Abstract 3841: Unique and redundant roles of Notch1 and Notch4 in retinal angiogenesis

Angiogenesis plays a vital role in the progression of cancer: the proliferation and metastasis of tumor cells relies on extending the vascular network that supplies oxygen and nutrients necessary for growth. Understanding the modulators and regulators of angiogenesis thus allows us insight into the mechanisms of a developing cancer. Notch signaling plays a critical role in the mediation of angiogenesis. Of the four Notch genes, the mechanisms of the Notch1 pathway are most well-understood, whereas the impact of Notch4 regulation on the vasculature is poorly studied and has shown contradictory phenotypes. In recent work, we have shown that an anti-Notch4 antibody, E7011, reduces tumor growth in multiple tumor models that express Notch4 in tumor endothelium and changes the number and phenotype of tumor associated macrophages. To better understand the role of Notch4 in angiogenesis, we have examined endothelial-specific loss of Notch4 during developmental retinal angiogenesis and determined that loss of Notch4 has substantially different effects than loss of Notch1. Loss of Notch4 is anti-angiogenic, characterized by reduced radial outgrowth of vessels and decreased vessel density, in contrast to the loss of Notch1, which results in hypersprouting and increased density. However, the structural similarity and common transcriptional targets of Notch1 and Notch4 suggest they have overlapping functions that are not evident until both genes are removed. To investigate the distinct and overlapping roles of Notch1 and Notch4, we generated mice with endothelial-specific knockouts of Notch1 (Notch1ECKO), Notch4 (Notch4ECKO), or both genes (Notch1Notch4ECKO). We harvested retina from mice at postnatal day (P)5, P10, and P12 timepoints to analyze vascular growth in the superficial and deeper plexus of the retina. Harvested retina were stained for Isolectin B4 to identify vasculature and Iba1 to identify microglia and analyzed for vascular growth measurements, including density, outgrowth, and tip cell counts. Quantitative analysis of the postnatal day 5 timepoint showed a highly abnormal double-mutant Notch1Notch4ECKO phenotype characterized by a significant reduction in radial outgrowth and a significant increase in tip cell density at the vascular front. Qualitative analysis suggested diminished vascular density near the center of the retina. Ongoing analysis is defining changes unique to Notch4ECKO, Notch1ECKO, and Notch1Notch4ECKO to clarify each gene’s individual function in angiogenesis. Citation Format: Nivedha G. Krishnan, Christie Kang, Taliha Nadeem, Braulio Aguilar, Krishna Thakkar, L.A. Naiche, Jan K. Kitajewski. Unique and redundant roles of Notch1 and Notch4 in retinal angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3841.