Blockade of Kca3.1: A Novel Therapeutic Target To Treat TGF-β1 Induced Fibrosis Associated With Glaucoma Filtration Surgery

Abstract

Purpose : Postoperative scarring (fibrosis) is a major complication after filtration surgery in glaucoma patients. The currently used drugs (Mitomycin C and 5-fluorouracil) extend short-term benefit but cause cytotoxicity and complications including damage to the corneal endothelium. The calcium-activated potassium channel Kca3.1 plays an important role in cellular proliferation, tissue remodeling, and fibrosis. However, the role of Kca3.1 channel in treating fibrosis seen after glaucoma filtration surgery remained unknown. We sought to explore the anti-fibrotic role of Kca3.1 channel using a selective blocker, TRAM34, in preventing TGF-β1 induced fibrosis seen in conjunctival fibroblasts after glaucoma filtration surgery using an in vitro model.

Methods : Primary human conjunctival fibroblast (HCF) cultures were generated from donor human conjunctival tissues, and fibrosis was produced by growing cultures in TGFβ1 (5ng/ml). PCR and immunofluorescence were performed to confirm Kca3.1 mRNA and protein expression in HCF. Anti-fibrotic effects of Kca3.1 was examined by growing HCF in +/- TGFβ1 (5ng/ml) and TRAM34 (0, 10, 25µM) for 72h under serum-free conditions and quantifying fibrotic gene expression by quantitative real-time PCR (qPCR) and western blot. Further, TRAM34 role in TGFβ1 signaling was analyzed by smad2/3 nuclear translocation using immunofluorescence.

Results : Significant Kca3.1 gene and protein levels were detected in HCF. The Kca3.1 gene expression increased in response to TGFβ1 treatment. TRAM34 showed no significant toxicity to HCF in histology or trypan blue assay (p<0.05). Kca3.1 channel inhibition by TRAM34 significantly attenuated TGFβ1-induced Smad2/3-dependent transcription of fibrotic markers, αSMA (p<0.01), fibronectin (p<0.05), collagen 1(p<0.001) and collagen III (p<0.001). Further TRAM34 also significantly inhibited TGFβ1-dependent αSMA protein expression (p<0.01) and smad2/3 nuclear translocation in HCF.

Conclusions : Our study suggests that TRAM34 (Kca3.1 inhibitor) could be a useful therapeutic option to treat TGFβ1-induced fibrosis in conjunctival fibroblasts and may improve filtration bleb fibrosis outcome. In vivo studies using rabbit model of glaucoma filtration surgery are underway.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.