The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Fibrosis is an end-stage response to tissue injury that is associated with loss of organ function as a result of excess extracellular matrix (ECM) production by fibroblasts. In skin, pathologic fibrosis is evident during keloid scar formation, systemic sclerosis (SSc), and morphea. Dermal fibroblasts in these fibrotic diseases exhibit increased Wnt/β-catenin signaling, a pathway that is sufficient to cause fibrosis in mice. However, in the context of this complex pathology, the precise pro-fibrotic consequences of Wnt/β-catenin signaling are not known. We found that expression of stabilized β-catenin in mouse dermal fibroblasts resulted in spontaneous, progressive skin fibrosis with thickened collagen fibres and altered collagen fibril morphology. The fibrotic phenotype was predominated by resident dermal fibroblasts. Genome-wide profiling of the fibrotic mouse dermis revealed elevated expression of matrix-encoding genes, and the promoter regions of these genes were enriched for Tcf/Lef family transcription factor binding sites. Additionally, we identified 32 β-catenin-responsive genes in our mouse model that are also over-expressed in human fibrotic tissues and poised for regulation by Tcf/Lef family transcription factors. Therefore, we have uncovered a matrix-regulatory role for stabilized β-catenin in fibroblasts in vivo and have defined a set of β-catenin-responsive genes with relevance to fibrotic disease.

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The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on twenty-one human choroids, eleven of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages, and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.