ABCA1 gene promoter DNA methylation is associated with HDL particle profile and coronary artery disease in familial hypercholesterolemia

High-density lipoproteins cholesterol (HDL-C) level, a strong coronary artery disease (CAD) clinical biomarker, shows significant interindividual variability. However, the molecular mechanisms involved remain mostly unknown. ATP-binding cassette A1 (ABCA1) catalyzes the cholesterol transfer from peripheral cells to nascent HDL particles. Recently, a differentially methylation region was identified in ABCA1 gene promoter locus, near the first exon. Therefore, we hypothesized that DNA methylation changes at ABCA1 gene locus is one of the molecular mechanisms involved in HDL-C interindividual variability. The study was conducted in familial hypercholesterolemia (FH), a monogenic disorder associated with a high risk of CAD . Ninety-seven FH patients (all p.W66G for the LDLR gene mutation and not under lipid-lowering treatment) were recruited and finely phenotyped for DNA methylation analyses at ABCA1 gene locus. ABCA1 DNA methylation levels were found negatively correlated with circulating HDL-C (r = -0.20; p = 0.05), HDL2-phospholipid levels (r = -0.43; p = 0.04), and with a trend for association with HDL peak particle size (r = -0.38; p = 0.08). ABCA1 DNA methylation levels were also found associated with prior history of CAD (CAD = 40.2% vs. without CAD = 34.3%; p = 0.003). These results suggest that epigenetic changes within the ABCA1 gene promoter contribute to the interindividual variability in plasma HDL-C concentrations and are associated with CAD expression. These findings could change our understanding of the molecular mechanisms involved in the pathophysiological processes leading to CAD.     

Thioredoxin-interacting protein promotes activation and inflammation of monocytes with DNA demethylation in coronary artery disease

Numerous studies have demonstrated that thioredoxin-interacting protein (TXNIP) expression of peripheral blood leucocytes is increased in coronary artery disease (CAD). However, the molecular mechanism of this phenomenon remained unclear. DNA methylation plays important roles in the regulation of gene expression. Therefore, we speculated there might be a close association between the expression of TXNIP and methylation. In this study, we found that compared with controls, DNA methylation at cg19693031 was decreased in CAD, while mRNA expressions of TXNIP and inflammatory factors, NLRP3, IL-1β, IL-18, were increased. Methylation at cg19693031 was negatively associated with TXNIP expression in the cohort, THP-1 and macrophages/foam cells. Furthermore, Transwell assay and co-cultured adhesion assay were performed to investigate functions of TXNIP on the migration of THP-1 or the adhesion of THP-1 on the surface of endothelial cells, respectively. Notably, overexpressed TXNIP promoted the migration and adhesion of THP-1 cells and expressions of NLRP3, IL-18 and IL-1β. Oppositely, knock-down TXNIP inhibited the migration and adhesion of THP-1 and expressions of NLRP3, IL-18. In conclusion, increased TXNIP expression, related to cg19693031 demethylation orientates monocytes towards an inflammatory status through the NLRP3 inflammasome pathway involved in the development of CAD.     

Common gene polymorphism in ATP-binding cassette transporter A1 and coronary artery disease: A genetic association study and a structural analysis

ATP-binding cassette transporter A1 (ABCA1) has a crucial role in removing intracellular cholesterol and plays a protective role against atherosclerosis. Therefore, genetic polymorphisms in this gene may alter the susceptibility to coronary artery disease (CAD). This study was aimed to examine the association of rs2230806 (c.1051 G > A; p.R219K) variation in the ABCA1 gene with CAD in a case-control design which was followed by a meta-analysis and in silico approach. In the case-control study, 300 subjects including 150 individuals with CAD and 150 healthy controls were recruited. The c.1051 G > A genotyping was done by polymerase chain reaction-restriction fragment length polymorphism method. In the meta-analysis, eligible studies were collected from PubMed, Google Scholar, and ScienceDirect databases and pooled odds ratio, heterogeneity, publication bias, and sensitivity analyses were carried. Finally, some bioinformatics tools were employed to assess the impacts of p.R219K variation on ABCA1 protein structure. Our case-control examination showed a statistically significant association between c.1051 G > A genetic polymorphism and CAD risk. In addition, the meta-analysis showed reliable significant associations between c.1051 G > A transition and risk of CAD in the Caucasian population. In silico analysis showed that the p.R219K substitution could alter the secondary structure, hydrophobicity pattern, and Ramachandran plot of ABCA1. These findings elucidate that the c.1051 G > A variation could be a genetic risk factor for CAD and it could be considered as a prognostic and predictive biomarker for susceptible individuals.     

