Astrocyte-specific molecular cues guiding retinal angiogenesis

摘要 血管生成是神经系统发育中的一个中心事件，需要在 视网膜的背景。在正常发育中，视网膜血管生成从视神经开始并进行 沿着由视网膜神经纤维和发育中的星形细胞网络组成的视网膜最内层。 星形细胞发育发生在血管生成之前，对指导发育中的血管系统至关重要。 随着血管生成波阵面的推进，被称为TIP细胞的特殊内皮细胞沿着星形细胞爬行， 所以血管结构与星形细胞的花纹相匹配。众所周知，星形胶质细胞分泌 血管内皮生长因子-A在缺氧区启动血管生成，但信号推动波前，并引导尖端细胞 沿着星形胶质细胞的模板，都是未知的。这项拟议的研究的目的是鉴定新的星形胶质细胞- 衍生出引导视网膜血管形成的分子线索。中心假设是星形胶质细胞提供 通过1)促进TIP细胞的产生和2)引导TIP细胞的生长从而促进血管生成的分子信号 这种血管结构采用星形细胞网络的模式。这项工作的基本原理是提供更深层次的 理解可能适用于整个神经系统的神经胶质介导的血管生成调节 同时还提供了针对诸如早产儿视网膜病变之类的视网膜病变的候选分子 (ROP)。具体的目标是：1)确定星形胶质细胞衍生的信号驱动血管生成的进展 波前。肾上腺髓质素(adrenomedullin，ADM)是一种分泌型多肽，以前被发现通过 它的受体，降钙素受体样受体(CLR)和受体活性修饰蛋白， RAMP2，由内皮细胞表达。初步的单细胞rna-seq数据显示 肾上腺髓质素基因在未成熟星形胶质细胞中高表达。为了测试这一点，它的血管生成特性将是 使用包括分析培养的小鼠的血管波前进展的功能分析进行测试 视网膜外植体应与表达ADM的HEK293细胞共培养。2)鉴定星形胶质细胞来源 这暗示着正在生长的血管。星形胶质细胞选择性表达基因是引导TIP细胞的候选基因 生长和血管生成。为了识别这些分子，神经纤维层中的每种细胞类型都将被提纯 ScRNA-seq.星形胶质细胞特有的基因编码细胞表面或分泌分子将被识别 生物信息学上的。与目标1一样，将使用视网膜外植体的功能测试来测试血管生成能力 推测的TIP细胞引导星形胶质细胞特异性基因。这项工作的完成将具有重大意义，因为它将 揭示视网膜中星形胶质细胞优先表达的血管生成的新成分和驱动因素。是这样的 这些信息将阐明视网膜血管生成的基本机制，同时也将确定重要的分子 在ROP等致病条件下可能参与血管生成的信号。

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.