Neslihan Çoban

Oxidative stress-mediated (sex-specific) loss of protection against type-2 diabetes by macrophage migration inhibitory factor (MIF)-173G/C polymorphism.

BACKGROUND The archetypical yet atypical cytokine macrophage migration inhibitory factor (MIF) fulfills pleiotropic immune functions in inflammatory diseases. Evidence emerging from both expression and functional studies implicates MIF in various aspects of cardiovascular diseases. We aimed to determine the covariates of MIF-173G/C polymorphism and its influence on type-2 diabetes risk in a sample representative of middle-aged Turks. METHODS Randomly selected 2250 Turkish adults (mean age; 49.7±11.9, 48.5% male) were genotyped for -173G/C polymorphism using hybridization probes in Real-Time PCR LC480 device. RESULTS The MIF-173CC genotype prevailed in 3.7% in men and 2.9% in women. C-allele carriage was associated linearly with wider waist girth, independently of fasting glucose, and was further related to higher apolipoprotein B (apoB) (p<0.05) in men, but not women. Logistic regression analysis showed the C-allele carriage to tend to predict new-onset diabetes (RR 1.51; [95% CI 0.98; 2.32]), additively to age and fasting glucose in men, but not in women. In contrast, risk for established (baseline) diabetes mellitus was lower (OR=0.49, 95% Cl 0.26-0.93, p=0.03) in heterozygotes, after adjustment for atherogenic dyslipidemia and other confounders. CONCLUSION MIF-173GC polymorphism independently contributes to abdominal obesity and is related to apoB concentrations apparently in men alone. Tendency of the -173C-allele carriage to predict new-onset diabetes independently was also confined to men. These gender-modulated associations suggest novel gene-gender-environmental interactions originating from a proinflammatory state.

Gender-specific associations of the APOA1 -75G>A polymorphism with several metabolic syndrome components in Turkish adults.

BACKGROUND Variations in the apolipoprotein A-1 (APOA1) gene, a determinant of plasma high-density lipoprotein cholesterol (HDL-C) and apoA-I levels, may contribute to cardiovascular diseases. We evaluated the effects of a promoter polymorphism (-75G>A) in the APOA1 gene on metabolic syndrome (MetS) components in a Turkish population sample. METHODS Randomly selected 1515 Turkish adults (age 49.9±11.8 years, 785 females) were genotyped for -75G>A polymorphism using hybridization probes in Real-Time PCR LC480 device. MetS and atherogenic dyslipidemia were defined using the criteria of ATP III. RESULTS The -75AA genotype prevailed in 3.9% of men and 2.4% of women, and was independently associated with significantly higher HDL-C concentrations. Independent associations with the -75GA genotype existed only in men: higher diastolic and systolic blood pressure (BP) levels (p<0.05) were observed in male -75GA heterozygotes. Logistic regression revealed that the GA genotype confers elevated risk for atherogenic dyslipidemia (OR=1.57, 95% Cl 1.06-2.3) after adjustment for associated risk factors. Independent associations with atherogenic dyslipidemia or elevated BP did not emerge in women. CONCLUSION APOA1 -75G>A polymorphism is independently related to HDL-C concentrations. Independent associations of the -75GA genotype with elevated BP and atherogenic dyslipidemia were confined to men. These gender-modulated associations suggest novel gene-gender-environmental interactions.

Sex- and Obesity-specific Association of Aromatase (CYP19A1) Gene Variant with Apolipoprotein B and Hypertension

BACKGROUND AND AIMS Gender differences in cardiovascular disease risk have been attributed to sex hormones. The CYP19A1 protein (aromatase) plays a critical role in estrogen biosynthesis and thus affects body fat distribution and regulation. We examined the relationship between polymorphism of the CYP19A1 gene and lipoproteins, body mass index (BMI), insulin levels and HOMA index. METHODS Randomly selected 2250 Turkish adults (aged 49.7 ± 11.9 years; 48.6% males) were genotyped for CYP19A1 rs10046 polymorphism using hybridization probes in Real-Time PCR LC480 device. RESULTS Distribution of the CYP19A1 rs10046 polymorphism was 28% (n = 630), 48.3 % (n = 1085) and 23.7% (n = 535) for the CC, CT and TT genotypes, respectively, and the T allele frequency was 0.48. In relation to apolipoprotein (apo)B levels, C homozygosity was associated with higher apoB in non-obese females, contrasting to being so in obese males only, and further in postmenopausal females. CC genotype in females was associated in linear regression analysis by 7.2 ± 3.3 mg/dL higher apoB than CT + TT genotypes, independent of age and BMI. Among premenopausal females, insulin levels (p = 0.007), BMI (p = 0.05) and HOMA index (p = 0.034) were higher in C homozygotes than in T-allele carriers. However, CYP19A1 TT genotype contributed to hypertension at an OR 1.80 (95% CI 1.12-2.91), independently of age, BMI and other confounders, in males alone. CONCLUSION The CYP19A1 rs10046 polymorphism is associated with cardiovascular risk factors such as circulating apoB, insulin resistance and hypertension in a sex- and obesity-specific manner.

