Abstract
Purpose :
Endothelial cell dysfunction and increased vascular permeability constituting the blood–retinal barrier (BRB) alteration is the hallmark of diabetic macular edema (DME). Using next generation sequencing (NGS), we investigated the gene expression and transcriptional regulatory network dynamics across human and mouse retinal endothelial cells to understand the molecular mechanisms of DME
Methods :
Retinal Endothelial (ECs) from the non-diabetic and diabetic (n=12) mouse retina were obtained through fluorescent activated cell sorting (FACS) uisng endothelial cell specific marker (CD31-FITC) staining. RNA sequencing of isolated E retinal ECs was done using paired-end Illumina sequencing (NovaSeq 6000 System). Single-cell RNA sequencing (snRNA) of in vitro human retinal endothelial microvessel model was performed under low and high- glucose conditions. DESeq2 analysis was performed to identify the differentially expressed genes (DEGs) set, followed by gene set enrichment analysis (GSEA) analysis and the top genes were identified using custom functional analysis tools.
Results :
Transcriptomic analysis of isolated retinal ECs from diabetic animals showed differential expression of genes linked to barrier maintenance and prosurvival. We also identified enrichment of divergent transcriptional clusters connected to cellular stress response pathway in the diabetic retinal EC. In vitro expression analysis of human retinal ECs revealed early upregulation of proinflammatory and angiogenic factors such as IL-6, ICAM-1, CCL2, ANG2, which are well documented to play a critical role in BRB alterations. Integrated analysis of human and mouse retinal endothelial cells revealed identification of novel upstream signaling mediators and transcription factors such as BRK1, PKIG, HEXIM1, AND DDIT4 involved in the regulation of proinflammatory and angiogenic factors involved in the retinal vascaulr permeability alterations. Further, using single-cell transcriptome analysis, we characterized retinal endothelial specific robust cell markers.
Conclusions :
Our study highlights the importance of endothelial cell-specific transcriptomic analysis to understand the pathogenesis of diabetic retinopathy. A detailed understanding of the transcriptional landscape of endothelial cells in diabetes will help in the development of novel molecular targets and therapies for DME.
This is a 2021 ARVO Annual Meeting abstract.