Benzalkonium Chloride Induces Subconjunctival Fibrosis Through the COX-2–Modulated Activation of a TGF-β1/Smad3 Signaling Pathway

Abstract

Purpose.: The purpose is to investigate the mechanism of subconjunctival fibrosis caused by benzalkonium chloride (BAC), which is the most common preservative in ophthalmic preparations.

Methods.: The left eyes of male Sprague-Dawley rats were topically treated with 0.01% BAC or PBS twice daily for 1 month. Primary conjunctival fibroblasts (CFs) were exposed for 24 hours to 0.00005% BAC, 0.000075% BAC, 0.000075% BAC + LY2157299 (a selective transforming growth factor β receptor type I inhibitor); 0.000075% BAC + NS-398 (a selective cyclooxygenase-2 inhibitor) and PBS, respectively. The pathological changes of the bulbar conjunctival tissue of rats were examined using hematoxylin-eosin (HE), Van Gieson's (vG), periodic acid-Schiff (PAS) stains, or immunohistochemisty (IHC). The expression of the extracellular matrix (ECM), the transforming growth factor β (TGF-β) signaling pathway–related molecules, and cyclooxygenase-2 (COX-2) in bulbar conjunctival tissues and CFs were detected using Western blot (WB) and quantitative real-time RT-PCR (qRT-PCR).

Results.: Rats treated with 0.01% BAC exhibited a slight increase of the fibroblast density and a more compact collagen deposition in the bulbar subepithelial connective tissues in comparison with rats treated with PBS. Western blot and qRT-PCR analyses showed that the expression of ECM, TGF-β signaling pathway–related molecules, and COX-2 were markedly increased in the bulbar conjunctival tissues of rats exposed to 0.01% BAC and in CFs exposed to 0.00005% and 0.000075% BAC. In conjunctival fibroblasts, BAC-induced ECM expression was clearly decreased by LY2157299, while the BAC-induced activation of the TGF-β1/Smad3 signaling pathway was greatly attenuated by NS-398.

Conclusions.: Subconjunctival fibrosis BAC-induced is a consequence of excessive ECM production of CFs through the COX-2–modulated activation of a TGF-β1/Smad3 signaling pathway.

Introduction

Benzalkonium chloride (BAC) is the most commonly used preservative in topical medication; nearly all antiglaucoma medications contain BAC, which is most often used at a concentration of 0.01% (ranging from 0.004%–0.02%).^1,2 The subconjunctival fibrosis, which is a potential risk of failure for further glaucoma surgeries, has been reported to be induced in patients treated for a long period of time with antiglaucoma medications containing BAC.^3,4 Some clinical studies have consistently demonstrated that richer collagen fibers are found in conjunctival biopsy specimens of patients receiving long-term glaucoma treatment,^5–7 and the rate of glaucoma surgery failure is increased in the patients with prolonged use of antiglaucoma medications containing BAC prior to surgery.^5,8 These studies indicate that BAC-induced subconjunctival fibrosis is related to cell injury and inflammation caused by the toxicity of BAC^3,6,8; however, it has also been reported that BAC-induced subconjunctival fibrosis is not due to the toxicity of BAC.⁹ Therefore, the mechanism that causes BAC-induced subconjunctival fibrosis is still unclear. 

Transforming growth factor β is a multifunctional cytokine that has potent profibrotic properties. Its profibrotic effects are numerous, including the induction of myofibroblasts, an increase of matrix synthesis, and an inhibition of collagen breakdown. Transforming growth factor β exerts its functions by binding to the TGF-β type II receptor (TGF-βR2) located in the cell membrane. The constitutively active TGF-βR2 recruits a TGF-β type I receptor (TGF-βR1) to form a heterotetrameric receptor complex in which TGF-βR2 phosphorylates the TGF-βR1. The activated TGF-βR1 then phosphorylates and stimulates mothers against decapentaplegic homolog2/3 (Smad2/3), which then form complexes with Smad4. The combined complexes are then translocated into the nucleus and interact with numerous TGF-β transcription factors to regulate the expression of TGF-β–responsive genes.^10,11 The effects of TGF-β on fibrosis have been demonstrated by many studies that show that regions of the increased matrix have increased the expression of TGF-β, especially the isoform TGF-β1, and that delivery of exogenous TGF-β1 by various means to a variety of tissues results in severe fibrosis in experimental animals.^12–14 Studies also demonstrate that the profibrotic effect of TGF-β requires the action of the Smads and that Smad3 is essential for the synthesis of many ECM, including collagen type I.^15,16 It has been illustrated that in mice, a Smad3 deficiency attenuates pulmonary fibrosis induced by bleomycin or mediated by an overexpression of TGF-β1, reduces the total collagen deposition, and suppresses the expression of ECM in the obstructed kidney.^17–20 Therefore, a TGF-β/Smad signaling pathway, particularly the TGF-β/Smad3 signaling pathway, plays a central role in the profibrotic effect of TGF-β. Fibrotic disease of the eye is quite similar to fibrotic disorders in other tissues of the body. The TGF-β/Smad signaling pathway has also been shown to be involved in corneal and conjunctival fibrosis^21,22; however, it remains unknown whether or not the activation of a TGF-β1/Smad3 signaling pathway is a mechanism that causes BAC-induced subconjunctival fibrosis. 

