Effects Of Metallothionein And Homocysteine On PAI-1 And Its MRNA Expression By Cultured Endothelial Cells

Coronary heart disease (CHD) is a common disease that threats the health of people seriously, the pathogenesis has not been understand sufficiently. The effects of hyperhomocysteinemia(HHcy) and endothelial dysfunction on atherosclerosis(As) have caused much attention in the past years. The imbalance of coagulaton and fibrinolysis which is characterized by an increase in coagulaton function and a decrease in fibrinolysis function is responsible for As. Homocysteine(Hcy) is a mid-product in methionine metabolization. It is generally believed that HHcy is an independent risk factors of cardiovascular disease. Hcy may accelerate the progression of As by many biological effects, such as inducing platelet aggregation, stimulating vascular smooth muscle cells (VSMCs) proliferation, jeopardizing vascular endothelial cells (VECs), etc. Investigation has shown that HHcy is responsible for endothelial dysfunction. Endothelial dysfunction is a systemic disorder, it is characterized by a reduction of the bioavailability of vasodilators, particularly nitric oxide(NO), and a increase of endothelium-derived contracting factors. This imbalance leads to an impairment of endothelium-dependent vasodilation. On the other hand, besides the decline in endothelium-dependent vasodilation, endothelial dysfunction reflects a decrease in endothelium-derived anticoagulation medium, such as plasminogen activator inhibitor- l(PAI-l) and tissue factor, and a proinflammationstate, which contribute to the formation of As. Once the normal function of VECs was affected, substances such as NO, PGI2, t-PA, which released from VECs decline gradually, whereas PAI-1, ET-1 and TXA2 increase sharply. Investigation has shown that the increase in plasma PAI-1 level plays an important role in the pathogenesis of As. Metallothionein (MT) is a sort of low molecular protein prolific of cysteine that can eliminate free radicals, restrain lipid oxidation and it has been regarded as one of the most intense elimator of籓H. The effect of MT as an exogenous cells protecting protein in ischemia-reperfusion injury has been reported and caused much attention recently.The objects of this study include: (1) To investigate the effects of homocysteine of different dosage on the activity of PAI-1 and the expression of PAI-1 mRNA by cultured human umbilical vein endothelial cells (HUVECs) after treating 6 hours. (2) After treated by 1 .0 mmol/L Hey with or without different dosage of MT , the difference of PAI-1 activity and the expression of PAI-1 mRNA of HUVECs wrer compared. HUVECs were cultured by DMEM supplemented with 15% PCS. Experments were performed on 6 to 10 passage cells. The HUVECs were seeded at a density of 10" cells per well in 96-wells plate and grow to confluence. Upon confluence the medium was replaced with DMEM for 24 hours to achieve a basal state. The experiment was devided into 2 stages. The effects of Hey on the activity of PAI-1 and the expression of PAI-1 mRNA were investigated after treating 6 hours in stage 1. The HUVECs were separate into 4 ~groups:(l) Control group(C group), with the medium lacking experimental agents. (2) Hey group I , with the medium containing Hey 0. 1 mmol/L.(3) Hey group II ,with the medium containing Hey 0.5mmol/L (4) Hey group III, with the medium containing Hey 1.0mmol/L. The protein and mRNA levels of PAI-1 secreted by HUVECs after being treated by 1.0 mmol/L Hcy with or without different dose of MT were compared in stage 2. The HUVECs were then seperated into 5 groups. (1 )Hcy group, with the medium containing Hcy 1.0mmol/L. (2)Hcy+MT, group, with the medium containing Hey 1.0 mmol/L plus MT 1 X 10-6 mmol/L. (3) Hcy+MT2 group, with the medium containing Hcy 1.0mmol/L plus MT 5 X 10"6 mmol/L. (4) Hcy+MT3group, with the medium containing Hcy 1.0 mmol/L plus MT 1 X 10"5 mmol/L. (5) Hcy+MT4 group, with the medium containing Hcy 1.0 mmol/L plus MT 5 10"5 mmol/L. The medium and cells from each well were collected after 6 hours. PAI-1 activity was detected by chromogem'c assay; and mRNA levels of PAI-1 expression we...