BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis in vivo

Authors

Ashish Tandon, Harry S. Truman Memorial Veterans’ Hospital
Ajay Sharma, Chapman UniversityFollow
Jason T. Rodier, Harry S. Truman Memorial Veterans' Hospital
Alexander M. Klibanov, Massachusetts Institute of Technology
Frank G. Rieger, Harry S. Truman Memorial Veterans’ Hospital
Rajiv R. Mohan, Harry S. Truman Memorial Veterans' Hospital

Document Type

Article

Publication Date

2013

Abstract

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 [a-smooth muscle actin (aSMA), 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 (26104 gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.6860.31 compared to 3.260.43 in control corneas; p,0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of aSMA (4665% p,0.001) and fibronectin proteins (4865% 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 TGFb demonstrated significantly enhanced pSmad-1/5/8 (95%; p,0.001) and Smad6 (53%, p,0.001), and decreased aSMA (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 TGFb1-mediated profibrotic Smad signaling.

Comments

This article was originally published in PLoS ONE, volume 8, issue 6, in 2013. DOI: 10.1371/journal.pone.0066434

Recommended Citation

Tandon A, Sharma A, Rodier JT, Klibanov AM, Rieger FG, Mohan RR. BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis in vivo. PLoS ONE. 2013;8(6):e66434. doi:10.1371/journal.pone.0066434.

Copyright

The authors

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.