KCa3.1 Ion Channel Inhibition Prevents Exuberant Corneal Wound Healing and Ameliorates TGFβ1-induced Corneal Fibrosis

Abstract

Purpose : KCa3.1, intermediate-conductance calcium-activated potassium channels, have been found to regulate fibrosis in several non-ocular tissues. This study examined the role of KCa3.1 in corneal wound healing and evaluated the therapeutic potential of a selective KCa3.1 inhibitor, TRAM-34, for ameliorating corneal fibrosis using an established in vitro human corneal fibrosis model and in vivo Kcnn4 (calcium-activated potassium channel subfamily-N-member-4 gene that encodes for KCa3.1) knockout mice.

Methods : Human corneal stromal fibroblasts (hCSFs) generated from donor human corneas and age- and sex-matched B6;129S1-Kcnn4^tm1Jemn/J mice (KCa3.1 knockout) mice were used. Corneal fibrosis in vitro was induced by TGFβ1 (5 ng/ml) and in vivo in mouse cornea by alkali-injury. TRAM-34 was purchased from Sigma. Quantitative RT-PCR, Western blot, H&E staining, and immunofluorescence were used to quantify mRNA and protein levels of profibrotic markers, alpha-smooth muscle actin (αSMA), collagen I, and collagen IV.

Results : Time-dependent analyses of alkali-wounded Kcnn4-/- corneas showed significantly reduced mRNA and protein expressions of the three selected profibrotic genes compared to alkali-wounded corneas of wild type mice suggesting direct involvement of KCa3.1 in corneal wound healing in vivo. Compared to wild type mice, KCa3.1 mice showed significantly reduced corneal haze after alkali trauma in live animals and αSMA+ cells in corneal sections (4h-21days; p<0.05, p<0.01, or p<0.001). TRAM-34 significantly inhibited TGFβ1-induced differentiation of CSFs to myofibroblasts (p<0.001) in an in vitro corneal fibrosis model.

Conclusions : KCa3.1 inhibition regulates CSF function and plays an important role in corneal wound healing. TRAM-34 has the potential to offer a pharmacological intervention for corneal haze treatment in vivo.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.