| Chapter1Association of genetic polymorphisms in ADMA metabolism enzymes with risk for Coronary heart disease in Chinese Han populationBackgroundCoronary heart disease (CHD) is one of the major cardiovascular diseases (CVDs), and is the main cause of death worldwide. The etiology of CHD include genetic factors and environmental factors.Atherosclerosis is the fundamental pathogenesis for CHD. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor, and can lead to endothelial dysfunction by inhibiting the sproduction of NO. ADMA can also accelerate the development of arteriosclerosis by decreasing NO availability stimulating inflammation.The majority of ADMA produced in the body is metabolized by ADMA metabolism enzymes, including dimethylarginine dimethylaminohydrolases1(DDAH1) and Alanine-glyoxylate aminotransferase2(AGXT2). The purpose of this study was to clarify the association of DDAH1and AGXT2genetic polymorphisms with risk for CHD in Chinese Han population, to explore the effects of genetic polymorphisms in these genes on plasma ADMA level in healthy subjects, and to identify new susceptibility loci for CHD in Chinese populationMethods1. Association between DDAH1and AGXT2genetic polymorphisms and CHD susceptibility with case-control studyBlood samples from1103healthy subjects and942CHD patients were collected for extraction of genomic DNA. AGXT2rs37369and DDAH1rs233113weren genetyped for all cases and controls by method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Chi-square test and Logistic regression were used for analyzing the association of the polymorphisms with risk for CHD.2. The effect of DDAH1and AGXT2genetic polymorphisms on plasma ADMA level in healthy subjectsPlasma ADMA concentration was detected by enzyme-linked immunosorbent assay (ELISA) in311healthy subjects with plasma available. One-way ANOVA and independent samples t-test were carried out to analyze the associations between genotypes and plasma ADMA levels. Results1. When adjusted for confounding factors using logistic regression, carriers of DDAH1rs233113AT genotype and T allele (AT+TT) showed a significantly decreased risk for CHD in (OR=0.79,95%CI:0.65-0.95,P＝0.013and OR=0.82,95%CI:0.69-0.98, P=0.028, respectively); In non-smokers, the rs233113AT genotype and carriers of the rs233113T allele (AT+TT) were also associated with decreased risk for CHD (OR=0.73,95%CI:0.58-0.92, P=0.009and OR=0.78,95%CI:0.63-0.98, P=0.030, respectively). When stratified by diabetes mellitus (DM), the rs233113AT genotype and T alleje (AT+TT) were associated with decreased risk for CHD in individuals without DM (OR=0.77,95%CI:0.63-0.94, P=0.011and OR=0.79,95%CI:0.65-0.96, P=0.015, respectively). When smoking status and diabetis status were considered concomitantly, the rs233113AT genotype and carriers of the rs233113T allele (AT+TT) showed significantly decreased risk for CHD in non-smokers without DM (OR=0.73,95%CI:0.56-0.93, P=0.013and OR=0.76,95%CI:0.60-0.96, P=0.023, respectively).2. The AGXT2rs37369GG (Val140Val) genotype was overrepresented in CHD cases than controls (18.5%vs14.8%, respectively, P=0.025). When logistic regression analysis was performed, carriers of rs37369GG genotype showed an increased risk for CHD in smokers as compared with carriers of the rs37369A allele (AA+AG)(OR=2.21,95%CI:1.24-3.92, P=0.007). At the same time, rs37369GG was associated with increased risk for CHD in smoking individuals with DM (OR=3.32,95%CI:1.14-9.67, P=0.028).3. In smokers, carriers of DDAH1rs233113AT genotype showed significant higher plasma ADMA level as compared rs233113AA and rs233113TT genotype (P=0.038and P=0.036, respectively); In non-smokers, carriers of AGXT2rs37369GG genotype showed significant higher plasma ADMA level when compared with carriers of the rs37369A allele (AA+AG)(0.45±0.05μM, n=43vs0.65±0.04μM, n=184, P=0.003, respectively), whereas an opposite result was observed in smokers (0.92±0.16μM, n=14vs0.51±0.05μM, n=70, P=0.004); Furthermore, rs37369GG homozygotes in smokers showed significantly higher plasma ADMA concentration compared with the same genotype in non-smokers (P=0.012).4. Carriers of both risk genotypes for DDAH1rs233113and AGXT2rs37369showed an increased risk for CHD; meanwile, cigarette