Regulation of retinal endothelial cell barrier function by the hydroxycarboxylic acid receptor 2 (HCAR2)/GPR109A

Abstract

Purpose : Loss of barrier integrity due to endothelial cell damage or dysfunction is common in retinal microvascular diseases such as diabetic retinopathy. We reported previously, the expression of the beta-hydroxybutyrate (BHB) receptor HCAR2/GPR109A in retina where it localizes robustly to the basolateral membrane of the retinal pigment epithelium (RPE) and regulates outer retinal barrier integrity. Our recent work confirms receptor expression also in retinal endothelial cells. However, its functional relevance in these cells is not well understood. Here, we examined relevance of receptor expression to endothelial cell dependent (inner)-blood retinal barrier integrity.

Methods : DasiRNA technology was used to modulate HCAR2/GPR109A expression in primary human retinal endothelial cells (HRECs). Scrambled control and Hcar2/Gpr109a-knockdown HRECs were grown on transwell filters for 4-6 weeks. qPCR was used to monitor HCAR2/GPR109A expression and electrical cell impedance sensing (ECIS) technology to evaluate barrier function. To investigate the relevance of HCAR2/GPR109A expression to barrier function in vivo, retinal inflammation was induced in wildtype and HCAR2/knockout mice using LPS (4 mg/kg). Single cell suspensions prepared from retinal homogenates obtained mice injected intraperitoneally LPS or PBS (control) +/- HCAR2/GPR109A agonist BHB were assessed by flow cytometry to monitor immune cell infiltration.

Results : Absence (KO) or reduction (siRNA) of HCAR2/GPR109A expression impaired barrier function (ECIS) significantly contributing to the increased infiltration of pro-inflammatory immune cells in retina as indicated by flow studies. BHB treatment preserved retinal barrier integrity and prevented increased immune cell infiltration in retina even in the presence of a pro-inflammatory stimulus (LPS), an effect that was receptor (HCAR2/GPR109A)-dependent.

Conclusions : Collectively, these data establish a role for HCAR2/GPR109A in regulating retinal endothelial cell barrier function in normal and inflammatory conditions. Thus, highlighting the therapeutic potential that targeting the receptor holds with respect to prevention and treatment of degenerative retinal diseases like diabetic retinopathy in which compromised barrier function is paramount.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.