Autophagy regulates corneal wound healing

Abstract

Purpose : Autophagy plays a key role in homeostatic maintenance by disposing of and recycling cytoplasmic proteins and dysfunctional organelles. The precise role of autophagy in corneal wound healing and fibrosis is poorly understood. This study investigated (a) the expression of autophagy-related genes (Atgs) in healthy and diseased human corneas and (b) autophagy's contribution to corneal wound healing involving fibrosis and neovascularization using donor human corneas and primary human corneal cells.

Methods : Donor healthy and diseased human corneas, normal and wounded rabbit corneas, and primary cultures of corneal stromal fibroblasts (HSFs), epithelial (HCE) and endothelial (HCN) cells generated from healthy human corneas were utilized for experiments. Cultures were grown in MEM medium with 1% or 10% FBS in +/- TGFβ1 or VEGF. 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, and other commercial assays were used for wound healing studies.

Results : Expression of LC3, BECN1, Atg5, DRAM1, SQSTM1, and PRKN2 genes was detected in the three selected corneal cells in vitro and in vivo in human and rabbit corneas. A significantly reduced level of autophagosomal (LC3-II) and lysosomal marker (LAMP1) genes was noted in cone and periphery regions of the corneal epithelium of keratoconus (KC) corneas compared to the healthy corneas. TGFβ1 or VEGF stimulation significantly altered mRNA and protein expression of six tested Atgs in vitro. Results of in vitro wound healing experiments are pending.

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

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.