Study On Role Of DDAH/ADMA Pathway In Regulation Of Endothelial Tissue Factor Expression

Acute coronary syndromes (ACS) are one of the most important causes for death of patients with cardiovascular diseases. Occlusive thrombus formation on atherosclerotic plaque is believed to be of prime importance in the cause of ACS. The activation of TF coagulating pathway plays a pivotal role in the pathophysiology of ACS. Endothelium plays a key role in maintenance of normal homeostasis. With endothelial cell activation or damage, TF expression and activity is significantly enhanced. Asymmetric dimethylarginine (ADMA) is a major endogenous inhibitor of NOS, which can inhibit NO production and induce endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) plays a key role in regulation of ADMA level in vivo. Recent several clinical studies demonstrated that the plasma levels of ADMA are higher in patients with ACS, and it has been suggested that ADMA may be a novel cardiovascular risk factor. The aims of the present study were to investigate the regulatory effects of the DDAH/ADMA pathway on endothelial TF expression and its possible mechanisms.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations (1-10μM) of ADMA for various periods (6 -12 h) or with lysophosphatidylcholine (LPC, 10μg/ml) for 24 h. Endothelial cells with overexpression of DDAH2 were prepared by transfecting hDDAH2-expressed plasmid into HUVECs. Cellular procoagulating activity was determined using a one-stage clotting assay. Messenger RNA (mRNA) levels of both TF and DDAH2 were determined by reverse transcription PCR (RT-PCR). High-performance liquid chromatography (HPLC) was employed to determine level of ADMA in cultured medium.Results: (1) Exogenous ADMA significantly increased both TF mRNA level and activity in a concentration- and time-dependent manner. Pretreatment with L-arginine (0.5 mM), the specific inhibitor of p38 mitogen-actived protein kinase (p38 MAPK) SB239063 (10μM) or intracellular antioxidant PDTC (10μM) could significantly attenuate ADMA-induced increases in both TF procoagulating activity and mRNA level.(2) Treatment with LPC (10μg/ml, 24 h) markedly increased both TF procoagulating activity and mRNA level in HUVECs. Such treatment also markedly elevated the level of ADMA in cultured medium. Overexpression of DDAH2 could attenuate LPC-induced increases in both TF procoagulating activity and mRNA level.(3) All-trans-retinoic acid (atRA, 1-10μM) concentration-dependently inhibited attenuate LPC-induced increases in both TF procoagulating activity and mRNA level. Moreover, atRA could markedly upregulate the mRNA level of DDAH2 of endothelial cells and decrease ADMA level of cultured medium in concentration-dependent manner.Conclusions: (1) ADMA increased endothelial TF procoagulating activity by upregulating TF mRNA expression via ROS-p38 MAPK-dependent pathway.(2) The DDAH/ADMA pathway mediates LPC-induced increase in TF expression.(3) The inhibitory effects of atRA on LPC-induced increase in endothelial TF expression may involve the upregulation of DDAH expression.