The Regulatory Effect Of Dimethylarginine Dimethylaminohydrolase On Function And Senescence Of The Endothelial Progenitor Cell: Role In Dysfunction Of Vessels In Diabetes Mellitus

Endothelial progenitor cells (EPCs), which can differentiate into mature endothelial cells and myocytes, promote neovascularization and improve cardiac function. EPCs participate in the reparation of vessles and angiogenesis, and are used for the treatment of ischemic diseases.Vascular endothelial growth factor (VEGF) is a potent factor regulating EPCs functions, including reendothelization and angiogenesis. The effects of VEGF have been shown to mediated by type 2 VEGF receptor (KDR).It is know that dimethylarginine dimethylaminohydrolase (DDAH) metabolizes asymmetric dimethylarginine (ADMA), which is endogenous inhibitor of nitric oxide synthase (NOS) activity and inhibits nitric oxide (NO) production. It has been demonstrated that the DDAH/ADMA pathway is involed in senescence of endothelial cell and angiogesis.SIRT1, human silent information regulator 2 (Sir2), is a potent NAD+-dependent protein deacetylase and plays an important role in the maintenance of gene silencing. SIRT1 inhibites senescence of endothelial cells through the DDAH2/ADMA pathway. It has been shown that SIRT1 is involved in the senescence of EPCs induced by high glucose.Based on the regulatory effect of DDAH/ADMA and SIRT1 on angiogenesis and cell senescence, and SIRT1-mediated the effect of DDAH/ADMA, therefore, in the present study we tested the effect of DDAH/ADMA and SIRT1 on senescenc and fucntion of EPCs.METHODSEPCs were isolated by density gradient centrifugation from human peripheral blood mononuclear cells, and cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 Single-Quots. On the 7th day, EPCs were characterized by dual staining for Dil-acetylated low-density lipoprotein (Dil-acLDL) and FITC-ulex europaeus agglutinin-1 (FITC-UEA-1), and by flow cell markers including CD 133, CD34, KDR, CD45 and von willebrand factor (vWF).To test the effect of DDAH on the differentiation and function of EPCs, mRNA expressions of DDAH1 and 2 were detected at 7th day, and the expressions of DDAH2 and SIRT1 mRNA and protein were detected at 7th and 17th days. To study the regulatory effect of DDAH2 on senescence and fucntion of EPCs, cells were transfected with pGCSIL-GFP-hDDAH2 shRNA to interrupt expression of DDAH2 protein. To study the regulatory effect of SIRT1 on DDAH2, cells were transfected with pGCSIL-GFP-hSIRT1 shRNA to interrupt expression of SIRT1 protein. The levels of DDAH1, DDAH2, VEGF, KDR and SIRT1 mRNA were analyzed by Real-time PCR. Protein expressions of DDAH2 and SIRT1 were detected by flow cytometry or Western Blotting. The level of ADMA was detected by high performance liquid chromatography (HPLC). Cell senescence, angiogenesis and adhesion were determined by senescence-associatedβ-galactosidase (SA-β-gal) activity assay, tube formation and fibronectin.RESULTSPeripheral blood EPCs predominantly expressed DDAH2 which was increased with EPCs differentiation. Interrupting DDAH2 expression induced EPCs senecence and dysfunction, including angiogenesis and adhesion. The expressions of VEGF and KDR mRNA were down-regulated. The expression of SIRT1 was increased with EPCs differentiation. Interrupting SIRT1 inhibited the expressions of DDAH2, VEGF and KDR, but had no effect on the level of ADMA.CONCLUSIONDDAH2 participated in the differentiation of EPCs and regulated the senescence and fucntion of EPCs through the VEGF/KDR pathway by the activation of SIRT1. Vascular disease is a main complication of diabetes mellitus. Clinical and animal researches have showed that diabetes accelareted EPCs senescence and attenuated EPCs function. And high glucose induced EPCs senescence and impaired angiogenesis, resulting in dysfunction of vessels and reduction of collateral circulation. These results suggest that EPCs senescence may be related with the development of cardiovascular diseases.ADMA, a major endogenous inhibitor of NOS, has been considered as a novel risk factor for cardiovascular diseases. It has been demonstrated that the plasma level of ADMA is elevated and NO concentration is decreased concomitantly with a reduction of expression and acitvity of DDAH in patients with diabetes. Clinical and animal researches have showed that dysfunction of EPCs is related with reduction of NO level in diabetes. Exogenous ADMA directly induced the damage of EPCs. Studies in vivo or in vitro have documented that high glucose treatment induced senescence and angiogenesis impairment of EPCs and down-regulation of SIRT1 expression. It has been reported that the regulatory effect of SIRT1 on endothelial cells senescnece is mediated by the DDAH2/ADMA pathway.In the present experiment, we explored the regulatory effect of DDAH2/ADMA on sensecence of EPCs in diabetic rats or EPCs treated with high glucose. Since SIRT1 plays an important role in the development of senescence of EPCs, we also tested the effect of SIRT1 on the DDAH2/ADMA pathway.METHODSForty control people and 40 T2DM patients were recruied, and control people were age and sex matched with T2DM patients. The level of ADMA in plasma was detected by HPLC. EPCs were isolated by density gradient centrifugation from human peripheral blood mononuclear cells. EPCs senescence was evaluated by SA-β-gal activity