Effect of Targeted Disruption of Profibrotic SMAD-Signaling Pathway on Myofibroblasts and Corneal Haze
Narrative Responses:
Purpose
Transforming growth factor beta (TGFb) produces active myofibroblasts via Smad signaling to do corneal healing following injury/infection. Hyper TGFb function causes corneal scarring. We tested Smad2/3 knockdown, Smad7 over-expression, or their combination as a novel approach to inhibit TGFβ function and treat corneal haze effectively in vivo.
Methods
Donor human corneas, primary human corneal fibroblasts (HCFs) and New Zealand white rabbits were used. Corneal fibrosis in HCF cultures was induced by TGFb1, and in rabbits by -9D Photorefractive Keratectomy. Smad2/3-RNAi and AAV-Smad7 DNA constructs were made with mammalian expression vectors. Gene transfer in cultures was done with Lipofectamine-2000 and in vivo with AAV. Corneal myofibroblast and fibrosis levels were analyzed measuring alpha smooth muscle actin (SMA) expression using immunofluorescence, western blotting and qPCR. Cellular viability and proliferation were determined with Trypan blue or MTT assay. Delivered-transgene expression was quantified with qPCR and/or slot blotting.
Results
HCFs grown in the presence of TGFb1 under serum-free conditions showed exceptionally high myofibroblast formation (89-94%; p<0.001). Gene transfer achieving Smad2/3 knockdown, Smad7 over-expression or their combination significantly reduced SMA mRNA (2-5-fold, p <0.01) and protein (45-69%; p<0.5), and collagen type-1 levels in HCFs in vitro. None of the treatments showed significant change in HCFs phenotype, viability (4-11%) or proliferation (6-19%). In vivo rabbit experiments and analysis are underway.
Conclusion
Disrupting TGFβ-driven profibrotic Smad signaling by Smad2/3 knockdown or Smad7 over-expression has potential to treat corneal haze without significant toxicity in vivo.