Wang Jie, Morita Ikuo, Onodera Mitsue, Murota Sei-Itsu
Section of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
J Cell Physiol. 2002 Feb;190(2):238-50. doi: 10.1002/jcp.10059.
Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.
凝血酶是一种多功能丝氨酸蛋白酶，在血管损伤部位生成。它不仅参与凝血级联反应，还能诱导许多与细胞有丝分裂和迁移相关的事件。在本研究中，我们调查了凝血酶对血管内皮生长因子（VEGF）诱导的内皮细胞增殖的影响。凝血酶以时间和剂量依赖的方式促进培养的牛颈动脉内皮细胞的增殖。此外，它显著增强了VEGF刺激的细胞生长。蛋白激酶C（PKC）或丝裂原活化蛋白激酶激酶（MAPKK）的抑制剂可降低这种刺激作用。凝血酶以时间依赖的方式诱导含激酶结构域受体（KDR）的mRNA水平显著增加，但不影响类tms酪氨酸激酶（Flt-1），刺激24小时后达到最大值。这种增加与KDR蛋白表达很好地吻合。荧光素酶测定表明，与对照相比，凝血酶诱导KDR启动子活性增加约7.5倍。PKC或MAPKK的抑制剂也消除了这种增强的KDR启动子活性。缺失分析表明，KDR启动子基因中-115至-97区域（包含Sp-1结合区域）是凝血酶和VEGF诱导KDR表达增强所必需的。此外，一氧化氮合酶（NOS）抑制剂消除了凝血酶和VEGF诱导的细胞增殖加速和KDR表达增加。这种抑制作用被半衰期长的NO供体DETA NONOate消除。这些发现表明，凝血酶可能通过增加VEGF受体KDR的水平来增强VEGF诱导的血管生成活性，其中涉及NO的产生。
