Molecular Characterization of Neovascular AMD in Human Eyes using Spatial Transcriptomics and Single Cell RNA Sequencing

Abstract

Purpose : Neovascular age-related macular degeneration (nvAMD) is a major cause of vision loss. Although very effective treatments exist for nvAMD, these require intervention after the rupture of Bruch’s membrane, require multiple treatments, and may be associated with increased risk of atrophy after prolonged treatments. Discovering how neovascular endothelial cells from human eyes differ from non-neovascular cells at the transcriptional level will provide new knowledge about the process of pathologic angiogenesis and may offer opportunities for prevention.

Methods : Three frozen sections were collected from a macula with an extensive type I neovascular membrane (death to freezing interval < 5 hours) and two frozen sections were collected from an age-matched control eye. The spatial transcriptome of each section was evaluated using the Visium platform. Gene expression profiles from each spot were integrated with an independent collection of single-cell expression data comparing the choroid of nvAMD maculas (n = 2), non-neovascular, early AMD maculas (n = 9), and control maculas (n = 9).

Results : Spatial transcriptomics identified three retinal layers, with distinct ganglion cell, interneurons/glia, and photoreceptor cell-associated genes in spots corresponding to ganglion cell layer, inner nuclear layer, and outer nuclear layer, respectively (Figure 1). Twenty four genes were identified with a log fold change of >0.2 between both (a) spots overlapping with neovascular membranes and those in the choroid outside of neovascular membranes and (b) nvAMD single-cells vs controls . These include genes involved in cytoskeleton (TUBA1A, TUBA1B), extracellular matrix (FN1) and stress responses (SOD2, GADD45B).

Conclusions : Spatial transcriptomic analysis of the macula reliably maps transcripts to their cellular sources. Cells from neovascular membranes have altered transcriptomes compared to both the peripheral choroid outside of the CNV lesion and from non-AMD maculas. These two experiments identify several genes enriched in neovascular AMD that may offer insight into pathogenic angiogenesis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Top: section used for spatial RNA-seq (top) where each spot is annotated by an unbiased cell type prediction. Bottom: higher mag, UEA-I labeled adjacent section showing type I CNVM.