smoking increased plasma ADMA level in individuals carrying the rs233113and rs37369risk genotypes concomitantly (P=0.003). Conclusions1. DDAH1rs233113polymorphism was associated with decreased risk for CHD in Chinese Han population, especially in non-smokers without DM.2. AGXT2rs37369polymorphism is associated with increased risk for CHD in Chinese population, especially in smokers and in individuals with T2DM, possibly through increasing plasma ADMA levels.3. The is a synergistic effects between AGXT2rs37369and DDAH1rs233113polymorphisms in modigying the risk of CHD in Chinese Han population. Chapter2Association of genetic polymorphisms in ADMA metabolism enzymes with risk for and prognosis of ischemic stroke in Chinese Han populationBackgroundStroke is the main cause of disability worldwide, and also was the second cause of death. Ischemic stroke (IS) or cerebral infarction is the most common type of stroke, accounting for60%to80%of stroke in clinic. The prevalence of stroke is rising in China, with high morbidity and mortality rates. Therefore, IS is becoming a seriousl health threaten and disease burden in China. The etiology of IS also includes environmental and genetic factors.Carotid atherosclerosis (AS) is the most important cause and risk factors of IS. As is closely related to the occurrence, development and recurrent, as well as sites of the infarction for IS. Asymmetric dimethylarginine (ADMA) inhibits nitric oxide production through inhibition of the activity of nitric oxide synthase (NOS), leading to endothelial dysfunction, while the latter is the initial step of atherosclerosis.The majority of ADMA formed in the body is metabolized by imethylarginine dimethylaminohydrolases1(DDAH1) and alanine-glyoxylate aminotransferase2(AGXT2). Studies have shown that genetic polymorphisms in genes encoding ADMA metabolic enzymes were associated with ADMA levels. The purpose of this study was to investigate whether DDAH1and AGXT2genetic polymorphisms are associated with the risk for and prognosis of IS in Chinese Han population.Methods1. Association between DDAH1and AGXT2genetic polymorphisms and IS susceptibility with case-control study1103healthy subjects and1012IS patients were collected for extraction of genomic DNA. AGXT2rs37369and DDAH1rs233113/rs233112genotype of all samples were determined by method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Chi-square test and logistic regression were carried out to analyze the association between the polymorphisms and risk for IS.2. Association between DDAH1and AGXT2genetic polymorphisms and prognosis of ischemic strokeIS patients with ischemic events occurred for the first-time in the study were followed-up by telephone of re-hospitalization after the first hospitalization. Major cardiovascular and cerebravascular events in cluding recurrent IS, myocardial infarction and death were recorded. Chi-square test and Kaplan-Meier analysis were performed to analyzed the effects of DDAH1and AGXT2genetic polymorphisms on IS prognosis.Results1.No significant difference in the distribution of AGXT2rs37369genotypes in IS cases and the controls was observed. The polymorphism was was not associated with risk for IS.2. DDAH1rs233113polymorphism was not associated with the risk for IS in the overall population or in individuals stratified by smoking status. When stratified by hypertension and diabetes mellitus (DM), the rs233113AT genotype was associated with significantly decreased risk for IS in individuals with DM (OR=0.73,95%CI=0.53-0.99, P=0.046).3. The DDAH1rs233112G allele (AG+GG) was overrepresented in IS cases than controls (71.6%vs66.7%, P=0.020). When adjusted for confounding factors, results of logistic regression indicated that carriers of the rs233112AG/GG genotypes and the G allele (AG+GG) showed significantly decreased risk for IS in the overall population (OR=0.78,95%CI=0.63-0.96, P=0.019; OR=0.76,95%CI=0.58-0.99, P=0.045'OR=0.77,95%CI=0.63-0.94, P=0.011). Rs233112AG/GG genotypes and G allele (AG+GG) were also associated with significantly decreased risk for IS in individuals with hypertension (OR=0.73,95%CI=0.57-0.92, P=0.007; OR=0.71,95%CI=0.52-0.96, P=0.026and OR=0.72,95%CI=0.58-0.90, P=0.003, respectively). Rs233112AG