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Praveen Kumar Balne, Ratnakar Tripathi, Suneel Gupta, Sabeeh Kamil, Prashant R. Sinha, Nishant Rajiv Sinha, Nathan Hesemann, Shyam S Chaurasia, Victor J. Thannickal, Rajiv R Mohan; Corneal stromal fibroblast-myofibroblast-fibroblast regulation via gene silencing. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2921.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal insult leads to scarring (fibrosis) and vision impairment. Corneal stromal fibroblasts (CSFs) differentiation to myofibroblasts (CMFs) is a dominant mechanism for corneal fibrosis. In pathological conditions, myofibroblast contribute to the continuous synthesis of ECM and leading to corneal fibrosis. We tested a novel hypothesis that MyoD (basic helix-loop-helix) gene silencing could regulate fibroblast-myofibroblast-fibroblast interchange using an established in vitro model.
Donor healthy and diseased human corneas, normal and wounded rabbit corneas, and primary cultures of hCSFs cells generated from human corneas were used. An in vitro model facilitating human corneal stromal fibroblast-myofibroblast-fibroblast interchange in +/- TGFβ1 was used. MyoD-RNAi gene silencing experiments were performed using Lipofectamine-3000. Immunofluorescence, confocal microscopy, western blotting, and qRT-PCR were used to analyze expression of fibroblast and myofibroblast specific markers.
Diseased human cornea showed significantly increased expression of MyoD mRNA and protein expression compared to the healthy human cornea (p≤0.05). Likewise, alkali injured rabbit corneas demonstrated significant up-regulation of MyoD mRNA and protein compared to the uninjured rabbit corneas (p≤0.05). TGFβ1 treatment facilitated stromal fibroblast-myofibroblast conversion in a time dependent manner from 6h-72h with a peak observed at 72h. A significant upregulation of MyoD and αSMA mRNA and protein expression was detected at 72h in hCSFs grown in +TGFβ1 compared to grown in -TGFβ1 (p≤ 0.01). MyoD-RNAi transfected CSFs showed no morphological changes and cytotoxicity. However, MyoD-RNAi transfected CMFs acquired phenotype similar to CSFs. A significantly decreased mRNA and protein levels of myofibroblast specific marker αSMA, and increased levels of CSFs specific marker fibroblast specific protein-1 (FSP1) were observed in CMFs transfected with MyoD-RNAi compared to controls (p≤ 0.01).
MyoD gene silencing has potential to control fibroblast-myofibroblast-fibroblast interchange involved in corneal fibrosis.
This is a 2020 ARVO Annual Meeting abstract.
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