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Marta Sloniecka, Patrik Danielson; Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix components production in an in vitro human corneal fibrosis model. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2290. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Acetylcholine (ACh) is a neurotransmitter present in corneal stroma and produced by keratocytes. It has been shown to play a role in processes important for wound healing. Based on literature and our previous studies, we hypothesize that ACh regulates expression of extracellular matrix (ECM) components that are overexpressed during fibrosis, such as collagens, proteoglycans, fibronectin and metalloproteinases, in a protective manner during corneal fibrosis, i.e. decreasing their expression.
Primary keratocytes were isolated from healthy human corneas obtained from the local cornea bank and grown in presence of 10% fetal bovine serum in order to obtain corneal fibroblasts. A corneal fibrosis in vitro model, in which fibroblasts are stimulated with transforming growth factor beta 1 (TGF-β1) and stable vitamin C, was used throughout this study. Contractile ability of myofibroblasts was tested using a cell contraction assay. Gene expression of ECM components (collagen I, collagen III, collagen V and lumican), markers of fibrosis (α-smooth muscle actin [α-SMA] and fibronectin), and metalloproteinases (MMP2, MMP9 and MMP12) were assessed by qRT-PCR. Intracellular production and secretion of pro-collagen I and lumican was determined by ELISA. α-SMA protein expression was assessed by western blot.
ACh decreased the contractile ability of the newly formed myofibroblasts. ACh significantly decreased gene expression of collagen I, collagen III and collagen V in myofibroblasts. Moreover, ACh treated cells produced and secreted less pro-collagen I. Gene expression of lumican was unaffected by ACh treatment up to day 2 but significantly decreased by day 4. However, no differences in lumican protein level, both intracellular and secreted, were found. ACh downregulated expression of both α-SMA and fibronectin genes. Additionally, α-SMA protein expression was also diminished in ACh treated cells. Furthermore, ACh treatment resulted in downregulation of MMP2, MMP9 and MMP12 genes.
Our results are consistent with our hypothesis that ACh regulates expression of various collagens, lumican, fibronectin and metalloproteinases during corneal fibrosis in vitro, in a way that it diminishes their expression, both on RNA and protein levels. In conclusion, ACh seems to provide protection against formation of fibrosis in human cornea.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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