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. Author manuscript; available in PMC: 2022 Jul 2.
Published in final edited form as: Exp Eye Res. 2017 Dec 12;167:140–144. doi: 10.1016/j.exer.2017.12.003

Blockade of KCa3.1: A novel target to treat TGF-β1 induced conjunctival fibrosis

Govindaraj Anumanthan a,b, Philip J Wilson c, Ratnakar Tripathi a,b, Nathan P Hesemann b,c, Rajiv R Mohan a,b,c,*
PMCID: PMC9250277  NIHMSID: NIHMS1817621  PMID: 29242028

Abstract

Postoperative conjunctival fibrosis is common in patients after glaucoma filtration surgery. The calcium activated potassium (KCa3.1) channel has been shown to inhibit fibrosis in many non-ocular tissues. However, its potential in treating ocular fibrosis remains unknown. We tested the anti-fibrotic potential of TRAM34, a selective blocker of KCa3.1 channel, in treating conjunctival fibrosis. Primary human conjunctival fibroblast (HCF) cultures derived from donor tissues. Myofibroblasts causing conjunctival fibrosis were generated by growing HCFs in the presence of TGFβ1 for 72 h. KCa3.1 mRNA and protein expression in HCF was examined with PCR and western blot. The anti-fibrotic potential of TRAM34 was examined by measuring fibrotic gene expression with quantitative PCR (qPCR), immunofluorescence, and western blotting in HCFs in ± TGFβ1 (5ng/ml) and TRAM34 (0–25 μM). The cytotoxicity of Tram34 was analyzed with trypan blue assay and its role in Smad signaling was studied with immunofluorescence. Expression of KCa3.1 mRNA and protein was detected in HCFs and TGFβ1 treatment to HCFs significantly increased expression of KCa3.1. TRAM34 treatment attenuated transcription of fibrotic markers, αSMA (p < .001), fibronectin (p < .05), collagen I (p < .001) and collagen IV (p < .001) in TGFβ1-induced HCFs. Further, TRAM34 significantly inhibited TGFβ1-stimulated αSMA protein expression (p < .01) and nuclear translocation of fibrotic Smad2/3 in HCFs and showed no significant cytotoxicity (p < .05). The KCa3.1 potassium channel plays a significant role in the prevention of conjunctival fibrosis and TRAM34 has potential to control post surgical bleb fibrosis in patients. In vivo studies are warranted.

1. Introduction

Glaucoma is a blinding disorder caused by progressive optic nerve damage leading to irreversible blindness. The main treatment modality for glaucoma is the maintenance of the intraocular pressure (IOP) in the normal range to prevent damage to the optic nerve (Weinreb et al., 2014). The medical management of glaucoma through drugs is generally insufficient and fails to control the progression of visual deterioration. Glaucoma filtration surgery (GFS) is a standard of care for glaucoma and halting irreversible vision loss by maintaining an acceptable level of IOP. However, postoperative scarring from excessive wound healing in the subconjunctival filtering bleb site, wound leakage, hypotony, and infection limit long-term bleb survival and IOP control after GFS (DeBry et al., 2002; Muckley and Lehrer, 2004; Anand et al., 2006; Palanca-Capistrano et al., 2009). Several therapeutic agents have been used for the prevention of conjunctival fibrosis after GFS. Currently, Mitomycin C (MMC) and 5-fluorouracil are commonly used to mitigate ocular fibrosis complication of GFS (Lama and Fechtner, 2003). Other therapeutic agents such as tumor growth factor beta antibodies, vascular endothelial growth factor antibodies, and Ologen® implants (Menda et al., 2015) also used for the prevention of postoperative ocular scarring. However, these agents were either ineffective or showed poor efficacy to replace MMC. In addition, these drugs demonstrated nonspecific cytotoxicity and complications including bleb leaks, cataracts, and endophthalmitis.

