Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, since there are no FDA-approved drugs with the claim of reducing light-scattering corneal haze. Clinical trials of anti-inflammatory steroids have not shown statistically significant benefits, and mitomycin C treatment carries the risk serious side effects. Cell culture studies, animal experiments, and recent clinical trials strongly indicate that connective tissue growth factor (CTGF), which is induced by transforming growth factor beta (TGFb), is the dominant scar-inducing growth factor in the cornea and in other tissues, including skin and kidney. CTGF stimulates corneal scar formation by up-regulating synthesis of irregular extracellular scar matrix and inducing transformation of quiescent keratocytes into activated fibroblasts and myofibroblasts, which combine to produce the majority of light scattering in corneal scars. Our overall goal is to understand the basic cellular and molecular regulation of corneal wound healing and apply that knowledge to develop therapies that effectively eliminate corneal scarring. In this grant, we propose four specific aims that further expand our basic knowledge of cell and molecular regulation of corneal wound healing and translate that information into new treatments that prevent corneal scarring.
Specific Aim #1 will further assess the roles of CTGF in regulating epithelial healing and stromal scarring and regression of corneal haze using systemic and corneal epithelial cell specific conditional CTGF knockout (KO) transgenic mice strains that we developed during the last grant. Epigenetic regulation of corneal wound healing is an important, but unexplored area. Using PCR arrays, we identified 44 microRNAs (miRs) that significantly change expression levels in mouse corneas at 30 minutes and at 24 hours after excimer ablation, including miR-133b and miR-22, which target mRNAs of key scarring genes including TGFb1, CTGF, TGFb1, TGFbRI, and collagen.
Specific Aim #2 will further characterize changes in patterns of miR expression at key times during corneal scar formation and resolution (days 3, 6, and 9 days) and will assess the effects of additional pre-miRs and anti-miRs that target key scarring genes using cultures of mouse corneal cells. Transitioning from cell culture studies to animal studies, Specific Aim #3 will compare the anti-scarring effects in excimer ablated rabbit corneas of treatment with the most effective miR drugs identified in Specific Aim #2 (e.g., pre- miR-133b or anti-miR-22). In addition, we will test a potent, triple combination of siRNAs we developed during the last grant that specifically targets TGFb, TGFbRII and CTGF mRNAs. Finally, we hypothesize that maximum reduction of corneal scarring will be achieved by using a combination of gene targeted drugs and broad gene targeting epigenetic drugs. To test this hypothesis, Specific Aim #4 will evaluate antiscarring effects in excimer ablated rabbit corneas using the optimal combination of a gene specific drug (siRNAs or ASOs) and a broad gene targeting epigenetic drug (miRs or SAHA, a histone deacetylase inhibitor).
Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, yet no drugs effectively reduce corneal scarring without the risk of serious side effects.(1),(2),(3) This grant will expand our baic understanding of molecular and cellular regulation of corneal wound healing using two conditional knockout mice strains for connective tissue growth factor (CTGF) we developed, and by identifying, for the first time, key microRNAs (miRs) that epigenetically regulate expression of multiple genes during corneal wound healing.(4) Finally, we will translate this basic knowledge into potential novel drug therapies that prevent and reverse corneal scarring without producing serious, long-term side effects using a combination of both gene-specific therapies (ASOs or siRNAs) and multi-gene epigenetic therapies (miRs or histone deacetylase inhibitor SAHA).
|Pi, Liya; Robinson, Paulette M; Jorgensen, Marda et al. (2015) Connective tissue growth factor and integrin ?v?6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice. Hepatology 61:678-91|
|Gibson, Daniel J; Pi, Liya; Sriram, Sriniwas et al. (2014) Conditional knockout of CTGF affects corneal wound healing. Invest Ophthalmol Vis Sci 55:2062-70|
|Sriram, Sriniwas; Gibson, Daniel J; Robinson, Paulette et al. (2014) Assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas. Exp Eye Res 125:173-82|
|Gibson, Daniel J; Tuli, Sonal S; Schultz, Gregory S (2013) The progression of haze formation in rabbit corneas following phototherapeutic keratectomy. Invest Ophthalmol Vis Sci 54:4776-81|
|Tandon, Ashish; Tovey, Jonathan C K; Waggoner, Michael R et al. (2012) Vorinostat: a potent agent to prevent and treat laser-induced corneal haze. J Refract Surg 28:285-90|
|Blalock, Timothy D; Gibson, Daniel J; Duncan, Matthew R et al. (2012) A connective tissue growth factor signaling receptor in corneal fibroblasts. Invest Ophthalmol Vis Sci 53:3387-94|
|Schultz, Gregory S; Davidson, Jeffrey M; Kirsner, Robert S et al. (2011) Dynamic reciprocity in the wound microenvironment. Wound Repair Regen 19:134-48|
|Secker, Genevieve A; Shortt, Alex J; Sampson, Edith et al. (2008) TGFbeta stimulated re-epithelialisation is regulated by CTGF and Ras/MEK/ERK signalling. Exp Cell Res 314:131-42|
|Liu, J; Saghizadeh, M; Tuli, S S et al. (2008) Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy. Mol Vis 14:2087-96|
|Liu, Jia; Lewin, Alfred S; Tuli, Sonal S et al. (2008) Reduction in severity of a herpes simplex virus type 1 murine infection by treatment with a ribozyme targeting the UL20 gene RNA. J Virol 82:7467-74|
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