One of two cyclooxygenase isoforms, COX-2, is a key enzyme in the formation of prostaglandins. It has a low level of basal expression in most tissues and cell types but is highly expressed in response to many stimuli, such as cytokines, growth factors, and xenobiotics.²³ In addition to being highly expressed at the site of inflammation, an aberrantly upregulated COX-2 expression is increasingly implicated in the embryonic development, the pathogenesis of a number of epithelial cell carcinomas, in Alzheimer disease, and possibly other neurologic conditions.^24,25 It has also been indicated that COX-2 is implicated in TGF-β production and tissue fibrosis. For example, the upregulated expression of COX-2 stimulates TGF-β production, and the effect is inhibited by a selective inhibitor of COX-2, NS-398.^26,27 The significantly upregulated expression of COX-2 is considered to contribute to the pathogenesis of oral submucous fibrosis induced by arecoline.²⁸ Serum levels of COX-2 increased in systemic sclerosis patients.²⁹ The inhibition of COX-2 by NS-398 could reduce collagen deposition in mice hearts after myocardial infarction.²⁶ Another selective COX-2 inhibitor, SC-236, had also been shown to significantly reduce liver fibrosis in carbon tetrachloride-treated mice³⁰; however, it is unclear whether or not the activation of a TGF-β1/Smad3 signaling pathway is also modulated by COX-2 in BAC-induced subconjunctival fibrosis. 

In this study, we investigated the mechanism that causes BAC-induced subconjunctival fibrosis by measuring the levels of ECM, COX-2, and TGF-β1/Smad3-related molecules both in vivo and in vitro. We found that in rats, subepithelial collagen deposition in the conjunctiva is indeed induced with exposure to 0.01% BAC for 1 month, and the TGF-β1/Smad3 signaling pathway played an important role in this pathological process. Furthermore, we found that the activation of a TGF-β1/Smad3 signaling pathway was modulated by an upregulated expression of COX-2 in BAC-induced subconjunctival fibrosis. 

Materials and Methods

Materials

Benzalkonium chloride was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). The antibodies used were mouse anti-rat CD68 (ED1; AbD serotec, Kidlington, Oxford, UK), rabbit anti-rat polymorphonuclear neutrophils (PMN; Fitzgerald Industries International, Acton, MA, USA), anti-fibronectin-1 (FN-1; Proteintech Group, Inc., Chicago, IL, USA), goat anti–alpha-1 type I collagen (Col1α1; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti–COX-2 (Abcam, Cambridge, England), rabbit antibodies against TGFB-β1, TGF-βR1, TGF-βR2, phosphorylated Smad3 (P-Smad3), Smad2/3 and Smad3 (Abzoom Biolabs, Inc., Dallas, TX, USA), horseradish peroxidase (HRP)-conjugated mouse anti-β-actin, HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated rabbit anti-goat IgG (Sigma-Aldrich Corp.). The inhibitors used were a COX-2 selective inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; Cayman Chemical Company, Ann Arbor, MI, USA), TGF-βR1 selective inhibitor 4-[5,6-dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide (LY2157299; Axon Medchem, Groningen, The Netherlands). Other materials used were a Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA); fetal bovine serum (FBS, Invitrogen); collagenase IV (Invitrogen); a cell counting kit-8 (CCK-8 Dojindo Molecular Technologies, Inc., Rockville, MD, USA); and a PAS staining system (Sigma-Aldrich Corp.). 

Animals and BAC Treatment

Adult male Sprague-Dawley rats (from 180–200 g, quarantined and purchased from Shanghai SLAC Laboratory Animal Center, Shanghai, China) were used in the study. Animal experiments were performed in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the experimental protocol was approved by the experimental animal ethics committee of Xiamen University (approval ID: XMUMC2011-03-3). All rats were confirmed to be free of ocular surface disease before the experiments. Forty-five rats were randomly assigned to three groups; 0.01% BAC (dissolved in PBS) was topically administered to the left eye of rats in the primary group (twice a day, 10 μL each). As a control, PBS was topically applied to the left eye of rats in the secondary group (twice a day, 10 μL each). After treatment for 1 month, these two groups of rats were euthanized. Their left eyes were enucleated and fixed with cold 4% paraformaldehyde and then embedded in paraffin wax for study via histology and IHC (n = 5), or bulbar conjunctival tissues dissection was performed to isolate RNA for qRT-PCR (n = 5) and extract protein for WB (n = 5). Rats in the third group were directly euthanized. Their left eyes were sterilely enucleated for the isolation of bulbar subconjunctival tissues and the culture of primary CFs. 

Culture of Primary CFs

Primary CFs were isolated and cultured as previously described with some modifications.³¹ Sterilely isolated bulbar subconjunctival tissues were rinsed three times with PBS (containing 100 U/mL penicillin and 0.1 mg/mL streptomycin) and cut into explants of approximately 2 × 2 mm.² These explants were placed into tissue culture dishes with DMEM containing 10% FBS and incubated in an incubator at 37°C with 95% humidity and 5% CO₂. The media were changed every 3 days until the primary CFs reached confluence. The conjunctival fibroblasts were subcultured with 0.25% trypsin and 0.02% EDTA digestion and used for experiments between passages 5 and 7. 

It is known that BAC is toxic to cells. In cultured cells, the concentrations of BAC with the threshold of toxicity inhibiting cell growth and inducing cell apoptosis are respectively approximately 0.0001% and 0.005%.^3,32,33 To investigate the effect of BAC at a lower concentration regarding the viability of CFs and to determine the concentration of BAC that is suitable to investigate the mechanisms that cause an ECM expression of CFs stimulated by BAC, CFs were plated at a density of 3 × 10³ cells per well in 96-well culture plates that were treated with a medium containing PBS or BAC at a concentration of 0.00001%, 0.000025%, 0.00005%, 0.000075%, 0.0001%, 0.00025%, 0.0005%, 0.00075%, and 0.001%. After 24 hours, a cell viability assay was performed with a CCK-8 kit. To investigate the effect of BAC on the expression of an extracellular matrix (Col1α1 and FN-1), TGF-β signaling pathway-related molecules (TGF-β1, TGF-βR1, TGF-βR2, Smad2/3, and P-Smad3) and COX-2 in CFs, the cells were plated at a density of 1 × 10⁶ cells per well in six wells that were treated with a medium containing 0.00005% BAC, 0.000075% BAC, 0.000075% BAC + 200 μM LY2157299, and 0.000075% BAC + 100 μM NS-398. The control CFs were treated with a medium containing PBS alone. After 24 hours, the cells were harvested to isolate RNA for qRT-PCR and extract protein for WB. 

