Notchl and its ligands are critical for blood vessel development and angiogenesis, as evidenced by the embryonic lethality observed when these genes are deleted in mice. While notch signaling is well charac- erized, """"""""reverse signaling"""""""", through the ligands delta and jagged, has only recently been recognized,and its role in the vasculature is completely unknown. We have found that the inflammatory mediator TNF induces agged-1 expression in endothelial cells (EC), and in addition, induces genes associated with an angiogenic ip cell phenotype. Furthermore, jagged is involved in bi-directional signaling that helps to establish tip and trunk cell phenotypes in developing sprouts. Jagged signals through notch to induce the bHLHtranscription factor HESR1, and the subsequent down regulation of its target gene VEGFR2. Jagged also signals cell autonomously to the nucleus where it induces genes associated with a tip cell phenotype, such as VEGFR2 and PDGF-B, and promotes sprouting. In addition, TNF-induces 0^3 expression, which reinforces these divergent phenotypes by synergizing with notch signaling in trunk cells. TNF initially blocks sprouting, both in vitro and in vivo, but once removed, sprouting is augmented suggesting that TNF """"""""primes"""""""" EC. Our preliminary data have led us, therefore, to the hypothesis that TNF and jagged cooperate to temporally coordinate the onset of angiogenesis with the resolution of acute inflammation. We will test this hypothesis and identify the critical mechanisms by addressing the following aims: (1) determine the mechanism underlying TNF induction of jagged-1 and the tip cell genes VEGFR2 and PDGF-B;(2) determine the mechanism(s) underlying jagged-1 induction of tip cell genes;and,(3) determine the mechanism underlying 3 integrin modulation of jagged-notch signaling. We will use a combination of in vitro angiogenesis models, including collagen gels, fibrin gels, aortic ring assays and embryoid bodies induced to sprout capillaries, as well as angiogenic sprouting into Matrigel plugs in vivo. Completion of this study will furnish us with adeeper understanding of jagged-notch interactions in angiogenesis and the role of TNF in coordinating these events. Lay Summary: The growth of new blood vessels is a critical process in development and cancer. The cells that line blooc vessels - the endothelial cells - talk to each other through cell surface molecules including a pair called notch and jagged. This study aims to understand why notch and jagged are so important for blood vesse development,. Completion of this study will not only provide us with fundamental knowledge about how the body works, but it will also provide us with new information that will help in the fight against cancer.