The multifunctional cytokine Transforming growth factor-β1 (TGFβ1) has been known to be involved in extra cellular matrix (ECM) protein expression and pathological tissue fibrosis (Mohan et al., 2011). TGF-β ligand binding to TGFβ1 type II receptor (TβRII), initiates the heteromeric complex formation and activates the downstream Smad dependent signaling pathways (Tandon et al., 2010). Smad dependent signaling is the main pathway extending TGFβ1 mediated differentiation of ocular (corneal and subconjunctival) fibroblasts to myofibroblasts, which is described by the excessive formation of α-smooth muscle actin (α-SMA), reorganization of actin cytoskeleton and incorporation of actin stress fibers (Vardouli et al., 2008). Myofibroblasts are considered as the primary effector of tissue fibrosis as they known to synthesize ECM and collagen. Thus, targeting profibrotic TGFβ1 cytokine and its downstream intracellular signaling pathways might offer a therapeutic strategy for disrupting postoperative fibrosis.

In recent years, KCa3.1 channel proteins were intensively studied as novel molecular targets in fibrosis treatment and was found over-expressed in many fibrotic tissues (Freise et al., 2015). Higher expression of KCa3.1 in ventricular fibroblasts was linked with excessive fibrosis and impaired heart function (Ju et al., 2015). Modification in KCa3.1 channel activity alters the ultrastructural membrane potential responsible for multiple cellular processes (Stocker, 2004). The KCa3.1 blockade was shown to inhibit several TGFβ1-dependent cell processes in the normal and fibrotic tissues (Roach et al., 2013; Huang et al., 2013; Freise et al., 2015). However, the expression of KCa3.1 and selective inhibitor TRAM34 in blocking ocular tissues fibrosis has not been studied yet. We hypothesized that pharmacological inhibition of KCa3.1 channel activity by the selective agent, TRAM34, regulates TGFβ1/Smad signaling dependent responses in ocular tissues and fibrosis development.

In this study, we investigated the therapeutic potential of TRAM34, to prevent conjunctival fibrosis development through the down regulation of the TGFβ1 and Smad pathway using an in vitro model of ocular fibrosis. We generated primary human conjunctival fibroblast (HCF) cultures, investigated TRAM34 toxicity, demonstrated the inhibitory effects of TRAM34 on TGFβ1-induced profibrotic mRNA and protein expression, and smad2/3 nuclear translocation.

2. Materials and methods

2.1. Primary conjunctival fibroblast culture (HCF)

Primary HCF cultures were generated from donor conjunctival tissues and fibroblast cells were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum and antibiotics. Cells were maintained in the logarithmic growth phase, and used within passages 2 to 6 in all experiments, which were performed at least three times. A stock solution of TRAM34 (Tocris Bioscience, Bristol, UK) at a concentration of 25 mM was prepared in DMSO and stored at −80 °C.

Myofibroblast cultures were generated by seeded HCFs in six-well plates using DMEM containing 10% fetal bovine serum initially. After 8–12 h cultures were switched to serum-free medium supplemented with TGFβ1 (5 ng/ml), and incubated for 72 h. The cultures were fed with fresh serum-free TGFβ1-containing medium every 24 h.

2.2. Trypan blue exclusion assay

The concentration dependent cytotoxicity of TRAM34 was evaluated via a trypan blue exclusion test according to manufacturer’s instructions. Briefly, 3 × 104 HCFs were plated and grown for 24 h, then cells were treated with or without TRAM34 (0,1, 5, 10, 25 or 50 μM) for 24 h. For time-dependent cytotoxicity assay, cells were treated by TRAM34 (25 μM) for up to 7 days. After trypan blue (Corning) treatment as per vendor’s instructions, stained, and unstained cells were counted using a hemocytometer. The percent cell viability was calculated by a formula: % cell viability = (viable cell count/total cell count) x100. Three independent experiments were performed.