Cell Viability Assay

Cell viability assays were performed as previously described³⁴ using a CCK-8 kit according to the manufacturer's instructions. The medium in each well of 96-well plates was removed and replaced with 100 μL of fresh medium containing 10 μL of a CCK-8 solution. After being incubated for 2 hours at 37°C, the absorbance was measured spectrophotometrically at 450 nm with a microplate reader (Bio Tek ELX800; Bio-Tek Instruments, Winooski, VT, USA). 

Western Blot

Western blot was performed as previously described.³⁵ The total proteins were extracted from bulbar conjunctival tissues, and the CFs were extracted using a cold radio immunoprecipitation assay (RIPA) buffer that contains a proteinase inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA) and a phosphatase inhibitor cocktail (Sigma-Aldrich Corp.). Equal amounts of conjunctival tissues and CFs were subjected to electrophoresis with 10% sodium dodecyl sulfate–polyacrylamide gels. The standard WB assay protocol was applied. The specific primary antibodies (anti-Col1α1, anti-FN-1, anti-COX-2 anti-TGF-β1, anti-TGF-βR1, anti-TGF-βR2, anti-Smad2/3, anti-Smad3, and anti-P-Smad3) and secondary antibodies (HRP-conjugated rabbit anti-goat IgG and HRP-conjugated goat anti-rabbit IgG) were used. We used HRP-conjugated mouse anti–β-actin for protein quantification. Finally, the specific bands were examined using an enhanced chemiluminescence reagent and recorded, and image intensity was calculated using the transilluminator (Bio-Rad Laboratories, Inc., Hercules, CA, USA). 

QRT-PCR

We performed qRT-PCR as previously described.³⁵ The total RNA from bulbar conjunctival tissues was extracted using a reagent (TRIzol; Invitrogen) according to the manufacturer's instructions and were reverse transcribed to cDNA using a commercial kit (RevertAid First Strand cDNA Synthesis Kit; Fermentas, Thermo Fisher Scientific, Pittsburgh, PA, USA). We performed quantitative PCR using a commercial detection system (StepOne Real-Time PCR; Applied Biosystems, Foster City, CA, USA) and a Taq Kit (SYBR Premix Ex Taq Kit; Takara Bio, Inc., Shiga, Japan) according to the manufacturer's instructions, and the primer sequences are summarized in the Table. The amplification program included an initial denaturation step at 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing and extension at 60°C for 30 seconds. The results of the relative qRT-PCR were analyzed using the comparative threshold cycle (Ct) method and were normalized to β-actin (as an internal control gene). 

Table.

 

Rat Primer Sequences Used for Quantitative Real-Time RT-PCR

Gene	Access Code	Nucleotides	Primer Sequence
Col1α1	NM_053304.1	769–788	F5′-GATGATGGGGAAGCTGGTAA-3′
		917–936	R5′-CTTAGGACCAGCAGGACCAG-3′
FN-1	NM_019143.2	3413–3432	F5′-GAAAGGCAACCAGCAGAGTC-3′
		3623–3642	R5′-CTGGAGTCAAGCCAGACACA-3′
TGF-β1	NM_021578.2	717–736	F5′-ATACGCCTGAGTGGCTGTCT-3′
		850–869	R5′-TGGGACTGATCCCATTGATT-3′
TGF-βR1	NM_012775.2	243–262	F5′-TCACCGAGACCACAGACAAA-3′
		352–371	R5′-AAGCAGACTGGACCAGCAAT-3′
TGF-βR2	NM_031132.3	1261–1270	F5′-TCACTAGGCACGTCATCAGC-3′
		1436–1455	R5′-AGGACAACCCGAAGTCACAC-3′
Smad3	NM_013095.3	526–545	F5′-CTGTGAGTTCGCCTTCAACA-3′
		688–707	R5′-CAGCGGGGAAGTTAGTGTTC-3′
β-actin	NM_031144.3	860–879	F5′-TCTTCCAGCCTTCCTTCCTG-3′
		1078–1097	R5′-CACACAGAGTACTTGCGCTC-3′

Histological Stains and IHC

Histological stains and IHC were performed on 4-μm paraffin sections of 4% paraformaldehyde-fixed samples of rat eyes. The sections were deparaffinized and rehydrated and then stained with HE for histopathological examination, vG—a simple and special method of differential staining of collagen and other connective tissues⁹—for collagen examination; and PAS for the examination of goblet cells according to standard procedures. We performed IHC for specific cell markers (CD68 for macrophages, PMN for neutrophils) as previously described.³⁶ The images of the histological stains and IHC were observed under a microscope (Nikon Corp., Tokyo, Japan), captured, and saved to a computer for comparison and analysis. 

Statistical Analysis

The summary data are presented as means ± SD. Group means were analyzed using the Student's t-test. The differences were considered significant at P < 0.05. The statistical analysis was conducted using commercial software (GraphPad Prism version 5.00 for Windows, GraphPad Software, Inc., La Jolla, CA, USA). 

