Marek Kiliszek

Altered Gene Expression Pattern in Peripheral Blood Mononuclear Cells in Patients with Acute Myocardial Infarction

Background Despite a substantial progress in diagnosis and therapy, acute myocardial infarction (MI) is a major cause of mortality in the general population. A novel insight into the pathophysiology of myocardial infarction obtained by studying gene expression should help to discover novel biomarkers of MI and to suggest novel strategies of therapy. The aim of our study was to establish gene expression patterns in leukocytes from acute myocardial infarction patients. Methods and Results Twenty-eight patients with ST-segment elevation myocardial infarction (STEMI) were included. The blood was collected on the 1st day of myocardial infarction, after 4–6 days, and after 6 months. Control group comprised 14 patients with stable coronary artery disease, without history of myocardial infarction. Gene expression analysis was performed with Affymetrix Human Gene 1.0 ST microarrays and GCS3000 TG system. Lists of genes showing altered expression levels (fold change >1.5, p<0.05) were submitted to Ingenuity Pathway Analysis. Gene lists from each group were examined for canonical pathways and molecular and cellular functions. Comparing acute phase of MI with the same patients after 6 months (stable phase) and with control group we found 24 genes with changed expression. In canonical analysis three pathways were highlighted: signaling of PPAR (peroxisome proliferator-activated receptor), IL-10 and IL-6 (interleukin 10 and 6). Conclusions In the acute phase of STEMI, dozens of genes from several pathways linked with lipid/glucose metabolism, platelet function and atherosclerotic plaque stability show altered expression. Up-regulation of SOCS3 and FAM20 genes in the first days of myocardial infarction is observed in the vast majority of patients.

Association between Variants on Chromosome 4q25, 16q22 and 1q21 and Atrial Fibrillation in the Polish Population

Background Genome-wide studies have shown that polymorphisms on chromosome 4q25, 16q22 and 1q21 correlate with atrial fibrillation (AF). However, the distribution of these polymorphisms differs significantly among populations. Objective To test the polymorphisms on chromosome 4q25, 16q22 and 1q21 in a group of patients (pts) that underwent catheter ablation of AF. Methods Four hundred and ten patients with AF that underwent pulmonary vein isolation were included in the study. Control group (n = 550) was taken from healthy population, matched for age, sex and presence of hypertension. All participants were genotyped for the presence of the rs2200733, rs10033464, rs17570669, rs3853445, rs6838973 (4q25), rs7193343 (16q22) and rs13376333 (1q21) polymorphisms. Results All the polymorphisms tested (except rs17570669) correlated significantly with AF in univariate analysis (p values between 0.039 for rs7193343 and 2.7e-27 for rs2200733), with the odds ratio (OR) 0.572 and 0.617 for rs3853445 and rs6838973, respectively (protective role) and OR 1.268 to 3.52 for the other polymorphisms. All 4q25 SNPs tested but rs3853445 were independently linked with AF in multivariate logistic regression analysis. In haplotype analysis six out of nine 4q25 haplotypes were significantly linked with AF. The T allele of rs2200733 favoured increased number of episodes of AF per month (p = 0.045) and larger pulmonary vein diameter (recessive model, p = 0.032). Conclusions Patients qualified for catheter ablation of AF have a significantly higher frequency of 4q25, 16q22 and 1q21 variants than the control group. The T allele of rs2200733 favours larger pulmonary veins and increased number of episodes of AF.

