Transcriptomic analysis of TGFß-mediated fibrosis in primary human Tenon's fibroblasts
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 TGFß1 induced changes in the transcriptome of human Tenon's fibroblasts (HTFs). RNA sequencing was performed on HTFs after 5 days of TGFß1 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, TGFß-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 SVIL, LTBP4,  and TNXB. 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. Overall design: HTFs were propagated from explanted subconjunctival Tenon's capsules collected during GFS performed in three human patients. HTFs were assigned to either the control (medium alone) or the TGFß1 (10 ng/mL) treatment group. RNA-sequencing was performed on samples after 5-days of treatment. RNA was extracted using the illustra RNAspin Mini Kit and sequenced using Novaseq 6000 with 100bp single-end sequencing at a depth of 20million reads per sample.