Results

Chronic Application of 0.01% BAC Induces Rat Bulbar Subconjunctival Fibrosis

Previous studies show that long-term use of antiglaucoma drugs or BAC caused significant increases in the thickness of conjunctival epithelium, a decrease in the number of conjunctival goblet cells, an infiltration of inflammatory cells, and subconjunctival fibrosis due to the toxic effect.^{3,6,8,37–41} To clarify whether or not chronic exposure to 0.01% BAC can cause these pathological changes in rat bulbar conjunctivas, we examined the sections of samples of rat eyes stained by HE, vG, PAS, and IHC. Staining with HE showed that there was a slight increase of the fibroblast density and more compact collagen deposition in the subepithelial part of bulbar conjunctivas in the 0.01% BAC-treated group in comparison with PBS-treated group (Figs. 1A–D). The increase of collagen deposition in the subepithelial part of bulbar conjunctivas was also clearly indicated on the sections stained with vG (Figs. 1E–H), which is a special stain for collagen.⁹ We examined fibrosis using vG staining instead of IHC because available antibodies against collagen are suboptimal on rat conjunctival tissues. These results demonstrate that chronic applications of 0.01% BAC could induce bulbar subconjunctival fibrosis; however, HE and PAS staining showed that there was no significant difference between the 0.01% BAC-treated group and the PBS-treated group (Figs. 1A–D versus 1I–L) in the morphology and the number of conjunctival epithelial cells and goblet cells, and IHC for CD68 and PMN also showed that there was no difference between these two groups in inflammatory cell infiltration (data not shown). Therefore, our results indicate that it is possible that the chronic application of 0.01% BAC-induced bulbar subconjunctival fibrosis is not caused by cell injury and inflammation due to the toxic effect of BAC. 

Figure 1

 

Histological stains of rat conjunctival tissues. The conjunctival tissue sections were stained with HE (A–D), vG (E–H), and PAS (I–L). Staining with HE showed a slight increase of the fibroblast density and more compact collagen deposition in the subepithelial part of bulbar conjunctiva in the 0.01% BAC-treated group (C, D) in comparison with the PBS control group (A, B). Compact collagen deposition was further confirmed by vG staining in the 0.01% BAC-treated group (E, F) in comparison with the PBS control group (G, H). Staining with PAS showed that there was no significant difference in the morphology or the number of conjunctival epithelial cells and goblet cells between the 0.01% BAC-treated group (K, L) and the PBS control group (I, J). The results are representative examples of five independent experiments. Scale bars: 250 μm (A, C, E, G, I, K); 50 μm (B, D, F, H, J, L).

Chronic Application of 0.01% BAC Promotes ECM Expression in Bulbar Conjunctiva Tissues

Fibrosis is characterized by a deposition of excessive collagen, especially collagen type I, and other ECM components, such as fibronectin.^15,42 It has been shown that there is a richer collagen deposition in conjunctival biopsy specimens from patients receiving long-term glaucoma treatment.^5–7 To confirm that the chronic application of 0.01% BAC-induced bulbar subconjunctival fibrosis was due to the excessive production of collagen and other ECM components in the bulbar conjunctival tissues, we examined the expression of Col1α1 and FN-1 in the bulbar conjunctival tissues of rats. The Western blot results show that the expression of Col1α1 and FN-1 were indeed significantly increased in the 0.01% BAC-treated group in comparison with the PBS-treated group (Figs. 2A, 2B). The QRT-PCR also showed that the expression of Col1α1 and FN-1 mRNA were also dramatically upregulated in the 0.01% BAC-treated group (Fig. 2C). 

Figure 2

 

The expression of ECM in rat bulbar conjunctival tissues. (A) The Western blot showed that the expression of Col1α1 and FN-1 were significantly increased in the 0.01% BAC treated group in comparison with the PBS control group. (B) A graphic representation of densitometry of Col1α1 and FN-1 expression is shown in (A). ***P < 0.0001. (C) Quantitative RT-PCR showed that the expression of Col1α1 and FN-1 mRNA were clearly increased in the 0.01% BAC-treated group in comparison with the PBS control group. The results are representative examples of five independent experiments.

Chronic Application of 0.01% BAC Stimulates the Expression of TGF-β1/Smad3 Signaling Pathway-Related Molecules in Bulbar Conjunctiva Tissues

It has been reported that TGF-β mediated conjunctival fibrosis and that the TGF-β/Smad3 signaling pathway plays a key role in fibrosis.^{15,17–19,22,42,43} To investigate whether or not the chronic application of 0.01% BAC-induced subconjunctival fibrosis is related to the activation of the TGF-β/Smad3 signaling pathway, we identified the expression of the TGF-β signaling pathway–related molecules in bulbar conjunctival tissues of rats using Western blot and qRT-PCR. The Western blot shows that the expression of TGF-β1, TGF-βR1, and Smad3 were markedly increased in the 0.01% BAC treatment group in comparison with the PBS-treated group, but there was no difference in the expression of TGF-βR2 and Smad2 between the two groups (Figs. 3A–D). To confirm that the activation of TGF-β signaling was through the TGF-β1/Smad3 pathway, we identified the level of P-Smad3 using the Western blot. The result shows that the expression of P-Smad3 was significantly augmented in the 0.01% BAC group in comparison with the PBS group (Figs. 3C, 3D). Quantitative RT-PCR also demonstrated that the mRNA expression of TGF-β1, TGF-βR1, and Smad3 were dramatically upregulated in the 0.01% BAC-treated group (Fig. 3E). These results indicate that the chronic application of 0.01% BAC could stimulate the activation of a TGF-β1/Smad3 signaling pathway in which ECM were excessively produced, which resulted in bulbar subconjunctival fibrosis. 

