Changes in organization and composition of the extracellular matrix underlying cultured endothelial cells exposed to laminar steady shear stress.
Changes in organization and composition of the extracellular matrix underlying cultured endothelial cells exposed to laminar steady shear stress.
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发表时间：
1995-10
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通讯作者：
O. Thoumine;R. Nerem;P. Girard
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作者：
O. Thoumine;R. Nerem;P. Girard
背景在血管中，内皮下面的细胞外基质（ECM）支持内皮细胞（EC）附着、扩散、迁移和增殖。ECM的结构和组成可能受到血流动力学切应力的调节，这可能在血管疾病如动脉粥样硬化的发病机制中起作用。实验设计在这项研究中，在体外流体剪切应力对EC ECM的影响进行了研究。培养的牛主动脉EC（BAEC）暴露于30达因/厘米2的稳定层流剪切应力从3到48小时，使用平行板流动室。在静态条件下维持平行对照培养物。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。使用二维凝胶电泳测定EC的脱氧胆酸盐不溶性ECM组分中存在的蛋白质的分布的变化，并通过Western印迹法测定Fn，Ln和Vn的水平。结果Fn、Ln和Col IV在细胞核周区呈颗粒状分布，在EC下方呈纤维状分布。暴露牛主动脉EC的剪切应力，Fn原纤维分组成较厚的纤维束，并有一种趋势，这些跟踪的原纤维，以对齐的流动方向。Ln和Col IV也分组为较厚的纤维，与Fn相反，它们是随机取向的。VN表现出弥散颗粒状的图案，这并没有改变响应剪切应力。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（mol wt/pI = 52/4.9、70/4.7、70/5.5和110/4.4）水平的一致增加。细胞单层暴露于流动后12小时，ECM中存在的Fn水平降低两倍，然后在24和48小时后增加。Ln的水平在24和48小时的流动后显示出两倍的增加，而Vn的水平没有被剪切应力改变。结论：在培养EC的ECM中观察到的组织和组成的这些变化可能在EC中剪切应力诱导的形态学改变中起重要作用，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。
BACKGROUND 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. EXPERIMENTAL DESIGN 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. RESULTS 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. CONCLUSIONS 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.