Role Of ADMA In Homocysteine-induced Apoptosis Of Vascular Smooth Muscle Cells

Role of ADMA in homocysteine-induced apoptosis of vascular smooth muscle cellsBAKGROUNDArtherosclerosis(AS)is one of the causes to induce cardiovascular diseases.Atheromatous plaque becoming unstable forming aortic aneurysms or calcification results in the complications of AS,which can be induced sufficiently by the apoptosis of vascular smooth muscle cells (VSMC)alone.There is substantive evidence to suggest that one mechanism of homocysteine(Hcy)inducing the progress of AS is the bioactivity of inducing the apoptosis of VSMC.Asymmetric dimethylarginine(ADMA)is an endogenous L-arginine analogue. Recently,it has bee reported that ADMA can induce the 'NOS uncounpling' to increase intracellular reactive oxidative species(ROS) and activate the ROS-p38 mitogen-activated protein kinases (MAPKs)-dependent signaling pathway to induce the apoptosis of endothelial cells.Dimethylarginine dimethylaminohydrolase(DDAH)is the main metabolic pathway of ADMA and Hey can inhibit the bioactivity of DDAH resulting in the accumulation of ADMA.Therefore, in the present study,we investigated the role of ADMA in Hey-induced VSMC apoptosis and its underlying mechanisms as well.METHODST/G HA-VSMC were cultured,pEGFP-Cl-hDDAH2 vector was constructed for transient transfection by Lipofectamin2000.The percentage of transfected cells were detected using fluorescence microscope;the expression of hDDAH2 of the transfected cells were measured by Western Blot;apoptosis of VSMC was detected using Hoechst33342 staining or flow cytometry(FCM)(Becton Dichinson) with AnnexinV+ Propidium Iodide(PI);the level of ADMA in cell culture was measured by high-performance liquid chromatography-mass spectrum(HPLC-MS);ROS was determined using fluorescent ROS detection kit;the phosphorylation of JNK and p38MAPK was determined by Western blot.RESULTS(1)Incubation of VSMC with Hcy(1 mM)for 48 h significantly induced cell apoptosis,concomitantly with a marked elevation of ADMA level in the medium.The elevation of ADMA level and cell apoptosis induced by Hcy in the cells with hDDAH2-overexpression was markedly attenuated compared with wild-type cells.Exogenous ADMA(10μM,48 h)markedly induced apoptotosis of wild-type VSMC.(2)ADMA(10μM,6 h)significantly increased the intracellular ROS production and activated JNK and p38MAPK.Pretreatment with antioxidant PDTC or L-arginine inhibited the percentages of VSMC-apoptosis induced by ADMA(10μM,24 h),attenuated the production of ROS as well as depressed phosphorylation of JNK and p38MAPK.(3)ADMA(10μtM,6 h)significantly activated JNK and p38MAPK in VSMC.Both JNK specific inhibitor SP600125(5μM)and p38MAPK specific inhibitor SB20358(10μM)significantly attenuated ADMA-induced cell apoptosis.CONCLUSIONS1.Apoptosis of VSMC induced by Hcy is related to elevation of ADMA.2.AMDA-induced VSMC apoptosis is mediated by ROS-JNK/ p38MAPK signaling pathways.