Abstract
Purpose :
The success of anti-VEGF therapies for treating diabetic retinopathy (DR) demonstrates the therapeutic efficacy of neutralizing agents in the vitreous that cause permeability of retinal blood vessels. In addition to VEGF, cytokines are elevated in the vitreous of patients with DR and promote leakage. The overall goal of this project is to develop an approach to prevent leakage driven by all of these pro-permeability agents. To this end, we sought to identify common permeability genes, which mediate permeability in response to both classes of pro-permeability agents (VEGF and cytokines).
Methods :
Our transcriptomics study consisted of two parts. In part one, we performed RNA-Seq analysis on primary human retinal endothelial cells (HRECs) cultured in high glucose (HG; 30 mM) conditions that were treated with vehicle, VEGF, TNFa, IL-1b, or IL-6 to identify genes which are commonly regulated by pro-permeability agonists. In part two, we compared the transcriptomes of HRECs treated with VEGF to those treated with VEGF then anti-VEGF to identify the VEGF-regulated genes that were also sensitive to anti-VEGF. Additionally, transendothelial electrical resistance (TEER) assays were used to study paracellular permeability.
Results :
Analysis of the RNA sequencing data revealed that VEGF, TNFa and IL-1b each altered expression of thousands of genes, whereas IL-6 altered expression of just 81 genes, 10 of which were also regulated by the other three agonists. To identify genes likely to govern permeability, we filtered this list of 10 genes for those that were governed by both VEGF and anti-VEGF. The final qPCR-based validation step identified NPR1 as the top candidate. Natriuretic peptide receptor 1 (NPR1) has been shown to complex with gamma secretases of the Notch pathway. Our RNA-Seq analysis found that several Notch target genes and pathway members are regulated by both VEGF and cytokines. We have previously found that Notch signaling is required for VEGF to sustain permeability. Here we report that Notch signaling is also required to maintain cytokine-induced permeability.
Conclusions :
These findings support the existence of common permeability genes and identify our top candidate as NPR1. Further studies will determine whether agonist-induced changes in NPR1 expression alter Notch signaling, leading to endothelial permeability.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.