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Michael K Fink, Suneel Gupta, Ratnakar Tripathi, govindaraj anumanthan, Prashant Rajiv Sinha, Matthew Faubion, Arkasubhra Ghosh, Nathan P Hesemann, Shyam S Chaurasia, Elizabeth A Giuliano, Rajiv R Mohan; Precision nanomedicine for eliminating corneal fibrosis and restoring vision with HGF-BMP7 gene transfer. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1175.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal fibrosis is a leading cause of blindness worldwide. Myofibroblast persistence in the stroma after wound healing contributes to corneal opacity. Hepatocyte growth factor (HGF) is shown to selectively eliminate myofibroblasts via apoptosis in multiple non-ocular tissues. Bone morphogenic protein-7 (BMP7) modulates Smad signaling to limit excessive corneal wound healing and fibrosis. We tested the postulate that nanoparticle-mediated HGF-BMP7 combination gene therapy is an innovative approach to eliminate corneal fibrosis in vivo using an established corneal fibrosis rabbit model.
In vivo corneal fibrosis in New Zealand White rabbits was created by a single topical 0.5N NaOH application following IACUC approved protocols and ARVO animal use guidelines. Stromal fibroblasts (HSF) were generated from donor human corneas and stromal myofibroblasts (HMF) were produced by growing HSF in serum-free conditions with 5ng/ml TGFβ1 for in vitro studies. HGF-BMP7 gene transfer was conducted using polyethylimine-DNA nanopaticles as reported previously. Serial slit lamp and stereomicroscopy evaluated ocular health and corneal opacity. Real-time quantitative PCR and immunoblotting were used to quantify delivered gene copies and the mRNA and protein of fibrosis markers. Immunofluorescence and TUNEL assays evaluated levels of fibrosis and apoptosis.
Nanoparticle treatment showed substantial HGF and BMP7 gene delivery into stroma in vivo (>104 gene copies/ug DNA). HGF-BMP7 gene therapy significantly decreased corneal opacity in vivo (Fantes scale 3.3 control vs 0.6 treated, p<0.001), and ocular lesion severity (Draize scale 30 control vs 5 treated, p<0.01; and modified McDonald-Shadduck scoring system 4.1 control vs 0.8 treated, p<0.01). Treated corneas contained significantly reduced amounts of α-smooth muscle actin, f-actin, collagen-I and collagen-IV mRNA (up to 15-fold; p<0.01). Immunofluorescence revealed significantly fewer myofibroblasts in the stroma of treated corneas (83%; p<0.001). HSF and HMF cultures exposed to HGF showed many TUNEL+ apoptotic cells in HMF but not in HSF (>15 TUNEL+ in HMF vs <2 in HSF).
In vivo studies demonstrated that combination HGF-BMP7 gene therapy would be an effective and safe approach to eliminate corneal fibrosis. In vitro studies suggest that HGF caused apoptosis only in corneal myofibroblasts. Future studies are warranted.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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