidogrel to inhibit platelet aggregation with variant genotypes of CYP2C19*2 and iT744C of P2Y12 compared to respective wild type genotype at 24 h.

Matrix metalloproteinase-3 (MMP-3) -1612 5A/6A promoter polymorphism in coronary artery disease in Indian population.

Matrix metalloproteinases (MMPs) play important role in the pathogenesis of coronary artery disease (CAD). 5A allele of -1612 5A/6A polymorphism of MMP-3 is associated with two fold higher activity than 6A allele. Present study was designed to analyse the association of this polymorphism with CAD in Indian population. Subjects included in the study were patients with stable angina (n=35), unstable angina (n=53), patients with recent event of myocardial infarction (MI) (MI Group-1, n=56) and patients at presentation of the acute MI (MI Group-2, n=49). Controls were healthy individuals (n=99). Genotyping of MMP-3 5A/6A polymorphism was carried out by PCR-based restriction digestion method. The genotype distribution of patient groups did not deviate from controls. Serum MMP-3 levels were significantly elevated at presentation of the acute MI by 36.8% (P=0.031) as compared to controls and more associated with 6A genotype suggesting discrepancy between in vitro transfection experiment and peripheral MMP-3 levels.

Levels of cathepsins in acute myocardial infarction.

AIMS/OBJECTIVE Over expression of matrix degrading enzymes have been implicated in plaque destabilisation and rupture. Cathepsins associated with extracellular matrix breakdown make them intriguing suspects. The aim of the study was to analyse peripheral levels of cathepsin B and cathepsin K and their inhibitor cystatin C during acute myocardial infarction (AMI). MATERIALS AND METHODS Study population included AMI patients at acute event (AMI group, n=48), stable angina patients (stable angina group n = 17), and healthy individuals (Control group, n=31). Cathepsin B, cathepsin K, cystatin C, and matrix metalloproteinases (MMP)-9 were analysed by enzyme-linked immunosorbent assay (ELISA) method. RESULTS Cathepsin B (45.9%) and cathepsin K (92.31%) at acute event of myocardial infarction (AMI group) increased (P=0.001) while cystatin C decreased marginally (12.5%) as compared to controls. Stable angina group, demonstrated only marginal reduction in all the parameters studied as compared to controls. CONCLUSION Cathepsin B and cathepsin K can be further evaluated as biomarkers in identifying high-risk individuals for AMI.

Study of C-Reactive Protein and Myocardial Infarction in the Indian Population

To analyse the association of high sensitivity C-reactive (hsCRP) protein levels and −717A/G single nucleotide polymorphism of CRP with acute myocardial infarction (AMI) in the Indian population. Study population included 100 MI cases wherein 32 patients had experienced previous MI (MI-Group-1), 68 MI cases were recruited at presentation (MI-Group-2) and equal number of age and gender matched healthy individuals. hsCRP levels were determined by ELISA and genotyping of −717A/G was carried out by polymerase chain reaction-based restriction digestion method. The −717A/G genotypes did not influence hsCRP level and their distribution did not differ between groups. However, in the present study hsCRP demonstrated significant correlation with BMI in controls of both the genders and with triglycerides in females of AMI at presentation who otherwise are with low risk profile. Identifying traditional risk factors associated with inflammation may help in controlling the acute event.

Circulating thrombotic and haemostatic components in patients with coronary artery disease

The study aimed to analyze the circulating levels of thrombotic and haemostatic components; tissue factor, tissue factor pathway inhibitor, tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with acute myocardial infarction at presentation (Group 1, n=49), unstable angina and Non-ST elevated MI after treatment (Group 2, n=22), stable angina (Group 3, n=18) and healthy individuals (Group 4, n=31). Significant finding was increase in tissue factor not only in Group 1 (2.0 fold, P=0.001), Group 2 (2.2 fold, P=0.015) but also in Group 3 (1.8 fold, P=0.018) as compared to controls. In Group 1 Plasminogen activator inhibitor-1 increased significantly (35.8%, P=0.02). Tissue factor pathway inhibitor and tissue plasminogen activator demonstrated increase in Group 1 of age<40 years while insignificant changes in elder patients. Increased thrombotic and decreased fibrinolytic conditions in acute myocardial infarction patients were observed. Increase TF in stable angina demonstrates procoagulant status in these patients as well.

Stem Cell Therapy for Acute Myocardial Infarction-Long Term 24 Months Follow-Up

Background: Intra-coronary injection of Autologous Bone Marrow-Stem Cells (ABMSC) has been demonstrated to improve Left Ventricular (LV) function after acute ST Elevation Myocardial Infarction (STEMI). After analyzing the safety and feasibility of this treatment, long term durability of the treatment effect is needed to be analyzed. The aim of the present study was to analyze the ventricular remodeling by LV function and clinical effects of patients undergone ABMSCs therapy following Acute Myocardial Infarction (AMI) at 24 months. Material and Methods: Twelve AMI patients with ABMSC therapy (ABMSC group) and seven AMI patients without this therapy (Controls) were followed up for 24 months. LV functions were analyzed by 2D Echocardiography (2D ECHO) while the long term effects of bone marrow infusion on any organ were assessed by clinical evaluation, pathological tests, ECG, Holter monitoring, chest X-ray and Sonography of abdomen and pelvic organs. Results: Long term follow-up of 24 months of AMI patients with ABMSC therapy demonstrated no adverse clinical effects on cardiac or extra cardiac organs. 2D ECHO of ABMSC group demonstrated increase in LVEF from baseline at 6 month (3.8%) which was sustained at 24 month (1.63% increase) with improvement in LV functions. However, in the control group there was marginal increase in LVEF (1.5%) at sixth month which was decreased by 7.3% at 24 month as compared to baseline, with no change in LV functions. Conclusion: This study demonstrated safety and feasibility of infusing ABMSCs in the culprit coronary artery. It also demonstrated better clinical course in stem cell therapy group as compared to patients without this therapy at 24 months follow-up.

