Vascular networks develop through several process including vasculogenesis, the de novo formation of primitive vessels, and angiogenesis, the sprouting of new vessels from pre-existing ones. Vascular endothelial growth factor-A (VEGF) and its receptors, Flt-1 and Flk-1, play critical roles in controlling these processes. VEGF signaling effects via Flk-1 have been well characterized, but the role of Flt-1 in regulating vessel formation remains unclear. Flt-1 regulates vessel branching, primarily by producing a soluble Flt-1 isoform that acts largely as a ligand sink. Furthermore, recent evidence suggests that Notch signaling interacts with VEGF pathways to guide developing vessels, although the exact nature of this interaction is not well understood. Thus, the hypotheses that will be tested are that Flt-1 regulates vessel morphogenesis by providing spatial cues to guide endothelial cell (EC) filopodia and sprout formation, and that Notch signaling modulates downstream Flt-1 activity to establish the proper pattern of sprout extension and in turn vessel branching. To achieve these goals, models of vessel formation in differentiated embryonic stem (ES) cell cultures and in mouse retina development will be utilized. In both models, VEGF signaling will be disrupted through genetic modification of Flt-1 expression. After characterizing the spatial distribution of Flt- 1 expression in these models, Flt-1 mutant and wild-type (WT) vessels will be compared with regard to EC filopodia extension, tip cell formation, vessel sprout guidance, and overall vessel branching and morphology. To characterize how Notch signaling regulates Flt-1 in guiding vessel sprouts, the location of Notch signaling relative to EC filopodia and vessel sprouts will be established in both the WT and Flt-1 mutant ES cell and retina vessel models. Notch signaling will be disrupted genetically and through the use of inhibitors or neutralizing antibodies. Developing vessels will be analyzed as described above. From these studies, knowledge will be gained regarding how Flt-1 regulates VEGF signaling such that proper EC filopodia extension, sprout formation and vessel branching occur, and how Notch signaling coordinates Flt-1 activity to yield properly branched vascular networks. Insights from this study will be essential in the development of therapeutic strategies to disrupt blood vessel growth in treating certain pathological conditions such as tumor growth and diabetic retinopathy. Furthermore, this study will be informative in the design of clinically relevant treatments for stimulating blood vessel formation in patients suffering from vascular occlusive diseases such as coronary heart disease and peripheral vascular disease.
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