Abstract
The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-β. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGβ1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.
Highlights
Visual clarity depends on a clear and transparent cornea
Corneal wound healing involves the differentiation of quiescent keratocytes to active fibroblasts and myofibroblasts which proliferate and secrete extracellular matrix proteins, thereby aiding wound contraction and closure [3,26–28]
Continued TGF-β stimulation in myofibroblasts can drive the development of fibrosis by inducing excessive collagen synthesis and extracellular matrix (ECM) deposition [29,30]
Summary
Visual clarity depends on a clear and transparent cornea. Trauma to the cornea due to corrective refractory surgical procedures such as photorefractive keratectomy (PRK) and mechanical or chemical injuries such as corneal tear, abrasion, burn, etc., can cause an aberrant corneal stromal wound repair response leading to haze or opacity that can distort vision [1,2]. In certain scenarios (e.g., flap detachment or post-LASIK ectasia), aberrant or uncontrolled wound healing leads to fibrosis (haze or scarring) with reduced corneal transparency or increased corneal opacity [11]. It is pertinent to understand the molecular mechanisms that modulate corneal wound healing at the early stages when stromal keratocytes are activated and undergo the differentiation to the repair cell types. Identification of such molecular factors could provide the putative targets to modulate the wound healing process and reduce the incidence of corneal fibrosis in the clinical scenario
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