Figure 3

 

The expression of TGF-β pathway–related molecules in rat bulbar conjunctival tissues. (A) The Western blot showed that the expression of TGF-β1 and TGF-βR1 were markedly increased in the 0.01% BAC-treated group in comparison with the PBS control group, but there was no significant difference in TGF-βR2 between the two groups. (B) A graphic representation of the densitometry of TGF-β1, TGF-βR1, and TGF-βR2 expression is shown in (A). #P > 0.05. ***P < 0.0001. (C) The Western blot showed that the expression of Smad3 and P-Smad3 were markedly increased in the 0.01% BAC-treated group in comparison with the PBS control group, but there was no difference in Smad2 between the two groups. (D) A graphic representation of the densitometry of Smad2, Smad3, and P-Smad3 expression is shown in (C). #P > 0.05. ***P < 0.0001. (E) Quantitative RT-PCR showed that the expression of TGF-β1, TGF-βR1, and Smad3 mRNA were all significantly upregulated in the 0.01% BAC-treated group in comparison with the PBS control group. **P < 0.001. The results are representative examples of five independent experiments.

Effects of BAC at a Lower Concentration on the Expression of ECM and TGF-β1/Smad3 Signaling Pathway-Related Molecules in Primary CFs

Fibroblasts secrete ECM and TGF-β.^15,44–47 To identify the potential mechanisms that cause 0.01% BAC-induced subconjunctival fibrosis, we examined the culture of rat primary CFs and investigated whether or not BAC can stimulate the expression of ECM and the TGF-β1/Smad3 signaling pathway–related molecules in CFs. 

The concentrations of BAC with the threshold of toxicity that inhibits cell growth and induces cell apoptosis are respectively approximately 0.0001% and 0.005%.^3,32,33 In topical medication, BAC is commonly used at an average concentration of 0.01% (range, 0.004%–0.02%)²; however, it has been reported that BAC can penetrate into conjunctival deeper structures³ and its concentration of BAC will be heavily diluted in the tear film once the topical medication is applied to the eyes.⁴⁸ Therefore, the concentration of BAC will be lower in the subconjunctival tissues. To investigate the effect of BAC at a lower concentration on the viability of CFs and to determine the concentration of BAC that is suitable to investigate the mechanisms stimulated by BAC that cause the expression of ECM and the TGF-β1/Smad3 signaling pathway in CFs, the CFs were treated for 24 hours with a medium containing PBS or BAC at a concentration of 0.00001%, 0.000025%, 0.00005%, 0.000075%, 0.0001%, 0.00025%, 0.0005%, 0.00075%, and 0.001%, and the cell viability was subsequently evaluated with a CCK-8 assay. As shown in Figure 4A, at concentrations higher than 0.0005%, BAC was toxic to CFs and markedly decreased the viability of CFs, whereas at the concentrations of 0.00005% and 0.000075%, BAC was nontoxic to CFs and could slightly stimulate the proliferation of CFs. Therefore, BAC at the concentrations of 0.00005% and 0.000075% was determined to be appropriate for the investigation of mechanisms that cause the expression of ECM and the TGF-β1/Smad3 signaling pathway stimulated by BAC in CFs. The Western blot results show that after CFs were exposed to BAC at a concentration above 0.00005%, especially at 0.000075% for 24 hours, the expression of Col1α1, FN-1 TGF-β1, TGF-βR1, and Smad3 as well as the level of P-Smad3 were all significantly increased (Figs. 4B–E). The results confirm that at a lower concentration, BAC not only was not toxic to CFs but also stimulated CFs to express ECM and TGF-β1/Smad3 signaling pathway–related molecules. 

Figure 4

 

Effects of BAC at a lower concentration on primary CFs. (A) The toxic effect of BAC at various concentrations of BAC (0.00001%, 0.000025%, 0.00005%, 0.000075%, 0.0001%, 0.00025%, 0.0005%, 0.00075%, and 0.001%) on CFs was identified using a CCK-8 assay. The results show that BAC at concentrations higher than 0.0005% was toxic to CFs and markedly decreased the viability of CFs and that concentrations lower than 0.00025% were nontoxic to CFs, whereas 0.00005% and 0.000075% concentrations of BAC could slightly stimulate the proliferation of CFs. (B) The Western blot showed that the expression of Col1α1 and FN-1 were markedly increased in the 0.00005% and 0.000075% BAC-treated groups in comparison with the PBS control group. (C) A graphic representation of the densitometry of Col1α1 and FN-1 expression is shown in (B). **P < 0.001. ***P < 0.0001. (D) The Western blot showed that the expression of the TGF-β1/Smad3 signaling pathway–related molecules TGF-β1, TGF-βR1, Smad3, and P-Smad3 were all clearly increased in the 0.00005% and 0.000075% BAC-treated groups in comparison with the PBS control group. (E) A graphic representation of the densitometry of TGF-β1, TGF-βR1, Smad3, and P-Smad3 expression is shown in (D). *P < 0.05. **P < 0.001. ***P < 0.0001. The results are representative examples of five independent experiments.

BAC at a Lower Concentration Stimulates CFs to Express ECM Through the Activation of a TGF-β1/Smad3 Signaling Pathway

To induce TGF-β gene expression in fibroblasts, Smad3 is required; and the genetic deletion of Smad3 reduces the total collagen deposition and suppresses the expression of ECM.^15,20 To investigate whether or not the expression of ECM is stimulated by BAC at lower concentrations due to the activation of a TGF-β1/Smad3 signaling pathway in CFs, we treated CFs with 0.000075% BAC and 0.000075% BAC + 200 μM LY2157299 and PBS, respectively. The Western blot shows that the expression of P-Smad3 was clearly increased in the 0.000075% BAC-treated CFs in comparison with the PBS-treated CFs, whereas the level of P-Smad3 was significantly decreased in 0.000075% BAC + 200 μM LY2157299-treated CFs in comparison with 0.000075% BAC-treated CFs (Figs. 5A, 5B). Similar to the expression of P-Smad3, the expression of Col1α1 and FN-1 were all clearly downregulated in 0.000075% BAC + 200 μM LY2157299-treated CFs in comparison with 0.000075% BAC-treated CFs (Figs. 5C, 5D). These data demonstrate that at a lower concentration, BAC stimulated ECM expression through the activation of a TGF-β1/Smad3 signaling pathway in CFs. 

