Abstract
Purpose :
Proliferative Vitreoretinopathy (PVR) is characterized by the proliferation of retinal pigment epithelial (RPE) cells. In PVR, RPE cells undergo transformation into fibroblast-like cells by epithelial to mesenchymal transition (EMT). The purpose of this study was to investigate the role of αB crystallin (αBC) peptide on epithelial EMT in experimental PVR.
Methods :
Subconfluent primary human RPE (hRPE) cells were stimulated by TGFβ2 (10 ng/ml) with or without αBC peptide (50 or 75 μg/ml) for 24h or 48h EMT/MET-associated markers were examined by RT-PCR and WB analysis. The effect of TGFβ2 on glycolysis and oxidative phosphorylation was determined in hRPE cells using Seahorse XF96 analyzer. PVR was induced in 6-8 week old male C57BL/6J mice by intravitreal administration of dispase (10 mg/ml) and PVR progression was studied on day 21. Expression of αSMA, E-cadherin (E-C), fibronectin (FN), and RPE65 in control and dispase-treated retina was determined by immunostaining on day 21.
Results :
The EMT-associated αSMA and vimentin showed a significant upregulation with TGFβ2 stimulation at the gene (3- and 1.5-fold) and protein (2.5-fold) level. The protein expression of these markers was significantly suppressed by co-treatment with αBC peptide (p < 0.05 vs TGFβ2 alone). The mesenchymal epithelial transition (MET)-associated genes and proteins, E-C and SIRT1 were significantly downregulated by TGFβ2 (> 2-fold) and were restored by αBC peptide cotreatment. Mitochondrial oxygen consumption rate (OCR, pmol/min/total DNA) increased with TGFβ2 treatment for 24h or 48h and αBC peptide co-treatment caused a further increase in OCR for both time points. An increased deposition of αSMA and FN and a decreased expression of E-C and migration of RPE65 was observed 3 weeks after dispase administration by immunofluorescence.
Conclusions :
Our findings suggest that αBC peptide may have therapeutic potential in preventing the proliferation of PVR by reversing the phenotype of EMT/MET and improving the mitochondrial function in RPE cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.