and the expression of DDAH2, SIRT1 mRNA was measured by Real-time PCR.EPCs were treated with glucose (10,20 or 30 mmol/L) for inducing senescence, and mannitol(10,20,30 mM) was used as osmotic pressure control. To study the effect of DDAH2 on senescence of EPCs, EPCs were transfected with pGC-FU-hDDAH2. ADMA(0,0.3,0.6,1μmol/L) was used to directly induce senscence of EPCs. The effect of resveratrol (RSV), a agonist of SIRT1, on senscence of EPCs was observed.Peripheral blood mononuclear cells were collected for isolating EPCs. EPCs senescence was evaluated by SA-β-gal activity. The expressions of DDAH2 and SIRT1 mRNA or protein were measured by Real-time PCR and by Wetern Blot, respectively. The level of ADMA was detected by HPLC, and the level of NO was detected by Griess.RESULTSIn T2DM patients, the percentage of EPCs senescence was increased, while the expressions of both DDAH2 and SIRT1 mRNA were down-regulated concomitantly with an increase of concentrations of ADMA in plasma.High glucose induced senescenc of EPCs in a dose-dependent manner. The mRNA and protein expression of DDAH2 were down-regulated, and level of NO was decreased while level of ADMA was increased in the presence of high glucose. Overexpression of DDAH2 reversed the effect of glucose, but did not affect NO concentration in cultured EPCs. ADMA induced the senescence of EPCs in a dose-dependent manner. High glucose treatment down-regulated the mRNA and protein of SIRT1, which an effect was attenuated by RSV. CONCLUSIONDDAH2/ADMA pathway participats in senescence of EPCs induced by high glucose, which is related with activation of SIRT1. Resveratrol (trans-3,5,4'-trihydroxystilbene) is a natural activator of SIRT1. Growing evidence suggests that SIRT1 regulates glucose and insulin secretion. It has been showed that in diabetic rats, RSV exerts multiple benificial effects including upregulating expression of VEGF and eNOS, inhibiting oxidative stress and mobilizing angiogenesis. In cultured EPCs, RSV can induce cell proliferation, migration and angiogenesis. Others have found that RSV reduced the injury of EPCs by TNF-a.(E)-3,5,4'-trimethoxystilbene (BTM-0512) is the methylated derivative of RSV. The absorbance rate of BTM-0512 is higher than RSV, and its half-life is extended.Based on the protective effect of RSV on senescence of endothelial cells and EPCs and the regulatory effect of SIRT1-DDAH2/ADMA pathway on EPCs function, therefore, we tested the influence of BTM-0512 on insulin resistance and senescence of EPCs in type 2 diabetic rats and cultured EPCs. METHODSType 2 diabetic rat were established by bonus injection of streptozotocin (STZ,35 mg/kg, intraperitonealy) and feeding high-fat/high-sucrose diet. Rats were divided into 4 groups:control, T2DM, T2DM plus BTM-0512(10 mg/kg) and T2DM plus BTM-0512(40 mg/kg). The animals were treated with drugs for 3 weeks.The concentrations of fasting blood glucose (FBG) and glycosylated hemoglobin (Hb1AC), the level of fast insulin (FIns) and oral glucose tolerance (OGTT) were detected. The values of HOME.IR, insulin activity index (IAI) and insulin secretion index (IS) were calculated to evaluate insulin resistance (IR). The level of ADMA was measured. Vasodilator responses to acetylcholine in aortic rings and the protein expression of expression of DDAH2 and SIRT1 in the isolated aortia were measured. EPCs were isolated by density gradient centrifugation from rat bone marrow. Cell senescence, angiogenesis, adhesion and migration were determined by SA-β-gal activity assay, tube formation, fibronectin and transwell assay respectively. The mRNA expressions of DDAH2 and SIRT1 were measured by Real-time PCR.EPCs from rat bone marrow were characterized by dual staining for Dil-acLDL and UEA-1 on the 7th day. EPCs were treated with glucose (30 mmol/L) for inducing senescence. To study the effect of BTM-0512 on senescence of EPCs, EPCs were pretreated with BTM-0512 (0-3 μmol/L). To study the role of SIRT1 in BTM-0512 effect, EPCs were pretreated with splitomicin (50μmol/L), the special inhibitor of SIRT1.EPCs senescence was evaluated by examining SA-β-gal activity. The mRNA expressions of DDAH2 and SIRT1 were measured by Real-time PCR. Protein expression was detected by Wetern Blot. The level of ADMA was detected by HPLC. The level of NO was detected by Griess.RESULTSBTM-0512 significantly decreased the level of FBG and Hb1 AC in a dose-dependent manner. BTM-0512 improved the HOME.IR and IAI, but had no effect on OGTT or IS. BTM-0512 improved vasodilator responses to acetylcholine in diabetic rats. BTM-0512 inhibited senescence of EPCs and up-regulated the expression of DDAH2 and SIRT1 protein of endothelial cells as well as the expression of DDAH2 and SIRT1 mRNA of EPCs.BTM-0512 attenuated senescence of EPCs induced by high-glucose in a dose-dependent manner. BTM-0512 also reversed high glucose induced the downregulation of SIRT1 and DDAH2 mRNA expression with increased the level of ADMA, which were reversed by splimtomicin (50μM), the sepecial inhibiotr of SIRT1. CONCLUSION1. BTM-0512 reduced fasting blood glucose and improved insulin resistance in type 2 diabetic rats. BTM-0512 also improved endothelial function in diabetic rats.2. BTM-0512 exerted the beneficial effects on high glucose induced EPCs senescence, and the effects of BTM-0512 was related to activation of SIRT1-DDAH2/ADMA pathway.