Figure 5

 

Molecule inhibitor LY2157299 reduces the expression of P-Smad3, Col1α1, and FN-1 in CFs. (A) The Western blot showed that LY2157299 significantly decreased the expression of P-Smad3 stimulated by the 0.000075% BAC treatment in CFs in comparison with the 0.000075% BAC treatment. (B) A graphic representation of the densitometry of P-Smad3 expression is shown in (A). ***P < 0.0001. (C) The Western blot showed that LY2157299 significantly reduced the expression of Col1α1 and FN-1 stimulated by the 0.000075% BAC treatment in CFs in comparison with the 0.000075% BAC treatment. (D) A graphic representation of the densitometry of Col1α1 and FN-1 expression is shown in (C). ***P < 0.0001. The results are representative examples of five independent experiments.

BAC at Lower Concentration Activates a TGF-β1/Smad3 Signaling Pathway Through the Upregulated Expression of COX-2 in CFs

Fibroblasts express COX-2^49,50 in addition to TGF-β.^15,47 The upregulated expression of COX-2 has been revealed to stimulate TGF-β production and to be implicated in tissue fibrosis.^26–30,51 The results above show that at a lower concentration, BAC stimulated CFs to express ECM through the activation of a TGF-β1/Smad3 signaling pathway. These data indicate that the upregulated expression of COX-2 can induce tissue fibrosis through the activation of a TGF-β1/Smad3 signaling pathway. To confirm that BAC can induce COX-2 expression and subsequently can activate the TGF-β1/Smad3 signaling pathway through upregulating the COX-2 expression in subconjunctival fibrosis, we first investigated the expression of COX-2 in BAC-treated rat conjunctival tissues and CFs using the Western blot. The results show that the expression of COX-2 was significantly upregulated in BAC-treated rat conjunctival tissues and CFs in comparison with PBS-treated conjunctival tissues and CFs (Figs. 6A–D). We then investigated whether or not the inhibition of COX-2 activity could attenuate the BAC-induced activation of a TGF-β1/Smad3 signaling pathway in CFs. The results show that after CFs were treated with 0.000075% BAC + 100 μM NS-398 for 24 hours, the expression of TGF-β1, TGF-βR1, Smad3, and P-Smad3 were significantly decreased in comparison with 0.000075% BAC treatment (Figs. 7A, 7B). These data demonstrate that the BAC-stimulated activation of the TGF-β1/Smad3 signaling pathway is through the upregulated expression of COX-2. 

Figure 6

 

Effect of BAC on the expression of COX-2 in bulbar conjunctival tissues and the CFs. (A) The Western blot showed that the expression of COX-2 was significantly increased in bulbar conjunctival tissues of rats with a chronic application of 0.01% BAC in comparison with the PBS treatment. (B) A graphic representation of the densitometry of COX-2 expression is shown in (A). ***P < 0.0001. (C) The Western blot showed that the expression of COX-2 was significantly increased in CFs with the 0.00005% and 0.000075% BAC treatment for 24 hours in comparison with the PBS treatment. (D) A graphic representation of the densitometry of COX-2 expression is shown in (C). **P < 0.001. ***P < 0.0001. The results are representative examples of five independent experiments.

Figure 7

 

NS-398 inhibits the expression of a TGF-β1/Smad3 signaling pathway–related molecules in CFs. (A) The Western blot showed that the 0.000075% BAC treatment markedly up-regulated the expression of TGF-β1, TGF-βR1, Smad3, and P-Smad3 in CFs in comparison with the PBS treatment, and NS-398 significantly downregulated the expression of TGF-β1, TGF-βR1, Smad3, and P-Smad3 stimulated by a 0.000075% BAC treatment in CFs. (B) A graphic representation of the densitometry of TGF-β1, TGF-βR1, Smad3, and P-Smad3 expression is shown in (A). **P < 0.001. ***P < 0.0001. The results are representative examples of five independent experiments.

Discussion

In this study, we demonstrate for the first time that BAC-induced subconjunctival fibrosis was caused by the COX-2–modulated activation of the TGF-β1/Smad3 signaling pathway. This conclusion is supported by substantial evidence. The chronic application of 0.01% BAC caused a slightly increased density of fibroblasts and a more compact collagen deposition in the bulbar subconjunctival tissue of rats. The chronic application of 0.01% BAC markedly upregulated the expression of ECM, TGF-β1/Smad3 signaling pathway–related molecules, and COX-2 in the bulbar conjunctival tissues of rats. At a lower concentration, BAC not only had no toxic effect on the cultured CFs but also stimulated the CFs to express ECM and COX-2 and to activate a TGF-β1/Smad3 signaling pathway. Expression of BAC-stimulated ECM was attenuated by the TGF-β R1 inhibitor, LY2157299. The benzalkonium chloride–stimulated activation of the TGF-β1/Smad3 signaling pathway was downregulated by the COX-2 selective inhibitor, NS-398. Our findings will contribute to the understanding of the mechanism that causes subconjunctival fibrosis induced by BAC and the development of new therapeutic agents for BAC-related ocular surface diseases. 

