Targeting the MRTF/SRF gene transcription pathway in conjunctival fibrosis in glaucoma

Abstract

BackgroundGlaucoma is the leading cause of irreversible blindness and it affects over 70 million people worldwide. Existing strategies to prevent postsurgical fibrosis in glaucoma are inadequate and have complications that could potentially lead to blindness. Despite recent evidence that the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway has a pivotal role in fibroblast activation, there are currently no published studies investigating its role in conjunctival fibrosis. MethodsWe prospectively collected 30 non-fibrotic and fibrotic conjunctival tissues from patients with glaucoma at the time of surgery at Moorfields Eye Hospital, London, UK, according to local ethics approval. We analysed the profibrotic marker expression on whole tissue sections using in-situ tissue immunohistochemistry, and compared the contractile properties between primary cultures of non-fibrotic and fibrotic human conjunctival fibroblasts. We also studied the effect of silencing the MRTF/SRF pathway on the function of fibrotic conjunctival fibroblasts and analysed the group differences using Student's t test. FindingsThe fibrotic human conjunctiva expressed increased markers of fibrosis such as α smooth muscle actin, tenascin C, vimentin, and fibronectin. The fibrotic conjunctival fibroblasts significantly increased matrix contraction (from 28% [SD 16] to 76% [25], p=0·0024), and the cell force (0·25 mN [SD 0 ·08] to 0·56 mN [0·05], p=0·0044) compared with the non-fibrotic fibroblasts. MRTF and SRF silencing with siRNAs markedly decreased matrix contraction in fibrotic conjunctival fibroblasts by 80% and 87%, respectively. Knocking down MRTF significantly reduced the fibroblast dynamic index from 0·66 to 0·19 (p<0·0001). In addition, MRTF silencing decreased matrix degradation from 0·87 to 0·33 (p=0·006) and downregulated the expression of key matrix metalloproteinases genes such as MMP1, MMP2, MMP9, and MMP14 (p<0·05). InterpretationOur study is the first, to our knowledge, to show that silencing the MRTF/SRF pathway significantly decreases matrix contraction, fibroblast protrusive behaviour, matrix degradation, and MMP gene expression in fibrotic human conjunctival fibroblasts. Our study suggests that inhibiting the MRTF/SRF pathway could become a potential new therapeutic approach to prevent conjunctival fibrosis in glaucoma. FundingNIHR Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Francis Crick Institute.