| BackgroundNitric oxide (NO) is an important signaling molecule playing crucial roles in maintaining vascular endothelial function and cardiovascular homeostasis. It exerts cardiovascular protective effects through direct vasodilation, preventing vascular smooth muscle cell proliferation, inhibiting platelet aggregation and adhesion, and inhibiting infiltration of inflammatory cells. NO is synthesized by NO synthase (NOS) from L-arginine in the body. Asymmetric dimethylarginine (ADMA) is an endogenous NOS inhibitor that can inhibit NO production competitively and thus results in endothelial dysfunction. Increased plasma ADMA level has been proved to be a novel risk factor for cardiovascular events and a direct predictor of the prognosis for cardiovascular diseases.The majority of ADMA formed in the body is hydrolyzed to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolases (DDAH1and DDAH2). DDAH1is the major enzyme responsible for the clearance of circulating ADMA, while DDAH2is important in the regulation of endothelium dependent relaxing factor (EDRF)-induced vasodilatation.Two transcript variants of DDAH1are deposited in the NCBI database, namely DDAH1-V1and DDAH1-V2. However, the contribution of the transcript variant in ADMA metabolism remains unknown. This study was designed to confirm the expression of DDAH1transcript variants, to elucidate the relationship between expression levels of DDAH1transcript variants, and to investigate the relationship between DDAH1transcript expression and ADMA metabolism in primarily cultured human umbilical vein endothelial cells (HUVECs) from healthy newborns. Methods1. Confirmation of the expression of DDAHl transcript variants in human DDAH1transcript variants specific RT-PCR primers were designed and used to detcect the expression of the transcript variants in primarily cultured HUVEC, HUVEC-C cell line, HepG2cell line and peripheral blood mononuclear cells (PBMC) from healthy volunteers.2. DDAH1transcript variants specific reverse transcription PCR (RT-PCR) primers were used to amplify DDAH1-V2cDNA including the coding sequence (CDS) region and5’-UTR. The RT-PCR products were underwent2%agarose electrophoresis, the DNA band on the gele were purified, and the PCR products were sequenced.3. Semi-quantitative determination of DDAH1transcript variants mRNA expression and determination of ADMA metabolizing activity in primary HUVECs.HUVECs were isolated from12human umbilical cords, DDAH1transcript variants mRNA expression were determined by real-time semi-quatitative PCR. ADMA metabolizing activity in cell lysates were determined by enzyme-linked immunosorbent assay (ELISA).Results1. The targed band for DDAH1-V2transcript was detected in all the cells studied. An additional slightly shorter band in electrophoresis agarose of the RT-PCR products for DDAH1-V2variant was observed. Sequencing of the band indicated a novel transcript variant which was designated as DDAH1-V3. The DDAHl-V3variant shared all other6exons but Exon2’with DDAH1-V2.2. In HUVECs isolated from12healthy newborns, DDAHl-V3mRNA expression correlated positively with both DDAH1-V2(R=0.811, P=0.000008, n=21) and DDAH1-V1(R=0.454, P=0.039, n=21) mRNA expression.3. In HUVECs from12neborns, the mRNA expression level of DDAH1-V1correlated positively with ADMA metabolizing activity of the cell lysates from the corresponding individuals (R=0.805, P=0.002). Ne association between either DDAH1-V2or DDAH1-V3mRNA expression and ADMA metabolizing activity of the cell lysates was observed.Conclusions1. DDAH-V3variant is a novel transcript variant of DDAH1.2. DDAH1-V2mRNA expression level correlated positively with DDAH1-V3mRNA expression, but showed no correlation with DDAH1-V1mRNA expression in primarily cultured HUVECs.3. DDAHl-V1mRNA expression correlated positively with ADMA metabolizing activity in primarily cultured HUVECs. BackgroundEndothelial dysfunction is suggested to be the pathophysiological basis and initiating factor for several cardiovascular diseases. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) which can results in endothelial dysfunction by competitively inhibits NO production. ADMA plays an important role in the development of cardiovascular diseases (CVDs) such as pulmonary arterial hypertension (PAH), atherosclerosis, stroke, and renal diseases. Increase in plasma ADMA level is regarded as a novel independent risk factor for CVDs, especially for thrombotic diseases such as acute coronary syndrome and stoke.About80%of ADMA in the body is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1and DDAH2). DDAH1is the key enzyme responsible for elimination of ADMA in plasma. Two human DDAH1transcript variants could are deposited in the National Center of Biotechnology Information (NCBI) database, one is widely studied in previous literatures, while one novel transcript variant has never been studied. In HUVECs isolated from healthy newborns, we confirmed the expression of DDAH1two transcript variants, and identified a novel transcript namely DDAH1-V3in the previous section. We observed that the mRNA expression of DDAH1-V3and DDAH1-V2correlated positively. However, only the mRNA expression of DDAHl-V1transcript variant correlated positively with intracellular ADMA metabolizing activity. DDAHl-V1transcript is reported to be important in the development of cardiovascular diseases (CVDs)We hypothesize, however, the significance of DDAH1-V1and DDAH1-V3variant in CVDs remains unknown. This study was designed to elucidate the correlationships between the mRNA expression levels of DDAH1transcript variants in PBMCs from healthy controls and in patients suffering from either acute ischemic stroke (AIS) or acute myocardial infarction (AMI).Methods1. Analysis the correlations between the expression of different DDAH1transcript variants mRNA in PBMCs from health volunteers. PBMCs from36health volunteers we isolated. RNA were extracted and cDNA were synthased. DDAH1transcript variants mRNA expression levels were determined by real-time quatitative PCR by transcript variant specific real-time PCR primers.2. Analysis the correlations between the expression of different DDAH1transcript variants mRNA in PBMCs from acute ischemic stroke (AIS) patients.PBMCs were isolated from35patients of acute ischemic stroke (AIS) within6hours after disease onset. DDAH1transcript variants mRNA, expression were determined by real-time quatitative PCR with transcript variant specific real-time PCR primers.3. Analysis the correlationships between the expression of different DDAH1transcript variants mRNA in PBMCs from the patients of acute myocardial infarction (AMI).PBMCs were isolated from35patients suffered from acute myocardial infarction (AMI) within6hours after disease onset. DDAH1transcript variants mRNA expression was determined by real-time quatitative PCR with transcript variant specific real-time PCR primers.4. Comparison of expression levels of DDAH1transcript variants in healthy controls and in patients suffering from AIS or AMI.The cycle threshold (CT) values of real-time PCR and ACT values of DDAH1transcripts relative to the endogenous control GAPDH mRNA (CTDDAHI transcripts-CTGAPDH) of each transcript variant in PBMCs with different disease status were calculated. Difference inACT values among groups were analyzed by one-way analysis of variance (ANOVA).Results1. Significant correlation between DDAH1-V2and DDAH1-V3mRNA expression was observed in PBMCs from healthy subjects as indicated by partial correlation (R=0.571,P=0.001, n=36)2. Significant correlations between pair-wise transcript variants were observed in AIS patients (V1and V2:R=0.700,P=0.00003; V1and V3: R=0.457,P=0.014; V2and V3:R=0.616, P=0.0005, n=35)3. Significant correlations between pair-wise transcript variants were observed in AMI patients (V1and V2: R=0.651, P=0.001; V1and V3: R=0.493, P=0.014; V2and V3:R=0.544, P=0.006, n=30)4. In PBMCs from healthy subjects, the mean CT values for DDAH1-V1,-V2and-V3transcripts were29.6±1.5,32.2±2.0and29.6±1.7, respectively. As for AIS and AMI patients, the corresponding values were28.7±1.1,32.2±2.3,29.7±1.3and28.7±1.5,32.1±2.3,29.3±1.5, respectively. CT values for DDAH1-V1were significantly lower than those of-V2and-V3in PBMCs regardless of disease status.Conclusions1. Significant correlations between pair-wise transcript variants in AIS patients2. Significant correlations between pair-wise transcript variants were in AMI patients3. The expression of DDAH1-V1were significantly lower than those of-V2and-V3in PBMCs regardless of disease status. BackgroundCardiovascular disease (CVD) is a class of diseases that becomes a major threaten to human health. CVDs include hypertension, atherosclerosis, coronary heart disease, heart failure and stroke. A lot of similarities in the pathogenesis of CVDs are reported. Inflammatory factors and pathological physiological changes they bring up play important roles in the development of these diseases. They may play the role of a bridge and intermediary between hypertension and AS, two major basic diseases for cardio-cebrovascular diseases.Changes of internal environment and many inflammatory cytokines produced by different cells, such as interferon-y (IFNy), tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β) will damage the morphology and function of endothelial cell. Endothelial dysfunction is due to destruction of the balance between vasodilatation and vasoconstriction. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthesis (NOS) inhibitor, which can inhibit eNOS activity and NO production. DDAH1play a major role in removal ADMA from vascular endothelial cells and blood. Different inflammatory cytokines and pathological/physiological changes caused by them could regulate the expression of DDAH1transcript variants. This may lead to changes in the level of ADMA in vivo, and thus the function of the cardiovascular system maybe affected.In this research, we studied changes in the expression of DDAHl transcript variants that caused by various inflammatory factors and physiological and pathological changes. To investigate the changes of the environment regulate DDAH1transcripts expression and its possible role in the molecular mechanisms. Methods1. Smoking and drinking impact the expression of DDAH1gene transcript variants in PBMC from health volunteers who carrying different DDAH1rs233113A/T genotype Isolate the PBMCs from36health volunteers. Then genotype DDAH1rs233113A/T locus. DDAH1transcript variants mRNA expressions were determined by real-time quantitative PCR and transcript variant specific Real-time PCR primers.2. Use DDAH1-siRNA transfected human umbilical vein endothelial cells to detect changes of different transcripts DDAH1expression Using different concentrations of three kinds DDAH1-siRNA transfected human umbilical vein endothelial cell line, and then DDAH1transcript variants mRNA expressions were determined by real-time quantitative PCR and transcript variant specific Real-time PCR primers.3. Cultured human umbilical vein endothelial cells, the cells were treated with different drugs, testing different transcripts DDAH1expression Treat cultured human umbilical vein endothelial cells with different concentrations of angiotensin II, tumor necrosis factor-a, high glucose, simvastatin for24h respectively. DDAH1transcript variants mRNA expressions were determined by real-time quantitative PCR.Results1. The expression of DDAH2-V3was significantly reduced in healthy smokers carry DDAH1rs233113AT type (AA, n=8; AT, n=4;*p<0.05);2.3kinds of DDAHl-siRNA(5、10、20、50nM) can significantly decrease DDAH1transcript variants mRNA expressions in HUVEC-C(V1、V2、V3、p<0.01, compare with mock);3. Angiotensin Ⅱ (10-5mol/L) can significantly decrease DDAH1transcript variants mRNA expressions in HUVEC-C (V1and V3, p<0.05, compare with control; p<0.05, compare with Ang Ⅱ10-7mol/L; V2,p<0.05, compare with control); 4. TNF-α (50、100ng/ml) can significantly decrease DDAH1transcript variants mRNA expressions in HUVEC-C (V1and V3, p<0.05, compare with control; p<0.05, compare with TNF-α25ng/ml; V2, p＜0.05, compare with control);5. In HUVEC-C, high concentration of glucose(HG)(40mmol/L) can significantly decrease DDAH1-V1mRNA expressions(p＜0.05, compare with mannitol);50mmol/L HG can significantly decrease DDAH1-V2mRNA expressions(p<0.05, compare with mannitol); HG can significantly decrease DDAH1-V3mRNA expressions(p<0.05,50mmol/L compare with mannitol;p<0.05,40mmol/L compare with mannitol);6. In HUVEC-C, simvastatin(2μM) can significantly decrease DDAH1-V2mRNA expressions (p<0.05, compare with DMSO, p＜0.05, compare with0.2μM) and simvastatin(1、2μM) can significantly decrease DDAH1-V3mRNA expressions (p<0.05, compare with DMSO)Conclusion1. The expression of DDAH2-V3was significantly reduced in healthy smokers carry DDAH1rs233113AT type;2. DDAH1-siRNA can significantly decrease DDAH1transcript variants mRNA expressions in HUVEC-C;3. Angiotensin II can significantly decrease DDAH1transcript variants mRNA expressions in HUVEC-C;4. TNF-α can significantly decrease DDAH1transcript variants mRNA expressions in HUVEC-C;5. High concentration of glucose can significantly decrease DDAH1-V1mRNA expressions in HUVEC-C;6. Simvastatin can significantly decrease DDAH1-V2、DDAH1-V3mRNA expressions in HUVEC-C... |
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