Regulation of Adhesion Mechanosignalling by Fibronectin Matrix Structure

Fibronectin is an extracellular matrix (ECM) component found in atherosclerotic lesions, which form in regions of disturbed shear stress and are associated with endothelial dysfunction. When shear stress is applied to the endothelium, forces are transmitted through the cytoskeleton of endothelial cells to focal adhesions, which are sites of integrin-dependent mechanotransduction that link the cytoskeleton and the ECM and are required for structural remodeling in response to shear stress. Integrin-mediated signaling is also required for fibronectin assembly, but how fibronectin assembly might in turn modulate integrin-mediated signaling under shear stress had not been addressed. We hypothesized that fibronectin assembly regulates shear stress-induced structural remodeling through regulation of integrin binding. To test this hypothesis we developed methods to control fibronectin assembly independent of cell density. Bovine aortic endothelial cells interacting with assembled or unassembled fibronectin were then subjected to a step increase from 0 to 15 dyn/cm2 unidirectional shear stress in a parallel plate flowchamber. We then measured how fibronectin assembly modulated shear stress-induced changes in motility, cytoskeletal
organization, and focal adhesion displacement in both single endothelial cells and confluent endothelial monolayers. We also evaluated the effects of fibronectin assembly and shear stress on the distribution and binding of a5b1 and avb3 integrins. In single endothelial cells, fibronectin assembly inhibited shear stress-induced downstream mechanotaxis and spreading. Fibronectin assembly also inhibited baseline stress fiber assembly, shear stress-induced ruffling, and shear stress-induced focal adhesion arrest. We also observed that fibronectin assembly promoted a5b1 binding over avb3, and by using blocking antibodies we showed that avb3 is important for shear stress-induced mechanotaxis. In confluent endothelial monolayers, fibronectin assembly slowed shear stress-induced monolayer alignment. Fibronectin assembly also promoted shear stress-induced stress fiber disassembly and inhibited shear stress-induced focal adhesion arrest. Finally, we observed an assembly dependent switch in integrin binding. Monolayers interacted with unassembled fibronectin through avb3, but interacted with assembled fibronectin through a5b1. These results demonstrate for the first time that fibronectin assembly inhibits endothelial mechanosensing under shear stress and alters the specificity of a5b1 versus avb3 binding. Inhibition of avb3 suggests that this integrin plays an important role in guiding mechanotaxis. Overall, our data suggest that fibronectin assembly-dependent regulation of a5b1 and avb3 binding results in activation of different downstream signaling pathways to control shear stress-induced structural remodeling.