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Rajiv R Mohan, govindaraj anumanthan, Tripathi Ratnakar, Suneel Gupta, Michael K Fink, Prashant Rajiv Sinha, Sally D Heil, Shyam S Chaurasia, Elizabeth A Giuliano, Nathan P Hesemann; SIRT1: Expression in the cornea and its role in corneal wound healing. Invest. Ophthalmol. Vis. Sci. 2017;58(8):144.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal scarring is frequent after injury. Our recent studies revealed that class I and II histone deacetylase (HDAC) inhibitors, Trichostatin, SAHA and ITF2357, inhibit corneal fibrosis, in vivo. However, the class III HDAC, sirtuins (SIRT1-7) role in modulating corneal wound healing and fibrosis remains unknown. This study tested the hypothesis that SIRT1 regulates proliferation and differentiation of corneal fibroblasts and extracellular matrix production by altering TGFβ1 signaling pathways. We sought to characterize levels and localization of SIRT1 mRNA and protein in the cornea, and its role in corneal wound healing.
Donor human corneas were used to generate corneal fibroblasts (HCF). Human and rabbit (normal and wounded) corneas and in vitro model of corneal fibrosis were used to characterize SIRT1 expression and role in the cornea and wound healing. Time-dependent experiments using primary HCFs analyzed SIRT1 mRNA and protein expression in +/- TGFβ1 (5ng/ml) at 0, 12, 24, 48 and 72 hours. SIRT1 overexpression by nanoparticles or gene silencing by RNAi technology were performed in HCF cells to analyze the fibrosis related gene expression by qPCR. Immunofluorescence, immunoblotting, and qPCR were used to confirm gene transfer and quantify mRNA and protein levels in cultures and in in vivo tissues.
All seven sirtuins were detected in HCF. High levels of SIRT1 was observed in all three major layers (epithelium, stroma and endothelium) of normal human and rabbit corneas, and significantly decreased levels in wounded human and rabbit corneas. HCF exposed to TGFβ1 demonstrated decreased SIRT1 mRNA and protein expression in a time-dependent manner. Gene transfer gain-of-function and loss-of-function experiments involving SIRT1 overexpression by nanoparticles or gene silencing by RNAi technology demonstrated SIRT1 role in the regulation of TGFβ1-mediated wound healing process (SIRT1 (p<0.01), αSMA (p<0.01), collagen I(p<0.01) and collagen IV (p<0.01)) in an in vitro model of corneal fibrosis.
SIRT1 modulates corneal wound healing and is a potential therapeutic target for developing gene therapy for treating corneal fibrosis in vivo. To the best of our knowledge, this is the first report showing the expression of SIRT1 in the cornea and its role in TGFβ1 driven fibrosis process in the cornea. More in vitro and in vivo studies are underway.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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