The Role Of DDAH2/ADMA Pathway In Mitochondrial Dysfunction Of Endothelial Progenitor Cells Induced By High Glucose And Its Mechanisms

Background:Vascular diseases are main complications of diabetes mellitus and are also the leading cause of diabetic-related disability and death. Diabetic vascular complications include macrovascular complications (such as hypertension and atherosclerosis) and microvascular complications (such as diabetic retinopathy and diabetic nephropathy). It has been considered that vascular endothelial dysfunction and impaired vascularization are contributed to the occurance and development of diabetic vascular complications. Endothelial progenitor cells (EPCs), a kind of precursor cells which can proliferate and differentiate into mature endothelial cells, promote endothelial repair and neovascularization. Clinical and animal research showed that the number of circulating EPCs in diabetic patients and diabetic animals reduced accompanied with impaired vascular formation ability, suggesting that the alteration of EPCs function may be related to initiation and development of diabetic vascular complications. Recent studies have demonstrated that mitochondrial dysfunction is closely related to diabetes and diabetic vascular complications. Decreased mitochondrial ATP generation and reduced mitochondrial oxidative phosphorylation were detected in skeletal muscle, liver, heart, retinal microvessel and adipose tissue in type1and type2diabetes mellitus animal and patients. Therefore, regulating mitochondrial function may become an important target for the treatment of diabetes and diabetic vascular complications. Up to now, it is still unclear that whether mitochondrial dysfunction of EPCs is related to the occurance and development of diabetic vascular complications. Voltage-dependent anion channel (VDAC), a key channel protein for the maintenance of mitochondrial function, plays important roles in regulating the exchange of the inside and outside of mitochondria in energy metabolism and substance metabolism. It was reported that VDAC1significantly increased in type1diabetes mellitus, type2diabetes mellitus and diabetic microvascualr complications. Based on the maintenance of vascular function and neovascularization by EPCs, the inverse relationship between the number of circulating EPCs and peripheral vascular complications of diabetes, and elevated VDAC1in diabetic animal models, the present study explored the effects of high glucose on mitochondrial function of EPCs and the relationship between VDAC1and EPCs mitochondrial dysfunction induced by high glucose. In addition, elevated endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) was reported to play important roles in insulin resistance, diabetes and diabetic vascular complications. ADMA, mainly metabolized by dimethylarginine dimethylaminohydrolase (DDAH), not only could inhibit the proliferation and migration of human umbilical vein endothelial cells, but also was related to the reduction of the number of circulating EPCs in streptozotocin (STZ)-induced diabetic rats. Furthermore, exogenous ADMA could inhibit senescence, proliferation and vessel formation of EPCs in dose-dependent manner. Recent studies have shown that ADMA was associated with mitochondrial dysfunction. Elevated endogenous ADMA concentration was closely related to the mitochondrial dysfunction of liver and heart in type1and type2diabetic rats, and exogenous ADMA could cause mitochondrial dysfunction of hepatic cells, suggesting that ADMA probably promotes the development of diabetes and its complications by interrupting the mitochondrial function of EPCs. Therefore, the present study observed the effects of high glucose on mitochondrial function of EPCs, and tested whether exogenous ADMA could also exert similar effects, and further explored whether ADMA-induced EPCs mitochondrial dysfunction was involved in the alteration of VDAC1expression.Methods:To test the effects of high glucose on EPCs mitochondrial function, EPCs were treated with glucose (10,20or30mmol/L) for12h,24h and48h, and mannitol (30mmol/L) was used as osmotic control. To explore whether DDAH/ADMA pathway is involved in mitochondrial dysfunction of EPCs induced by high glucose, EPCs were treated with ADMA (3,10,30μmol/L) for12h,24h and48h. To further investigate the role of VDAC1in mitochondrial dysfunction of EPCs stimulated by ADMA, EPCs were pretreated with VDAC inhibitor4,4’diisothiocyanatostilbene-2,2’dsulfonicacid (DIDS)(2,10or50μmol/L) for1h, then treated with ADMA (30μmol/L) for12h.EPCs were isolated by density gradient centrifugation from rat bone marrow. On the7th day, EPCs were identified by dual staining for Dil-acetylated low-density lipoprotein (Dil-acLDL) and FITC-ulex europaeus agglutinin-1(FITC-UEA-1). The mitochondrial function of EPCs was evaluated by mitochondrial membrane potential (flow cytometry) and ATP content (firefly luciferase method). The cell apoptosis were determined by Hoechst. The expression of DDAH1, DDAH2and VDAC1mRNA were analyzed by Real-time PCR. The protein expression of VDAC1was detected by Western Blotting and cell immunofluorescence. The levels of ADMA were measured by high performance liquid chromatography (HPLC). The levels of intracellular reactive oxygen species were detected by fluorescent probe of DCFH-DA.Results:High glucose significantly decreased mitochondrial ATP generation and promoted apoptosis of EPCs in dose-dependent manner, accompanied by elevated ADMA, decreased DDAH2mRNA expression and upregulated VDAC1mRNA and protein expression, but had no effect on DDAH1mRNA expression. The treatment with mannitol (30mmoI/L) had no effect on EPCs function. In cultured EPCs, exogenous ADMA markedly upregulated VDAC1mRNA and protein expression, increased intracellular ROS generation, lowered mitochondrial membrane potential, reduced ATP generation, and led to apoptosis of EPCs. VDAC inhibitor DIDS significantly inhibited the effects of ADMA on EPCs function.Conclusions:DDAH2/ADMA pathway is involved in mitochondrial dysfunction of EPCs induced by high glucose via upregulation of VDAC1expression.