ABSTRACT Angiogenesis is a central event in nervous system development that requires further elucidation within the context of the retina. In normal development, retinal angiogenesis initiates from the optic nerve and proceeds along the innermost layer of retina consisting of retinal nerve fibers and the developing astrocytic network. Astrocytic development occurs prior to angiogenesis and is critical for guiding the developing vasculature. Specialized endothelial cells called tip cells crawl along the astrocytes as the angiogenic wavefront advances, so that the vasculature matches the pattern of astrocyte arbors. It is well understood that astrocytes secrete VEGF-A in hypoxic zones to initiate angiogenesis, but the signals that advance the wavefront, and guide tip cells along the astrocyte template, are unknown. The objective of this proposed study is to identify novel astrocyte- derived molecular cues that guide retinal vascularization. The central hypothesis is that astrocytes provide molecular cues that promote angiogenesis by 1) enhancing tip cell production; and 2) guiding tip cell growth so that vasculature adopts the pattern of the astrocyte network. The rationale for this work is to provide a deeper understanding of glial-mediated regulation of angiogenesis that may be applicable to the entire nervous system while also providing candidate molecules to target in retinal pathologies such as retinopathy of prematurity (ROP). The specific aims are: 1) Identify astrocyte-derived cues driving progression of the angiogenic wavefront. Adrenomedullin (ADM) is a secreted peptide that was previously found to mediate angiogenesis via its receptor, calcitonin receptor-like receptor (CLR) in conjunction with receptor activity modifying protein, RAMP2, which are expressed by endothelial cells. Preliminary single-cell RNA-seq data shows that the adrenomedullin gene is highly expressed by immature astrocytes. To test this, its angiogenic properties will be tested utilizing a functional assay that consists of analyzing vasculature wavefront progression in cultured mouse retinal explants that shall be co-cultured with ADM-expressing HEK293 cells. 2) Identify astrocyte-derived cues that pattern growing vessels. Genes selectively expressed by astrocytes are candidates to guide tip cell growth and angiogenesis. To identify such molecules, each cell type in the nerve fiber layer will be purified for scRNA-seq. Astrocyte-specific genes encoding cell-surface or secreted molecules will be identified bioinformatically. As in Aim 1, a functional assay using retinal explants will be used to test angiogenic capabilities of putative tip cell-guiding astrocyte-specific genes. Completion of this work will be significant because it will reveal novel components and drivers of angiogenesis expressed preferentially by astrocytes in the retina. Such information will elucidate basic mechanisms of retinal angiogenesis, while also pinpointing important molecular cues that may be involved in angiogenesis during pathogenic conditions such as ROP.

PROJECT NARRATIVE Retinal vascularization in humans occurs in gestation, reaching completion at 36 weeks, and it is a crucial developmental event involving precisely timed and localized events. Defects in any step of the process or halting it before its full completion will lead to mispatterned or incomplete vascular coverage of the retina, which are hallmarks observed in retinopathologies such as retinopathy of prematurity (ROP). Completion of this work will elucidate unknown mechanisms of glial regulation of retinal angiogenesis, while also pinpointing important molecular cues that may be involved in angiogenesis during pathogenic conditions.