Gene expression profiling reveals potential prognostic biomarkers associated with the progression of heart failure

BackgroundHeart failure (HF) is the most common cause of morbidity and mortality in developed countries. Here, we identify biologically relevant transcripts that are significantly altered in the early phase of myocardial infarction and are associated with the development of post-myocardial infarction HF.MethodsWe collected peripheral blood samples from patients with ST-segment elevation myocardial infarction (STEMI): n = 111 and n = 41 patients from the study and validation groups, respectively. Control groups comprised patients with a stable coronary artery disease and without a history of myocardial infarction. Based on plasma NT-proBNP level and left ventricular ejection fraction parameters the STEMI patients were divided into HF and non-HF groups. Microarrays were used to analyze mRNA levels in peripheral blood mononuclear cells (PBMCs) isolated from the study group at four time points and control group. Microarray results were validated by RT-qPCR using whole blood RNA from the validation group.ResultsSamples from the first three time points (admission, discharge, and 1 month after AMI) were compared with the samples from the same patients collected 6 months after AMI (stable phase) and with the control group. The greatest differences in transcriptional profiles were observed on admission and they gradually stabilized during the follow-up. We have also identified a set of genes the expression of which on the first day of STEMI differed significantly between patients who developed HF after 6 months of observation and those who did not. RNASE1, FMN1, and JDP2 were selected for further analysis and their early up-regulation was confirmed in HF patients from both the study and validation groups. Significant correlations were found between expression levels of these biomarkers and clinical parameters. The receiver operating characteristic (ROC) curves indicated a good prognostic value of the genes chosen.ConclusionsThis study demonstrates an altered gene expression profile in PBMCs during acute myocardial infarction and through the follow-up. The identified gene expression changes at the early phase of STEMI that differentiated the patients who developed HF from those who did not could serve as a convenient tool contributing to the prognosis of heart failure.

Investigating the effects of statins on cellular lipid metabolism using a yeast expression system.

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.

Does a blanking period after pulmonary vein isolation impact long-term results? Results after 55 months of follow-up.

BACKGROUND The aims of the study are 1) to assess antiarrhythmic prophylaxis efficacy during the first 2 months after radiofrequency ablation (ARF) due to AF; 2) to define risk factors for early AF recurrence (EAFR) after ARF; 3) to determine the long-term follow-up results and risk factors for late AF recurrence (LAFR). METHODS A total number of 210 consecutive patients who had undergone ARF due to AF were analyzed. Patients were randomized into three groups: Group 1 (G1), without any anti-arrhythmic drug (AAD); Group 2 (G2), with amiodarone or sotalol; Group 3 (G3), with last ineffective AAD. The study was designed to analyze two periods: short-term observation, the first 2 months after ARF; and at least 2 years of long-term follow-up. RESULTS After 2 months, clinical data were collected from 171 patients (123 males, mean age of 50.3 years; persistent AF in 19.8%; lone AF in 36.6%). Sinus rhythm (SR) was maintained in 84 (49.1%) patients; 35 (20.4%) patients presented with a single episode of AF, 39 (23%) patients experienced a reduction in number of AF episodes, and 13 (7.5%) patients showed no improvement. No predisposing factor for early recurrence was found. After a mean follow-up of 55 months, clinical data were collected in 137 patients, of which 47 (34%) maintained SR. Those more likely to sustain SR were: males (82.9% vs. 62.2%; p = 0.018), younger patients (44.8 ± 12.7 vs. 52.5 ± 9.9; p = 0.0001), patients with smaller left atrium diameter (4.05 ± ± 0.49 cm vs. 4.25 ± 0.51 cm; p = 0.04), and those without any AF recurrence during the first 2 months after ARF (78.7% vs. 35.6%; p < 0.0001). In the multivariable analysis, the independent risk factors for LAFR were hypertension (p < 0.001) and persistent AF (p = 0.014). CONCLUSIONS Antiarrhythmic prophylaxis does not affect the number of AF recurrences during the first 2 months after ablation. SR maintenance during a blanking period after AF ablation is a positive prognostic factor in long-term follow-up. Persistent AF and hypertension are independent risk factors for late AF recurrence after pulmonary vein isolation.

Low-density lipoprotein reduction by simvastatin is accompanied by angiotensin II type 1 receptor downregulation, reduced oxidative stress, and improved endothelial function in patients with stable coronary artery disease.