A preliminary study of human paraoxonase and PON 1 L/M55–PON 1 Q/R 192 polymorphisms in Turkish patients with coronary artery disease

Paraoxonase 1 (PON 1) is a high‐density lipoprotein (HDL)‐associated enzyme with antioxidant function protecting low‐density lipoprotein (LDL) from oxidation. PON 1 has two amino acid polymorphisms in coding region; L/M 55 and Q/R 192. These polymorphisms modulate paraoxonase activity of the enzyme. PON 1 activity decreases in coronary artery disease (CAD). In the present study, distribution of PON 1 L/M 55 and Q/R 192 polymorphisms and the effect of these polymorphisms on the activities of PON 1, and on the severity of CAD in 277 CAD (+) patient and 92 CAD (?) subjects were examined. PON 1 L/M 55 and Q/R 192 genotypes were determined by PCR, RFLP and agarose gel electrophoresis techniques. Genotype distributions and allele frequencies for PON 1 Q/R 192 polymorphism were not significantly different between controls and CAD (+) patient group (p > 0.05), but in genotype and allele distribution of PON 1 L/M55 polymorphism, there was significantly difference among groups (p < 0.05). Genotype distributions for both polymorphisms were not significantly different between subgroups of single‐vessel disease (SVD), double‐vessel disease (DVD) and triple‐vessel disease (TVD). Serum PON 1 activity was lower in CAD (+) group than in controls and this was also statistically significant (p < 0.001). In both groups, the highest PON activities were detected in LL and RR genotypes. In summary, our results suggest that there is an association between the PON 1 L/M 55 polymorphism of paraoxonase and CAD in Turkish patients but not with PON 1 Q/R 192 polymorphism. However, it is hard to correlate these polymorphisms and severity of CAD. Copyright © 2009 John Wiley & Sons, Ltd.     

Increased plasma HB-EGF associated with obesity and coronary artery disease

The mechanism by which the obese subjects are more associated with vascular disease remains unclear. We reported that the adipose tissues produce and secrete many bioactive molecules, conceptualized as adipocytokines. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), produced locally by vascular macrophages and smooth muscle cells, has been suggested to induce the migration and proliferation of vascular smooth muscle cells. The current study reveals that (1) HB-EGF mRNA is abundantly expressed in human adipose tissue, (2) HB-EGF mRNA increases in the fat tissues of obese mice, (3) plasma HB-EGF levels increase in parallel with fat accumulation in human, and (4) the subjects with coronary artery disease have higher plasma HB-EGF levels, associated with fat accumulation. These results suggest that increased plasma HB-EGF derived from the accumulated fat contributes to the higher incidence of vascular disease in obesity, proposing HB-EGF as an adipocytokine directly linking adipovascular axis.     

Effects of cholesterol ester transfer protein Taq1B gene polymorphism on serum lipoprotein levels in Turkish coronary artery disease patients

To evaluate the effect of cholesterol ester transfer protein (CETP) Taq1B gene polymorphism on serum lipid profile in Turkish coronary artery disease (CAD) patients, we investigated Taq1B gene polymorphism of CETP and serum lipid levels in 111 controls and in 173 CAD patients with myocardial infarction. There were no significant differences in the allele distribution at this polymorphic locus between the population sample and patients with coronary artery disease with myocardial infarction. To detect the association between the Taq1B RFLP and serum lipid levels, we determined the serum concentrations of total cholesterol, triglycerides and high density lipoprotein cholesterol (HDL-C) in the subjects studied and correlated the results to the Taq1B RFLP. Patients with Taq B1B1 genotypes had lower HDL-C levels than patients with B2B2 genotype (p = 0.003). Also in control subjects with Taq B1B1 genotype, lower HDL-C levels (p = 0.05) and higher triglyceride levels (p = 0.017) and body mass index (p = 0.05) were observed compared with control subjects with the B1B2 genotype. It was observed that in our population the distribution of CETP Taq1B genotypes is similar to other populations (except Greeks). The present study demonstrates that CETP Taq1B gene polymorphism may be responsible for low HDL cholesterol levels in patients with CAD and in healthy controls in Turkey.     

Serum Metrnl is associated with the presence and severity of coronary artery disease

Meteorin‐like (Metrnl) is a novel adipokine that is highly expressed in white adipose tissue. Metrnl stimulates energy expenditure and improves glucose tolerance in rodents. However, whether Metrnl plays a role in coronary artery disease (CAD) remains to be elucidated. The present study aimed to investigate the association of serum Metrnl with CAD in Chinese patients. A total of 193 patients with CAD and 156 control subjects were enrolled in this study. Serum Metrnl concentration was measured by enzyme‐linked immunosorbent assay. Anthropometric phenotypes, fasting glucose, serum lipids, and inflammatory cytokines were measured. Serum Metrnl was lower in CAD patients when compared to those controls (132.41 vs 173.17 pg/mL, P < 0.001). Serum Metrnl was negatively correlated with metabolic parameters, including body mass index, total cholesterol, and low‐density lipoprotein cholesterol as well as inflammatory markers including high‐sensitivity C‐reactive protein, IL‐1β, and IL‐11 even after adjustment for potential confounding variables (P < 0.05). In multivariable logistic regression analyses, compared to those in the highest tertile of serum Metrnl levels, subjects in the lowest tertile had the highest risks for CAD (adjusted OR = 2.63, 95% CI = 1.46‐4.27, P = 0.001). After adjustment for potential confounding variables, serum Metrnl was also decreased as the number of stenosed vessels increased (P < 0.001). Furthermore, decreased Metrnl level was negatively correlated with the severity of CAD quantified by the Gensini score. This first case‐control study shows significant associations of serum Metrnl with the presence and severity of CAD, suggesting Metrnl might be a new promising therapeutic target for CAD.     

MiRNA-1271-5p regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting forkhead box O1 (FOXO1)

Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-1271-5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR-1271-5p. The expression levels of FOXO1 and Runx2 were detected by RT-qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT-qPCR. Our results showed that miR-1271-5p was downregulated during osteogenic induction. And the expression levels of miR-1271-5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR-1271-5p and FOXO1 in osteoporotic tissues. Overexpression of miR-1271-5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR-1271-5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR-1271-5p on osteogenic differentiation. In conclusion, miR-1271-5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR-1271-5p as a target in the treatment of osteoporosis and other bone metabolic diseases.     

Genetic variation in vitamin D receptor gene (Fok1:rs2228570) is associated with risk of coronary artery disease

Objective: The Fok1 polymorphism (rs2228570) in vitamin D receptor gene appears to be the only polymorphism influencing size of translated protein. Investigations into its association with coronary artery disease (CAD) are sparse.

Methods: Male patients (n?=?98) with verified CAD were recruited alongside age- and sex-matched controls (n?=?55). Genotyping was performed by PCR-RFLP and plasma 25-Hydroxyvitamin D levels were assessed by HPLC-UV.

Results: The C-variant (mutant) was predominantly expressed in patients compared to controls (68.9% versus 55.5%; p?=?0.025). The observed genotypes were not associated with 25-Hydroxyvitamin D levels.

Conclusion: This study presents Fok1 polymorphism as a potential genetic marker for CAD.     

Myeloperoxidase gene-463G > A polymorphism and premature coronary artery disease

We investigated the association between myeloperoxidase gene -463G > A polymorphism and premature coronary artery disease (CAD) in two Chinese population samples: 229 patients and 230 controls. Genotypes were determined by ligase detection reaction-polymerase chain reaction sequencing and the grouping technique. We found lower frequencies of both the A/A genotype and the A allele in patients (p < 0.05). Multivariate logistic regression showed that the risk of premature CAD in subjects carrying the AA genotype was reduced by 83% in relation to individuals carrying the G/G genotype (OR = 0.172, 95% CI: 0.057-0.526, p = 0.002). Our results indicate that -463G > A polymorphism of the myeloperoxidase gene is associated with premature CAD in Chinese individuals, suggesting that the AA genotype is a protective factor against premature CAD.     

Two- and three-locus haplotypes of the paraoxonase (PON1) gene are associated with coronary artery disease in Asian Indians

Introduction

In view of the reported association of SNPs in the paraoxonase (PON1) gene with coronary artery disease (CAD), and the absence of conclusive data from India, we investigated the relationship of three SNPs at different loci (‐108C/T, L55M and Q192R) of the PON1 gene and their haplotypes with CAD among people residing in the northern plains of India.

Materials and methods

One hundred and seventy-eight healthy controls and two hundred and four angiographically-proven CAD patients were genotyped using PCR-RFLP.

Results

Of the three SNPs, only the R allele of Q192R polymorphism was associated with CAD (p < 0.05). Two locus haplotypes QT (OR 0.55, p = 0.0004, 95% CI 0.39–0.77, significant) and LQ (odds ratio 0.73, p = 0.03, 95% CI 0.55–0.97, trend) showed protective effects, while haplotypes MR (OR = 5.36, p = 0.0001, 95% CI 2.045–14.049) and MC (OR = 2.71, p = 0.011, 95% CI 1.221–6.046) were associated with increased risk of CAD. MRT, a minor three-locus haplotype also displayed significant association (OR 4.93, 95% CI 1.7–13.5) with the disease. Significance was assessed after applying Bonferroni's correction.

