BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In VivoReport as inadecuate

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This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles PEI2-GNPs transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis α-smooth muscle actin αSMA, F-actin and fibronectin, immune reaction CD11b and F4-80, keratocyte apoptosis TUNEL, calcification alizarin red, vonKossa and osteocalcin, and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and-or real-time PCR. Human corneal fibroblasts HCF and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo 2×104 gene copies-ug DNA. Localized BMP7 gene therapy showed a significant corneal haze decrease 1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05 in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA 46±5% p<0.001 and fibronectin proteins 48±5% p<0.01. TUNEL, CD11b, and F4-80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1-5-8 nuclear localization >88%; p<0.0001, and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1-5-8 95%; p<0.001 and Smad6 53%, p<0.001, and decreased αSMA 78%; p<0.001 protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.

Author: Ashish Tandon, Ajay Sharma, Jason T. Rodier, Alexander M. Klibanov, Frank G. Rieger, Rajiv R. Mohan



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