Abstract
Purpose :
To investigate the function of endoplasmic reticulum stress in the development of fibrotic cataract and its underlying mechanisms.
Methods :
C57BL/6 mice were punctuated with a 26-gauge needle in the central lens capsule as the fibrotic cataract model. Primary rabbit lens epithelial cells (LEC) were exposed to TGF-β2. eIF2α inhibitor ISRIB was used to treat LEC both in vivo and in vitro, ER stress inhibitor 4-PBA, inducer thapsigargin and tunicamycin, IRE1α inhibitor 4μ8C were used to treat LEC in vitro. LECs were transfected with siRNA to knockdown ATF4. Western blot assays and immunofluorescence staining were carried out to measure changes in protein expression such as BIP, eIF2α, IRE1α, LC3-II, p62, FN, α-SMA, etc. mRNA expression was investigated by RT-PCR. Autophagosome was observed by electron microscopy (TEM). Immunostaining and mRFP-GFP-LC3 reporter were applied to indicate autophagy flux. Clinical parameters were measured using slit-lamp bio-microscopy.
Results :
ER stress was triggered during fibrotic cataract and TGFβ2-induced EMT of LEC, which in turn enhanced the EMT process. PERK/eIF2α/ATF4 branch of unfolded protein response is selectively required for EMT. Anterior chamber injection of ISRIB in fibrotic cataract mouse model showed significantly improved clinical parameters and limited capsular opacities. Interestingly, we found ISRIB decreased LC3-II, along with impaired the turnover of p62 in a time-dependent manner. Besides, TEM showed abnormally autophagic vacuoles under TGFβ2 stimulation, which was largely abolished by the cotreatment with ISRIB. To further investigate the crosstalk between ER stress and autophagy during fibrotic cataract, autophagic inducer rapamycin and lysosomal inhibitor chloroquine was used. Our results indicated that the suppression of ISRIB on mesenchymal gene expression was attenuated by rapamycin, but augmented by CQ, indicating that the regulation of autophagic flux by ISRIB is implied in the anti-fibrotic process.
Conclusions :
Our research for the first time suggests that the activation of ER stress in fibrotic cataract both in vivo and in vitro. PERK/eIF2α/ATF4 branch selectively regulates EMT process in LEC through autophagy. eIF2α inhibitor, ISRIB, is highly effective at suppressing the development of lens subcapsular plaque and keeping the transparency of lens. Thus, ISRIB may be a potential therapeutic target to reduce EMT and fibrotic diseases.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.