Shear Stress Activation of Endothelial Membrane Function
NIH R37 HL040696
The goal is to understand the molecular mechanisms of mechanochemical transduction.
The overall goal of our research is to determine the molecular basis of mechanochemical signal transduction in endothelial cells. We have established that temporal gradients in shear stress and steady shear stress represent two distinct mechanical signals that are transduced by two different signaling pathways. The temporal gradient in shear stress has been shown to be a potent stimulator of mitogenic and pro-atherogenic signal and gene expression, and is mediated by the Gq heterotrimeric G protein. In contrast, steady shear stress appears to dose-dependently stimulate anti-atherogenic signals such as prostacyclin and nitric oxide.
We hypothesize that mechanoreception of these two mechanical signals occurs at different spatial locations on the cell: temporal gradients in shear stress are perceived at the cell–cell junctions and steady shear is sensed over the luminal membrane. It is the objective of this proposal to elucidate the molecular events that lead to mechanoreception and mechanochemical transduction. Our first two specific aims are to elucidate the molecular associations and sequence of signaling events of Gq and endothelial nitric oxide synthase activation that occur at the cell-cell junctions as endothelial cells are subjected to temporal gradients in shear stress. The third specific aim is to investigate the spatial and temporal pattern of shear stress-induced changes in membrane microviscosity. To accomplish this, we will develop a technique to image membrane microenvironmental changes (in real time) in endothelial cells subjected to both temporal gradients in shear and steady shear stress. The fourth specific aim will determine if spatial gradients in shear stress do in fact stimulate a proatherogenic phenotype in human endothelial cells.
This investigation will test a comprehensive hypothesis on the mechanism of mechanochemical transduction in endothelial cells. If successful, it will provide a fundamental understanding of how the endothelium senses hemodynamic forces in both normal physiology and in vascular disease.
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-1’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.
Funding
Principal Investigator: John A. Frangos, Ph.D.
Agency: NIH