(Verbatim from the application): Cellular activation by a wide variety of stimuli results in the formation of the bioactive lipid sphingosine-l-phosphate (SPP), which is a ligand for the endothelial cell derived G-protein-coupled receptor EDG-1. Indeed, SPP signaling via the EDG-l and -3 receptors results in the induction of angiogenic response in vitro and in vivo. Furthermore, deletion of the EDG-l gene in the mouse results in embryonic lethality due to a vascular maturation defect, suggesting that SPP is a critical angiogenic modulator. The central hypothesis of this proposal is that activation of the sphingosine kinase enzyme (SK) results in the formation of SPP, stimulation of the EDG-l family of receptors, which ultimately regulate endothelial cell migration and morphogenesis, critical events of angiogenesis.
Specific Aim 1 will explore the molecular mechanisms involved in SPP release and receptor access. The effect of physiological regulators of angiogenesis on SK activity, export of SPP and receptor activation will be assessed. Structure-function analysis of the SK enzyme will be performed to develop dominant negative reagents.
Specific Aim 2 will test the concept that SK activity is rate-limiting during angiogenesis. HEK293 cells inducibly transfected with the SK enzyme will be used to study in vivo angiogenesis and vascular maturation.
Specific Aim 3 will focus on the ability of SPP to induce endothelial cell migration. The effect of SPP on the avb3 integrin-dependent focal contact assembly will be defined in detail. Interactions with the uPA, uPAR, PAl-1 system will be defined.
Specific Aim 4 will define the in vivo correlates of the SKISPP/EDG signaling system in angiogenesis and vascular homeostasis in transgenic models. Platelet-derived SK will be targeted for inducible antisense expression and the effect on vascular homeostasis and angiogenesis will be determined In addition, SK will be inducibly overexpressed in endothelial cell-specific manner and the effect on physiological and pathological angiogenesis will be studied. These efforts will critically assess the central hypothesis that SKIEDG signaling system is a major regulator of angiogenesis.
Showing the most recent 10 out of 49 publications
