Abstract
Purpose :
Macroautophagy is a vital cellular mechanism, activated in response to oxidative stress. Aim of this study is to understand the pathogenesis of keratoconus with respect to oxidative damage in patient samples and an in-vitro model
Methods :
After informed consent and Institutional Ethics approval, 50 corneal epithelia were collected for the study including 30 KC patients (Amsler Krumeich grades 1-3; 10 patients per grade) undergoing topography guided photorefractive keratectomy(T-PRK) or corneal cross-linking and 20 were controls subjects undergoing photorefractive keratectomy (PRK) for the correction of refractive errors. We performed microarray, Quantitative PCR and Western blot analysis from different grades of KC patients and control epithelium respectively. For the in-vitro model, human corneal epithelial cells exposed to chronic hyperoxic condition was assessed by qPCR, WB, fluorescence imaging and flowcytometry (Cyto-ID, Lysotracker Red, tfLC3)
Results :
In KC patients, the micro array and qPCR analysis revealed increasing fold expression of autophagy pathway genes, including those involved in induction, maturation and degradation process compared to controls. Further validation of protein levels showed accumulation of autophagsosmes (microtubule-associated protein-1 light chain 3- LC3A/B) and autolysosomes (lysosomal associated membrane protein- LAMP-1) that correlated with increasing grades of KC. In addition, human corneal epithelial cells exposed to chronic oxidative stress showed imbalanced expression of autophagy related proteins including LC3A/B and LAMP-1 when compared to controls. Interestingly, the oxidatively stressed corneal epithelial cells and KC epithelium showed decreased levels of phosphorylated RPS6KB (p70S6 Kinase), a downstream target of mTOR pathway
Conclusions :
All together our data suggests impaired autophagy regulation due to oxidative damage in cornea which may be driving the mechanism and progression of keratoconus. Targeting the mTOR and autophagic lysosomal pathway may open new avenues to develop therapeutic approaches for the treatment of Keratoconus
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.