The main objective of this proposal is to elucidate the molecular regulation of endothelial cell behavior during development of the early cardiovascular system. Defects in the development of the cardiovascular system often lead to embryonic lethality, and are the most common cause of birth defect related deaths in the United States (American Heart Association). I hypothesize that a tight balance of FoxOI activity is required for the coordinated regulation of endothelial cellular function during vascular remodeling in vivo.
The specific aims focus on testing this hypothesis by assessing the role of FoxOI loss- and gain-of-function in regulating endothelial cell morphology and behavior during early vessel development.
Specific Aim 1 : To determine the endothelial cellular response to loss of FoxOI activity during vascular remodeling I will use an endothelial cell specific conditional knockout mouse to analyze FoxOI loss-of-function in regulating cell morphology and behavior during vessel remodeling. Endothelial cell migration and proliferation will be examined in vivo by crossing the Flk1-H2B::YFP reporter line to the conditional knockout and performing time lapse confocal imaging. Westem blot and qRT-PCR will be used to examine changes in expression of implicated downstream apoptotic and cell cycle genes regulated by FoxOI.
Specific Aim 2 : To assess the effect of FoxOI gain-of-function on endothelial cell morphology and behavior during vascular remodeling In order to assess FoxOI gain-of-function, I will inhibit the P13K/Akt pathway, a negative regulator of FoxOI, using Resveratrol and LY294,002HCI in vivo. 1 will test whether inhibition of this pathway affects vascular remodeling. I will use time lapse confocal microscopy as in Aim 1 to assess endothelial cell migration and proliferation in drug treated embryos. 1 will use western blot analysis to detect changes in phosphorylation levels of FoxOI and other downstream targets regulated by PI3K/Akt. Finally, 1 will assess changes in endothelial morphology, cell death and proliferation by performing immunofluorescence. By more fully defining the molecular regulation of dynamic endothelial cell function in vivo, I will come to a better understanding of the cellular mechanisms that regulate blood vessel development. In the future, I can apply what I learn about the cellular dynamics of endothelial cells during vascular development and stabilization to health problems associated with cardiovascular disease states.
