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R.R. Mohan, W.M. Stapleton, S. Sinha, M.V. Netto; Decorin Gene Transfer Into Keratocytes Inhibits Alpha Smooth Muscle Actin (SMA) Expression and Myofibroblast Transformation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1815.
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© ARVO (1962-2015); The Authors (2016-present)
TGF beta plays an important role in development of haze in the cornea. Decorin gene therapy has been shown to prevent TGF beta–induced fibrosis in lungs, kidneys and skin. The goal of this study was to examine whether decorin gene therapy has the potential to be used to treat corneal haze in vivo.
Rabbit keratocytes cultures were prepared by growing decorin–transfected and un–transfected normal rabbit corneal fibroblasts (RSF) under serum–free conditions in the presence or absence of TGFß1 (1ng/ml). Decorin–transfected RSF clones were generated by transfecting decorin cDNA cloned into mammalian expression vector (pcDNA3.1/HisA) into primary rabbit corneal fibroblasts using the calcium phosphate method. G418 was used to select stable clones. Decorin cDNA was generated using RT–PCR and cloned into pcDNA3.1/HisA vector using standard molecular biology techniques. Nucleotide sequence of the construct was confirmed by restriction analysis and DNA sequencing. Decorin–transfected clones were characterized using phase contrast microscopy, real–time PCR, western blotting and immunocytochemistry. Expression of SMA was determined by western blotting and immunocytochemistry. Statistical analysis was performed using analysis of variance (AVOVA) with the Bonferonni–Dunn adjustment for repeated measures.
Keratocyte cultures generated from decorin–transfected clones showed high levels of decorin mRNA and protein, whereas un–transfected RSF showed extremely low levels of decorin. Decorin–transfected rabbit keratocytes grown in the presence of TGFß1 under serum–free conditions showed a statistically significant decrease in SMA expression (80–83%, p value < 0.01) compared to the un–transfected normal keratocytes. Conversely, no expression of SMA was detected in keratocytes grown in the absence of TGFß1.
Decorin over–expression inhibits SMA expression and transformation of keratocytes to myofibroblasts in vitro. Tissue–targeted selective decorin gene therapy is a promising approach to prevent or reduce corneal haze formation in vivo.
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