2.3. Quantification of mRNA by real-time PCR

HCFs were grown in a 60-mm tissue culture dish and incubated for 0, 24, 48 and 72 h in the presence or absence of TGFβ1 (Peprotech, Inc. Rocky Hills, NJ) in serum-free medium. Total RNA was extracted using RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Briefly, first strand cDNA was synthesized by reverse transcriptase enzyme (Promega, Madison, WI). The expression of KCa3.1, αSMA, collagen Iα1, and Collagen IV mRNA was determined by qPCR using the One Step Plus Real-Time PCR system (Applied Biosystems, Carlsbad, CA). Twenty microliters (μl) reactions containing 1 μl cDNA, 2 μl forward and 2 μl reverse primers, 10 μl iQ™ SYBR® Green Super Mix (Bio-Rad Laboratories, Hercules, CA) were run following PCR parameters: 95 °C for 5min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1min. The fluorescence threshold value (Ct) was calculated to detect differences in signal associated with an exponential increase of PCR products in the log linear phase. Relative expression/fold change over the corresponding values for the control were calculated by the 2−ΔΔCt method. RT-PCR reactions were run in triplicates for each sample, and the average fold changes were calculated.

Qualitative conventional polymerase chain reaction was performed using 50 μl reaction mixtures containing 10 μl buffer (5x green GoTaq ® Flexi Buffer, Promega, Madison, WI), 8 μl MgCl2 (Promega, Madison, WI), 1 μl dNTP mix (Promega, Madison, WI), 1 μl forward and 1 μl reverse primers (0.4 μM each), 0.25 μl GoTaq®Flexi DNA (Promega, Madison, WI), 26.75 μl DEPC treated water and 2 μl of cDNA. Cycle details were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min, 95 °C for 1 min, and a final cycle of 72 °C for 10 min. β-actin was used as a housekeeping gene.

2.4. Immunofluorescence staining

Cells were fixed with ice-cold methanol for 15 min. Cells were then washed and blocked in 5% normal donkey Serum (Jackson Research Laboratories, Inc., West Grove, PA) for 1 h at room temperature, followed by mouse monoclonal αSMA antibody (1:200 dilution, Dako, Carpinteria, CA) for 90 min. Cells were rinsed and then incubated in secondary antibodies Alexa Flour ® 488 donkey anti-mouse (Invitrogen, Eugene, OR) for 1 h at room temperature. The cells were washed three times with PBS, mounted in Vectashield containing 4′−6-diamidino-2-phenylindole (DAPI; Vector Laboratories), and photographed with a Leica DM 4000B fluorescent microscope (Leica, place, state, country) equipped with a digital camera (SpotCam RT KE).

For Smad2/3 staining, HCF cells (1.8 × 103 in 300 μl medium/well) were grown to sub-confluency in each well of 4-well Nunc Lab-Tec chamber slides (Nunc, Rochester, NY). Cells were treated with or without TRAM34 (25 μM) for 24 h. The cultures were washed and treated with TGFβ−/+ (5 ng/ml) for 1 h. Cells were fixed with ice-cold methanol for 15 min and immunohistochemistry for smad2/3 protein was performed using mouse monoclonal anti-smad2/3 antibody (1:100 dilution in PBS; Santa Cruz Biotechnology, Santa Cruz, CA) followed by the incubation with secondary antibody Alexa Flour® 488 donkey anti-mouse (Invitrogen, Eugene, OR) was used, respectively.

2.5. Immunoblotting

For western blot analysis, HCFs were grown to confluence in 60-mm culture dishes and then treated with TGFβ1, TGFβ1+TRAM34 (10 and 25 μM) two different concentrations of TRAM34 for 72 h. Cells were washed with PBS, lysed directly on plates with radioimmunoprecipitation assay protein lysis buffer (RIPA) containing protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN). Samples were suspended in 4 × NuPAGE LDS buffer containing a reducing agent (Invitrogen, Carlsbad, CA), centrifuged for 5 min at 10,000 g, and then heated at 70 °C for 10 min. Protein samples were resolved by 4–12% SDS-PAGE on NuPAGE Novex Bis-Tris mini gels (Invitrogen, Carlsbad, CA) and transferred onto a 0.45-μm pore size PVDF membrane (Invitrogen, Carlsbad, CA). The PVDF membrane was incubated with αSMA (1:500 dilution; DAKO, Carpinteria, CA), or GAPDH primary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 3 h followed by alkaline phosphatase conjugated anti-mouse secondary antibody for 1.5 h at room temperature. The PVDF blot was developed using the nitro blue tetrazolium/5-bromo-4-chloro-3-in-dolylphosphate (BCIP/NBT) method. Three separate western blots were performed and β-actin was used as housekeeping for normalization of data.

