June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
SIRT1: A novel target to regulate corneal myofibroblast formation
Author Affiliations & Notes
  • Ratnakar Tripathi
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
  • Lynn M. Martin
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
  • Sabeeh Kamil
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
  • Nishant Rajiv Sinha
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
  • Alexandria Hofmann
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
  • Sydney Green
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
  • Praveen Balne
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
  • Suneel Gupta
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
  • Prashant R. Sinha
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
  • Nathan Hesemann
    Mason Eye Institute, School of Medicine, University of Missouri, Columbia, Missouri, United States
  • Frederick W Fraunfelder
    Mason Eye Institute, School of Medicine, University of Missouri, Columbia, Missouri, United States
  • Shyam S Chaurasia
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
  • Rajiv R Mohan
    Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Ratnakar Tripathi, None; Lynn Martin, None; Sabeeh Kamil, None; Nishant Sinha, None; Alexandria Hofmann, None; Sydney Green, None; Praveen Balne, None; Suneel Gupta, None; Prashant Sinha, None; Nathan Hesemann, None; Frederick Fraunfelder, None; Shyam Chaurasia, None; Rajiv Mohan, None
  • Footnotes
    Support  NEI/NIH R01EY030774 and RO1EY17294, VA 1I01BX000357 Merit & RCS and Ruth M. Kraeuchi Missouri Endowment Chair Ophthalmology Fund.
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2605. doi:
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      Ratnakar Tripathi, Lynn M. Martin, Sabeeh Kamil, Nishant Rajiv Sinha, Alexandria Hofmann, Sydney Green, Praveen Balne, Suneel Gupta, Prashant R. Sinha, Nathan Hesemann, Frederick W Fraunfelder, Shyam S Chaurasia, Rajiv R Mohan; SIRT1: A novel target to regulate corneal myofibroblast formation. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2605.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Corneal scarring/fibrosis is common after trauma, infection, or refractive surgery in the eye. Our recent studies revealed that histone deacetylase (HDAC) inhibitors, Trichostatin-A, SAHA, and ITF2357, inhibit corneal fibrosis in vivo. We hypothesized that class III HDAC, sirtuins are involved in corneal wound healing and fibrosis regulation. The aims of the study were to (1) characterize levels and localization of SIRT1 mRNA and protein in the cornea, and (2) study its role in corneal wound healing modulation.

Methods : Donor human corneas and naive/wounded corneas of New Zealand White rabbits collected at different times were used. The qRT-PCR, Immunofluorescence, and immunoblotting techniques were employed to measure the level and localization of gene and protein in cell cultures and corneal tissues. Primary human corneal stromal fibroblasts (hCSF) were grown in the presence and absence of TGFβ1 (5ng/ml) at different time points (0, 12, 24, 48, and 72 hours) for time-dependent experiments aimed to analyze SIRT1 mRNA and protein expression. Gene transfer gain-of-function and loss-of-function experiments were also explored, involving nanoparticles delivered SIRT1 overexpression and siRNA mediated gene silencing.

Results : Normal human and rabbit corneas showed SIRT1 expression in the epithelium, stroma, and endothelium. A significantly (p<0.001) decreased levels of SIRT1 were observed in wounded human and rabbit corneas. hCSFs exposed to TGFβ1 demonstrated decreased SIRT1 mRNA (p<0.001) and protein (p<0.001) levels in a time-dependent manner. Gene transfer gain-of-function shows significant (p<0.01) decreased level of αSMA (key regulator of fibrosis) and siRNA driven gene silencing shows significant (p<0.01) increase in αSMA in vitro. Gain-of-function and loss of function experiments involving SIRT1 overexpression and gene silencing demonstrated regulation of the TGFβ1-mediated wound healing process by SIRT1 in an in vitro model of corneal fibrosis.

Conclusions : To the best of our knowledge, this is the first report to show the expression of SIRT1 in the cornea and its role in TGFβ1 driven corneal fibrosis. More studies are underway.

This is a 2020 ARVO Annual Meeting abstract.

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