Elucidating the developmental and molecular mechanism of chorioretinal anastomoses in a model of type 3 neovascular age-related macular degeneration

项目摘要 新生血管性年龄相关性黄斑变性（AMD）是老年人视力丧失和失明的主要原因。 老人在新生血管性AMD的亚型中，3型新生血管形成（3型NV）与 保护视力预后，并以视网膜和脉络膜中的血管增生为独特特征。 晚期3型NV的一个定义特征是发展为脉络膜视网膜病变（CRA），异常视网膜病变， 视网膜和脉络膜循环之间的直接联系。不幸的是，CRA可以作为“热点”， 复发性渗出，对现有治疗有弹性。 CRA的发生机制尚不清楚。我们建立了3型NV的鼠模型， 其中携带IV型胶原（Col 4a 1）突变的小鼠发展出人类疾病的特征，包括 浆液性视网膜脱离、出血、萎缩和明显的CRA。与患者一样，Col 4a 1突变型CRA 小鼠在成年期形成并成熟为视网膜和脉络膜循环之间的大的从头融合， 让人联想到高流量动静脉（AV）分流。最近，AV规范已经成为一个强有力的 血管模式的调节因子，以及控制AV特性的关键基因的失调诱导AV的形成。 分流器值得注意的是，正常化AV规格已被证明可以逆转AV分流，尽管它们的高流量 在某些动物模型中。阐明AV规范在CRA中的作用可能会为两者提供新的见解 疾病的发病机制和治疗策略。我们的中心假设是Col 4a 1突变小鼠中的CRA 是AV分流，具有来自视网膜或脉络膜的保守发育起源。我们最近 能够直接通过有色眼睛的巩膜对脉络膜进行多光子成像， 多光子显微术因此，这项跨学科研究提案的目标是： 1)利用经巩膜多光子显微镜优化脉络膜视网膜血管动力学的活体成像; 2） 确定Col 4a 1突变小鼠中CRA的起源、血流和动静脉特性;以及3）鉴定 使用单细胞RNA测序在内皮中驱动CRA的候选途径。 这个职业发展研究项目的长期目标是为研究人员提供一个跨- 先进的活体成像和单细胞转录组学的学科技能，使他能够 研究眼血管发育不全的基本机制，以发现新的治疗方法， 血管疾病

PROJECT SUMMARY Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. Among the subtype of neovascular AMD, type 3 neovascularization (type 3 NV) is associated with a guarded visual prognosis and is uniquely characterized by vascular proliferation in both the retina and choroid. A defining feature of advanced type 3 NV is the development of chorioretinal anastomoses (CRA), abnormal direct connections between the retinal and choroidal circulations. Unfortunately, CRA can act as ‘hot spots’ for recurrent exudation which are resilient to available therapies. The developmental mechanism of CRA remains unclear. We established a murine model for type 3 NV, wherein mice harboring a mutation of type IV collagen (Col4a1) develop features of human disease including serous retinal detachment, hemorrhage, atrophy, and pronounced CRA. As in patients, CRA in Col4a1 mutant mice form in adulthood and mature into large de novo fusions between retinal and choroidal circulations, reminiscent of high-flow arteriovenous (AV) shunts. Recently, AV specification has emerged as a potent regulator of vascular patterning, and dysregulation of key genes governing AV identity induces formation of AV shunts. Notably, normalizing AV specification has been shown to reverse AV shunts despite their high-flow state in certain animal models. Elucidating a role for AV specification in CRA may offer novel insights into both disease pathogenesis and therapeutic strategies. Our central hypothesis is that CRA in Col4a1 mutant mice are AV shunts with a conserved developmental origin from either the retina or choroid. We have recently enabled multiphoton imaging of the choroid directly through the sclera of pigmented eyes using optimized multiphoton microscopy. The objectives of this inter-disciplinary research proposal are therefore to: 1) optimize intravital imaging of chorioretinal vascular dynamics with transscleral multiphoton microscopy; 2) determine the origin, blood flow, and arteriovenous identity of CRA in Col4a1 mutant mice; and 3) identify candidate pathways driving CRA in the endothelium using single cell RNA sequencing. The long-term goal of this career development research project is to provide the investigator with a cross- disciplinary skillset in advanced intravital imaging and single-cell transcriptomics that will allow him to investigate fundamental mechanisms of oculovascular dysgenesis toward the discovery of new therapies in vascular disease.