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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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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.
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.
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.
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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