Effects Of Nicotine On The Expression Of T-PA And PAI-1 In Cultured Human Umbilical Vein Endothelial Cells And Its Related Mechanism

BackgroudEpidemiological surveys have shown that cigarette smoking has an influence on both arterial-type and venous-type thrombosis. It could damage the vascular endothelial cells, lead to vascular endothelial cells dysfunction, as well as trigger fibrinolysis imbalance, then increasing the risk of thrombosis. As the major anticoagulation system in the body, fibrinolytic system relies heavily on the activity of tissue-type plasminogen activator(t-PA)and tissue-type plasminogen inhibitor-1(PAI-1) released by vascular endothelial cells. A majority of experimental studies associated with smoking and coagulation-fibrinolysis of endothelial cells focus on the coagulation system. But studies about cigarette smoking on fibrinolytic activity of vascular endothelial cells are rare, and its related mechanism is entirely unclear.Protein kinase C (PKC) is an important biological intracellular signal transduction pathway in the body. Staurosporine (STS), a specific inhibitor, can block PKC expression. Nicotine, one of the main harmful substances of smoking, can damage the fibrinolytic function of endothelial cells. But the mechanism of nicotine on the fibrinolytic damage of endothelial cells is unclear. Whether the PKC signaling pathway plays a role in fibrinolytic dysfunction caused by nicotine needs to be investigated.ObjectiveTo study the effect of nicotine on the expression of secreting t-PA and PAI-1 in Human Umbilical Vein Endothelial Cells (HUVECs) on the different experimental conditions in order to select the best time and concentration of nicotine. STS was used to investigate the possible role of PKC in the process of t-PA or PAI-1 protein and mRNA expression in HUVECs induced by nicotine.Methods(1) HUVECs were cultured in 24-well tissue-culture plates and randomly divided into the control group and nicotine treated group.①Concentration-dependent effect of nicotine on the expression of t-PA and PAI-1:HUVECs were incubated with 0,0.1,1,10,100μmol/L nicotine for 12 hours respectively.②Time-dependent effect of nicotine on the expression of t-PA and PAI-1:The cells were exposured to 100μmol/L nicotine for 0,4,6,8,12,24 hours respectively. The cells of control groups were exposured to the same volume of PBS instead of nicotine at each time points. The supematants were collected respectively. The expression of t-PA and PAI-1 were determined by the enzyme linked immunosorbent assay (ELISA).(2) STS intervention trials. HUVECs were cultured in 25 cm×25cm culture flasks and randomly divided into the control group, 100μmol/L nicotine group, 100nmol/L STS group,and nicotine+STS group. The cells and supernatants of each group were collected after 12 hours cultured.The expression of t-PA and PAI-1 protein were measured by ELISA and the expression of t-PA and PAI-1 mRNA were determined by RT-PCR.Results(1) Compared with control group,the expression levels of PAI-1 protein of 100μmol/L nicotine treated group were increased significantly (P<0.01).However, the PAI-1 protein expression of other concentrations nicotine group had no significant difference (P>0.05, respectively).The stimulated with 100μmol/L nicotine for 0,4,6,8,12,24 hours, the levels of PAI-1 protein increased over time and reached the peak at 12 hours, which were significantly higher than those of control groups (P＜0.05, respectively).But the expression levels of t-PA protein had no significant levels of t-PA protein of each group had no significant difference (P>0.05, respectively).After difference than those of control groups (P>0.05, respectively).(2) Compared with control group (0.729±0.100, (13.388±0.927) ng/ml), the PAI-1 mRNA and protein expression of nicotine group (1.321±0.198, (21.084±0.829) ng/ml) were increased significantly (P＜0.01). Compared with nicotine group, the PAI-1 mRNA and protein expression of nicotine+STS group (1.074±0.099, (16.190±2.149) ng/ml) were decreased significantly, but still higher than control group (P＜0.01, respectively).Compared with control group (0.782±0.152), the t-PA mRNA (0.193±0.073) expression of nicotine group were decreased significantly (P＜0.01), However, the decrease of t-PA protein expression was no significant (P＞0.05). Compared with nicotine group, the t-PA mRNA expression of nicotine+STS group(0.394±0.076) were increased significantly, but still lower than control group (P＜0.01, respectively), but no significant on the t-PA protein expression (P>0.05).Conclusions(1) Nicotine can increased the expression of PAI-1 mRNA and protein in HUVECs, then inhibits the fibrinolytic function of endothelial cells.(2) Nicotine had no significant effect on the expression of t-PA protein.But increasing expression of PAI-1 can disturb the balance of t-PA/PAI-1 and affecting the fibrinolytic activity of endothelial cells.(3) PKC-dependent pathway may play a partial role on the fibrinolytic disorders of endothelial cells that induced by nicotine.