Molecular Mechanisms in Endothelial Cell Differentiation

Abstract: Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays.The role of the signaling protein H-Ras in FGF-2-induced in vitro angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. In vitro angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-? (TNF-?). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation.In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation.