Id Genes: Key Regulator of Fibroblast Transdifferentiation to Myofibroblast In The Cornea

Abstract

Purpose : Differentiation of corneal fibroblasts to myofibroblasts is a major underlying mechanism for corneal scarring development. Inhibitor of differentiation (Id) genes regulate cellular proliferation, differentiation and fibrosis in many tissues. Recently, we characterized Id genes expression in human cornea, and their role in corneal wound healing. This study tested the hypothesis that Id2 or Id3 over-expression into human corneal fibroblasts effectively blocks differentiation of corneal fibroblasts to myofibroblasts and offers an innovative gene therapy method to treat corneal fibrosis in vivo.

Methods : Primary human corneal fibroblast (HCF) and corneal myofibroblast (HMF) cultures generated from donor human corneas were used. HMF were produced by growing HCF in 5ng/ml TGFβ1 for 72h under serum-free conditions. Lipofectamine-3000 and G418 were used for gene transfer and stable clone selection, respectively. Immunofluorescence, immunoblotting, and qPCR were used to confirm gene transfer and quantify mRNA and protein levels of profibrotic markers in cultures. New Zealand White rabbits were used for in vivo studies.

Results : Several Id2 or Id3 overexpressing HCF clones were characterized. Stably transfected HCF clones having Id2 or Id3 gene copies >10⁶ showed phenotype, viability, and proliferation similar to non-transfected normal HCFs. Id2 or Id3 gene transfer significantly inhibited TGFβ1-mediated HCF differentiation to HMF in vitro. Immunofluorescence detected significant decrease in alpha smooth muscle actin (75-87%, p<0.01) and fibronectin (76-82%, p<0.01) proteins. The qPCR demonstrated significantly attenuated mRNA levels of fibrotic markers, smooth muscle actin (4.3-4.6±0.05 fold, p<0.1), fibronectin (2.4-2.8±0.4 fold, p<0.01), collagen I (2.9-3.3±0.6 fold, p<0.01), and collagen IV (2.1-2.5±0.05 fold, p<0.01). Results of in vivo rabbit studies are pending.

Conclusions : Id2 and Id3 gene therapy given topically using identified safe and efficacious nanoparticle or adeno-associated virus (AAV) vector has potential to treat corneal fibrosis in vivo.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.