Description
Inflammation is a major driver of fibrosis. In the eye, Mitomycin-C (MMC) is used in the clinic to prevent scarring following surgery to manage glaucoma. High inflammation levels before surgery is a significant risk factor for the development of fibrosis and subsequent treatment failure. Furthermore, in the presence of local inflammation, MMC is less efficient in modulating scarring, despite higher doses often used to compensate. We have developed a co-culture system to study how macrophages promote conjunctival fibroblast-mediated matrix contraction, as a model for post-surgical scarring. We show that macrophages stimulate fibroblast-mediated gel contraction through an increase in fibroblast spreading and protrusive activity, leading to a more elongated cell phenotype. Consistent with the clinical phenotype, MMC-arrested fibroblasts still contract gels efficiently in the presence of macrophages, despite being unable to do so following serum stimulation. Macrophage-conditioned medium partially recapitulated the effect of macrophage co-culture on gel contraction. In a wound scratch assay, while both baseline serum-free migration and serum-stimulated migration were inhibited in MMC-treated cells, MMC treatment had little effect on fibroblast migration in the presence of macrophage-conditioned medium. Both EGF and VEGF receptor inhibition reduced fibroblast migration and contraction. Increased Rab5 levels in the presence of macrophage-derived signals suggest enhanced receptor trafficking. These findings suggest that macrophage-derived growth factors promote fibroblast activation through growth factor-dependent signalling, influencing migration, polarity, and contractility independently of MMC arrest.
Lay Abstract
Glaucoma is a leading cause of blindness, and surgery is often needed to reduce increased pressure in the eye. A drug called Mitomycin C (MMC) is applied during surgery to prevent scarring, but it can have some severe side effects, and in patients with high levels of inflammation, scarring still occurs, and the surgery fails. We wanted to understand why. Using our model, we grew eye tissue cells (fibroblasts) alongside immune cells (macrophages) in gels made of collagen, the same protein found in scar tissue. We found that macrophages release signals that cause fibroblasts to change shape, become more active, and pull on the collagen around them, mimicking scar formation. Importantly, even when fibroblasts were treated with MMC to stop them from growing, macrophage signals still made them contract and move, meaning MMC alone is not enough to stop scarring when inflammation is present. We have now identified some of the molecules through which macrophages activate fibroblasts' movement and contraction, and have begun characterising how they work in fibroblasts. Understanding these pathways could lead to new, more specific and less toxic, anti-scarring treatments, to improve surgical outcomes for glaucoma patients.
| Lay Title | Macrophages and macrophage-conditioned medium activate contraction and migration in Mitomycin C-arrested fibroblasts |
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| Role | PhD Student |