Gender specific association of ABCA1 gene R219K variant in coronary disease risk through interactions with serum triglyceride elevation in Turkish adults.

OBJECTIVE ATP binding cassette transporter A1 (ABCA1) controls the reverse cholesterol transport. Some ABCA1 variants are correlated with serum high-density lipoprotein cholesterol (HDL-C) and other lipid concentrations. We aimed to explore the relationship of ABCA1 gene with both the lipid profile and coronary heart disease (CHD) risk. METHODS Selected 627 individuals of the Turkish Adult Risk Factor Study were genotyped for ABCA1 R219K polymorphism using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Students t-test, one-way ANOVA, Chi-square test, linear and logistic regression was used for statistical analysis. RESULTS We demonstrated a gender-specific effect of the R219K polymorphism on plasma lipids and CHD. In men, while homozygosity of the K allele was associated with increased plasma low-density lipoprotein cholesterol (LDL-C) (p<0.05) and total cholesterol concentrations (p<0.05), carriage of this allele was associated with higher HDL-C concentrations (p<0.05) after adjustment for associated risk factors, but not with CHD. In women, however, without being related to HDL-C levels, each 219K allele was associated with 10% higher triglycerides (TG) concentrations (p<0.05). R219K heterozygosity in women independently doubled (95% CI 1.00; 3.80) the odds ratio for CHD risk in regression models, after adjustment for several variables. Interaction of TG elevation (>140 mg/dL) with CHD was demonstrated in female 219RK genotype carriers. CONCLUSION R219 allele of the ABCA1 gene independently confers CHD risk in heterozygote Turkish women, not via reduced HDL-C, but interacting with elevated TG expressed by the 219K allele, but not in men.

Gender- and obesity-specific effect of apolipoprotein C3 gene (APOC3) –482C>T polymorphism on triglyceride concentration in Turkish adults

Abstract Background: Apolipoprotein C3 (APOC3) gene polymorphisms are associated with cardiometabolic risk factors, varying in ethnicities. This study aimed to investigate such association between the APOC3 –482C>T polymorphism and cardiometabolic risk factors in the turkish adult risk factor (TARF) study cohort, stratifying by gender and obesity. Methods: Randomly selected 1548 individuals (757 male and 791 female, mean age 49.9±11.8 years) were genotyped for –482C>T polymorphism using hybridization probes in a Real-Time PCR LC480 device. Results: The –482TT genotype prevailed 9.9% in men and 11.5% in women. Association between 482C>T polymorphism and dyslipidemia (p=0.036, OR=1.42, 95%Cl=1.02–1.97) was found only in men. Analysis of variance showed that anthropometric and metabolic variables were not differently distributed in APOC3 –482C>T genotypes in the study population. In relation to dyslipidemia and obesity, the –482C>T polymorphism showed significant gender-by-genotype interactions (p<0.01). When the study population was stratified according to gender and obesity, homozygotes for the T allele were associated strongly with (by 45%) elevated fasting triglyceride concentrations in obese men (p=0.009) and homeostatic model assessment (HOMA) index in non-obese women (p=0.013). Furthermore, in the same subgroups, the associations of the fasting triglyceride concentrations and HOMA index with the TT genotype remained after adjustment for risk factors (p<0.05). Conclusions: APOC3 –482TT genotype is independently associated with elevated fasting triglyceride concentrations in obese men. Presence of obesity seems to be required for this genotype to induce markedly elevated triglycerides. Furthermore, it is associated with the dyslipidemia in men, without requirement of obesity.

