Identification of novel predictive factors for post surgical corneal haze

Nimisha R Kumar,Arkasubhra Ghosh,Ankit Sharma,Anuprita Ghosh,Rajani Kanth Vangala,Valsala Gopalakrishnan Abilash,Rajiv R Mohan,Vishal Jhanji,Rohit Shetty,Naren Shetty,Natasha Pahuja,Pooja Khamar

doi:10.1038/s41598-019-53123-3

Abstract

Molecular factors altered in corneas that develop haze post refractive surgery have been described, but pre-existing factors that predispose clinically normal corneas to aberrant fibrosis post surgery and the role of the corneal epithelium remains unknown. We analyzed the global gene expression in epithelium collected intraoperatively from subjects undergoing photorefractive keratectomy. Subjects were grouped into those that developed haze 12 months post surgery (n = 6 eyes; haze predisposed) and those that did not develop haze in a similar follow up duration (n = 11 eyes; controls). Ontological analysis of 1100 upregulated and 1780 downregulated genes in the haze predisposed group revealed alterations in pathways associated with inflammation, wnt signaling, oxidative stress, nerve functions and extra cellular matrix remodeling. Novel factors such as PREX1, WNT3A, SOX17, GABRA1and PXDN were found to be significantly altered in haze predisposed subjects and those with active haze(n = 3), indicating their pro-fibrotic role. PREX1 was significantly upregulated in haze predisposed subjects. Ectopic expression of PREX1 in cultured human corneal epithelial cells enhanced their rate of wound healing while its ablation using shRNA reduced healing compared to matched controls. Recombinant TGFβ treatment in PREX1 overexpressing corneal cells led to enhanced αSMA expression and Vimentin phosphorylation while the converse was true for shPREX1 expressing cells. Our data identify a few novel factors in the corneal epithelium that may define a patient’s risk to developing post refractive corneal haze.

Highlights

With reasonable success but the safety of this drug with reference to the cytotoxic effects on stromal keratocytes and corneal endothelium remains a concern[9,10]
They are responsible for secreting the factors such as IL1α, PDGF, etc. which are required for activating the stromal keratocytes to initiate a wound healing response[23]
A scratch wound assay was performed and (a) microphotographs were taken at 0, 8, 16 and 24 hr. (b) Analysis of wound closure area in empty control vs PREX1 ORF. (c) A representative western blot showing PREX1 protein and α-tubulin served as endogenous control. q-PCR analysis showing fold changes of (d–g) PREX1, FN, ACTA2 and connective tissue growth factor (CTGF)

Summary

Introduction

With reasonable success but the safety of this drug with reference to the cytotoxic effects on stromal keratocytes and corneal endothelium remains a concern[9,10]. If the corneal epithelium secretes unbalanced levels of regulatory factors, it may contribute to abnormal fibrotic response in the stromal cells by driving excessive myofibroblast formation, aberrant collagen deposition and extracellular matrix remodelling[16]. Corneal epithelium could serve as repository of factors that may predispose clinically normal subjects undergoing refractive surgery to develop haze. There is a distinct lack of prior knowledge regarding molecular and tissue factors that predispose clinically healthy human eyes to develop haze post refractive surgery. Animal models of corneal haze adopt acute damage models such as 9D PRK20 and alkali burns[21] (1 N NaOH) which precipitate an immediate, robust pro-fibrotic response, which precludes the study of pre-existing tissue specific factors that tilt the balance of the wound healing response in certain human corneas post insult. This study reveals, for the first time, a set of factors whose pre-existing levels in the corneal tissue may possibly interact or influence known fibrotic mechanisms in corneal tissue post surgery leading to aberrant wound healing and haze

Methods

Results

Conclusion