凝血酶及凝血因子Xa诱导血管平滑肌细胞中植物凝集素样氧化型低密度脂蛋白受体-1表达的实验研究

目的研究血管平滑肌细胞中凝血酶及凝血因子xa对新型的氧化型低密度脂蛋白的清道夫受体,即植物凝集素样氧化型低密度脂蛋白受体-1（LOX-1）表达的影响。方法培养的牛主动脉平滑肌细胞,予凝血酶及凝血因子xa刺激后,用鼠抗LOX-1单克隆抗体,对细胞裂解液和浓缩的培养基进行Western blot分析,观察LOX-1表达的变化。结果在凝血酶2．0U／ml及凝血因子Xa 50 nmol／L时,可观察到细胞膜结合型LOX-1表达明显增加。凝血酶3．0U／ml和凝血因子Xa100nmol／L刺激14h后,细胞培养基中可溶型LOX-1表达明显升高。对平滑肌细胞给予1．0U／ml凝血酶及100nmol／L凝血因子xa刺激,4h后LOX-1表达开始增加,12h后达高峰。AGl478是表皮生长因子受体相关酪氨酸激酶抑制剂。用指定浓度AGl478预刺激后,再予凝血酶和凝血因子xa,然后对细胞裂解液进行Western blot分析。AGl478可显著抑制凝血酶及凝血因子xa导致的LOX-1表达增加。结论凝血酶及凝血因子Xa可诱导LOX-1表达增加,此作用由表皮生长因子受体介导。

Tyrosine kinase dependent lectin-like oxidized LDL receptor-1 upregulation by thrombin and factor Xa in vascular smooth muscle cells

Objective Thrombin and factor Xa are key players in the process of arterial thrombi formation and lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a cell surface endocytosis receptor for atherogenic oxidized LDL (ox-LDL). Here we investigate whether thrombin and factor Xa can induce LOX-1 protein expressions in cell-associated forms and soluble forms in cultured bovine aortic smooth muscle cells (BSMCs).Methods BSMCs were treated with thrombin or factor Xa in the presence or absence of AG1478, an epidermal growth factor (EGF) receptor-associated tyrosine kinase inhibitor. Total cell lysates and concentrated culture medium were then analyzed by Western blot using a mouse anti-LOX-1 monoclonal antibody. Results LOX-1 protein levels in cell lysates and culture medium were significantly increased by thrombin and factor Xa in a concentration- and time-dependent manner. Upregulation of LOX-1 protein expressions in cell lysates and concentrated culture medium was observed at concentrations above 2.0 and 3.0 U/ml of thrombin and 50 and 100 nmol/L of factor Xa, respectively. Increased LOX-1 protein expressions in cell lysates and cell culture medium were detectable as early as 4 h and peaked at 12 h after treatment with thrombin or factor Xa. LOX-1 expression induced by thrombin and factor Xa could be blocked by pretreatment with AG1478. Conclusions Thrombin and factor Xa can act as LOX-1 inducers via tyrosine kinase activation.