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.

Fig 1. KCa3.1 is expressed in human cornea.

RT-PCR (A) and immunofluorescence (B) showing KCa3.1 expression in normal healthy human cornea. β-actin was used as internal control. HCE: corneal epithelium, HSF: corneal fibroblast, HCN: corneal endothelium.

Fig 2. Loss of functional KCa3.1 channels suppresses pro-fibrotic gene expression.

Comparison of RT–PCR in WT and KCa3.1^-/- mice cornea showed significantly suppressed mRNA expression levels of (A) α-SMA (p<0.001) and (B) collagen I (p<0.001), Collagen IV (p<0.001), and TGFβ1 (p<0.001). Results are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Fig 3. Loss of KCa3.1 reduced corneal haze in mice after alkali wounding.

Representative stereomicroscopic images showing corneal haze in Wild type (A, B, C), and KCa3.1^-/- mice (D, E, F). Representative examples of Naïve (A, D), 3 days (B, E) and 7 days (C, F) alkali wounding are shown. A decrease in corneal haze was observed in KCa3.1^-/- mice.

Fig 4. Effect of KCa3.1 loss on corneal fibrosis.

Corneal tissue immunostaining of mouse cornea showing levels of α-SMA expression in WT mice at 0, 3 and 7 days (A-C) and KCa3.1^-/- mice at 0, 3 and 7 days (D-F) in alkali-induced corneal fibrosis. Blue: DAPI-stained nuclei and green: α-SMA staining. KCa3.1^-/- mouse corneas showed a decrease in α-SMA expression in the stroma compared with that in control WT corneas.

Fig 5. Effect of TRAM-34 on HCF cell viability.

No significant difference in cell viability was noted between time points in the controls. A. Cell viability in TRAM-34 exposed cultures up to 7 days of continuous treatment, showing a minimal reduction after 7 days. Concentration dependent changes in cell viability (B). Results are expressed as mean ± SEM.

Fig 6. TRAM-34 suppresses TGFβ1 induced pro-fibrotic gene expression.

Real-time RT-PCR showed that TRAM-34 treatment in primary human cornea fibroblast cells significantly suppressed TGFβ1 induced mRNA expression levels of (A) α-SMA (p<0.001) and (B) collagen I (p<0.001) at 24 hours (C) and 72 hours (D). Results are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Fig 7

Western blot analysis (A) and quantification RT-PCR showing the effect of TRAM-34 treatment on α-SMA protein expression. A prominent α-SMA band was detected in the cellular lysate of TGFβ1 treated HCFs. TRAM-34 treatment attenuated TGFβ1 induced α-SMA expression. Corresponding densitometry analysis of the Western blot is presented in (B). Representative immunostaining images (a, b, c) and their quantification, showing the effect of TRAM-34 treatment on α-SMA protein expression in HCFs. Sparse α-SMA staining (green) can be seen in (a) control (no treatment) cultures compared with cultures grown in the presence of (b) TGFβ1 (+TGFβ1). TRAM-34 (25 μM) +TGFβ1 treatment resulted in a significant decrease in α-SMA (c). Scale bar = 50 μm. The quantitation graph demonstrates a 66% increase in the number of α-SMA positive cells (*p<0.001) when compared with no treatment control, which was significantly attenuated with TRAM34 treatment (p<0.001). (α-SMA = green, DAPI = Blue).

Fig 8. Cell migration scratch assay.

The microscopic appearance of the scratch at 0 hours is shown in Figs. A, B and C, and at 24 hours after TRAM-34 treatment in Figs. D, E and F. Composite phase-contrast microscopy image showing HCF taken 24 hours after initiation of treatment with TRAM-34 (25 μM) or TGFβ1 (5 ng/ml). TRAM-34 treatment effectively reduced cellular migration as compared to the TGFβ1 treated control.