Flow-induced Reorganization of Laminin-integrin Networks Within the Endothelial Basement Membrane Uncovered by Proteomics
Flow-induced Reorganization of Laminin-integrin Networks Within the Endothelial Basement Membrane Uncovered by Proteomics
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DOI：
10.1074/mcp.ra120.001964
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发表时间：
2020-07-01
影响因子：
7
通讯作者：
van den Biggelaar, Maartje
中科院分区：
文献类型：
--
作者：
Beguin, Eelke P.;Janssen, Esmee F. J.;van den Biggelaar, Maartje
血管壁不断地暴露在血液流动产生的血液动力中。内皮机械传感器通过细胞信号通路感知机械信号，并将其转化为控制内皮发育、表型和功能的生物过程。为了在全系统水平上评估血流动力学对血管内皮细胞的影响，我们应用了结合细胞表面化学足迹的定量质谱仪方法。SILAC标记的内皮细胞分别承受0、24或48h的流动诱导剪应力，然后用非膜透性生物素标记表面蛋白质，并用LC-MS/MS分析整个蛋白质组和细胞表面蛋白质组。这些研究表明，在>；5000个量化蛋白中，有104个蛋白发生了改变，这些蛋白高度富含细胞外基质蛋白和参与细胞-基质黏附的蛋白。细胞表面蛋白质组学表明，LAMA4在流动暴露时被蛋白质降解，这与免疫印迹观察到的LAMA4质量减少相对应。免疫荧光显微镜研究强调，在流动暴露时，内皮细胞基底膜发生了剧烈的重塑。我们观察到LAMA4和LAMA5呈网状分布，这与层粘连蛋白黏附分子ITGA6和ITGB4的定位相对应。此外，对血流暴露的适应不影响对肿瘤坏死因子α的炎症反应，表明炎症和血流触发了基本不同的内皮信号通路，但相互作用和协同作用有限。综上所述，这项研究揭示了血流诱导的基底膜重塑，并强调了内皮下基底膜在血管动态平衡中的重要性。
The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. To assess the hemodynamic effects on the endothelium on a system-wide level, we applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting. SILAC-labeled endothelial cells were subjected to flow-induced shear stress for 0, 24 or 48 h, followed by chemical labeling of surface proteins using a non-membrane permeable biotin label, and analysis of the whole proteome and the cell surface proteome by LC-MS/MS analysis. These studies revealed that of the > 5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor alpha, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis.