European Journal of Pharmaceutical Sciences 58 (2014) 13–19

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate/e jps

Effect of pirfenidone on proliferation, TGF-b-induced myofibroblastdifferentiation and fibrogenic activity of primary human lung fibroblasts

http://dx.doi.org/10.1016/j.ejps.2014.02.0140928-0987/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Address: Dipartimento di Biomedicina Clinica e Moleco-lare, Sezione di Medicina Respiratoria, Università di Catania, Policlinico Universi-tario, Via Santa Sofia 78, 95123 Catania, Italy. Tel.: +39 39 095 378 1254; fax: +39 39095 378 1427.

E-mail address: econte@unict.it (E. Conte).

Enrico Conte ⇑, Elisa Gili, Evelina Fagone, Mary Fruciano, Maria Iemmolo, Carlo VancheriDepartment of Molecular and Clinical Biomedicine, University of Catania, 95123 Catania, Italy

a r t i c l e i n f o

Article history:Received 24 September 2013Received in revised form 28 January 2014Accepted 25 February 2014Available online 12 March 2014

Keywords:PirfenidoneIPFLung fibroblastsProliferationFibrogenic activity

a b s t r a c t

Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis inanimal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits welldocumented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets andmechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidoneon proliferation, TGF-b-induced differentiation and fibrogenic activity of primary human lung fibroblasts(HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-b-induced a-smooth muscleactin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-b-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-b pathway. Together, theseresults demonstrate that pirfenidone modulates HLF proliferation and TGF-b-mediated differentiationinto myofibroblasts by attenuating key TGF-b-induced signaling pathways.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitiallung disease characterized by patchy fibrotic areas with mesenchy-mal cell proliferation and aberrant deposition of extracellular ma-trix (ECM) proteins that results in irreversible distortion of the lungarchitecture and loss of function (Noble et al., 2012). Activatedfibroblasts/myofibroblasts are the key effector cells in fibrosisand typical pathological features of IPF include scattered fibroblastfoci (aggregates of fibroblast/myofibroblasts juxtaposed to dam-aged alveolar epithelium) associated with the progression of fibro-sis (Flaherty et al., 2003). Myofibroblasts produce excessiveamounts of collagen and ECM proteins and have a contractilephenotype that is characterized by the presence of alpha-smoothmuscle actin (a-SMA) stress fibers. The underlying etio-pathologi-cal mechanisms through which mesenchymal expansion occursand excessive ECM proteins are deposited in fibrotic lesions arenot fully understood. Nevertheless, TGF-b-induced differentiationof human lung fibroblasts into myofibroblasts is recognized as aprimary event in the pathogenesis of IPF. Molecular pathways in-volved in this process are both Smad-dependent and -independent

(Wilkes et al., 2005; Martin et al., 2007; Munshi et al., 2004; Dery-nck and Zhang, 2003).

Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a smallmolecule that inhibits fibrosis progression in a variety of animalmodels of lung, kidney, hepatic and cardiac fibrosis (reviewed inSchaefer et al. (2011)) as well as in clinical trials (reviewed in Azu-ma (2012), Richeldi (2012)). In vitro studies have shown that pirf-enidone inhibits proliferation and/or activation of a wide range ofcell types including rat renal fibroblasts (Hewitson et al., 2001), hu-man Tenon’s fibroblasts (Lin et al., 2009), human myometrial andleiomyoma cells (Lee et al., 1998), human T cells (Visner et al.,2009), rat hepatic stellate cells (Di et al., 2002), and rat cardiacfibroblasts (Shi et al., 2011). In addition, pirfenidone inhibits prolif-eration, migration and epithlial–mesenchymal transition of humanlens epithelial cell line (Yang et al., 2013), decreases levels of inter-cellular adhesion molecule-1 in cultured human synovial fibro-blasts (Kaneko et al., 1998), inhibits heat shock protein 47expression in human lung fibroblasts (Nakayama et al., 2008), re-stricts Th2 differentiation in vitro and limits Th2 response in exper-imental liver fibrosis (Navarro-Partida et al., 2012).

In this in vitro study in HLFs, we investigated specific effects ofpirfenidone on critical aspects of the fibrotic process, such as fibro-blast proliferation and fibrogenic activity as well as TGF-b-induceddifferentiation into myofibroblasts. We also evaluated pirfenidoneeffects on the relevant Smad-dependent and -independent signaltransduction pathways underlying these processes.

14 E. Conte et al. / European Journal of Pharmaceutical Sciences 58 (2014) 13–19

2. Materials and methods

2.1. Reagents and chemicals

Unless otherwise stated, all compounds were obtained fromSigma–Aldrich Company Ltd. (Milan, Italy). Pirfenidone was kindlyprovided from InterMune (Brisbane, CA, USA) and dissolved inDMSO such that the final DMSO concentration in cell-based assaysdid not exceed 0.1%. All other stock solutions, were prepared innon-pyrogenic saline (0.9% NaCl; Baxter). RPMI medium and fetalcalf serum (FCS) were purchased from Life Technologies Europe

Fig. 1. Pirfenidone had no cytotoxicity in HLFs. Following treatments with different conceperformed to cytotoxicity. Data represent mean ± SEM of cytotoxicity % calculated in celltreatment.

Fig. 2. Anti-proliferative effect of pirfenidone on HLFs. In three separate experiments, pror 96 wells plates, respectively, and incubated for 24 h in serum-free RPMI medium. Aftpirfenidone at various concentrations (0.01–0.3 mg/ml, equivalent to 0.054–1.6 mM), thexperiments, determined by optical microscopy, are reported as percent of baseline (meacells per well. (B) BrdU absorbance are reported as percent of control (untreated cells, meone-way ANOVA with raw data of each experiment. The asterisk indicates a P value <0.

(Monza, Italy). Recombinant human TGF-b was from Merck Milli-pore (Milan, Italy).

2.2. Cell cultures

Primary HLF cultures were established as previously described(Conte et al., 2013) and used at passage eight or less. Prior to treat-ments, cells were plated and incubated for 24 h in serum-free RPMImedium. The media was subsequently replaced with RPMI supple-mented with 2% FCS and cells were treated with pirfenidone (0.03–0.3 mg/mL; 0.16–1.6 mM) in DMSO or DMSO alone to the same

ntrations of pirfenidone (0, 0.01, 0.03, 0.1 and 0.3 mg/ml) for 48 h, an LDH assay wass from three independent donors. No significant cytoxic effect was observed for any

imary HLFs from three different donors were plated in triplicate or quintuplicate 24erward, cells were grown for 48 h in 2% FCS medium in the absence or presence ofen counted (24 wells) or subjected to BrdU assay (96 wells). (A) Cell counts in allns ± SD). Baseline counts, following the 24 h starvation, ranged from 4.4 to 8.2 � 104

ans ± SD). Statistical significance of results across groups was determined using the05 in all comparisons with untreated and/or DMSO controls.

E. Conte et al. / European Journal of Pharmaceutical Sciences 58 (2014) 13–19 15

final concentration. TGF-b (5 ng/mL) was added at the same timeas pirfenidone treatment and cells were grown for 48 h.

2.3. Cell proliferation assay

Cell proliferation was evaluated by determining cell numberswith optical microscopy as well as evaluating 5-bromo-20-deoxy-uridine (BrdU) incorporation. Viable cells were counted in tripli-cate on a hemocytometer (Burker chamber) after trypan bluestaining at time 0 (after 24 h serum starvation) and after 48 htreatment. A mean of four fields was used to calculate the averagenumber of cells. The BrdU proliferation assay was performed witha commercially available kit (BrdU Cell Proliferation Kit, MerckMillipore) following manufacturer’s instructions.

2.4. Cytotoxicity assay

Following treatments with different concentrations of pirfeni-done (0, 0.01, 0.03, 0.1 and 0.3 mg/ml) the release of lactate dehy-drogenase (LDH) was evaluated by the Colorimetric CytotoxicityDetection Kit (Roche) according to the manufacturer’s instructions.Cytotoxicity was indicated as percentage of LDH release, expressedas the ratio between the mean absorbance of treated culturesminus the absorbance of untreated control cells, and the meanabsorbance of high positive control (maximum LDH release by celllysis) minus the absorbance of untreated cultures.

