扰流诱导内皮细胞vWF分泌和血栓形成的机制研究

Thromboembolism most frequently occurs as a consequence of atherosclerosis. Platelet aggregation and thrombosis often takes place in the downstream of atherosclerotic plaque where irregular hemodynamics, or disturbance of the blood flow normally shown. This observed phenomenon is likely due to the change of hemostasis in the endothelial cell-derived procoagulant and anticoagulant molecules. Among these molecules, von Willebrand factor (vWF), which is located in the Weibel-Palade body (WPB) of endothelial cells, is a key factor that mediates platelet adhesion to damaged endothelium. However, the detailed mechanisms of how disturbed flow regulates vWF secretion have not been well understood. It has been reported that vesicular transport regulatory proteins, VAMP3 and SNAP23, mediate exocytosis of WPB.in endothelial cell. We have also found that disturbed flow increases the expressions of and physical interaction between VAMP3 and SNAP23 (PNAS, 2017). Therefore, we hypothesized that disturbed flow induces arterial thrombosis by promoting VAMP3 and SNAP23-mediated vWF secretion. We will use fluid shear stress loading apparatus and animal models in which FeCl3-induced thrombosis is combined with the creation of disturbed flow to investigate the role of VAMP3 and SNAP23 in mediating the shear stress-induced vWF secretion, platelet adhesion, and aggregation, thrombosis in endothelial cells. Besides, we will explore the mechanotransduction by which VAMP3 and SNAP23 are activated by shear stress. The results from the present study will help to elucidate the pathophysiology of atherosclerosis-associated thrombosis and provide putative targets for the diagnosis and treatment of vascular occlusive diseases.

动脉粥样硬化斑块下游扰流区域是血小板聚集和血栓生成的易发部位，可能由于这些部位内皮源性促凝和抗凝血分子水平的改变。位于内皮细胞特有细胞器Weibel-Palade小体（WPB）中的血管性血友病因子（vWF）是介导血小板粘附到受损内皮的关键。但扰流对内皮细胞vWF分泌的调控机制尚不明确。有报道称囊泡运输调节蛋白VAMP3和SNAP23可介导内皮细胞WPB胞吐，而前期工作中我们发现扰流上调VAMP3和SNAP23的表达。在此我们提出研究假设：扰流通过促进VAMP3和SNAP23介导的vWF分泌诱导动脉血栓形成。我们将利用流体剪切力加载模型和扰流合并FeCl3诱导血栓的动物模型，探讨这两个基因在剪切力调控的内皮细胞vWF分泌、血小板粘附和聚集、在血栓生成中的意义，并探索剪切力激活VAMP3与SNAP23的力信号转导途径。研究结果将增进对血栓生成机制的了解，为血管闭塞性疾病的诊疗提供新的靶标。

狭窄动脉下游部位的扰流通过促进局部血管性血友病因子（Von Willebrand factor, VWF）积聚，诱发血小板聚集和血栓生成，然而扰流促进动脉血栓形成尤其是扰流促进内皮细胞VWF分泌的机制还未阐明。我们开发了一个小鼠扰流模型研究血流调控的动脉血栓生成，在这个模型中结扎小鼠左侧颈外动脉，通过小动物超声多普勒以及核磁共振成像确认了颈外动脉结扎诱导颈总动脉内扰流产生。此外发现左侧颈外动脉结扎小鼠血浆中VWF水平以及VWF多聚体的数量显著增加。采用三氯化铁孵育诱导颈总动脉血栓生成，发现颈外动脉结扎小鼠颈总动脉内血栓生成时间显著低于未结扎小鼠。体外对内皮细胞施加模拟狭窄血管下游的振荡型剪切力（Oscillatory shear, OS, 0.5 ± 4 dynes/cm2）和模拟狭窄血管直部的脉冲型剪切力（Pulsatile shear, PS, 12 ± 4 dynes/cm2），发现OS通过调节内皮细胞内SNAREs蛋白（Soluble NSF Attachment Protein, 可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体）囊泡相关膜蛋白3（intracellular localization of vesicle-associated membrane protein 3，VAMP3）和突触相关蛋白23（synaptosomal-associated protein 23，SNAP23）的细胞内定位促进内皮细胞VWF分泌以及内皮细胞条件培养液诱导的血小板聚集。内皮细胞内细胞骨架波形蛋白（Vimentin）在OS诱导的SNAP23细胞膜定位中发挥重要作用，Vimentin的解聚消除了OS诱导的SNAP23细胞膜转位。内皮细胞内VAMP3和SNAP23的敲低降低了内皮细胞VWF分泌。动物实验中腹腔注射雷帕霉素（Rapamycin）系统性抑制小鼠血管内皮细胞VAMP3和SNAP23表达显著延缓颈外动脉结扎小鼠FeCl3诱导的动脉血栓形成。通过颈总动脉内孵育VAMP3和SNAP23腺病毒诱导颈总动脉内皮细胞VAMP3和SNAP23的过表达，显著加重颈外动脉结扎小鼠FeCl3诱导的动脉血栓生成。上述结果表明VAMP3 和SNAP23在扰流加速的动脉血栓形成发挥重要作用，VAMP3 和SNAP23可以作为干预扰流加重的动脉血栓形成中的潜在靶点。