Noria S, Cowan D B, Gotlieb A I, Langille B L
Vascular Research Laboratory, The Toronto Hospital Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Circ Res. 1999 Sep 17;85(6):504-14. doi: 10.1161/01.res.85.6.504.
Endothelial cells exhibit profound changes in cell shape in response to altered shear stress that may require disassembly/reassembly of adherens junction protein complexes that mediate cell-cell adhesion. To test this hypothesis, we exposed confluent porcine aortic endothelial cells to 15 dyne/cm(2) of shear stress for 0, 8.5, 24, or 48 hours, using a parallel plate flow chamber. Cells were fixed and stained with antibodies to vascular endothelial (VE) cadherin, alpha-catenin, beta-catenin, or plakoglobin. Under static conditions, staining for all proteins was intense and peripheral, forming a nearly continuous band around the cells at cell-cell junctions. After 8.5 hours of shear stress, staining was punctate and occurred only at sites of continuous cell attachment. After 24 or 48 hours of shear, staining for VE-cadherin, alpha-catenin, and beta-catenin was intense and peripheral, forming a band of "dashes" (adherens plaques) that colocalized with the ends of stress fibers that inserted along the lateral membranes of cells. Staining for plakoglobin was not observed after 24 hours of shear stress, but returned after 48 hours. Western blot analysis indicated that protein levels of VE-cadherin, alpha-catenin, and plakoglobin decreased, whereas beta-catenin levels increased after 8.5 hours of shear stress. As cell shape change reached completion (24 to 48 hours), all protein levels were upregulated except for plakoglobin, which remained below control levels. The partial disassembly of adherens junctions we have observed during shear induced changes in endothelial cell shape may have important implications for control of the endothelial permeability barrier and other aspects of endothelial cell function.
内皮细胞会因剪切应力改变而呈现出深刻的细胞形态变化，这可能需要介导细胞间黏附的黏附连接蛋白复合物进行拆卸/重新组装。为了验证这一假设，我们使用平行板流动腔将汇合的猪主动脉内皮细胞暴露于15达因/平方厘米的剪切应力下0、8.5、24或48小时。细胞固定后，用针对血管内皮（VE）钙黏蛋白、α-连环蛋白、β-连环蛋白或桥粒斑蛋白的抗体进行染色。在静态条件下，所有蛋白质的染色都很强烈且位于周边，在细胞间连接处围绕细胞形成几乎连续的条带。在剪切应力作用8.5小时后，染色呈点状，仅出现在细胞连续附着的部位。在剪切24或48小时后，VE-钙黏蛋白、α-连环蛋白和β-连环蛋白的染色强烈且位于周边，形成与沿着细胞侧膜插入的应力纤维末端共定位的“短划线”（黏附斑）带。在剪切应力作用24小时后未观察到桥粒斑蛋白的染色，但在48小时后恢复。蛋白质印迹分析表明，在剪切应力作用8.5小时后，VE-钙黏蛋白、α-连环蛋白和桥粒斑蛋白的蛋白质水平下降，而β-连环蛋白水平升高。随着细胞形态变化完成（24至48小时），除桥粒斑蛋白外所有蛋白质水平均上调，桥粒斑蛋白仍低于对照水平。我们在剪切诱导的内皮细胞形态变化过程中观察到的黏附连接的部分拆卸可能对内皮通透性屏障的控制和内皮细胞功能的其他方面具有重要意义。
