Endothelial Notch signaling drives essential and diverse functions in angiogenesis. Abnormal Notch signaling is a significant contributor to many cardiovascular diseases; from blindness, stroke, and ischemic disease, to arteriovenous malformation (AVM) and tumor angiogenesis. Notch genes encode transmembrane receptors which undergo ligand-dependent cleavage of the intracellular domain; the intracellular domain then translocates to the nucleus, where it assembles transcriptional complexes to regulate the expression of downstream targets that carry out Notch function. Notch receptors respond to two classes of membrane bound ligands, Delta-like (Dll) and Jagged (Jag). Dll4 signals through Notch in normal and pathological angiogenesis to restrict sprout formation and promote perfusion, whereas Jag1 may have both Notch activating and inhibitory roles in endothelial cells. The transcriptional targets of Notch signaling drive many cellular functions that are critical for proper angiogenesis, yet our knowledge of Notch targets is currently limited. Defining these targets in the endothelium is needed to obtain a deeper knowledge of Notch angiogenic function and identifying the novel effectors will impact our ability to understand healthy vessels and human disease. Our preliminary studies highlight that Notch regulates several endothelial proteins that function in GPCR signaling, an unexplored area of endothelial Notch signaling. Our overall goal is to define endothelial genes regulated by Notch and use this knowledge to identify Notch effectors that regulate GPCR signaling and their mode of action in the vasculature. In this proposal, we will stimulate endothelial Dll4- or Jag1-Notch mediated transcription in vitro and identify Notch targets by RNAseq. A RiboTag mouse model will determine Notch-responsive endothelial genes in vivo. Combining data from the in vivo and in vitro analysis will lead to selection of candidate Notch targets that function in GPCR pathways and these will be validated as direct transcriptional targets. Using in vitro co- culture angiogenesis assays, we will determine if Notch-induced GPCR signaling molecules are required in order for Notch to regulate sprouting angiogenesis. One such candidate, Rnd1, a constitutively active GTPase, will be analyzed to test the hypothesis that Notch induces Rnd1 to regulate Rap-p120RasGAP- Ras signaling in endothelial cells. Rnd1, and other GPCR proteins induced by Notch, will be studied in mice to determine their roles in Notch regulated sprouting angiogenesis, hypoxia-induced retinopathy and arteriovenous malformation (AVM).
The Notch signaling pathway is fundamental to proper vascular development and angiogenesis. We will study how Notch regulates angiogenesis and promotes proper formation and function of blood vessels. This will allow an understanding of how angiogenesis occurs in healthy individuals and human disease, and development of rationale strategies to repair retinal vascular disorders, inhibit tumors, control inflammatory conditions, and promote wound healing.
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