Conclusions

Our study revealed that only one SNP at a single locus but several haplotype combinations of PON1 coding and promoter-region polymorphisms were associated with the risk of or protection against CAD. Thus, haplotype analysis brought better insights into the association of PON1 gene polymorphisms with CAD in Asian Indians.     

Genome wide DNA methylation profiling for epigenetic alteration in coronary artery disease patients

Background

The alteration in the epigenome forms an interface between the genotype and the environment. Epigenetic alteration is expected to make a significant contribution to the development of cardiovascular disease where environmental interactions play a key role in disease progression. We had previously shown that global DNA hypermethylation per se is associated with coronary artery disease (CAD) and is further accentuated by high levels of homocysteine, a thiol amino acid which is an independent risk factor for cardiovascular disease and is also a key modulator of macromolecular methylation.

Results

We have identified 72 differentially methylated regions (DMRs) that were hypermethylated in CAD patients in the background of varying homocysteine levels. Following deep bisulfite sequencing of a few of the selected DMRs, we found significantly higher methylation in CAD cases. We get six CpG sites in three DMRs that included the intronic region of C1QL4 gene and upstream region of CCDC47 and TGFBR3 genes.

Conclusion

To the best of our knowledge, this is the first study to identify hypermethylated regions across the genome in patients with coronary artery disease. Further validation in different populations is necessary for this information to be used for disease risk assessment and management.     

A potential oral microbiome signature associated with coronary artery disease in Tunisia

The coronary artery disease (CAD) is a chronic inflammatory disease involving genetic as well as environmental factors. Recent evidence suggests that the oral microbiome has a significant role in triggering atherosclerosis. The present study assessed the oral microbiome composition variation between coronary patients and healthy subjects in order to identify a potential pathogenic signature associated with CAD. We performed metagenomic profiling of salivary microbiomes by 16S ribosomal RNA (rRNA) next-generation sequencing. Oral microbiota profiling was performed for 30 individuals including 20 patients with CAD and ten healthy individuals without carotid plaques or previous stroke or myocardial infarction.We found that oral microbial communities in patients and healthy controls are represented by similar global core oral microbiome. The predominant taxa belonged to Firmicutes (genus Streptococcus, Veillonella, Granulicatella, Selenomonas), Proteobacteria (genus Neisseria, Haemophilus), Actinobacteria (genus Rothia), Bacteroidetes (genus Prevotella, Porphyromonas), and Fusobacteria (genus Fusobacterium, Leptotrichia). More than 60% relative abundance of each sample for both CAD patients and controls is represented by three major genera including Streptococcus (24.97 and 26.33%), Veillonella (21.43 and 19.91%), and Neisseria (14.23 and 15.33%).Using penalized regression analysis, the bacterial genus Eikenella was involved as the major discriminant genus for both status and Syntax score of CAD. We also reported a significant negative correlation between Syntax score and Eikenella abundance in coronary patients’ group (Spearman rho = −0.68, P=0.00094).In conclusion, the abundance of Eikenella in oral coronary patient samples compared with controls could be a prominent pathological indicator for the development of CAD.     

MPO129A／G基因多态性与冠心病严重程度相关研究

目的：探讨髓过氧化物酶129A／G基因多态性与冠心病严重程度关系。方法：入选住院冠心病患者267例,均行冠脉造影检查。用比色法测定血清髓过氧化物酶活性;用聚合酶链反应．限制性片段长度多态性（PCR-RFLP）确定各研究对象的基因型。根据冠脉造影结果分别采用冠脉病变支数和Gensini评分来评估冠心病严重程度。结果：MPO129位点A、G等位基因频率分别为0．107和0．893。不同基因型组间血清髓过氧化物酶活性无明显差异,不同基因型在Gensini评分组间的分布无明显差异,基因型GG患者冠脉病变倾向于多血管病变。结论：髓过氧化物酶129A／G基因多态性与血清髓过氧化物酶活性无显著相关,其不能作为预测冠心病病变严重程度的理想指标。     

miRNA 206 and miRNA 574-5p are highly expression in coronary artery disease

Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide. Innovative diagnostic biomarkers are a pressing need for this disease. miRNAs profiling is an innovative method of identifying biomarkers for many diseases and could be proven as a powerful tool in the diagnosis and treatment of CAD. We performed miRNA microarray analysis from the plasma of three CAD patients and three healthy controls. Subsequently, we performed quantitative real-time PCR (qRT-PCR) analysis of miRNA expression in plasma of another 67 CAD patients and 67 healthy controls. We identified two miRNAs (miR-206 and miR-574-5p) that were significantly up-regulated in CAD patients as compared with healthy controls (P<0.05). The receiver operating characteristic (ROC) curves indicated these two miRNAs had great potential to provide sensitive and specific diagnostic value for CAD.     

