KCa3.1 ion channel: A novel therapeutic target for corneal fibrosis

Abstract

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

Histone Deacetylases Enhance Ca2+-Activated K+ Channel KCa3.1 Expression in Murine Inflammatory CD4+ T Cells

“…The contribution of HDAC2 to tissue fibrosis is attracting increasing attention [ 48 , 49 ]. Recent studies showed that K Ca 3.1 inhibitors are a potential treatment option for renal, liver, kidney, corneal, and pulmonary fibrosis [ 50 , 51 , 52 , 53 , 54 ]. It currently remains unclear whether epigenetic modifications by HDAC2/3 are involved in these K Ca 3.1-related disorders; therefore, further studies are needed to clarify the therapeutic effectiveness of selective HDACis in various K Ca 3.1-related disorders.…”

Inhibition of Interleukin 10 Transcription through the SMAD2/3 Signaling Pathway by Ca2+-Activated K+Channel KCa3.1 Activation in Human T-Cell Lymphoma HuT-78 Cells

“…Recent studies showed that K Ca 3.1 blockade attenuated pulmonary, renal, and corneal fibrosis by mediating Smad2/3 signaling (Huang et al, 2013(Huang et al, , 2018Roach and Wulff, 2014;Roach et al, 2015;Anumanthan et al, 2018). The resultant decreases in Ca 21 influx prevented the phosphorylation of Smad2/3 and nuclear translocation of P-Smad2/3 in fibroblasts, and thus, the expression levels of the downstream target genes (a-smooth muscle actin and collagen type I) were decreased.…”

Cell-Free Biological Approach for Corneal Stromal Wound Healing

“…Its expression has been reported in the development of fibrosis of the cornea and different organs (Huang et al, 2013;Roach et al, 2015). Treatment with the KCa3.1 inhibitor, TRAM-34, reduced the expression of pro-fibrotic markers, such as αSMA, in the corneal stroma (Anumanthan et al, 2018).…”