genotype and carriers of the G allele (AG+GG) also showed significantly decreased risk for IS in individuals with DM (OR=0.59,95%CI=0.42-0.84, P=0.003'OR=0.65,95%CI=0.47-0.90, P=0.010).4. Carriers of both risk genotypes for DDAHl rs233112and rs233113showed an increased risk for IS (OR=1.30,95%CI=1.05-1.62, P=0.017). With the risk haplotype rs233113A/rs233112A as the reference, frequencies of the haplotypes rs233113A/rs233112G and rs233113T/rs233112A were significantly lower in the IS cases (P=0.017and P=0.026, respectively).5. None of the SNPs for rs37369, rs233113and rs233112was associated with the prognosis of IS. However, carriers of the rs233113T allele (AT+TT) trended to be overrepresented in those patients with events during follow up period (P=0.087and P=0.097, respectively).Conclusions1. DDAH1rs233113and rs233112were associated with reduced risk for IS, especially in individuals with diabetes and hypertension. 2. There is synergistic effects for DDAH1rs233113and rs233112in affecting IS risk.3. The AGXT2rs37369polymorphism was not associated with the risk for IS.4. None of the polymorphisms in AGXT2rs37369, DDAH1rs233113and rs233112was associated with IS prognosis. Chapter3Regulation of DDAH1expression by miR-455-5p and the effects of DDAH1polymorphismsBackgroundWith the results from the previous chapters, we foud out that DDAHl rs233113polymorphism was associated with the risk of coronary heart disease (CHD), while DDAH1rs233113and rs233112polymorphisms were also associated with the risk of ischemic stroke (IS). Previous studies in our group have also showed difference in DDAH1expression, ADMA metabolic activity of cell lysates and intracellular ADMA concentration in primary cultured human umbilical vein endothelial cells (HUVEC) among rs233113and rs233112genotypes. However, it is not known as to whether these SNPs are functional.MicroRNAs (miRNAs) are a class of non-coding RNA with about22nucleotides, which regulate the gene expression by interacting with the3’untranslated region (untranslated regions, UTR) of target protein encoding genes.Previous study by our group has indicated that DDAHl expression could be regulated by miRNA such as miR-21. MiR-455-5p is a kind of inflammation-related miRNA, with high expression in inflammatory status. Bioinformatics analysis indicated two miRNA recognition elements (MREs) of miR-455-5p in human DDAH13’UTR. The MRE1locates at about100bp upstream of rs233113, while the rs233112was resided in MRE2. Based on the previous two chapters and related backgrounds, we hypothesized that DDAH1rs233112and rs233113polymorphisms may affect the binding/or function of miR-455-5p on DDAH1, and thus affect the expression of DDAH1and risk for arteriosclerotic cardiovascular and cerebrovascular diseases. The aims of this study was to clarify whether DDAH1is a target of miR-455-5p, and to identify the influence of rs233112and rs233113polymorphisms on miR-455-5p mediated regulation of DDAH1expression.Methods1. Effects of miR-455-5p on DDAH1mRNA and protein expression in cultured HUVEC-CHUVEC-C was transfected with different concentrations (20,40,80nM) of miR-455-5p mimics for24hours. miR-455-5p and DDAH1mRNA expression were detected by real-time PCR, DDAHl protein expression was detected by western-bloting.2. Analysis of relationship between miR-455-5p and DDAHl mRNA expression level in peripheral blood mononuclear cells (PBMC) from healthy subjects and the effects of rs233113and rs233112polymorphisms PBMC were isolated from venous samples of healthy volunteers, DDAH1rs233113and rs233112genotypes were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. MiR-455-5p and DDAH1mRNA expression were determined by real-time PCR. Pearson correlation analysis was performed to analyze the relationship between miR-455-5p and DDAHl mRNA expression levels.3. Analysis of relationship between miR-455-5p and DDAH1mRNA expression, ADMA metabolic activity and intracellular ADMA levels in primary HUVEC and the effects of rs233113and rs233112polymorphismsPrimary HUVECs were isolated from umbilical veins of newborns and cultured. The4th generation of HUVECs were cultured in6-well plates for24hours. Real-time PCR was used to detect DDAH1mRNA expression, western-blot