PROJECT SUMMARY Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the western world. While the etiology of AMD is multifactorial, dysfunction of the choroidal vasculature, which provides the majority of oxygen to retinal photoreceptors, has been implicated as the earliest detectable event in AMD. An important contributor to choroidal vascular dysfunction is complement activation. Complement is a multiprotein component of the innate immune system that results in the formation of a lytic structure known as the membrane attack complex (MAC). While systemic vascular beds remain free from MAC formation, the superficial choroidal capillary system known as the choriocapillaris uniquely accumulates the MAC with advancing age. Such MAC accumulation is proposed to cause lytic injury to choriocapillaris endothelial cells and the downstream sequelae of AMD. However, the mechanisms that lead to choriocapillaris MAC accumulation are widely unknown. We hypothesize that unique transcriptional networks drive choriocapillaris development, which make these endothelial cells particularly susceptible to age-related molecular changes and MAC-mediated cell death. To test this hypothesis, we propose the following three specific aims: 1. Identify molecular features that make the choriocapillaris uniquely susceptible to MAC formation. We will differentiate choriocapillaris, arterial, and venous endothelial cells from human induced pluripotent stem cells and identify transcriptomic drivers of the choriocapillaris cell fate. After single-cell RNA sequencing, we will functionally validate choriocapillaris-enriched genes that influence MAC formation in vitro. 2. Identify molecular changes in age and AMD that increase choriocapillaris MAC accumulation and MAC- mediated damage. We will identify choroidal endothelial cell gene expression differences between young, adult (5th - 6th decade), elderly (8th - 9th decade) and AMD human donors. We will test the degree to which differentially expressed targets in aging choriocapillaris influence formation of the MAC in vitro. 3. Determine the role of RGCC in response to the MAC in health and AMD. We will quantify RGCC, a gene recently identified to be highly expressed in human choriocapillaris and implicated in MAC-mediated endothelial dysfunction, at the RNA and protein level using human choroids from healthy and AMD donors. We will also functionally assess the degree to which RGCC mediates endothelial cell death and dysfunction in both choriocapillaris and non-ocular endothelial cell lines. When successful, these studies will identify novel molecular features that lead to MAC accumulation and choriocapillaris damage. Further understanding how choriocapillaris cells accumulate and respond to the MAC may lead to new therapeutic targets for AMD, supporting the Mission, Goals, and Objectives of the NEI.