Choroidal endothelial and macrophage gene expression in atrophic and neovascular macular degeneration

Abstract

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.

Intercellular communication analysis of the human retinal pigment epithelial and choroidal cells predicts pathways associated with aging, cellular senescence and age-related macular degeneration

“…To the best of our knowledge, there are currently no other public datasets thatcontain more samples suitable for our analysis. For example, although a recent scRNA-seq dataset from Voigt et al (2022) contains more eye samples, it is not suitable for our intercellular communication study. Specifically, as cells have been sorted based on CD31 expression to enable the capture of endothelial cells, only a low number of RPE cells are present in the dataset and they are moreover confounded with photoreceptors (Voigt et al, 2019(Voigt et al, , 2022.…”

Role of Erythropoietin Receptor Signaling in Macrophages or Choroidal Endothelial Cells in Choroidal Neovascularization

“… EPOR mRNA expression in RPE/choroids from adult human donor eyes. EPO mRNA was detected in ( A ) macular and ( B ) extramacular RPE/choroids that were isolated from donors with AMD (red dots), including dry AMD (green dots) and wet AMD (purple dots), or without AMD (blue dots), and EPOR mRNA was detected in ( A ) macular or ( B ) extramacular RPE/choroids that were isolated from donors with AMD (black dots), including dry AMD (brown dots) and wet AMD (dark blue dots), or without AMD (orange dots) that were collected in a previous publication [ 26 ]; ( C ) EPOR mRNA was also detected in single cells dissociated from RPE/choroids from patients without AMD (green dots) or with dry AMD (red dots) or wet AMD (blue dots) that were collected in a previous publication [ 28 , 29 ]. …”

“…We found that EPOR mRNA is expressed in several cell types in RPE/choroids isolated from human donors, including macrophages and choriocapillaris endothelial cells using a publicly available database [ 29 ]. We previously observed that the RPE/choroids of mice with overactive EPOR signaling had more macrophages prior to laser-induced injury, greater concentrations of inflammatory cytokines three days after laser-induced injury, and increased laser-induced CNV seven days after laser-induced injury compared to littermate wildtype control mice [ 10 ].…”

“…We, therefore, wanted to confirm the expression of EPOR mRNA in specific cell populations. To address this consideration, we analyzed single-cell RNA-sequencing data from macular RPE/choroids of donors with AMD or donors without AMD as controls from a publicly available website (https://singlecell-eye.org (accessed on 12 April 2022)) and publication [28,29]. EPOR mRNA was expressed in macrophages (e.g., infiltrative and resident) and choriocapillaris cells isolated from all donors included in the study (Figure 1C).…”

“…(C) EPOR mRNA was also detected in single cells dissociated from RPE/choroids from patients without AMD (green dots) or with dry AMD (red dots) or wet AMD (blue dots) that were collected in a previous publication [28,29].…”