Single Cell RNA Sequence Analysis of Retinal Microvasculature Cells Reveals Novel Molecular Signature Underlying Blood-Retinal Barrier (BRB) alteration in Diabetic Retinopathy

Abstract

Purpose : Pericyte and endothelial cells dysfunction constituting the blood–retinal barrier (BRB) (retinal capillary) is the hallmark of increased vascular permeability and diabetic macular edema (DME). Using next generation cell specific RNA sequencing technique, we examined unique expression signatures across retinal endothelial and pericyte cells that could significantly advance our understanding of DME.

Methods : Endothelial (ECs) and pericyte cells (PC) from the non-diabetic (n=12) and diabetic (n=12) mouse retina were isolated through fluorescent activated cell sorting (FACS). RNA sequencing of isolated ECs and PCs was done using paired-end Illumina sequencing (NovaSeq 6000 System). Gene expression signature in the retinal endothelial and pericyte cells was identified using differentially expressed genes (DEGs) set through gene set enrichment analysis (GSEA). We established an in vitro human retinal endothelialized microvessel model with pericyte coverage and performed single-cell RNA sequencing (snRNA) using low glucose and high- glucose treatment conditions.

Results : Gene expression analysis of in vivo isolated mice retinal endothelial and pericyte cells revealed distinct transcriptional profiles between non-diabetic and diabetic conditions. The transcriptional clusters enriched in the diabetic retinal EC and PCs were significantly associated with the activators of angiogenesis and inflammatory pathway. Strikingly, both the cell types from diabetic mice have pronounced expression of signaling molecules that amplifies the activation of the apoptotic signaling pathway. Distinct gene set related to endothelial barrier maintenance such as Sav1, Gpc6, and Gpc4 from diabetic EC and PCs were significantly downregulated. Importantly our analysis demonstrated that the single-cell RNA sequence analysis of endothelial and pericyte cells in diabetic conditions exhibit similar changes in the gene expression related to specific pathology of BRB alterations.

Conclusions : RNA sequence analysis of the single-cell population from diabetic microvasculature (EC and PC) has identified novel genes and pathways contributing to the BRB alterations. Our study is unique in its cell-specific transcriptomic analysis with a specific focus on the identification of common cellular pathways and genes to identify an exclusive target for DME.

This is a 2020 ARVO Annual Meeting abstract.