Subconjunctival fibrosis, which most likely results from an increase in fibroblast density and collagen deposition in the subconjunctival tissue,³ is a common side effect of topical medication containing BAC for glaucoma patients. Its pathogenesis is still unclear. Some studies reported that the BAC in topical medications had direct toxic effects on ocular tissues and caused significant increases in the thickness of conjunctival epithelium and a decrease in the number of conjunctival goblet cells. The toxicity to the ocular surface may further result in chronic inflammation and infiltration of inflammatory cells, which are linked to subconjunctival fibrosis.^{3,6,8,37–41} Mietz et al.⁹ investigated the effect of 0.3% metipranolol and 2% pilocarpine with and without 0.01% BAC and 0.004% cetrimonium chloride, respectively, on rabbit conjunctiva after treatment for three months and found that there were no signs of acute or chronic inflammation or alteration of the epithelium and goblet cells in conjunctiva in four treated groups with the exception of a slight increase in the thickness of subepithelial collagen in the conjunctiva that was present in both groups treated with medication and preservatives. Furthermore, Pauly et al.⁵² applied 0.01%, 0.1%, 0.25%, and 0.5% BAC to rat corneas for 11 days and found that 0.01% BAC groups did not present any signs of inflammation, but the 0.1% BAC-treated rats exhibited stromal inflammation. Consistent with their findings, in the present study, we found that there were no differences in inflammatory cell infiltration or the alteration of epithelium and goblet cells in conjunctival tissues between the 0.01% BAC- and PBS-treated groups, although our previous studies also show that high concentrations of BAC (0.1%–0.2%) indeed had ocular surface toxicity and caused obvious inflammatory infiltration in mouse models.^53,54 The reasons our findings are similar to those of Mietz et al.⁹ and Pauly et al.,⁵² but are inconsistent with those of other studies,^{3,6,8,37–41} is difficult to analyze due to the differences in the medication and preservative concentration in the topical medication used and the treatment period. At concentrations of 0.001% to 0.01%, BAC appears to elicit a defense response by increasing expressions of Ki67, a cell marker of proliferation, in the reconstructed 3D model of human corneal epithelial cells.³ Therefore, a 0.01% concentration of BAC that would induce the increase of collagen deposition in the subepithelial part of conjunctiva is an unlikely response to the toxic effect of BAC. Mietz et al.⁹ suggested that preservatives may have an additional adverse effect on the conjunctiva. It is possible that at a lower concentration, BAC just acts as a xenobiotic to stimulate CFs to express ECM through the activation of a TGF-β1/Smad3 signaling pathway modulated by COX-2 but not as a toxicant causing cell injury and inflammation, which might be the additional adverse effect of BAC on the conjunctiva. 

In vitro, a cell culture system is one useful alternative to an in vivo animal model in the study of the toxic effects of BAC, although the monocharacteristic of the cell monolayer that cannot mimic a more complex structure similar to a tissue is one major limitation. The cultured cells have been widely used for demonstrating and investigating some pathophysiological aspects of BAC on human conjunctiva. Some objective biological criteria, such as cellular viability, chromatin condensation, the production of reactive oxygen species, and transmembrane mitochondrial potential, are used to evaluate the toxicity of BAC in cultured cells.^3,48 It is noteworthy that the concentration of BAC used in those studies is higher. It has been reported that the concentrations of BAC with the threshold of toxicity to inhibit cell growth and induce cell apoptosis are respectively approximately 0.0001% and 0.005%.^3,32,33 The nontoxic effect of BAC on cultured cells at the concentration lower than the threshold of toxicity had been explored previously by Michée et al.,³² in which BAC at a concentration of 0.00001% was shown to have a direct stimulating effect on macrophages regarding migration, cytokine production, phagocytosis and expression of cell markers.³ Ye et al.⁵⁵ also showed that although the DNA strand of human corneal epithelial cells are damaged after treatment with BAC at concentrations ranging from 0.00005% to 0.001% for 30 minutes, there was no significant loss of cell viability in BAC-treated groups in comparison with the control groups after BAC was removed from cell cultures and cells were allowed to recover for 24 hours. It has been demonstrated that BAC can penetrate conjunctival deeper structures.³ Moreover, Friedlaender et al.⁴⁸ have reported that the concentration of BAC dilutes eight times in 30 seconds, 16 times in 1 minute, and 36 times in 2 minutes because of a tear when topical medication is applied to the eyes. The concentration of BAC will be very low when it penetrates subconjunctival tissue, although the most commonly used concentration of BAC in topical medication ranges from 0.004% to 0.02%.² Hence, CFs in subconjunctival tissue are bound to be affected by BAC at a concentration lower than the threshold of toxicity. To identify the nontoxic effect of BAC on CFs at a concentration lower than the threshold of toxicity, we exposed the primary cultured CFs to BAC at a concentration of 0.00001%, 0.000025%, 0.00005%, 0.000075%, 0.0001%, 0.00025%, 0.0005%, 0.00075%, and 0.001% for 24 hours. Similar to the findings of Michée et al.,³² we showed that 0.00005% and 0.000075% BAC concentrations not only had no toxic on CFs but also slightly stimulated the proliferation of CFs. Therefore, it is reasonable that long-term exposure to low concentrations of BAC should be considered as a stimulating factor responsible for CFs. 

