June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
SIRT1: Expression in the cornea and its role in corneal wound healing
Author Affiliations & Notes
  • Rajiv R Mohan
    Mason Eye Institute and VMTH, University of Missouri-Columbia, Columbia, Missouri, United States
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • govindaraj anumanthan
    VMTH, University of Missouri, Columbia, Missouri, United States
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • Tripathi Ratnakar
    VMTH, University of Missouri, Columbia, Missouri, United States
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • Suneel Gupta
    VMTH, University of Missouri, Columbia, Missouri, United States
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • Michael K Fink
    VMTH, University of Missouri, Columbia, Missouri, United States
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • Prashant Rajiv Sinha
    VMTH, University of Missouri, Columbia, Missouri, United States
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • Sally D Heil
    VMTH, University of Missouri, Columbia, Missouri, United States
  • Shyam S Chaurasia
    VMTH, University of Missouri, Columbia, Missouri, United States
  • Elizabeth A Giuliano
    VMTH, University of Missouri, Columbia, Missouri, United States
  • Nathan P Hesemann
    Ophthalmology, Truman VA Hospital, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Rajiv Mohan, None; govindaraj anumanthan, None; Tripathi Ratnakar, None; Suneel Gupta, None; Michael Fink, None; Prashant Sinha, None; Sally Heil, None; Shyam Chaurasia, None; Elizabeth Giuliano, None; Nathan Hesemann, None
  • Footnotes
    Support  Veteran Health Affairs Merit 1I01BX00035701 (RRM) grant from Washington DC, The University of Missouri Ruth M. Kraeuchi Missouri Endowment Chair Ophthalmology Fund (RRM), and partially from the RO1EY17294 (RRM) National Eye Institute, NIH, Bethesda, Maryland, USA.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 144. doi:
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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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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