Post-operative fibrosis of the filtering bleb limits the success of glaucoma filtration surgery (GFS). To minimise subconjunctival scarring following GFS, treatment with antimetabolites such as Mitomycin C (MMC) has become standard practice; however, their use is associated with considerable side effects. This study aimed to investigate the anti-scarring properties of 3′,4′-dihydroxyflavonol (DiOHF). GFS was performed in New Zealand white rabbits who received eye drops of DiOHF three times daily and vehicle eye drops after surgery (n = 5) or a single intraoperative treatment of MMC (n = 5). Blebs were imaged immediately following surgery and on days 7, 15, 21, and 28 for clinical examination. On day 28, eyes were harvested to assess collagen deposition, expression of α-SMA, oxidative stress, angiogenesis, fibroblast activity, and inflammation in the conjunctiva/Tenon’s layer. At 7 and 28 days post-GFS, MMC-treated blebs were more ischaemic than DiOHF- or vehicle-treated blebs. On day 28, DiOHF treatment significantly suppressed collagen accumulation, CD31 expression, Vimentin expression, and CD45 expression compared to the vehicle control. No difference was observed in 3-Nitrotyrosine or αSMA expression between treatment groups. Treatment with DiOHF reduced conjunctival scarring and angiogenesis in rabbits with GFS, which was comparable to MMC. DiOHF may be a safer and more effective wound-modulating agent than conventional antifibrotic therapy in GFS.

Glaucoma filtration surgery (GFS) is performed to slow down disease progression in glaucoma, a leading cause of irreversible blindness worldwide. Following surgery, pathological wound healing may lead to conjunctival fibrosis and filtering failure. Myofibroblasts are the key cells responsible for postoperative conjunctival scarring. This study aims to further understand the molecular mechanisms of conjunctival fibrosis following GFS. We utilised RNA-sequencing (RNA-seq) to delineate the TGFβ1 induced changes in the transcriptome of human Tenon’s fibroblasts (HTFs). RNA sequencing was performed on HTFs after 5 days of TGFβ1 treatment. Following quality control, 3,362 differentially expressed genes were identified, of which 1,532 were upregulated and 1,820 were downregulated. We identified signaling pathways associated with the pathogenesis of conjunctival fibrosis. The DEGs (differentially expressed genes) were enriched in pathways including myofibroblast differentiation, TGFβ-signaling, collagen and extracellular matrix organization, epithelial to mesenchymal transition, and cell cycle regulation. The results of this study identified the transition from HTF to myofibroblast is characterised by the upregulation of key genes including LDLRAD4, CDKN2B, FZD8, MYOZ1 , and the downregulation of SOD3, LTBP4 and RCAN2 . This unprecedented insight into the transcriptional landscape of HTFs and myofibroblast differentiation is essential to understand the pathophysiology of conjunctival scarring and develop new therapeutic agents.

Background: Glaucoma filtration surgery (GFS) is performed to slow down disease progression in glaucoma, a leading cause of irreversible blindness worldwide. Following surgery, pathological wound healing may lead to conjunctival fibrosis and filtering failure. Myofibroblast transition of human Tenon’s fibroblast (HTF) is responsible for postoperative conjunctival scarring, with TGFβ signalling playing a key role in this process. However, the detailed molecular mechanism underlying myofibroblast transition in HTF remain understudied. Methods: In this study, we performed transcriptomic analysis to delineate the TGFβ1-induced changes in the transcriptome of HTF. HTF was isolated from three patients following GFS and treated with TGFβ1 for 5 days, followed by RNA-seq for transcriptomic profiling. Results: Our results identified 3362 differentially expressed genes (DEGs) in HTF following TGFβ1 treatment, of which 1532 were upregulated and 1820 were downregulated. Using gene ontology analysis, we identified signalling pathways associated with the pathogenesis of conjunctival fibrosis, including pathways involved in myofibroblast differentiation, TGFβ-signalling, collagen and extracellular matrix organisation, epithelial to mesenchymal transition, and cell cycle regulation. This study provided detailed characterisation of the transition from HTF to myofibroblast, and highlighted key genes that are upregulated (LDLRAD4, CDKN2B, FZD8, MYOZ1) and downregulated (SOD3, LTBP4 and RCAN2) in this myofibroblast transition process. Conclusions: Overall, this study provided insights into the transcriptional landscape of HTFs and myofibroblast differentiation, which has important implication to understanding the pathophysiology of conjunctival scarring and development of new therapeutic agents.