Abstract
Purpose :
Leakage of retinal blood vessels, which is an essential element of diabetic retinopathy (DR), is driven by chronic elevation of vascular endothelial growth factor (VEGF). VEGF quickly (within minutes) relaxes the endothelial cell barrier by triggering signaling events that post-translationally modify junctional proteins such as VE-cadherin. VEGF also changes expression of genes, some of which are known to regulate endothelial cell barrier function. The purpose of this project is to identify effectors by which VEGF and anti-VEGF control the endothelial cell barrier in cells that are chronically exposed to VEGF (hours instead of minutes).
Methods :
Electric cell-substrate impedance sensing (ECIS)- and FITC dextran-based approaches were used to quantify barrier function of high glucose-treated primary human retinal endothelial cells. RNAseq and qRT-PCR were used to identify VEGF/anti-VEGF-regulated genes. Pharmacological approaches were used to activate or antagonize the RAAS.
Results :
VEGF both activated VEGFR2, and relaxed the barrier within minutes. While anti-VEGF quickly inactivated VEGFR2, it took much longer to reclose the barrier. Furthermore, increasing the duration of exposure to VEGF resulted in a corresponding increase in how long it took anti-VEGF to re-establish barrier function. Because reversal of post-transcriptional changes did not fully explain how anti-VEGF reestablished barrier function in cells chronically exposed to VEGF, we considered additional contributors. Pre-clinical and clinical studies have demonstrated that RAAS contributes to leakage of retinal vessels and progression of DR. However, the mechanism has not been investigated. We considered if VEGF/anti-VEGF acted via RAAS to control barrier function. Indeed, VEGF increased expression of angiotensin converting enzyme (ACE) and anti-VEGF reversed this change. Furthermore, antagonizing either ACE, or angiotensin II receptor type 1 attenuated VEGF-induced permeability. Finally, activating RAAS with exogenous ACE or angiotensin II impaired anti-VEGF’s ability to re-close the barrier that was opened with VEGF.
Conclusions :
RAAS is one of the effectors that relax the endothelial barrier in cells that are chronically exposed to VEGF. These discoveries provide a plausible mechanistic explanation for the long-standing appreciation that antagonizing RAAS is beneficial for patients with DR.
This is a 2020 ARVO Annual Meeting abstract.