Differential mechano-signaling in vascular endothelium by varying degrees of mechanical stretch - Resubmission 01

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Differential mechano-signaling in vascular endothelium by varying degrees of mechanical stretch Excessive mechanical forces imposed on the microvascular endothelium lead to increased microvascular permeability accompanying life-threatening conditions such as stroke, pulmonary hypertension, or ventilator induced lung injury, to name a few. We have previously characterized signaling pathways induced in vascular endothelium by high magnitude cyclic stretch (CS) and described a key role of Rho GTPase in high CS- induced endothelial barrier dysfunction. However, cellular mechanotransduction complexes which transform mechanical signals to cellular responses remain to be characterized. Our unpublished pilot studies indicate that Rap1 signaling is activated by physiologically relevant low magnitude (5%) CS and promotes re-assembly of tight junctions disrupted by cell preconditioning at pathologically relevant high magnitude (18%) CS. The preliminary data also suggest that low CS-induced EC barrier restoration is associated with accumulation of adaptor protein cingulin at the tight junctions and formation of cingulin-GEF-H1 complex. The central hypothesis tested in this application is that recovery of vascular endothelial barrier after pathologic mechanical stress may be accelerated by cell exposure to physiologic CS levels and involves Rap1-dependent reassembly of endothelial tight junctions, recruitment of cingulin to the tight junctions, and stimulation of cingulin - GEF-H1 interaction. These events lead to inhibition of GEF-H1 nucleotide exchange activity, suppression of Rho- dependent barrier disruptive mechanisms, and accelerated recovery of the vascular endothelial barrier. The following questions will be addressed: Aim-1 will study cingulin-dependent mechanisms of endothelial barrier regulation by physiologic mechanical stretch. Aim-2 will study the role of cingulin in downregulation of the Rho pathway in mechanically stimulated microvascular endothelium after a switch from pathologic to physiologic CS amplitude. Aim-3 will evaluate cingulin-dependent vascular protective mechanisms in a mouse model of mechanical ventilation.