2.6. Statistical analysis

Data were analyzed by one-way ANOVA using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA). The value of p < .05 was considered to be statistically significant.

3. Results

3.1. Detection of KCa3.1 expression in primary human conjunctival fibroblast cells

To demonstrate the gene and protein expression of KCa 3.1 in HCFs, we used RT-PCR and western blot analysis. HCF cells express KCa3.1 gene analyzed by RT-PCR (Fig. 1A) and protein expression by western blot analysis (Fig. 1B). Further, analysis of KCa3.1 mRNA expression shown to be upregulated in response to TGFβ1 treatment (Fig. 1C).

Fig. 1.

Fig. 1.

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KCa3.1 are expressed in human conjunctival fibroblast cells. Western blot. (A) and RT-PCR (B) showing expression in normal human conjunctival fibroblasts. β-actin was used as internal control. TGF β1 induced KCa3.1 mRNA expression at various time points was shown (C). Average of each KCa3.1 gene expression data by qPCR was plotted.

3.2. Effect of TRAM34 on human conjunctival fibroblasts viability

TRAM34 exposure on HCF cell viability data demonstrated that HCFs tolerated a 25 μM dose of TRAM34 for up to 7 days (Fig. 2A), while cell viability data showed a moderate decrease at the 50 μM dose tested (Fig. 2B) (p < .05).

Fig. 2.

Fig. 2.

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Effect of TRAM34 on human conjunctival fibroblasts viability. No significant difference in cell viability was noted in the controls. TRAM34-exposed cultures up to 7 days of continuous treatment (A). Concentration dependent changes in cell viability (B) shows moderate decrease at tested concentration at 50 μM TRAM34 (P < .05). Results are expressed as mean ± SEM (*P < .05, **P < .01, ***P < .001).

3.3. KCa 3.1 blockade inhibits fibrotic gene expression induced by TGFβ1

To explore the inhibitory role of TRAM34 on TGFβ1 induced profibrogenic gene expression, HCF cells were treated with TGFβ1 in the presence and absence of TRAM34 for 24 h. and subjected to qRT-PCR. TRAM34 treatment significantly suppressed mRNA expression levels of Fibronectin 1.5 ± 0.25 fold decrease (p < .05), αSMA 6.0 ± 0.5 fold decrease (p < .001), Collagen I 6.5 ± 0.4 fold decrease (p < .001), and Collagen type-IV 18.0 ± 1.25 fold decrease (p < .001) at 25 μM concentration in HCFs treated with TGFβ1 for 24 h (Fig. 3A–D).

Fig. 3.

Fig. 3.

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TRAM34 suppresses TGF β1 induced pro-fibrotic gene expression: Real-time RT–PCR showed that TRAM34 treatment in primary human conjunctival fibroblast cells significantly suppressed TGF β1 induced mRNA expression levels of (A) Fibronectin (p < .05), (B) αSMA (p < .001), (C) Collagen 1 (p < .001) and (D) collagen type-IV (p < .001). Results are expressed as mean ± SEM (*P < .05, **P < .01, ***P < .001).

3.4. KCa3.1 channel inhibition prevents TGFβ induced myofibroblast proliferation

The anti-fibrotic role of TRAM34 treatment was further investigated by α-SMA immunostaining (Fig. 4). HCFs grown without TGFβ1 treatment demonstrated low αSMA expression (A). Stimulation of HCF with TGFβ1 (5 ng/ml) for 72 h significantly increased αSMA expressing myofibroblast proliferation (B) over control cells grown in the absence of TGFβ1 (P < .001). However, KCa3.1 blockade with TRAM34 (25 nM) in the presence of TGFβ1 significantly inhibited the fibroblasts differentiation to myofibroblasts (C). Corresponding quantitation of α-SMA positive cell count shown in Fig. 4D (P < .001).

