July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Id3 gene over-expression in corneal stromal fibroblasts blocks TGFβ-driven fibrosis in an in vitro model
Author Affiliations & Notes
  • Mikayla Fraunfelder
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Suneel Gupta
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Michael K. Fink
    Mason Eye Institute, University of Missouri, Columbia, Missouri, United States
  • Praveen Balne
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Ratnakar Tripathi
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Prashant R. Sinha
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Sally Heil
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Elizabeth A Giuliano
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Nathan P. Hesemann
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    Mason Eye Institute, University of Missouri, Columbia, Missouri, United States
  • Shyam S Chaurasia
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Rajiv R Mohan
    Harry S. Truman Memorial Veteran Hospital, Columbia, Missouri, United States
    College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Mikayla Fraunfelder, None; Suneel Gupta, None; Michael Fink, None; Praveen Balne, None; Ratnakar Tripathi, None; Prashant Sinha, None; Sally Heil, None; Elizabeth Giuliano, None; Nathan Hesemann, None; Shyam Chaurasia, None; Rajiv Mohan, None
  • Footnotes
    Support  Primarily by 1I01BX00035701 Veteran Health Affairs, Washington DC USA and partially by the RO1EY17294 National Eye Institute, NIH, Bethesda, Maryland, USA and the Ruth M. Kraeuchi Missouri Endowment Chair Ophthalmology Fund.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4674. doi:
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      Mikayla Fraunfelder, Suneel Gupta, Michael K. Fink, Praveen Balne, Ratnakar Tripathi, Prashant R. Sinha, Sally Heil, Elizabeth A Giuliano, Nathan P. Hesemann, Shyam S Chaurasia, Rajiv R Mohan; Id3 gene over-expression in corneal stromal fibroblasts blocks TGFβ-driven fibrosis in an in vitro model. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4674.

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

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Abstract

Purpose : Trauma, infection and injury cause corneal fibrosis. Previously, we demonstrated the role of inhibitor of differentiation (Id) genes in corneal wound healing. This study tested whether Id3 over-expression in corneal stromal fibroblasts (CSFs) could block their differentiation into myofibroblasts and thus be used for treating corneal fibrosis.

Methods : Primary human corneal stromal fibroblasts (hCSF) were generated from donor human corneas. Human corneal myofibroblasts (hCMF) were produced by growing hCSFs in 5ng/ml TGFβ1 for 72h in serum-free conditions. Gene transfer was accomplished using PEI2-GNPs nanoparticles and stable-clone selection by G418. Immunofluorescence, western blotting, qPCR, 2D/3D confocal microscopy, and slot blots were used to quantify levels of mRNA, protein, and gene-delivery.

Results : Stable hCSF-Id3 clones demonstrated phenotype, viability, and proliferation similar to non-transfected hCSFs. The hCSF-Id3 clones grown in serum-free conditions with TGFβ1 exhibited a significant decrease in mRNA of fibrotic genes such as α-SMA (3.3; p<0.05), fibronectin (2.1; p<0.01), collagen I (1.7; p<0.005) and collagen IV (1.4; p<0.05) compared to the hCSF-naked clones under similar conditions. Furthermore, hCSF-Id3 clones displayed significantly reduced expression of these four fibrotic proteins in immunofluorescence and western blot (71-83%; p<0.05, p<0.01 or p<0.001) compared to hCSF-naked clones. Ongoing mechanistic studies indicate that Id3 regulates hCSF differentiation via bHLH region of TATA box E protein.

Conclusions : Manipulation of the Id3 gene offers a potentially novel gene therapy to treat corneal fibrosis. Rabbit in vivo studies are warranted.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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