Modulation of autophagy-related genes in healthy, keratoconus and injured corneas

Abstract

Purpose : Autophagy plays a key role in cellular homeostatic maintenance by discarding and recycling cytoplasmic proteins and dysfunctional organelles. Recently, we identified the involvement of autophagy in corneal wound healing and fibrosis. This study investigated (a) the expression of autophagy-related genes (Atgs) in healthy and diseased human and rabbit corneas and (b) autophagy's response to select treatments and contribution to corneal wound healing using donor human corneas and primary human corneal stromal fibroblast (hCSFs) cells.

Methods : Donor healthy and diseased human corneas, normal and wounded rabbit corneas and primary cultures of hCSFs cells generated from healthy human corneas were utilized for experiments. Cultures were grown in MEM medium with 1% or 10% FBS in +/- TGFβ1 or +/- starvation (FBS), +/- chloroquine or +/- rapamycin treatment. Serial corneal tissue sections prepared from healthy and diseased tissues (human and rabbit) were used to study differential Atg expression. Immunofluorescence, 2D/3D confocal microscopy, western blotting, qPCR, MTT, TUNEL, LDH, wound-scratch, TEM and other commercial assays were used for wound healing studies.

Results : Expression of LC3, BECN1 and SQSTM1 Atgs was detected in the selected human and rabbit corneas in vitro and in vivo. Starvation conditions decreased SQSTM1 and BCN1 and increased LC3-II protein expression in vitro as compared to the control (untreated hCSFs). TEM identified autophagosomal structures within primary hCSFs. Analysis of mRNA for treatment conditions and TGFβ1 stimulation regarding the levels of Atgs in hCSFs compared to the control (untreated hCSFs) is underway. Results of in vivo wound healing experiments are pending.

Conclusions : Autophagy mechanisms are used by the cornea for cellular homeostasis and become altered during corneal wound repair. Ongoing additional studies will provide more specific functional roles of Atgs in corneal wound healing and disease modulation.

This is a 2020 ARVO Annual Meeting abstract.