ObjectivesWe tested the hypothesis that low-density lipoprotein-cholesterol induces angiotensin II type 1 receptor upregulation that, in turn, accounts for enhanced oxidative stress, and the subsequent endothelial dysfunction in patients with coronary artery disease. MethodsBrachial artery flow-mediated vasodilation, serum 8-iso-prostaglandin F2&agr; (8-isoprostane), and angiotensin II type 1 receptor density on platelets were measured in 19 patients with coronary artery disease, at entry and after 12 weeks of simvastatin therapy, 40 mg/day. ResultsAt entry there was a significant linear correlation between: angiotensin II type 1 receptor density and plasma low-density lipoprotein-cholesterol; plasma 8-isoprostane and angiotensin II type 1 receptor density; and flow-mediated vasodilation and 8-isoprostane. Simvastatin therapy reduced low-density lipoprotein-cholesterol, downregulated angiotensin II type 1 receptor, decreased 8-isoprostane, and improved flow-mediated vasodilation. The slopes of the presimvastatin and the postsimvastatin angiotensin II type 1 receptor/low-density lipoprotein relationships did not significantly differ, indicating that simvastatin caused a downregulation of angiotensin II type 1 receptor that could be predicted by the low-density lipoprotein reduction. In addition, simvastatin-mediated changes in 8-isoprostane could be predicted by angiotensin II type 1 receptor downregulation, and flow-mediated vasodilation improvement by changes in 8-isoprostane. A significant correlation existed between simvastatin-mediated changes in 8-isoprostane and angiotensin II type 1 receptor. ConclusionThe results of this study are consistent with the hypothesis that in coronary artery disease, the impairment of endothelial function is strongly associated with oxidative stress, oxidative stress with cellular angiotensin II type 1 receptor density, and the angiotensin II type 1 receptor density with low-density lipoprotein-cholesterol, suggesting cause–effect relationships between these variables. In support for this notion, these baseline associations were not significantly disturbed by low-density lipoprotein-lowering therapy with simvastatin.

A1166C polymorphism of the angiotensin AT1 receptor (AT1R) gene alters endothelial response to statin treatment.

Abstract Background: The function of vascular endothelium is influenced by several factors: low-density lipoprotein (LDL) cholesterol, oxidative stress and the reninangiotensin system. Methods: We tested the hypothesis that polymorphisms A1166C of the angiotensin AT1 receptor (AT1R) gene, C242T and A640G of the pphox22 gene (p22 phox is an essential component of NADH/NADPH oxidases) and G894T of the endothelial nitric oxide (NO) synthase (eNOS) gene influence endothelial function and its reaction to statin treatment. In 44 patients with coronary artery disease or hypercholesterolemia (not on lipid-lowering treatment), lipid profile and endothelial function (brachial artery flow-mediated dilation, FMD) were measured at baseline and after treatment with statins for 8–12 weeks. All subjects were genotyped for the above-mentioned polymorphisms. Results: None of the polymorphisms significantly predicted baseline FMD. Patients with the C allele of A1166C showed smaller changes in FMD in comparison with patients with the AA genotype (−0.044±0.439% vs. 0.386±0.599%; p=0.016). None of the other polymorphisms significantly influenced changes in FMD. Conclusions: The C allele of AT1R A1166C is associated with significantly lower endothelial response to statin treatment. Clin Chem Lab Med 2007;45:839–42.

The rs12526453 Polymorphism in an Intron of the PHACTR1 Gene and Its Association with 5-Year Mortality of Patients with Myocardial Infarction

