Flow-induced Reorganization of Laminin-integrin Networks Within the Endothelial Basement Membrane Uncovered by Proteomics

Abstract

<jats:p>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 &gt;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 α, 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.</jats:p>

Computational Characterization of the Dish-In-A-Dish, A High Yield Culture Platform for Endothelial Shear Stress Studies on the Orbital Shaker

“…To overcome this issue, while retaining high throughput, an annular dish culture setup has been proposed [20][21][22]. This is an easy-to-implement platform in which a small standard culture dish is mounted into a larger standard dish.…”

The circle of Willis revisited: Forebrain dehydration sensing facilitated by the anterior communicating artery

“…Taking into account that several morphologic variations of the ACoA were described in humans, [ 94 ] and that atherosclerotic stenosis of the ICA can trigger flow increase in the ACoA and possibly precipitate flow‐induced vessel remodeling, [ 28,80,81 ] we suggest that certain anatomical configurations and/or altered hemodynamic conditions of the ACoA could lead to suboptimal functioning of the forebrain sensory CVO.…”

“…[ 12,28,29 ] Atherosclerotic stenosis is a slow progressing pathologic process which theoretically gives blood vessels enough time to adapt to an increased flow and remodel accordingly. [ 26,79–81 ] Endothelial mechanoceptors transduce the perceived increase in blood flow into intracellular signaling. [ 80,82,83 ] This triggers reorganization of adhesion proteins in the basement membrane.…”

“…[ 26,79–81 ] Endothelial mechanoceptors transduce the perceived increase in blood flow into intracellular signaling. [ 80,82,83 ] This triggers reorganization of adhesion proteins in the basement membrane. [ 80 ] The ACoA does not normally accommodate significant blood flow, [ 12–15 ] so this flow increase would lead to flow‐induced intramural stress, endothelial dysfunction and aneurysm formation.…”

“…[ 80,82,83 ] This triggers reorganization of adhesion proteins in the basement membrane. [ 80 ] The ACoA does not normally accommodate significant blood flow, [ 12–15 ] so this flow increase would lead to flow‐induced intramural stress, endothelial dysfunction and aneurysm formation. [ 84,85 ] Taking into account that the ACoA is hemodynamically insignificant under physiologic conditions, and that atherosclerosis is primarily a disease associated with aging and the human lifestyle, [ 26,27 ] this compensatory function could not be a source of selection pressure for emergence and persistence of the ACoA in non‐human amniotes.…”