The overall goal of our research is to determine the molecular basis of mechanochemical signal transduction and the endothelium's ability to discriminate between shear, temporal gradients in shear, strain, and rate of strain. We have previously demonstrated that temporal gradients in shear stress and high strain rates rapidly activate the G protein, Gaq. We also have documented that Gaq and endothelial nitric oxide synthase (eNOS) are preferentially localized at endothelial cell-cell junctions through binding to PECAM-1 (CD31). The importance of PECAM-1 in transducing temporal gradients in shear has been shown in preliminary studies of flow dependent dilation and response to exercise in mice deficient in PECAM-I. It is our overall hypothesis that the junctional localization of Gaq and eNOS maintained by binding to the cytoplasmic scaffolding domain of PECAM-1 confers the ability of the endothelium to sense temporal gradients in shear stress and strain rate. To test this overall hypothesis, we are taking an entirely integrative approach, using molecular biology, cell biology and biochemistry, vascular and exercise physiology. The first specific aim is to investigate the critical sites involved in PECAM-I's interaction with G,q and eNOS by means of molecular manipulation. The second specific aim will investigate the role of PECAM in the mechanochemical transduction of shear stress, temporal gradients in shear stress, strain and strain rate in endothelial cells from PECAM knockout and wild type mice. The third specific aim will investigate the flow and myogenic responses in isolated arterioles from PECAM knockout and wild type mice. The fourth specific aim will study the role of PECAM-1 in the vascular adaptation to exercise. This investigation will test the comprehensive hypothesis concerning the role of PECAM-1, Gaq and eNOS in mechanochemical transduction of clinically important hemodynamic forces in endothelial cells. If successful it will provide the mechanistic basis on how endothelial cells sense flow in both normal physiology and in vascular disease.
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