Novel approaches for corneal haze/fibrosis elimination

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Abstract Corneal fibrosis (haze) after injury, infection, or trauma causes blindness in 1.3 million Americans each year and accounts for 7% of the world’s blindness. Current drugs often fail to cure severe and established corneal haze. At present, cornea transplant surgery is the standard of care. A normal clear cornea do not contain myofibroblasts. After insult, quiescent transparent keratocytes are activated by transforming growth factor b (TGFb) to become corneal stromal fibroblasts (CSFs), which then transdifferentiate into corneal myofibroblasts (CMFs) to do corneal repair. Excessive and prolonged formation of CMFs during early-stages and persistence of CMFs in stroma in late-stages of wound healing are the primary causes of haze in vivo. Recently, we uncovered 2 novel mechanisms regulating this process: (a) haze production involves the intermediate- conductance Ca2+-activated K+ channel, KCa3.1 whereas (b) haze elimination in vivo can be induced via selective apoptosis in CMFs by a dual therapy. This study provided the first evidence that haze can be cleared by directing the fate of CMFs in vivo. CMFs are not terminally differentiated cells and can be reverted to CSFs in vitro. Myofibroblast formation, reversal, and de-differentiation are tissue-specific events, and no cornea-specific data exists currently. This premise led to a central hypothesis that novel epigenetic approaches used to direct the fate of CMFs in vivo will eliminate established and severe haze in vivo, and provide a non-surgical means of vision restoration. This project aims optimizing non-surgical method to treat haze pursuing 3 specific aims. Aim-1 tests a hypothesis that TRAM-34 (a KCa3.1 specific inhibitor) limits CMF formation in early-stages of wound healing in vivo by inhibiting KCNN4 gene transcription via upregulation of Restrictive element-1 silencing transcription factor (REST) reducing AP1 (Activator protein-1). Aim-2 tests a hypothesis that epigenetic reprogramming in late-stages of wound healing by Sodium butyrate (NaB) will efficiently de-differentiate CMFs to precursor CSFs in vitro and keratocytes in vivo by enhancing DNA methyl transferases (DNMTs), methyl CpG binding protein 2 (MeCP2) and DNA methylation of CpG islands on the promoter on alpha-smooth muscle actin gene. Aim-3 tests a hypothesis that TRAM-34+NaB dual therapy will fully abolish severe/established haze in vivo in rabbits without significant side effects by (a) limiting TGFb-driven excessive and prolonged CMFs formation in early-stage wound healing by reducing KCa3.1 by TRAM-34, and (b) promoting CMFs reversal to precursor cells in late-stage wound healing by NaB via epigenetic reprogramming. Our pilot studies strongly support hypotheses. Proposed studies will be accomplished employing established in vitro human and in vivo (rabbit and KCa3.1-/- mice) corneal fibrosis models, clinical eye exams, molecular, and cellular assays and following our published method. The successful conclusion of project will fill key knowledge gaps and significantly advance the corneal field.