Abstract
Purpose :
Corneal neovascularization (CN) is a major cause of blindness worldwide. We previously showed that endothelial mineralocorticoid receptor (MR) contributed to CN in mice and that MR antagonists (MRA) reduced corneal angiogenesis, edema and inflammation in a rat model of CN. We thus aimed to investigate the mechanisms of action of MRA in this model. As MRA upregulated the expression of glucocorticoid receptor (GR), we also investigated the potential synergistic effects of MRA and glucocorticoid (GC).
Methods :
CN was induced in Lewis rats by total corneal de-epithelialization and limbal scratching. Spironolactone (SPL, 25 mg/kg) was administered daily. Control eyes received vehicle. Rats were euthanized at day 3 and 7, and corneas were dissected for qPCR analysis of MR, GR, genes of growth factors and extracellular matrix components. Proteins extracted from corneas at day 7 were used for western blot. Corneal innervation was evaluated by immunostaining of TUBB3 on corneal flat mounts on day 16. To investigate synergistic effects of MRA and GC, rats were treated with SPL or triamcinolone acetonide (TA, 1 mg/kg) alone, or co-administered with both. Corneal morphology and CN were assessed in vivo by Micron III optical coherence tomography and fluorescein angiography on day 14. Corneas were also dissected on day 7 for transcriptomic analysis.
Results :
SPL upregulated Dcn, Col1a1, Timp2 and downregulated Lrg1 at day 3, and induced Vegfa and Pedf at day 7. An increase in DCN protein by SPL was confirmed by western blot. SPL increased the density of corneal subbasal nerve fibers. Coadministration of SPL and TA significantly reduced CN compared to SPL alone, and improved corneal re-epithelialization delayed by TA. The transcriptomic analysis identified 373 differentially expressed genes in the group of coadministration compared to 320 in TA and 75 in SPL groups. Of note, 172 genes are specifically induced by combined treatment. Gene set enrichment analysis highlighted pathways including wound healing and differentiation, inflammatory and immune responses, hypoxia, angiogenesis and neuroprotection.
Conclusions :
MRA regulates genes/proteins involved in anti-inflammatory and anti-angiogenic processes and improves corneal re-innervation in CN model. Coadministration of MRA and GC has synergistic effects on CN and epithelial wound healing.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.