Transcriptomic Responses of Human Retinal Vascular Endothelial Cells to Inflammatory Cytokines

Abstract

Purpose Molecular profiling of human retinal endothelial cells provides opportunities to understand the roles of this cell population in maintenance of the blood-ocular barrier, and its involvements in diverse retinal vasculopathies. We aimed to generate a transcriptome of human retinal endothelial cells in the unstimulated state, and following treatment with inflammatory cytokines linked to cell dysfunction. Methods Endothelial cells were isolated from retinae of five human cadaveric donors, and treated for 60 minutes and 24 hours with interleukin-1β or tumor necrosis factor-α, or exposed to medium alone for the same intervals. Expression of intercellular adhesion molecule-1 was measured by RT-qPCR to confirm cytokine-induced activation of the cells. RNA was sequenced on the Illumina NovaSeq 6000 platform. Reads were aligned to the human GRCh38 genome, and reads that aligned to Ensembl-annotated genes were counted. Quality control of sequencing was performed with FastQC, and sequences were classified by Kraken. Results A human retinal endothelial cell RNA-sequencing dataset with mean of 99% reads aligned to the human genome was produced as raw RNA sequence data (FASTQ files) and processed read data (XLSX files). Multidimensional scaling analysis showed a strong donor effect, which was readily controlled by ComBat. Conclusions Our dataset may be useful for human retinal endothelial cell transcriptomic assemblies, functional gene annotating and/or gene expression and enrichment analyses, as well as cross-dataset harmonization. Translational Relevance The molecular profile of the human retinal endothelium is a source of candidate biologic targets for retinal vasculopathies.

Selective Transcription Factor Blockade Reduces Human Retinal Endothelial Cell Expression of Intercellular Adhesion Molecule-1 and Leukocyte Binding

“…To identify transcription factors that might promote leukocyte interactions with activated human retinal endothelial cells, we interrogated a previously published transcriptomic dataset generated from cell isolates that had been activated by brief exposure to inflammatory cytokines [ 18 ]. Differential expression analysis comparing five primary human retinal endothelial cell isolates, treated with IL-1β or TNF-α versus no cytokine for 60 min, demonstrated a 2-fold or more increase in expression of 87 genes by IL-1β and 68 genes by TNF-α at a false discovery rate (FDR) of less than 0.05 ( Supplementary Table S1 ).…”

“…Thus, for valid translation, it was important we employed a human system, although this necessarily required in vitro assays. In general, published research with human retinal endothelial cells uses a single isolate [ 18 ]. However, interindividual variation is an important consideration: for one of five human retinal endothelial cell isolates we studied, IRF1 silencing did not significantly reduce leukocyte binding, even while C2CD4B silencing did.…”

“…An RNA-sequencing dataset that represented the transcriptome of human retinal endothelial cells in resting state, and TNF-α- and IL-1β-activated states (Gene Expression Omnibus, National Center for Biotechnology Information Series link: GSE144785) [ 18 ] was the source of transcription factor candidates. Using the normalized read counts, adjusted for donor effect as presented in the description of this dataset, differential gene expression analysis was performed in the EdgeR v3.26.5 package [ 49 ].…”

Human retinal endothelial cells express functional interleukin-6 receptor

“…Methodological differences may contribute to variations in findings across groups that work in the area of IL-6 signaling in vascular endothelium. Most research on human retinal endothelial cells has been conducted with single samples, sourced commercially [ 11 ]. Our results show that IL-6R is present at variable levels across multiple primary human retinal endothelial cell isolates, and in agreement with Montgomery [ 24 ], that it is present at a relatively low level.…”

“…Complexes of IL-6 and mIL-6R associate with cell-surface gp130 or CD130, which mediates classic intracellular signal transduction in these cells. However, gp130 is expressed on all cells, including retinal endothelial cells [ 11 ], and can bind complexes of IL-6 and soluble IL-6 receptor (sIL-6R). The sIL-6R is shed from leukocytes during inflammation to trigger intracellular signaling more generally, referred to as trans-signaling.…”