Low HDL Cholesterol and ABCA1 in Healthy Individuals

Peripheral cells obtain their cholesterol from combination of synthesis and uptake from low density lipoproteins (LDL). Most peripheral cells are not able to catabolize cholesterol but transport excess cholesterol from peripheral tissues back to liver, the process called “reverse cholesterol transport” (RCT). This is the only way by which cholesterol homeostasis is maintained in vivo The first step of RCT is uptake of cellular cholesterol by the high density lipoprotein (HDL). A key regulator of this uptake is ATP-Binding Cassette transporter A1 (ABCA1) [1,2]. It mediates the cellular efflux of phospholipid and cholesterol across cell membranes to lipid-poor Apolipoprotein A1 (ApoA1), the major Apo in HDL. Thus it plays a significant role in HDL metabolism and lipid clearance thereby decreasing the risk of atherosclerosis [3,4]. ABCA1 mRNA is expressed in leukocytes and macrophages, besides placenta, liver lung and adrenal gland [5].

Identification of a single nucleotide polymorphism indicative of high risk in acute myocardial infarction

Background & objectives: Acute myocardial infarction (AMI) is a major health concern in India. The aim of the study was to identify single nucleotide polymorphisms (SNPs) associated with AMI in patients using dedicated chip and validating the identified SNPs on custom-designed chips using high-throughput microarray analysis. Methods: In pilot phase, 48 AMI patients and 48 healthy controls were screened for SNPs using human CVD55K BeadChip with 48,472 SNP probes on Illumina high-throughput microarray platform. The identified SNPs were validated by genotyping additional 160 patients and 179 controls using custom-made Illumina VeraCode GoldenGate Genotyping Assay. Analysis was carried out using PLINK software. Results: From the pilot phase, 98 SNPs present on 94 genes were identified with increased risk of AMI (odds ratio of 1.84-8.85, P=0.04861-0.003337). Five of these SNPs demonstrated association with AMI in the validation phase (P<0.05). Among these, one SNP rs9978223 on interferon gamma receptor 2 [IFNGR2, interferon (IFN)-gamma transducer 1] gene showed a significant association (P=0.00021) with AMI below Bonferroni corrected P value (P=0.00061). IFNGR2 is the second subunit of the receptor for IFN-gamma, an important cytokine in inflammatory reactions. Interpretation & conclusions: The study identified an SNP rs9978223 on IFNGR2 gene, associated with increased risk in AMI patient from India.

Peripheral Blood Mononuclear Cell ABCA1 Transcripts and Protein Expression in Acute Myocardial Infarction

ATP binding cassette transporter‐A1 (ABCA1) facilitates the formation of high density lipoprotein (HDL). HDL due to its anti‐atherosclerotic, anti‐inflammatory and anti‐thrombotic activities provides protection against atherothrombosis or myocardial infarction (MI). The aim was to investigate the role of peripheral blood mononuclear cell (PBMNC) ABCA1 expression in MI.

Single nucleotide polymorphisms associated with myocardial infarction in patients from Western India: A genome wide association study

incidence of cardiovascular complications and death in patients with ACS and increased severity of coronary artery lesion. Among these biomarkers some have emerged to be potentially useful in early risk assessment of patients with ACS, such as CRP, B type natriuretic peptide, troponin T/I, interleukin 6, and white blood cell (WBC) count and among these WBC count is the simplest. Although total leukocyte count is an established prognostic marker of ACS, there are limited studies for differential neutrophil count and prognosis in ACS. Methods: 202 consecutive patients admitted with a diagnosis of ACS were evaluated by history and physical examination. Venous blood was drawn from all the patients at the time of admission and was analysed for CK-MB, Troponin T and Total leukocyte count and differential leukocyte count. The neutrophil counts were assigned into three categories, N1 (<70%), N2 (70-90%) andN3 ( 90%): Coronary angiography was carried out within 24 hours of admission. Results: The Neutrophil count ranged from 45% to 98%. The median neutrophil count was 78%; Smokers had a higher neutrophil count than non-smokers. Neutrophil count also tended to be higher in patients who died within 7 days (P<0.001). The development of new CHF or shock was associated with a higher Neutrophil count (P<0.001) Patients with a closed infarct-related artery on angiography (TIMI grade 0 or 1 flow) had a higher neutrophil count than did patients with an open artery (P<0.001). The presence of angiographically apparent thrombus was associated with a higher Neutrophil count than those without thrombus. (82%, n1⁄428 versus 68%, n1⁄472) (p<.01) Conclusion: The results of the present study confirm previous observations that relate elevated WBC count to adverse clinical outcomes in patients with ACS and further explore the pathophysiology that underlies this relationship. In addition to the worse clinical outcome, reduced patency and greater thrombus burden seen in patients with an elevated Neutrophil count, these patients had poorer downstream microvascular perfusion as assessed with TIMI perfusion grade. It is possible that this impaired myocardial perfusion reflects neutrophilemediated endothelial dysfunction andmicrovascular plugging, as described in animal models of ischemia-reperfusion.