Fig. 4.

Fig. 4.

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TRAM34 suppresses TGF β1 induced differentiation of myofibroblasts. Representative immunostaining images (A–C) and their quantification (D) showing the effect of TRAM34 treatment on α-SMA protein expression in human conjunctival fibroblast cells. Sparse α-SMA staining (green) can be seen in (A) control (no treatment) cultures compared with cultures grown in the presence of (B) TGF β1 (+TGF β1). TRAM34 treatment TRAM34 (25 μM + TGF β1) resulted in significant decrease α-SMA (C). Scale bar = 50 μm. The graph (D) demonstrates a 72% increase in the number of αSMA positive cells (P* < 0.001) when compared with no treatment control HCFs, which was significantly attenuated (49%) with TRAM34 treatment (P < .001). (α-SMA = green, DAPI = Blue).

3.5. KCa3.1 inhibition attenuates TGFβ1-induced αSMA protein expression

The effect of TRAM34 on TGFβ1 induced differentiation of HCF to myofibroblast was further demonstrated by measuring the level of expression of αSMA protein (Fig. 5A). TGFβ1 treatment induced robust myofibroblast formation as shown by the increase in expression of αSMA protein. Whereas, HCF’s without TGFβ1 treatment showed minimal αSMA expression. The TRAM34 (10 and 25 μM) treatment in presence of TGFβ1 remarkably inhibited αSMA protein levels as indicated suggest that TRAM34 significantly decreased TGFβ1 -induced αSMA protein expression. Corresponding western blot quantitation data provided in Fig. 5B.

Fig. 5.

Fig. 5.

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Western blot analysis (A) and quantification (B) showing the effect of TRAM34 treatment on αSMA protein expression. A prominent αSMA band was detected in the cellular lysate of TGF β1 treated HCF’s. TRAM 34 treatment attenuated TGF β1-induced αSMA expression dose dependently. Corresponding densitometry analysis of the Western blot showed (B).

3.6. TRAM34 blocks TGFβ1 induced smad2/3 nuclear translocation

Because TGFβ1-dependent induction of αSMA protein expression is also inhibited by KCa3.1 blockers, we investigated the effects of TRAM34 on TGFβ1 signal transduction. Immunofluorescence staining showed that TRAM34 suppressed nuclear translocation of Smad2/3 (Fig. 6). In no treatment control, the Smad2/3 expression is widely distributed throughout the cells (Fig. 6A–C). In the absence of TRAM34, TGFβ1 increase the translocation of Smad2/3 from cytosol was detected in cell nuclei at 1.0 h post-TGFβ1 exposure (Fig. 6D–F); whereas in the presence of TGFβ1 and TRAM34 (25 μM), Smad2/3 nuclear translocation was significantly abolished (Fig. 6G–J).

Fig. 6.

Fig. 6.

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Smad2/3 nuclear translocation blocked by TRAM34. TGFβ1 induced increase in nuclear translocation of Smad2/3 from cytosol in primary conjunctival fibroblasts. No treatment control (A–C), a significant increase in nuclear localization of smad2/3 staining following TGFβ1 stimulation compared to control (D–F). TRAM34 blocked nuclear translocation of smad2/3 (TGFβ1+TRAM34) (G–I)

4. Discussion

Kca3.1 is known to express in multiple cell types and plays a significant role in cell cycle progression, migration and epithelial transport. KCa3.1 channel is the major contributor to Ca2+ influx in cells (Wulff and Castle, 2010). Further, KCa3.1 has been implicated in fibrotic disorders of the kidney (Grgic et al., 2009), liver (Freise et al., 2015), lungs (Roach et al., 2013) and myocardium (Zhao et al., 2015). In addition, a selective KCa3.1 inhibitor, TRAM34, has been shown to inhibit TGFβ1 activated smad2/3 pathway. Studies have shown that TRAM34 is highly lipophilic and can readily cross the cell membrane to act on KCa3.1. These reports led us to postulate that blocking of the KCa3.1 channel would inhibit myofibroblast formation and conjunctival fibrosis following GFS and had potential to offer a novel and efficient approach for treating post-surgical fibrosis. The present study demonstrates that HCFs express the high KCa3.1 gene and protein levels, and inhibition of KCa 3.1 channels by a highly specific efficient blocker TRAM34 potently attenuates TGFβ1-dependent pro-fibrotic gene transcription and de-differentiation of HCFs to myofibroblasts and pro-fibrotic activity.

