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 [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.
Highlights
Trauma, infection, or chemical/surgical injury to the cornea can cause fibrosis/scarring resulting in loss of corneal transparency
Our results show that PEI2-GNPs-mediated BMP7 gene delivery attenuates laser ablation-induced corneal fibrosis in vivo in a rabbit model
BMP7 expression in most organs declines with age during adulthood and no BMP7 expression could be detected in the adult mouse cornea [32]
Summary
Infection, or chemical/surgical injury to the cornea can cause fibrosis/scarring resulting in loss of corneal transparency. Though corneal scarring is the third leading cause of blindness worldwide and affects over one million Americans every year [1], no effective therapy is yet available to treat corneal scarring. Mitomycin C is commonly used in clinic to treat laser surgery-induced corneal scarring [4,5,6]. Its use has been associated with serious side effects both in preclinical and clinical studies [7,8,9,10,11]. Due to the lack of effective and safe drugs, many cases of corneal scarring require corneal transplantation. Despite its high success rate, corneal transplantation poses the challenges of postsurgical complications and the limited availability of high quality donor corneas. There is a need for newer and effective treatments for corneal scarring
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