was used to detect DDAH1protein expression, the intracellular and extracellular ADMA concentration as well as ADMA metabolic activity was determined by ELISA. The genotype of rs233113and rs233112for the umbilical vein tissue were determined by PCR-RFLP. Pearson correlation analysis was also carried out to analyze the relationships.4. Influence of tumor necrosis factor-a (TNF-a) on miR-455-5p, DDAH1mRNA and protein expression in cultured HUVEC-C HUVEC-C were treated with different concentrations of TNF-a (25,50,100ng/ml) for24hours. MiR-455-5p and DDAH1mRNA expression was detected by real-time PCR, DDAH1protein expression was detected by western-blot.5. The effects of miR-455-5p mimics on reporter gene activity of different plasmids in HUVEC-C and HEK293cellsSequences containing two MREs for miR-455-5p in DDAH13’-UTR were amplified by PCR and were then construted into the dual-reporter gene plasmid pmirGLO. Four plasmids in consideration of the four rs233113/233112haplotypes (rs233113A-rs233112A, rs233113A-rs233112G, rs233113T-rs233112A and rs233113T-rs233112G) were constructed. Plasmid with alternative mutation for the two MREs sites in combination with rs233113and rs233112polymorphisms [MRE1-A-A (wild-type), MRE1-A-MRE2mut, MRE1-T-MRE2mut, MRE1mut-A-A, MRE1mut-A-G and MRE1mut-A-MRE2mut] were also constructed. After co-transfection of miR-455-5p mimics (10,20,40nM) or negative control (20nM) with different plasmids in HUVEC-C and HEK293cells for24hours, luciferase activity of the cell lysates were detected by dual-luciferase report assay and compared the effect of miR-455-5p mimic on the reporter gene activity in different plasmids.Results 1. Transfection of miR-455-5p in to HUVEC-Cs were with different concentrations (20,40,80nM) increased the intracellular miR-455-5p expression increased in dose-dependent manner (P<0.05), while mRNA expression of three DDAH1transcript variants and the DDAH1protein were decreased significantly (P<0.05).2. Treatment of HUVEC-C cells with100ng/ml TNF-a reduced mRNA expression of DDAHl transcript variants significantly (P<0.05), TNF-a at concentrations of50ng/ml and100ng/ml reduced DDAH1protein expression significantly (P<0.05).3. In PBMC with rs233113T allele, miR-455-5p mRNA expression was correlated negatively with DDAH1-V1mRNA expression significantly (R=-0.636, P=0.026, n=12). In primarily cultured HUVECs, a significantly negative correlation between miR-455-5p and DDAH1-V1mRNA expression levels was also observed (R=-0.512, P=0.018, n=21). When analyzed according to DDAH1genotypes, the significant negative correlation was observed in HUVEC carrying the rs233113T allele or rs233112G allele (R=-0.764, P=0.017, n=9and R=-0.660, P=0.010, n=14, respectively).4. In primarily cultured HUVECs, miR-455-5p expression level correlated negatively with ADMA metabolizing activity of the cell lysates (R=-0.60, P=0.036, n=12), but correlated positively with intracellular ADMA levels (R=0.628, P=0.029, n=12). 5. In both HUVEC-C and HEK293cells, cotransfection of miR-455-5p mimic (10,20,40nM) decreased the reporter gene activity of the plasmids bearing the rs233113A-rs233112A, rs233113A-rs233112G and rs233113T-rs233112A haplotype; but for plasmid bearing the rs233113T-rs233112G haplotype, only20nM and40nM miR-455-5p mimic showed inhibitory effect on reporter gene activity in HUVECs, and only40nM miR-455-5p mimic showed inhibitory effect on reporter gene activity in HEK293.6. In HUVEC-C cells, miR-455-5p mimic at all three concentrations (10,20,40nM) decreased the reporter gene activity of MRE1-A-A and MRE1-A-MRE2mut plasmid,20nM and40nM miR-455-5p mimic decreased the reporter gene activity of the MRE1mut-A-A plasmid. MiR-455-5p mimic had no effect on the reporter gene activity of MRE1-T-MRE2mut, MRE1mut-A-G or MRE1mut-A-MRE2mut plasmids.Conclusions1. DDAH1is the target gene of miR-455-5p, miR-455-5p suppresses DDAH1expression through the two MRE in DDAH13’-UTRo2. TNF-a decreases DDAH1expression possible through up-regulation of miR-455-5p expression.3. Inhibition of DDAH1by miR-455-5p was affected by rs233113and rs233112polymorphisms.4. There is synergistic effects of DDAH1rs233113and rs233112polymorphism DDAH1regulation. |
PDF Full Text Request