THE PRO-INFLAMMATORY ROLE OF G-PROTEIN COUPLED RECEPTOR 4 IN AN ACIDIC MICROENVIRONMENT

The endothelium of the microvascular system serves an ideal mediator between circulating blood and tissues in order to regulate systemic homeostasis. During vascular disease , the normal function and phenotype of endothelial cells is altered and leads to endothelial dysfunction. In turn , vascular disease disrupts blood flow to downstream tissues and leads to tissue hypoxia and acidification. Localized acidification commonly exists in inflammatory tissues due to altered glycolytic metabolism , hypoxia and insufficient tissue perfusion. How the body initially detects and responds to an acidic microenvironment is crucial for endothelial metabolism , cytoskeletal alterations , and cell survival. G-protein receptor 4 (GPR4) is a proton-sensing G-protein coupled receptor highly expressed in vascular endothelial cells. GPR4 can initiate various signaling pathways based on its activation by extracellular protons. This dissertation focuses on acidosis-mediated GPR4 signaling and its role as a positive regulator of inflammation using in vitro and in vivo models. We microscopically evaluated permeability and cytoskeletal components , including fibrous actin rearrangements and vascular endothelial-cadherin alterations under acidic stress conditions in endothelial cells. In vitro data demonstrated that GPR4 is a key mediator for endothelial permeability by targeting one of the G-protein signaling pathways , G12/13 , to initiate a cascade in order to regulate endothelial permeability. Additionally , we employed a widely established tourniquet-based hindlimb ischemia murine model to initiate a localized ischemic milieu followed by a reperfusion (release of cuff) event. To evaluate the biological role associated with GPR4 , we utilized a genetic GPR4 knockout mouse and a small molecule inhibitor to abolish GPR4 signaling. Our immunohistochemical data show less serum immunoglobulin G leakage and inflammatory response in the GPR4 knockout mice compared to the wild type mice. These and other data suggest GPR4 deficiency reduces hindlimb edema , exudate quantity , and leukocyte migration to the loose connective tissue due to endothelial permeability alterations mediated by GPR4 signaling. This work highlights many GPR4-dependent properties from promoting endothelial permeability for leukocyte trafficking to eliciting a cytoskeletal response by the G12/13 G-protein signaling pathway cascade. Understanding how vascular endothelial cells sense and respond to an acidic microenvironment is critical for identifying therapeutic targets for cancers , cardiovascular , and ischemic diseases