Advances in Bioscience and Biotechnology, 2013, 4, 47-55 ABB http://dx.doi.org/10.4236/abb.2013.410A4005 Published Online October 2013 (http://www.scirp.org/journal/abb/ ) Reduction of corneal scarring in rabbits by targeting the TGFB1 pathway with a triple siRNA combination Sriniwas Sriram 1 , Daniel Gibson 2 , Paulette Robinson 2 , Sonal Tuli 3 , Alfred S. Lewin 4 , Gregory Schultz 1 1 Department of Biomedical Engineering, University of Florida, Gainesville, USA 2 Institute for Wound Research, Department of Obstetrics and Gynecology, University of Florida, Gainesville, USA 3 Department of Ophthalmology, University of Florida, Gainesville, USA 4 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, USA Email: vass87@gmail.com Received 30 August 2013; revised 19 September 2013; accepted 5 October 2013 Copyright © 2013 Sriniwas Sriram et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in sev- eral tissues including skin, liver, kidney and the cor- nea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2 (TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on reducing alpha smooth muscle actin (SMA) and cor- neal scarring (haze) in excimer laser ablated rabbit corneas. Methods: Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the epithelial and endothelial cell layers of normal and excimer ablated rabbit corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring models were utilized to assess the effects of the triple siRNA combination on corneal scarring. In the first model, rabbit corneas were unevenly ablated creating a mesh pattern then treated immediately with the triple siRNA combina- tion. After 1 day the ablated areas of corneas were collected and levels of mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein im- munolocalized in frozen sections. In the second model, rabbit corneas were uniformly ablated to a depth of 155 microns followed by three daily doses of the triple combination of siRNA. After 14 days, corneas were photographed and images were analyzed using Image J software to assess corneal scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always significantly higher in endothelial cells than in epithelial cells (10 to 30 fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs increased approximately 20-fold in both epithelial and endothelial cells, and further in- creased approximately 60-fold in 2 days. In the first therapeutic experiment with a single siRNA dose, two of three rabbits showed substantial reductions of all three target genes after 1 day with a maximum knock down of 80% of TGFb1, 50% reduction of TGFBR2 and 40% reduction of CTGF mRNA levels and reduc- ed SMA mRNA at day 14. In the second therapeutic experiment with multiple doses of siRNA treatment, both rabbits showed a ~22% reduction in scar forma- tion at day 14 as calculated by image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA expression. Conclusion: These results de- monstrate that both TGFB1 and CTGF dramatically increase in rabbit corneal epithelial and endothelial cells after injury. Treatment of excimer ablated rab- bit corneas with a triple combination of siRNAs effec- tively reduced levels of the target genes and SMA, lead- ing to reduced corneal scarring at 14 days, suggesting that this triple siRNA combination may be an effective new approach to reducing scarring in cornea and other tissues. Keywords: RNA Interference; siRNA Combination; Corneal Scarring; TGFB1; CTGF 1. INTRODUCTION Corneal scarring remains a serious complication that can ultimately lead to functional vision loss. In an injured cornea, a cascade of molecular events is initiated by pro- longed, elevated levels of transforming growth factor beta (TGFB1) which then combines with the Transform- ing Growth Factor Receptor II (TGFBR2) inducing the synthesis of Connective Tissue Growth Factor (CTGF) causing excessive scarring (corneal haze) that impairs vi- OPEN ACCESS