Yu Wai Man, CYOL; (2017) Targeting the Myocardin-related transcription factor / serum response factor (MRTF/SRF) pathway in conjunctival fibrosis. Doctoral thesis , UCL (University College London).

Abstract

Glaucoma is the leading cause of irreversible blindness and its prevalence is estimated to reach 79.6 million people by 2020. Wound contraction and scarring are the principal causes of blockage of aqueous flow at the drainage site in glaucoma filtration surgery. The cytotoxic antimetabolites, mitomycin-C and 5-fluorouracil, are widely used but have potentially blinding complications. There is thus a large unmet need for alternative agents with more targeted physiological effects and less cytotoxicity. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a master regulator of cytoskeletal gene expression and thus represents a promising therapeutic target to prevent fibrosis. The MRTF/SRF pathway in human conjunctival fibroblasts responds to actin dynamics but as human conjunctival fibroblasts have constitutively high MRTF-A and MRTF-B nuclear concentrations, they show a relatively small response to serum stimulation and actin binding drugs, such as latrunculin B and cytochalasin D. The localisation of MRTF-A and MRTF-B is also conserved across species in mouse and rabbit conjunctival fibroblasts. As the MRTF/SRF pathway is a master regulator of many key cytoskeletal genes in fibrosis, we next studied the effects of downregulating the MRTF/SRF pathway in human conjunctival fibroblasts using three-dimensional in vitro collagen contraction assays. MRTF-A and MRTF-B siRNA silencing significantly decreased collagen matrix contraction, the cell protrusive activity, matrix degradation, and the expression of key matrix metalloproteinase genes. Liposome-peptide-siRNA nanoparticles also represent an efficient and safe siRNA delivery system for MRTF silencing in conjunctival fibrosis. We further compared the contractility of fibrotic and non-fibrotic human conjunctival fibroblasts from glaucoma patients with and without previous glaucoma surgery, respectively. Fibrotic human conjunctival fibroblasts were significantly more contractile than non-fibrotic human conjunctival fibroblasts, and the increased contractility was linked to the upregulation of alpha smooth muscle actin, a marker of myofibroblast differentiation. We finally validated in vivo the effects of inhibiting the MRTF/SRF pathway in conjunctival fibrosis using a rabbit model of experimental glaucoma filtration surgery. We have identified a new MRTF/SRF pathway inhibitor CCG-222740, which was more potent in the fibroblast-mediated collagen contraction assay, less cytotoxic, and a more potent inhibitor of alpha smooth muscle actin expression than inhibitor CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a rabbit model of conjunctival fibrosis significantly increased the long-term success of the surgery and decreased scar tissue formation histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG- 203971 caused any epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous and serum. In conclusion, our in vitro and in vivo results support that inhibiting the MRTF/SRF pathway represents a potential new therapeutic target to prevent conjunctival fibrosis in glaucoma and other contractile scarring conditions in the eye.

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