Elucidating the signaling pathways that control angiogenesis allows new therapies to be designed for treatment of diseases where blood vessels are contributors, including blindness, inflammation, and cancer. Notch1 signaling generally acts as a negative regulator of angiogenic sprouting. We hypothesize that Notch4, a Notch receptor primarily expressed in endothelium, has both overlapping roles with Notch1 and distinct endothelial functions. We demonstrated that Notch4 promotes angiogenesis and possibly inflammation of blood vessels and surrounding cells. By removing Notch4 expression in blood vessels of mice or blocking Notch4 using an inhibitor, we discovered that Notch4 promotes angiogenesis in the developing retina. Notch4 is expressed in blood vessels of many types of tumors and we demonstrated that blocking Notch4 inhibits tumor growth when Notch4 is in tumor vessels. We found that endothelial Notch4 appears to function to alter both vessels and adjacent stromal tissue. Analysis of Notch4-regulated genes led us to discover that specific cytokine signaling proteins are regulated by Notch4, and not Notch1, a possible mechanism for Notch4 broad influence on both vessels and surrounding tissue. In these studies, we will define the unique signaling mechanisms regulated by Notch4 to understand how Notch4 promotes developmental, inflammatory-driven, and tumor angiogenesis and evaluate anti-Notch4 treatment for therapeutic benefit. We will use a new technique called CUT&RUN to see which genes are directly activated by Notch4 or Notch1 and use a novel RiboFlag translational analysis to understand expression control by each Notch protein. We will determine if newly discovered Notch4-regulated proteins promote angiogenesis and inflammation in cultured endothelial cells and remove Notch4 expression from endothelial cells in mice to determine if this causes reduced blood vessel growth, reduces the response to inflammation and reduces tumor growth. We will separately remove Notch1 or Notch4 genes from endothelium or remove both genes and describe result of Notch loss on developmental and pathological angiogenesis, establishing unique and overlapping roles for these genes. A new Notch inhibitor allows us to investigate pre- clinical therapeutic potential for treatment of proliferative retinopathy, an inflammatory eye disease, and in tumors that have Notch4 in the endothelial cells. We will investigate the stromal changes in tumors caused by genetic or pharmacologic blockade of Notch4 using flow cytometry and focused single cell RNA sequencing. In all of these studies, we will determine if select cytokines are regulated by Notch4 in healthy and disease-associated blood vessels, with the goal of understanding the mechanisms by which Notch4 impacts inflammation of blood vessels and surrounding tissue or tumor. Our goals for this proposal will be to discover the unique ways that Notch4 works to build normal blood vessels and contributes to diseased vessels, an...