The K+ Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis

Background\ud \ud Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that KCa3.1 K+ channel-dependent cell processes contribute to IPF pathophysiology.\ud Methods\ud \ud KCa3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of KCa3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).\ud Results\ud \ud Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed KCa3.1 channel mRNA and protein. KCa3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. KCa3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. KCa3.1 was expressed strongly in IPF tissue. KCa3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca2+.\ud Conclusions\ud \ud KCa3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking KCa3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic

The Role of the K⁺ Channel KCa3.1 in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a common disease with a median survival of only 3 years. There is no effective treatment. IPF is characterized by myofibroblast accumulation and progressive lung scarring. The Ca²⁺-activated K⁺ channel KCa3.1 modulates the activity of several structural and inflammatory cells which play important roles in model diseases characterized by tissue remodelling and fibrosis. We hypothesise that KCa3.1-dependent cell processes are a common denominator in IPF. KCa3.1 expression and function were examined in human myofibroblasts derived from IPF and non-fibrotic (NFC) donors. Myofibroblasts grown in vitro were characterised by western blot, immunofluorescence, RT-PCR and patch clamp electrophysiology to determine KCa3.1 channel expression. Wound healing, collagen secretion and contraction assays were performed using the pro-fibrotic mediators TGFβ1 and bFGF and two specific KCa3.1 blockers (TRAM-34, ICA-17043 [Senicapoc]). Both NFC and IPF myofibroblasts expressed KCa3.1 channel mRNA and protein. Using the KCa3.1 channel opener 1-EBIO, KCa3.1 ion currents were elicited in 59% of NFC and 77% of IPF myofibroblasts tested (P=0.0411). These currents were blocked by TRAM-34 (200 nM). The 1-EBIO-induced currents were significantly larger in IPF cells compared to NFC cells (P=0.0078). TGFβ1 and bFGF increased KCa3.1 channel expression. TRAM-34 and ICA-17043 dose-dependently attenuated wound healing, TGFβ1-dependent collagen secretion and bFGF- and TGFβ1-dependent contraction. We show for the first time that human lung myofibroblasts express the KCa3.1 K⁺ channel. KCa3.1 channel block attenuates pro-fibrotic myofibroblast function. These findings raise the possibility that blocking the KCa3.1 channel will inhibit pathological myofibroblast function in IPF, and thus offer a novel approach to IPF therapy

The K[superscript: +] channel K[subscript: Ca]3.1 as a novel target for idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that K[subscript: Ca]3.1 K[superscript: +] channel-dependent cell processes contribute to IPF pathophysiology. METHODS: K[subscript: Ca]3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of K[subscript: Ca]3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct K[subscript: Ca]3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]). RESULTS: Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed K[subscript: Ca]3.1 channel mRNA and protein. K[subscript: Ca]3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. K[subscript: Ca]3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. K[subscript: Ca]3.1 was expressed strongly in IPF tissue. K[subscript: Ca]3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca[superscript: 2+]. CONCLUSIONS: K[subscript: Ca]3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking K[subscript: Ca]3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic

Characterization of human lung myofibroblasts by immunofluorescence.

<p>Primary human lung myofibroblast cultures between passages 4 and 5 were stained with myofibroblast markers. Representative images are shown for: anti-fibroblast surface protein (FSP) and the mouse isotype control IgM; anti-fibroblast antigen which recognises the fibroblast antigen (thy-1/CD90) and the mouse isotype control IgG1; α-smooth muscle actin, and the isotype control IgG2a; collagen type 1 antibody and rabbit isotype control IgG; CD68 cell staining was negative; and corresponding isotype control IgG3, indicating that there is no contamination of monocytes or macrophage cells; CD34 antibody shows negative staining as does the appropriate isotype control IgG1. Nuclei are stained with DAPI. </p

Myofibroblast proliferation is inhibited by K_Ca3.1 channel block.