Lipoprotein(a) level and MIF gene variant predict incident metabolic syndrome and mortality.

Owing to the scarcity of available information, we aimed to assess the association of migration inhibitory factor (MIF)-173 G/C genotypes and serum lipoprotein(Lp)(a) with incident metabolic syndrome (MetS) and all-cause mortality, respectively. In population based, middle-aged adults (n=1297), stratified by gender and presence of MetS, we used Lp(a) quintiles to identify non-linear associations with outcomes using Cox regression models, adjusted for MIF genotype, age, smoking status, high density lipoprotein cholesterol, and systolic blood pressure. After 5.2 years of follow-up, 151 cases of incident MetS and 123 deaths were recorded. For incident MetS, adjusted HRs increased in each gender across four declining quintiles, starting from the highest quintile in men and from quintile 4 in women. The MIF CC-GC genotype appeared to contribute to the risk estimates in men. Similarly adjusted models in the whole sample disclosed that all-cause mortality tended to be inversely associated with Lp(a) quintiles and yielded an HR (2.42 (95% CI 1.03 to 5.81)) in men in quintile 2, whereas the MIF genotype additively predicted mortality (HR 1.79 (95% CI 1.01 to 3.18)) only in men. Excess risk of death was additively conferred on Turkish men by the MIF CC-GC genotype and by apparently reduced circulating Lp(a) assays, supporting the notion that ‘low’ serum Lp(a), mediating autoimmune activation, is a major determinant of metabolic disease risk and death. Damaged MIF protein and more complex autoimmune activation in women may be responsible from lack of relationship to MetS/mortality.

CYP19A1, MIF and ABCA1 genes are targets of the RORα in monocyte and endothelial cells†

RORα is a member of nuclear receptor superfamily of transcription factors, which has a vital role in the regulation of various physiological processes. Cholesterol is a known ligand of RORα and is one of the key components that take part in cardiovascular diseases such as atherosclerosis. Therefore, it is possible that RORα might have a role in the development of atherosclerosis. To test this hypothesis, we investigated the presence of novel RORα response elements (ROREs) located in the promoter of CYP19A1, MIF and ABCA1 genes. Briefly, the occupancy of RORα in the promoter regions of these genes was demonstrated in THP‐1 and HUVEC cell lines by ChIP analysis. In order to modulate RORα activity, THP‐1 and HUVEC cells were treated with specific RORα ligands (CPG 52608 and SR1001) and then the expression levels of target genes were analysed. In the next step, we tested whether RORα activity in THP‐1 macrophages was influenced by the presence of simvastatin, a cholesterol lowering drug. We found that in the presence of simvastatin the expression of the investigated target genes were down regulated and that this regulation was partially prevented by CPG 52608 and SR1001. Results of this study suggest that CYP19A1, MIF and ABCA1 are the direct target genes of RORα. In conclusion, it is important to demonstrate that certain genes involved in the development of atherosclerosis could be modulated by an inducible transcription factor. Therefore, these results offer a potential therapeutic approach for the treatment of atherosclerosis.

Lower circulating migration inhibitory factor protein is associated with metabolic syndrome and diabetes

AIM The controversial relationship between macrophage migration inhibitory factor (MIF) and the likelihood of cardiometabolic diseases was investigated. Results/methodology: Assayed MIF protein from 1225 adults was cross-sectionally analyzed. MIF was independently inversely associated with age, total testosterone and positively with high-density lipoprotein-cholesterol. In men MIF correlation with age, testosterone and waist circumference converted from inverse in the upper to positive in the bottom MIF third. Both metabolic syndrome and diabetes were significantly associated, in combined gender, with the intermediate (vs the highest) MIF tertile at an odds ratio 1.6. Coronary heart disease was not significantly related with MIF in either gender. DISCUSSION/CONCLUSION Findings are consistent with oxidative damage to MIF protein and its involvement in autoimmune activation, likely more extensive in women.

