Study on mechanism of thrombosis and hemostasis in endothelium

为了探讨血管内皮细胞血栓形成和止血的调控机制，我们进行了体外实验。血流动力调节内皮细胞的各种功能，即使在基因调控下存在细胞因子的情况下也是如此。用改进的锥板式粘度计研究了剪切力对培养的人脐静脉内皮细胞(HUVECs)在细胞因子或脂多糖(LPS)扰动下凝血和纤溶系统的影响。我们发现，细胞因子诱导的组织因子表达随着血流的增加而减少，而组织纤溶酶原激活物的分泌在细胞因子的存在下在剪应力下增加，纤溶酶原激活物抑制物-I的分泌在细胞因子的存在下增加，但在剪切力下减少。此外，我们还研究了切应力…作用下人脐静脉内皮细胞中各种因子的表达。更多的是在细胞因子存在的情况下，采用免疫细胞化学、EIA法和激光流式细胞术。组织因子途径抑制物(TFPI)是作为组织因子调节因子的丝氨酸蛋白酶，不受炎性细胞因子、肿瘤坏死因子和切应力的刺激，但受肿瘤促进剂PMA的影响。下一步，已经开发了几种针对人鼻粘膜微血管的单抗。针对炎症部位白细胞与内皮细胞黏附的重要分子E-选择素和MEL-CAM的两种黏附糖蛋白，已研制出抗E-选择素和MEL-CAM的单抗。静息状态下未刺激的HUVECs未检测到E-选择素的表达，而肿瘤坏死因子刺激3-6h后，细胞表膜E-选择素的表达增加。而静息状态下未刺激的HUVECs明显表达MEL-CAM，而培养上清液中MEL-CAM的释放量在肿瘤坏死因子刺激后增加，在剪切力作用下减少。这些结果提示血栓形成和止血的机制可能受复杂的网络调控。剪切力和细胞因子调节机制的相互作用，至少作为一部分，是血栓形成和抗血栓形成的重要调节器。较少

To investigate the regulatory mechanism of thrombosis and hemostasis in endothelium, in vitro experiments have been undertaken. Hemodynamic forces modulate various endothelial cell functions even in the presence of cytokines under gene regulation. We have investigated the effect of shear stress on coagulation and fibrinolysis system in cultured human umbilical vein endothelial cells (HUVECs) perturbed by cytokines or lipopolysaccharide (LPS), using modified cone-plate type viscometer, in which well controlled and defined shear forces were generated. We have revealed that cytokine-induced tissue factor expression was decreased in response to flow, whereas secretion of tissue plasminogen activator was augmented under shear stress in the presence of cytokines, and the secretion of plasminogen activator inhibitor-I was increased in the presence of cytokines, but, decreased under shear stress. Furthermore, we investigated the expression of various other factors in HUVECs under shear stress … More in the presence of cytokines, using immunocytochemistry, EIA method, and laser flow-cytometry. Tissue factor pathway inhibitor (TFPI), serine protease that acts as a regulator for tissue factor, was not affected by stimulation of inflammatory cytokines, TNF, nor shear stress, but decreased by tumor promoting activator, PMA. Next, several monoclonal antibodies have been developed against human nasal-mucous microvessels. Monoclonal antibodies have been produced against two adhesive glycoproteins for E-selectin and Mel-CAM that act as important molecules for leucocyte adhesion to endothelium under inflammatory site. E-selectin was not detected in resting unstimulated HUVECs, whereas, surface membrane expression of E-selectin was increased 3-6 hours after the stimulation of TNF. In contrast, Mel-CAM expression was clearly observed in resting unstimulated HUVECs, whereas the release to culture supernatant was increased after TNF stimulation and decreased under shear stress. These results indicate that the mechanism of thrombosis and hemostasis might be regulated by complicated net-work. The cross-talk of regulatory mechanism for shear forces and cytokines, at least as a part, is important modulator for thrombogenecity and anti-thrombogenecity. Less