Oxidative status and reduced glutathione levels in premature coronary artery disease and coronary artery disease

Identifying patients at risk of developing premature coronary artery disease (PCAD) which occurs at age below 45 years old and constitutes approximately 7–10% of coronary artery disease (CAD) worldwide remains a problem. Oxidative stress has been proposed as a crucial step in the early development of PCAD. This study was conducted to determine the oxidative status of PCAD in comparison to CAD patients. PCAD (<45 years old) and CAD (>60 years old) patients were recruited with age-matched controls (n?=?30, each group). DNA damage score, plasma malondialdehyde (MDA) and protein carbonyl content were measured for oxidative damage markers. Antioxidants such as erythrocyte glutathione (GSH), oxidised glutathione (GSSG), and glutathione peroxidase activity (GPx), superoxide dismutase (SOD) and catalase (CAT) were also determined. DNA damage score and protein carbonyl content were significantly higher in both PCAD and CAD when compared to age-matched controls while MDA level was increased only in PCAD (p<.05). In contrast, GSH, GSH/GSSG ratio, α-tocotrienol isomer, and GPx activity were significantly decreased, but only in PCAD when compared to age-matched controls. The decrease in GSH was associated with PCAD (OR?=?0.569 95%CI [0.375???0.864], p?=?.008) and cut-off values of 6.69?μM with areas under the ROC curves (AUROC) 95%CI: 0.88 [0.80–0.96] (sensitivity of 83.3%; specificity of 80%). However, there were no significant differences in SOD and CAT activities in all groups. A higher level of oxidative stress indicated by elevated MDA levels and low levels of GSH, α-tocotrienol and GPx activity in patients below 45 years old may play a role in the development of PCAD and has potential as biomarkers for PCAD.     

C-reactive protein gene polymorphisms affect plasma CRP and homocysteine concentrations in subjects with and without angiographically confirmed coronary artery disease

Human C-reactive protein (CRP) is a reactant involved in the acute phase response and one of the many molecular factors involved in pathogenesis of coronary artery disease (CAD). CRP gene variants potentially mediate CRP plasma concentrations and the development of CAD. 220 Croatian subjects with angiographically confirmed CAD and 132 control subjects were included in the study. CRP gene polymorphisms 1059G/C and -717G/A were determined by RFLPs, using MaeIII and KspI endonuclease, respectively. Plasma concentrations of CRP and homocysteine were determined by immunoturbidimetry and FPIA, respectively. CRP 1059G/C gene variants were significantly associated with CAD (OR = 0.50; 95% CI = 0.27, 0.94; P = 0.032). Wild GG genotype and rare allele C carrier genotypes were 184 and 22 in CAD(+) group, and 101 and 24 in CAD(−) group, respectively. Multivariate analysis with age, gender, BMI, smoking status, hypertension and diabetes as covariates showed that 1059C carriers had lower CRP concentrations in CAD(−) (P = 0.010) and CAD(+) subjects (P = 0.028). This allele was also significantly associated with lower plasma homocysteine concentrations in both groups (P = 0.018 for CAD(−) and 0.002 for CAD(+). There was no significant difference between CAD(+) and CAD(−) subjects in absolute frequencies for CRP -717A/G gene variant, but multivariate analysis showed that carriers of the rarer G allele had significantly higher CRP plasma concentrations in CAD(−) subjects (P = 0.031) and higher homocysteine concentrations in CAD(+) group (P < 0.001). Atherosclerosis is an inflammatory disease resulting from different genetic and environmental factors. Results presented here support the contribution of CRP genetic variations in the development of CAD.     

冠心病患者运动致QRS延长的临床意义

为探讨冠心病病人由于急性心肌缺血对ＱＲＳ持续时间变化的影响及其意义。本文收集了５３例病人,均进行运动试验和冠脉造影,其中２０例经冠脉造影排除心病,其他３３例经冠脉造影确定为冠心病。结果运动使冠脉正常者ＱＲＳ持续时间缩短,而使冠心病者ＱＲＳ持续时间延长。