Transforming growth factor β, a multifunctional cytokine, is a major contributor to the pathogenesis of the tissue fibrosis in various organ systems^42,43; TGF-β1 plays a key role through a Smad3 signaling pathway in fibrotic diseases of the eye, liver, lung, heart, kidney, and other diseases of the organs, such as the ocular cicatricial pemphigoid, cirrhosis, pulmonary fibrosis, cardiac fibrosis, glomerulosclerosis, and systemic sclerosis.^15,16,18 Transforming growth factor β is commonly considered to be an inflammatory cell-derived cytokine linking inflammation to fibrosis,^{14–16,22,56} but it is also secreted by fibroblasts.^15,47 In the present study, we found that although there was no difference in inflammatory cell infiltration between 0.01% BAC and PBS treatments, chronic application of 0.01% BAC could still markedly stimulate the expression of TGF-β1, TGF-βR1, Smad3, and P-Smad3 in rat conjunctival tissues. Furthermore, we also found that BAC at concentrations of 0.00005% and 0.000075% could markedly stimulate the expression of TGF-β1, TGF-βR1, Smad3, and P-Smad3 in cultured CFs. Our findings indicate that TGF-β1 can be at least partially directly produced by CFs when conjunctiva was chronically stimulated by a 0.01% concentration of BAC, although the possibility that TGF-β1 can be secreted by other cells, such as conjunctival epithelial cells or resident inflammatory cells, cannot be completely excluded. Therefore, our findings help explain the cause of subconjunctival fibrosis induced by topical medications containing BAC. 

Cyclooxygenase 2 is one of two isoforms of cyclooxygenase that is a rate-limiting enzyme in catalyzing the conversion of arachidonic acid to prostaglandins.^23,26,57 Unlike COX-1, which is constitutively expressed in almost all tissues, COX-2 is normally expressed at low levels, but its expression is highly inducible by many stimuli, such as cytokines, growth factors, and xenobiotics.^49,56,58,59 In addition to its central role in inflammation, the upregulated expression of COX-2 functions as a modulator in embryonic development, in the pathogenesis of a number of epithelial cell carcinomas, and in the diseases of the nervous system by catalyzing the conversion of arachidonic acid to prostaglandins.^23–25 Previous studies show that the upregulated expression of COX-2 stimulates TGF-β production and is implicated in tissue fibrosis.^26–30,60 In the present study, we found that the upregulated expression of COX-2–induced fibrosis was due to the activation of a TGF-β1/Smad3 signaling pathway. Our data indicated that there was a correlation between BAC, COX-2 and TGF-β1/Smad3 signaling pathway. However, the molecular mechanisms that cause BAC-stimulated COX-2 production and the COX-2–modulated TGF-β expression remain unclear. It has been reported that the expression of COX-2 can be induced by interleukin-1β (IL-1β).^58,61–63 Interleukin-1β is the common cytokine mainly produced by mononuclear cells and is secreted by the conjunctivas of patients receiving antiglaucoma medications containing BAC and CFs.^64–66 Moreover, IL-1β is one of the cytokines that are identified as important targets in a variety of fibrotic diseases, and is shown to promote TGF-β1 production and trigger fibrosis.^67,68 Some studies have demonstrated that transgenic mice with lung overexpression of IL-1β develop highly progressive pulmonary fibrosis, and IL-1β–deficient mice show attenuated fibrosis of lungs, liver, kidney, and heart.^67,68 Interestingly, IL-1β is also one of the cytokines that are induced by BAC to release.^32,69 It had been previously shown that BAC could dose dependently induce the release of IL-1β by cultured conjunctival epithelial cells in an irritational rather than truly “toxic” fashion.⁶⁹ Therefore, it is possible that BAC first induces the release of IL-1β by conjunctival epithelial cells in an irritational rather than truly “toxic” fashion when eye drops containing BAC is instilled onto the ocular surface, and IL-1β then induces the expression of COX-2 and stimulates CFs to express ECM through the activation of a TGF-β1/Smad3 signaling pathway modulated by COX-2. It might be the mechanism of subconjunctival fibrosis caused by BAC as a xenobiotic. 

Currently, it has been shown that IL-1β–induced COX-2 expression involves the production of reactive oxygen species (ROS) and the activation of protein kinase C (PKC) and multiple signaling pathways, including the extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, P38 mitogen-activated protein kinase (P38 MAPK), and nuclear factor-κB signaling pathways.^61–63 However, the molecular mechanisms that cause BAC-induced IL-1β expression and IL-1β–triggered fibrosis have still not been revealed. The regulating relations between COX-2 and TGF-β also remain unclear, and the results in the literature are inconsistent. Some reports show that the upregulated expression of COX-2 stimulates the production of TGF-β, and the effect is inhibited by a COX-2 selective inhibitor,^26,27,60 whereas others show that COX-2 expression is downregulated or upregulated by TGF-β.^11,70–74 Cyclooxygenase 2 is also shown to inhibit activation of Smad2/3 by TGF-β through the PGE₂/EP₂ pathway.^11,73 Therefore, the exact molecular mechanisms that cause BAC-stimulated COX-2 production and the COX-2-modulated TGF-β expression must be studied further. 

In summary, we clearly show that BAC-induced subconjunctival fibrosis is caused by the activation of a TGF-β1/Smad3 signaling pathway modulated by an upregulated expression of COX-2 in CFs. Our findings suggest that the development of BAC-free topical medication will be necessary for glaucoma patients and also contribute to the development of new therapeutic agents for BAC-related ocular surface diseases. 

Acknowledgments

Supported by National Key Basic Research Program of China (2013CB967502) and National Natural Science Foundation of China (No. 81370991, 81070075, 81270978, 81330022, U1205025). 

Disclosure: C. Huang, None; H. Wang, None; J. Pan, None; D. Zhou, None; W. Chen, None; W. Li, None; Y. Chen, None; Z. Liu, None 

Footnotes

 CH and HW contributed equally to the work presented here and should therefore be regarded as equivalent authors.