内皮细胞糖萼在冠状动脉分叉病变血管重构中的作用及机制研究

Coronary arterial sclerosis(CAS) is a chronic inflammative and proliferative disease,featured by vascular remodeling and proliferation of vascular smooth muscle cells,resulting in coronary arterial stenosis and ischemia.CAS commonly localizes at the site where there is a reduced endothelial shear stress (ESS), with resultant more frequent coronary bifurcation lesions. There are several sensors sensoring ESS signal,without known which one is the most direct sensor yet. Glycocalyx, a thinner layer structure separating endothelial cells and blood flow, participates in the sensoring and transduction of ESS signals. However, the role of glycocalyx in the development of coronary bifurcation lesions still remained to be unclear.We found that the timing of degradation of glycocalyx after exposured to lower ESS was significantly earlier than that of change of cellular configuration and eNOS-Thr 495. Accordingly, we proposed that endothelial glycocalyx would be the most direct sensor responsive to the ESS signals. As a result, we will investigate the mechanisms by which glycocalyx directly senses the dynamic change of ESS using cultured endothelial cells, electro microscopy, 3-D reconstruction of cells, parallel flow chamber, OCT, RT-qPCR, Western-Blot, immunochemistry, systemic circulating glycocalyx and distribution of glycocalyx on micro vessels, from the cultured endothelial cells, and patients having true coronary bifurcation diseases. The current study will explore the possible mechanisms attributive to the development of coronary bifurcation lesions and occurence of In-stent restenosis after stenting coronary bifurcation lesions. The final results will be potential of providing innovative ideas and therapeutical targets to coronary bifurcation lesions.

冠状动脉粥样硬化是以血管重构为标志、平滑肌细胞增生为特征的慢性炎性疾病，造成管腔狭窄和心肌缺血，常见于分叉外侧、弯曲处内侧及植入支架的分支开口等内皮剪切力(ESS)减低的区域，故易于形成分叉病变和再狭窄。内皮细胞表面有多个感知ESS信号的感受器，但不清楚何为最直接感受器。糖萼是位于内皮细胞最表面的糖蛋白，参与感知转导ESS信号，但在冠状动脉分叉病变中的作用未见报道。我们预实验发现低ESS作用于细胞后，糖萼被降解的时间显著早于细胞形态及内皮型—氧化氮合酶的变化，据此我们提出糖萼是ESS信号的最直接感受器这一假设。为此，本课题运用相性光学成像、免疫组化、细胞培养、电子显微镜、平行板流动腔、RTq-PCR及蛋白印迹等技术，以冠脉内皮细胞、冠脉分叉病变患者为对象，明确糖萼作为直接感受器感知ESS信号、形成分叉病变和再狭窄的机制。本研究的结果将为有效防治冠脉分叉病变提供新思路和新靶点。

冠状动脉粥样硬化是以血管重构为标志、平滑肌细胞增生为特征的慢性炎性疾病，造成管腔狭窄和心肌缺血，常见于分叉外侧、弯曲处内侧及植入支架的分支开口等内皮剪切力(ESS)减低的区域，故易于形成分叉病变和再狭窄。糖萼是位于内皮细胞最表面的糖蛋白，参与感知转导ESS信号。. 应用平板流动腔体外模拟血管低剪切力(LSS，2 dyn/cm2)观察LSS对人脐静脉内皮细胞糖萼层透明质酸(HA)表达和eNOS多位点磷酸化水平的影响。结果发现LSS 激活eNOS-Ser1177和eNOS-Thr495，抑制eNOS-Ser633. LSS明显下调内皮HA表达。外源性透明质酸酶降解内皮糖萼层透明质酸后eNOS-Ser633被抑制时间点提前。LSS 明显增加HYAL1和HYAL2的蛋白表达和活性。SiRNA-HYAL2转染后内皮细胞膜HA表达不能被LSS抑制，但不影响LSS降低胞浆内HA水平，eNOS-Ser633水平也较LSS 30min组明显增加，说明LSS主要是通过增加HYAL2的表达和活性，降解细胞膜糖萼层HA，继而下调eNOS-Ser633水平。. 本研究的发现提示阻断LSS激活HYAL2是防止内皮损伤的一个重要途径