Transforming growth factor-beta (TGFβ) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFβ are known in mammals and the human cornea expresses all of them. TGFβ1 has been shown to play a central role in scar formation in adult corneas whereas TGFβ2 and TGFβ3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFβ in the cornea have been shown to follow SMAD dependent as well as SMAD-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFβ expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFβ is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFβ is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFβ-mediated corneal scarring are under investigation. These include blocking of TGFβ, TGFβ receptor, TGFβ function and/or TGFβ maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFβ has also been demonstrated recently.

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.

This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNP) in the human cornea in vitro and rabbit cornea in vivo. PEI2-GNP with nitrogen-to-phosphorus (N/P) ratios of up to 180 exhibited significant transgene delivery in the human cornea without altering the viability or phenotype of these cells. Similarly, PEI2-GNP applied to corneal tissues collected after 12 h, 72 h, or 7 days exhibited appreciable gold uptake throughout the rabbit stroma with gradual clearance of GNP over time. Transmission electron microscopy detected GNP in the keratocytes and the extracellular matrix of the rabbit corneas. Additionally, slitlamp biomicroscopy in live animals even 7 days after topical PEI2-GNP application to the cornea detected no inflammation, redness, or edema in rabbit eyes in vivo, with only moderate cell death and immune reactions. These results suggest that PEI2-GNP are safe for the cornea and can be potentially useful for corneal gene therapy in vivo.