| Summary |
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. |
