Vasoactive compounds released after endotoxin exposure or vascular damage can transiently reprogram endothelial cells, converting them from quiescent cells that manifest a nonthrombogenic lumenal surface to activated cells promoting coagulatory events. This proposal focuses upon the receptors, transducers, and effector proteins that mediate endothelial cell responses to thrombin and the proinflammatory cytokines, interleukin-1beta (IL-1) and tumor necrosis factor alpha (TNF). Cells derived from different vascular beds-sheep pulmonary artery, vein, and microvascular endothelial cells as well as human umbilical vein endothelial cells - will be studied. The first goal of this proposal is to characterize IL-1 and TNF receptor activation of guanine nucleotide-binding (G) proteins and determine which signaling pathways activated by these cytokines are regulated by G proteins. Cytokine-induced guanine-nucleotide exchange, isolation of receptor-G protein complexes, as well as cytokine modulation of phospholipase A2 and adenylyl cyclase will be studied. The second specific aim is to determine how thrombin, IL-1, and TNF modulate signaling through the mitogen-activated protein kinase (MAPK) cascade, a pathway capable of integrating information from numerous transduction systems activated by extracellular signaling molecules. The MAPK cascade appears to act as a switch, promoting either cell proliferation or differentiation. The components of the MAPK cascade which are regulated by thrombin- or cytokine-activated second messenger pathways in endothelial cells, how thrombin sustains signaling when it serves as a mitogen, and how IL-1 and TNF prevent proliferation while activating MAPK will be determined.
The third aim i nvestigates thrombin, IL-1, and TNF priming of endothelial cells for migration. Maintenance of the vasculature depends critically upon the capacity of endothelial cells to respond rapidly to agents that indicate vessel damage. Wound repair requires, at athe very least, the activation and migration of endothelial cells. The signaling processes activated by thrombin and the cytokines promoting endothelial cell reorganization of the actin cytoskeleton will be examined, focusing on the regulation of the GTP- binding proteins Rac and Rho, heat shock protein 27, and athe myristoylated alanine-rich C kinase substrate. Rigorous control of transduction networks is vital for the maintenance of normal endothelial cell functions. These investigations will provide a framework for future studies delineating the altered regulation of the endothelium by thrombin and cytokines that manifests as endothelial cell dysfunction. The proposed research will provide information necessary for defining the molecular basis of the pathological changes in the endothelium that are observed in septic shock, acute respiratory distress syndrome, and many forms of heart disease.