Objective The rs12526453 (C/G) is a single nucleotide polymorphism in an intron of the PHACTR1 gene (phosphatase and actin regulator 1). The C allele is associated with increased risk of coronary artery disease in an unknown mechanism. We investigated its association with long-term overall mortality in patients with ST-elevation myocardial infarction (STEMI) treated invasively. Methods Two independent groups of patients with STEMI were analyzed: a derivation group (n= 638) and a validation one (n=348). Genotyping was performed with the TaqMan method. The analyzed end-point was total long term mortality. Additionally, transcriptomic analysis was performed in mononuclear blood leukocytes from rs12526453 CC monozygotes or G allele carriers. Results In the study group (mean age 62.3 ± 11.9 years; 24.9% of females, n=159), percentages of CC, CG, and GG genotypes were 45.3% (n=289), 44.7% (n=285), and 10% (n=64), respectively. In the 5-year follow-up 105 patients died (16.46%). CC homozygotes had significantly lower mortality compared to other genotypes: 13.1% (n=38) vs. 18.3% in G-allele carriers (n=67), (p=0.017, Cox`s F test). In the validation group 47 patients died within 3 years (13.5%). We confirmed lower mortality of CC homozygotes: 10.1 % (n=18) vs. 16.95% in G-allele carriers (n=29), (p=0.031, Cox`s F test). Transcriptomic analysis revealed a markedly higher expression of NLRP-2 in CC homozygotes. Conclusions The rs12526453 CC homozygotes (previously associated with increased risk of myocardial infarction) showed, in 2 independent samples, better long-term survival. The finding of such high effect size, after appropriate validation, could potentially be translated into clinical practice.

miR-22-5p revealed as a potential biomarker involved in the acute phase of myocardial infarction via profiling of circulating microRNAs

Acute myocardial infarction (AMI) is a life-threatening episode of coronary artery disease. Recently, circulating myocardial-derived microRNAs (miRNAs) have been reported as potential biomarkers of infarction. The present study aimed to identify differentially expressed miRNAs in patients with ST-segment elevation myocardial infarction that could be potentially dysregulated in response to early myocardial damage. miRNA expression profile analysis was performed using the Serum/Plasma Focus miRNA Polymerase Chain Reaction (PCR) panel of Exiqon A/S (Vedbaek, Denmark) on plasma samples of patients on the first day of AMI (admission) and on samples from the identical patients collected six months following AMI. Selected miRNAs were validated by reverse transcription‑quantitative PCR (RT‑qPCR) using independent patients with AMI and a control group of patients with a stable coronary artery disease. Thirty‑two species of plasma miRNA were differentially expressed (P<0.05) on admission compared with six months following AMI. Subsequent validation in an independent patient group confirmed that miR‑133b and miR‑22‑5p were significantly up‑regulated in the serum of patients with AMI. The receiver operating characteristic (ROC) curve analysis demonstrated a diagnostic utility for miR-22-5p, which has not previously been reported to be associated with AMI. Among the selected miRNAs, miR‑22‑5p represents a novel promising biomarker for the diagnosis of AMI.

The 4q25, 1q21, and 16q22 polymorphisms and recurrence of atrial fibrillation after pulmonary vein isolation.

Introduction The efficacy of pulmonary vein isolation (PVI) in atrial fibrillation (AF) is well documented. Several single nucleotide polymorphisms (SNPs) are associated with AF, mainly in the 4q25 locus, but also in 16q22 and 1q21. The aim of our study was to test the association between those SNPs and short- and long-term results of PVI. Material and methods Patients with AF who underwent PVI between 2006 and 2009 were included in the study. Pulmonary vein isolation was performed using a 4-mm non-irrigated ablation catheter, circular mapping catheter, and the LocaLisa system. All patients were genotyped for the 4q25, 16q22, and 1q21 SNPs. Results Two-hundred and thirty-eight patients were included. The median follow-up was 45 months. Six-month efficacy was 59.7%. None of the polymorphisms was linked with the risk of AF recurrence after 6 months in univariate analysis. In multivariate analysis rs2200733 in the recessive model was linked significantly with AF recurrence (odds ratio 1.87, p = 0.008). None of the polymorphisms predicted AF recurrence in long-term follow-up. Conclusions There is a trend in the relationship between TT genotype of the rs2200733 polymorphism and increased rate of AF recurrence after PVI in short-term (6 months) follow-up. None of the tested SNPs 4q25, 16q22, and 1q21 correlated with the results of a single AF ablation in long-term follow-up.