Post-operative wound healing process in scleral and conjunctival tissues plays a critical role in the success of GFS. The amount of scar tissue generated after glaucoma filtering surgery determines the levels of aqueous humor outflow and IOP (Roy Chowdhury et al., 2015). Thus, the wound-healing events following GFS have been studied extensively. An intra-operative use of MMC or 5-FU is shown to inhibit fibrosis and improve surgical outcomes, however excessive scar formation and other complications are observed in large patient population despite their use. We noted low toxicity or cell growth inhibition in HCFs at tested TRAM34 doses. Literature shows that TRAM34 inhibitory effects are dose-dependent and cell-specific (Freise et al., 2015). Further, literature showing blockade of KCa3.1 with clotrimazole and TRAM34 prevented TGFβ1 induced proliferation of airway SMCs (Perez-Zoghbi et al., 2009) and proliferation of renal fibroblasts (Huang et al., 2014) provide additional evidence that Ca2+ channels alter cell cycle progression by regulating cellular proliferation via increasing intracellular Ca2+ signaling. In vivo dose-dependent studies are warranted in a rabbit model of GFS to identify therapeutic doses of TRAM34 and risk of cytotoxicity.

TGFβ1 plays a dominant role in a complex wound healing process characterized by the differentiation of fibroblasts to myofibroblasts. TGFβ1 is shown to potently promote proliferation of conjunctival fibroblasts via a downstream signaling largely mediated by Smads. A non-ocular in vivo study investigating the role of KCa3.1 in renal fibrosis found that TRAM34 blocks TGFβ1 induced expression of fibrotic proteins (Grgic et al., 2009). Findings of this and other studies suggest that suppression of fibroblast proliferation locally at the site of injury/surgical site may halt fibrosis development by limiting the production of scar-generating cells. Recently, great efforts have been made to understand the mechanisms of GFS-induced fibrosis and identify pharmacological molecules. Accumulating literature suggests that TRAM34 treatment significantly inhibits profibrotic gene and protein expression by antagonizing TGFβ1 mediated downstream signaling corroborates that KCa3.1 contributes to TGFβ1-driven fibrosis. Our finding supports the notion that TRAM34 effectively attenuates TGFβ-induced Smad signaling in HCFs. Simultaneous treatment with TRAM34 specifically inhibited TGF-β-induced mRNA and protein expression of myofibroblast markers validates the functional role of TRAM34 in anti-fibrosis via the KCa3.1 blockade. Kca3.1 is known to inhibit TGFβ1-dependent cell processes in normal and fibrotic tissues (Roach et al., 2014; Huang et al., 2013; Roach et al., 2015). Moreover, functional loss of KCa3.1 gene deletion also shown to prevent fibrosis by targeting myofibroblasts, leading to decreased fibroblast proliferation and collagen synthesis (Grgic et al., 2009).

In conclusion, we demonstrate that pharmacological inhibition of KCa3.1 attenuates TGFβ mediated profibrotic events in HCFs in vitro. Furthermore, our results demonstrated the existence of Kca3.1 mediated additional protective mechanism through the down regulation of the TGFβ signaling. This study suggests, TRAM34 is a promising therapeutic approach to control postoperative fibrosis in patients undergoing glaucoma filtration surgery, whether sufficient long-term anti-fibrotic effects could be achieved by subconjunctival injection of TRAM34 remains to be elucidated.

Acknowledgements

This work was primarily supported by the Ruth M. Kraeuchi Missouri Endowed Chair Ophthalmology Fund (RRM), and partial support from the NIH/NEI R01EY017294 (RRM) and Veterans Health Affairs 1I01BX00357 (RRM) grants.

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