Role of simvastatin and RORα activity in the macrophage apoptotic pathway

Objective: Atherosclerosis is a chronic inflammatory condition and is one of the main causes of death worldwide. Macrophages play important roles in the formation of atherosclerotic plaques. Apoptosis is progressively observed while plaques develop, although the precise mechanisms and outcomes of apoptosis in atherosclerosis development and progression are still contradictory. This study was conducted to explore the effects of simvastatin and retinoic acid receptor-related orphan receptor alpha (RORa) ligands on apoptosis in human acute monocytic leukemia (THP-1) macrophage cells. Methods: Briefly, the occupancy of RORa in the promoter regions of apoptotic pathway genes was demonstrated in THP-1 cell lines using chromatin immunoprecipitation (ChIP) analysis. In order to modulate RORa activity, THP-1 macrophage cells were treated with specific ligands (CPG52608 and SR1001) and then viability as well as count of THP-1 macrophage cells were analyzed. Results: We observed that simvastatin and both RORa ligands had a tendency to decrease THP-1 macrophage cell viability in culture. When compared with non-treated controls, simvastatin significantly decreased cell viability (p=0.04) and cell count (p=0.03). However, this negative effect of simvastatin seemed to be partly prevented by RORa ligands. In addition, bioinformatics analysis of ChIP-on-chip data demonstrated that several genes that are involved in the apoptotic pathway were likely RORa target genes. These genes were involved in the regulation of apoptosis through various pathways. Conclusion: In summary, our study suggest that simvastatin-mediated macrophage apoptosis might be modulated by SR1001 administration. However, involvement of RORa in this modulation through potential apoptotic target genes remains elusive.OBJECTIVE Atherosclerosis is a chronic inflammatory condition and is one of the main causes of death worldwide. Macrophages play important roles in the formation of atherosclerotic plaques. Apoptosis is progressively observed while plaques develop, although the precise mechanisms and outcomes of apoptosis in atherosclerosis development and progression are still contradictory. This study was conducted to explore the effects of simvastatin and retinoic acid receptor-related orphan receptor alpha (RORα) ligands on apoptosis in human acute monocytic leukemia (THP-1) macrophage cells. METHODS Briefly, the occupancy of RORα in the promoter regions of apoptotic pathway genes was demonstrated in THP-1 cell lines using chromatin immunoprecipitation (ChIP) analysis. In order to modulate RORα activity, THP-1 macrophage cells were treated with specific ligands (CPG52608 and SR1001) and then viability as well as count of THP-1 macrophage cells were analyzed. RESULTS We observed that simvastatin and both RORα ligands had a tendency to decrease THP-1 macrophage cell viability in culture. When compared with non-treated controls, simvastatin significantly decreased cell viability (p=0.04) and cell count (p=0.03). However, this negative effect of simvastatin seemed to be partly prevented by RORα ligands. In addition, bioinformatics analysis of ChIP-on-chip data demonstrated that several genes that are involved in the apoptotic pathway were likely RORα target genes. These genes were involved in the regulation of apoptosis through various pathways. CONCLUSION In summary, our study suggest that simvastatin-mediated macrophage apoptosis might be modulated by SR1001 administration. However, involvement of RORα in this modulation through potential apoptotic target genes remains elusive.

Investigating the role of ceramide metabolism-associated CERS5 (LASS5) gene in atherosclerosis pathogenesis in endothelial cells

OBJECTIVE Ceramide, the backbone of sphingolipids, is the key component affecting atherosclerotic changes through its important second-messenger role. Previous studies have demonstrated protective role of AMP-activated protein kinase (AMPK) genes in regulating atherosclerosis and hypertension. Ceramide synthase 5 (LASS5 or CERS5) gene has function in de novo synthesis of ceramide, and has indirect effect on AMPK gene. Aim of the present study was to identify role of LASS5 gene in atherosclerosis. METHODS LASS5 gene-specific small interfering RNA (siRNA)-mediated gene silencing was performed in human umbilical vein endothelial cells (HUVEC) and differential expression of LASS5, AMPK-alpha and AMPK-alpha target genes were analyzed. HUVEC cells were then treated with AMPK activator in order to examine relationship of change in gene expression levels to AMPK activity. RESULTS Novel physiological function of LASS5 was identified. Downregulation of LASS5 was found to attenuate ceramide production and increase expression of some AMPK target genes in HUVEC. CONCLUSION This is the first study to demonstrate that LASS5 was involved in negative regulation of atherosclerosis-related genes, such as AMPK-alpha. These preliminary findings provide insight into molecular mechanism of atherosclerosis and are important for development of potential therapeutic agents in the treatment of atherosclerosis.