Differential effects of acetylsalicylic acid and mitomycin C on cytokine-induced Tenon?s capsule myofibroblast transdifferentiation and activity: Implications for glaucoma surgery

Inflammation-driven scarring is a major contributor to surgical failure after subconjunctival bleb forming glaucoma surgery. The current gold standard anti-scarring adjuvant mitomycin C (MMC) has variable effectiveness and is associated with significant risks. Acetylsalicylic acid (ASA), when delivered locally, repurposes the typically pro-inflammatory cyclooxygenase (COX-2) signaling for the resolution of inflammation and mitigating inflammation-mediated fibrosis. The aim of this study is to compare the effects of ASA and MMC in an in vitro model of subconjunctival scarring. Glaucoma patient-derived Tenon's capsule fibroblasts (HTCFs) were treated with TGF81 (2 ng/mL) plus or minus ASA (1600 mu g/ml), or MMC (0.05, 0.1, 0.2 mg/mL). In vitro collagen contraction, MTT, LDH, immunofluorescence, and Western blot assays were performed. To elucidate the mechanistic effects of ASA in TGF81induced HTCFs, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify and measure pro-inflammatory and pro-resolving lipid mediator secretion. ASA was at least as effective as MMC in reducing TGF81-induced HTCF-mediated collagen contraction, metabolic activity, and pro-fibrotic protein expression, with less cytotoxicity. Within cytokine-activated HTCFs, ASA significantly impaired secretion of pro-inflammatory lipid mediators prostaglandin E2 and 6-keto-prostaglandin F1 alpha and significantly increased secretion of the pro-resolving mediators 5-hydroxyeicosatetraenoic acid (HETE), 15-HETE and 18-hydroxyeicosapentaenoic acid (HEPE).ASA reduces cytokine-induced myofibroblast transdifferentiation in HTCFs, being non-inferior to MMC in vitro.ASA's effects are associated with a unique lipid mediator expression profile, suggesting that the ASA-induced resolution of inflammation may be a promising strategy to mitigate inflammation-mediated scarring and could offer a novel alternative as a surgical adjuvant.