<div><p>Constitutive unstimulated wound healing is not altered by K_Ca3.1 blockade, and growth factor-stimulated healing is not inhibited by TRAM-7 or TRAM-85 .</p> <p><b>a</b>) Myofibroblast proliferation was increased following 48h of stimulation with FBS and significantly reduced by TRAM-34 (200 nM). <b>b</b>) An example of the wound created in a confluent monolayer of myofibroblasts in the wound healing assay and how it heals over the 48 hours. <b>c</b>) This graph displays that over the time course of the wound healing assay no significant differences were found between NFC and IPF donors in response bFGF, similar results were seen with FBS but results are not shown. <b>d</b>) TRAM-34 does not inhibit wound healing in the absence of mitogenic stimulation. <b>e</b>) FBS significantly increases wound healing (*<i>P</i>=0.0168, Paired t-test) but the molecules TRAM-7 and TRAM-85 which are structurally related to TRAM-34 do not have K_Ca3.1 channel-blocking activity and do not inhibit mitogen-dependent wound healing. Data represent mean±SEM for all figures a, c, d and e.</p></div

K_Ca3.1 inhibition TGFβ1 and bFGF-induced myofibroblast contraction.

<p><b>a</b>) Myofibroblast collagen gel contraction was increased following TGFβ1 stimulation and this was inhibited by TRAM-34 200 nM in both IPF (n=3) and NFC (n=3) donors (data shown is pooled IPF and NFC which did not differ, n=6)(<i>P</i>=0.0014, repeated measures ANOVA, <i>P</i>=0.0023 for TGFβ1 compared to control, <i>P</i>=0.0026 for TRAM-34 compared to TGFβ1 corrected by Bonferroni’s multiple comparisons test). <b>b</b>) TGFβ1-dependent myofibroblast collagen gel contraction was also inhibited by ICA-17043 100 nM (n=6) (All groups; repeated measures ANOVA, <i>P</i>=0.0002, TGFβ1 versus control, <i>P</i>=0.0002 and for ICA-17043 versus TGFβ1, <i>P</i>=0.0006, corrected by Bonferroni’s multiple comparisons test). <b>c</b>) Similarly, myofibroblast collagen gel contraction was increased following bFGF stimulation and was also inhibited with 24h pre-treatment with TRAM-34 200 nM (All groups; repeated measures ANOVA <i>P</i><0.0001, for bFGF compared to control, <i>P</i><0.0001, and for TRAM-34 compared to bFGF, <i>P</i><0.0001 (corrected by Bonferroni’s multiple comparisons test). <b>d</b>) Similarly, 24h pre-treatment with ICA-17043 100 nM significantly reduced bFGF-dependent myofibroblast collagen gel contraction (<i>P</i>=0.0007, repeated measures ANOVA) (<i>P</i>=0.0005 for bFGF versus control and <i>P</i>=0.0053 for ICA-17043 versus bFGF, corrected by Bonferroni’s multiple comparisons test). Data represented as mean±SEM for all the above figures.</p

K_Ca3.1 channel mRNA is expressed by myofibroblasts and upregulated following TGFβ1 stimulation.

<p><b>a</b>) Products from quantitative real-time PCR for K_Ca3.1 were visualized on a 1.5% agarose gel to confirm that only one product was amplified and that it was the correct size (130 bp). β-Actin was used as the normalizing control (310bp). <b>b</b>) In preliminary experiments we examined the mRNA expression levels of K_Ca3.1 in the NFC donors at passages 2, 4 and 7 and again found no significant differences. To be sure that passage number had no effect all experiments were performed between passages 4 and 5. Data represent mean±SEM. <b>c</b>) Quantitative real-time PCR showed that K_Ca3.1 mRNA expression was greater in NFC donors (n=5) than IPF donors (n=5), <i>P</i>=0.0262 (unpaired t test). <b>d</b>) K_Ca3.1 mRNA expression increased after TGFβ1 stimulation (All groups; 1-way ANOVA, <i>P</i><0.0001), NFC donors (n=5), IPF donors (n=5). There was a highly significant increase in IPF myofibroblasts following 24h of TGFβ1 stimulation. <i>P</i><0.0001 (corrected by Bonferroni's multiple comparison test). <b>e</b>) Quantitative real-time PCR demonstrating the relative fold increase in NFC and IPF myofibroblasts after stimulation with TGFβ1. Following normalization with β-actin there was a relative fold increase in K_Ca3.1 expression in all IPF donors. Results were calculated using the δδCT method. Data represented as ±SEM for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085244#pone-0085244-g001" target="_blank">Figure 1b,c and d</a>.</p

TGFβ1 induces a rise in intracellular calcium which is inhibited by pharmacological blockade of K_Ca3.1.

<p>Upon stimulation with TGFβ1 (10 ng/ml) both NFC (n=4 donor, n=28 cells) and IPF (n=3 donors, n=33 cells) donors showed a significant rise in [Ca²⁺]i concentration as displayed the ratio 340/380 nm, <i>P</i><0.0001 for both, paired t-test. <b>b</b>) In IPF only (n=3 donors and n=19 cells), TRAM-34 (200 nM) was added 5 minutes prior to treatment with TGFβ1, which inhibited the rise in [Ca²⁺]i typically seen upon TGFβ1 stimulation. <b>c</b>) The difference in [Ca²⁺]i following the different treatments were calculated and TRAM-34 significantly decreased the change in [Ca²⁺]i, <i>P</i><0.0001, Mann Whitney t-test. </p

Functional K_Ca3.1 channels demonstrate greater expression in IPF myofibroblasts compared to NFC myofibroblasts and channel expression is increased by pro-fibrotic growth factors.

<p><b>a</b>) The mean percentage of IPF myofibroblasts per donor developing a K_Ca3.1 current in response to 1-EBIO was significantly higher than in NFC myofibroblasts (<i>P</i>=0.0285, unpaired t-test). Data presented as mean±SEM. <b>b</b>) The whole-cell current at +40 mV before and after the addition of 1-EBIO in all responding NFC and IPF human lung myofibroblasts. Data presented as median and IQR. <b>c</b>) The subtracted (1-EBIO minus baseline) 1-EBIO-dependent K_Ca3.1 current at +40 mV was significantly larger in IPF cells than in NFC cells (<i>P</i>=0.0054, Mann Whitney test). Data presented as median and IQR. <b>d</b>) The mean percentage of NFC and IPF myofibroblasts expressing K_Ca3.1 currents increased after stimulation with TGFβ1 and bFGF (All groups; 1-way ANOVA, <i>P</i>=0.0013). The proportion of IPF cells responding to 1-EBIO after TGFβ1 stimulation was significantly higher (*<i>P</i>=0.0336, corrected by Bonferroni’s multiple comparisons test). Significantly more NFC cells responded to 1-EBIO following bFGF stimulation (**<i>P</i>=0.0035, corrected by Bonferroni’s multiple comparisons test). Data presented as mean±SEM.</p

Blocking K_Ca3.1 with TRAM-34 and ICA-17043 induces a dose-dependent attenuation of myofibroblast wound healing.

<p><b>a</b>) and <b>b</b>) Myofibroblasts stimulated with 10% FBS and 0.1% DMSO vehicle control, showed accelerated wound healing in comparison to 0.1% DMSO alone (<i>P</i>=0.002, and P=0.004 respectively, paired t-test). There was a dose-dependent decrease in FBS-induced wound healing over 48h in the presence of either <b>a</b>) TRAM-34 (20 nM and 200 nM) (<i>P</i><0.0001, repeated measures ANOVA) or <b>b</b>) ICA-17043 (10 nM and 100 nM) (<i>P</i>=0.0095, repeated measures ANOVA). <b>c</b>) Myofibroblast wound healing in response to 10 ng/ml bFGF and 0.1% DMSO stimulation at 48 h was increased in comparison to media alone (*<i>P</i>=0.002, paired t-test). There was a dose-dependent decrease in wound healing over the 48 h in the presence ICA-17043 (<i>P</i>=0.0076, Repeated measures ANOVA). Data represented as mean±SEM for all the above figures.</p