June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Expression and Functional Role of a Novel Restrictive Element-1 Silencing Transcription Repressor (REST) in Cornea
Author Affiliations & Notes
  • Rajiv R Mohan
    Mason Eye Institute and VMTH, University of Missouri-Columbia , Columbia, Missouri, United States
    Truman VA Hospital, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Rajiv Mohan, None
  • Footnotes
    Support  NEI/NIH R01EY030774, NEI/NIH RO1EY17294, VA 1I01BX000357 merit & RCS, and Ruth M. Kraeuchi Missouri Endowed Chair Fund
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3549. doi:
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      Rajiv R Mohan; Expression and Functional Role of a Novel Restrictive Element-1 Silencing Transcription Repressor (REST) in Cornea. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3549.

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

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Abstract

Purpose : Previously, we reported role of epigenetics in corneal wound healing modulation and haze inhibition using HDAC inhibitors (SAHA, TSA and ITF2357). What molecular factors control this process remain largely unknown? This study tested a novel hypothesis that the Restrictive Element-1 Silencing Transcription repressor (REST) is expressed in the cornea and plays role in TGFβ-mediated fibrotic signaling events. Thus, we examined (a) expression of REST mRNA and protein in donor human corneas, (b) healthy and fibrotic rabbit corneas, and (c) its functional role in corneal fibrosis using an established in vitro model.

Methods : New Zealand White rabbits and donor human corneas were used. A corneal scar in rabbit eye was created with alkali, and unwounded and wounded corneas were collected 1- 3-, 7-, and 14-day after wounding. Serial sections of human and rabbit corneas were prepared and used. Primary corneal stromal fibroblast (hCSFs) were generated from donor human corneas. Human corneal myofibroblasts (hCMFs) were generated by growing hCSFs in the presence of TGFβ1 for 72h in serum-free conditions. Alpha smooth muscle actin (aSMA) was used to gauze formation of myofibroblast and fibrosis. Western blotting, immunofluorescence, confocal microscopy, and qRT-PCR were used to quantify mRNA and protein levels.

Results : Expression of REST was observed in human and rabbit corneas. The alkali-wounded fibrotic rabbit corneas showed substantially less REST mRNA (2-5-fold) and protein (30-55%) levels compared to the unwounded rabbit corneas collected on day-1, day-3, day-7, and day-14 post-wounding. The hCSFs grown in presence of TGFβ1 under serum-free conditions showed a significant upregulation of REST mRNA at early times (4h; p>0.001) then return back to normal in a time dependent manner in 72h whereas a consistently increasing aSMA mRNA levels were up to 72h in a time dependent manner. More experiments probing role of REST are underway.

Conclusions : This study first time reports expression of REST in the cornea and its involvement in corneal fibrosis. Further studies to characterize functional roles of REST in corneal fibrosis are warranted.

This is a 2020 ARVO Annual Meeting abstract.

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