MODULATION OF AUTOPHAGY FLUX AND CLEAR NETWORK IN HUMAN CORNEA IN VITRO AND RABBIT CORNEA IN VIVO

Abstract

Purpose : Earlier, we reported the expression of autophagy related proteins in human corneal epithelium, fibroblast, and endothelium and their modulation in corneal fibroblasts -/+ injury in vitro and normal and keratoconus human corneas from patients. This study investigated if autophagy flux and clear network during corneal repair and remodeling can be targeted with pharmacological agents in vitro and in vivo.

Methods : New Zealand White rabbits and healthy donor human corneas were used for in vivo and in vitro studies, respectively. Topical alkali was used for corneal injury. Rabbits were divided into 3 groups: Group-1 had alkali alone, Group-2 had alkali+inhibitor (chloroquine), and Group-2 had alkali+activator (rapamycin). Naïve corneas of other study and contralateral corneas of this study served as negative controls. Drugs were administered topically twice a day for 7 days post-alkali. Corneas were collected 14- and 28-day post-alkali. Human corneal fibroblasts (hCSFs) generated from donor human corneas were used for in vitro corneal wound healing model. Cells were grown in -/+ transforming growth factor beta 1 (TGFβ1; 5ng/ml) for 24-72h. Serial corneal sections, protein lysates and cDNAs were used to study in autophagy flux and CLEAR network (ATGs, LC3I/II, SQSTM1/p62, LAMP1, LAMP2, mTORC1, TFEB, vATPase).

Results : An expected corneal haze in rabbits was observed in vivo. The clinical slitlamp exams and in vivo confocal imaging showed remarkably different levels of corneal haze, inflammation, and opacity in rabbit eyes of three groups in a time-dependent manner (p<0.01, <0.05, <0.001). The qRT-PCR and immunoflurecscence studies observed significantly different levels of ATGs, LC3I, LC3II, SQSTM1/p62, LAMP1, and LAMP2 mRNA and protein levels in chloroquine and rapamycin administered eyes compared to the alkali-injury alone and naïve corneas. In vitro investigations found a remarkably different autophagy flux measured by measuring mRNA and protein expression of ATGs, LC3I/II, SQSTM1/p62, LAMP1, LAMP2, mTORC1, TFEB, and/or vATPase in hCSFs -/+ TGFβ1. Additional in vivo and in vitro investigations are ongoing.

Conclusions : Autophagy modulates corneal wound healing and can be potentailly used as a novel target to promote scarless corneal repair.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.