Thoumine O, Nerem R M, Girard P R
Bioengineering Center, Georgia Institute of Technology, Atlanta, USA.
Lab Invest. 1995 Oct;73(4):565-76.
In blood vessels, the extracellular matrix (ECM) underlying the endothelium supports endothelial cell (EC) attachment, spreading, migration, and proliferation. The structure and composition of the ECM may be modulated by hemodynamic shear stress, which may play a role in the pathogenesis of vascular diseases such as atherosclerosis.
In this study, in vitro effects of fluid shear stress on the ECM of EC were investigated. Cultured bovine aortic EC (BAEC) were exposed to a steady laminar shear stress of 30 dyn/cm2 from 3 to 48 hours, using a parallel-plate flow chamber. Parallel control cultures were maintained under static conditions. The organization of fibronectin (Fn), laminin (Ln), collagen type IV (Col IV), and vitronectin (Vn) was analyzed by immunofluorescence microscopy. Changes in the profile of proteins present in the deoxycholate-insoluble ECM fraction of EC were determined using two-dimensional gel electrophoresis, and the levels of Fn, Ln, and Vn were determined by Western blotting.
Fn, Ln, and Col IV exhibited both a granular pattern in cell perinuclear areas and a fibrillar pattern localized underneath EC. On exposure of bovine aortic EC to shear stress, Fn fibrils grouped into thicker tracts of fibrils, and there was a tendency for some of these tracks of fibrils to align with the direction of flow. Ln and Col IV also grouped into thicker fibers, which, in contrast to Fn, were randomly oriented. Vn exhibited a diffuse granular pattern, which did not change in response to shear stress. Consistent increases in the levels of four unidentified acidic proteins (mol wt/pI = 52/4.9, 70/4.7, 70/5.5, and 110/4.4) were observed after 3 to 6 hours of exposure to flow. The level of Fn present in the ECM was decreased twofold 12 hours after exposure of the cell monolayer to flow, and then increased after 24 and 48 hours. The level of Ln showed a twofold increase after 24 and 48 hours of flow, whereas the level of Vn was not altered by shear stress.
These changes in organization and composition observed in the ECM of cultured EC may play a significant role in shear stress-induced morphologic alterations in EC and may represent relevant events in the initiation of atherosclerotic lesions by influencing both EC and smooth muscle cell function.
在血管中，内皮细胞下方的细胞外基质（ECM）支持内皮细胞（EC）的附着、铺展、迁移和增殖。ECM的结构和组成可能会受到血流动力学剪切应力的调节，而血流动力学剪切应力可能在动脉粥样硬化等血管疾病的发病机制中起作用。
在本研究中，研究了流体剪切应力对EC的ECM的体外影响。使用平行板流动腔，将培养的牛主动脉内皮细胞（BAEC）暴露于30达因/平方厘米的稳定层流剪切应力下3至48小时。平行对照培养物在静态条件下维持。通过免疫荧光显微镜分析纤连蛋白（Fn）、层粘连蛋白（Ln）、IV型胶原（Col IV）和玻连蛋白（Vn）的组织情况。使用二维凝胶电泳确定EC的脱氧胆酸盐不溶性ECM部分中存在的蛋白质谱的变化，并通过蛋白质印迹法测定Fn、Ln和Vn的水平。
Fn、Ln和Col IV在细胞周核区域呈现颗粒状模式，在EC下方呈现纤维状模式。将牛主动脉内皮细胞暴露于剪切应力后，Fn纤维聚集成更粗的纤维束，并且这些纤维束中的一些有沿着流动方向排列的趋势。Ln和Col IV也聚集成更粗的纤维，与Fn不同的是，它们是随机取向的。Vn呈现弥漫性颗粒状模式，对剪切应力无反应。在暴露于流动3至6小时后，观察到四种未鉴定的酸性蛋白质（分子量/等电点=52/4.9、70/4.7、70/5.5和110/4.4）水平持续升高。细胞单层暴露于流动12小时后，ECM中Fn的水平降低了两倍，然后在24小时和48小时后升高。Ln的水平在流动24小时和48小时后增加了两倍，而Vn的水平未受剪切应力影响。
在培养的EC的ECM中观察到的这些组织和组成变化可能在剪切应力诱导的EC形态改变中起重要作用，并且可能通过影响EC和平滑肌细胞功能而代表动脉粥样硬化病变起始中的相关事件。