Fig. 3. Inhibitory effects of pirfenidone on a-SMA expression. (A) Results of qRT-PCR anal0.3 mg/ml (0.16–1.6 mM), in the absence or in the presence of TGF-b (5 ng/ml) stimulatTGF-b-induced conditions. Reported values are means ± SD of the relative fold inductionswith three different primary cell isolates. A P value <0.05, is indicated with one asterisk, <SMA expression, demonstrating a substantial or significant lowering of protein levels bGraphs in the bottom panel report for each lane the corresponding means ± SD of target

2.5. Western blot analysis

Cell lysates were subjected to denaturating SDS gel electropho-resis followed by electroblotting and incubation with monoclonalmouse anti-human a-SMA Ab (1:1000, Dako Cytomation, Den-mark), or monoclonal mouse anti-human GAPDH (1:1000, MerckMillipore), monoclonal mouse anti-human pospho(p)-Akt(Ser473, 1:500; Merck Millipore), monoclonal mouse anti-humanCOL-I (1:200, Merck Millipore), polyclonal rabbit anti-humanpSmad3 (1:200, Santa Cruz Biotechnologies), monoclonal mouseanti-human p38 (1:200, Santa Cruz Biotechnologies). The mem-branes were then thoroughly washed and incubated with biotin-conjugated anti-mouse or anti-rabbit secondary antibodies(1:5000; Life Technologies) and developed with the Quantum Dotdetection system (Invitrogen). The band area and intensityof ex-posed film were analyzed by densitometric scanning and meanintensity quantified using Image J (NIH, free share) software. Nor-malization of each target protein was performed by calculating theratio of target to GAPDH mean intensity.

2.6. RNA extraction, reverse transcription and quantitative real timePCR(qRT-PCR)

Total RNA was extracted by using TRIZOL (Invitrogen, UK),quantified by UV spectroscopy (BIO-photometer, Eppendorf, Ger-many), and treated with DNase (Invitrogen). cDNA was generated

ysis of a-SMA mRNA expression after 48 h treatment with pirfenidone at 0.03, 0.1 orion, showing a dose-dependent inhibition exerted by pirfenidone in both basal and

(calculated on the level of untreated cells) observed in three separate experiments0.01 with two. (B) Representative western blots of both basal and TGF-b-induced a-

y the highest pirfenidone dose in basal and TGF-b-induced conditions respectively./control gene ratios obtained by densitometric analysis of each experiment results.

16 E. Conte et al. / European Journal of Pharmaceutical Sciences 58 (2014) 13–19

using Superscript II Reverse Transcriptase (Invitrogen) and randomhexamer primers (Invitrogen) according to the manufacturer’sinstructions. Quantitative PCR (qPCR) was performed by usingthe commercially available Taqman assays for human a-SMA(ACTA2), Col-I (COL-1A1), and the housekeeping GAPDH gene (QIA-GEN, Germany) following the manufacturer’s instructions. Amplifi-cation conditions were identical for all genes: 94 �C, 5 min and 45cycles of 2 steps: 95 �C, 15 s and 60 �C, 30 s. Relative quantificationof target gene levels was performed by comparing DCt as describedelsewhere (Conte et al., 2008, 2013).

2.7. Statistical analysis

Statistical significance across treatment groups was determinedby using the one-way ANOVA with Tukey’s multiple-comparisonpost hoc test (Statgraphic Centurion XV software, StatPoint Tech-nologies Inc., ADALTA, Italy). A P value <0.05 or <0.001, which indi-cate a statistically significant difference, was designated with oneor two asterisks, respectively.

3. Results

3.1. Pirfenidone cytotoxicity

To rule out the possibility that in our experiments the putativeeffects of pirfenidone were mediated by cytotoxicity we prelimi-narily performed a cytotoxicity assay measuring the LDH release

Fig. 4. Inhibitory effects of pirfenidone on Col-I expression. (A) Results of qRT-PCR analywith pirfenidone at 0.03, 0.1 or 0.3 mg/ml (0.16–1.6 mM), in the absence or in the presblocking of TGF-b-induced COL-1 over-expression. Reported values are means ± SD of thecell isolates. (B) Representative western blots of both basal and TGF-b-induced expressexpression exerted by the pirfenidone highest dose. In the panel bottom, graph reportsanalysis of each experiment results.

after administration of pirfenidone at the time period and differentconcentrations we used it. Results indicated that in our experimen-tal setting all pirfenidone doses had no significant cytotoxic effecton cultured HLFs (Fig. 1).

3.2. Effects of pirfenidone on HLF proliferation

In independent experiments with cells from three donors,0.3 mg/ml pirfenidone (1.6 mM) significantly inhibited cell prolif-eration compared to untreated cells and pertinent control vehicle(0.1% DMSO) when assessed by either direct cell counting(Fig. 2A) or BrdU incorporation (Fig. 2B).

3.3. Effects of pirfenidone on a-SMA expression

Myofibroblast differentiation is considered to be a key step inIPF. Since the expression of a-SMA is a hallmark of myofibroblastdifferentiation, the effects of pirfenidone on TGF-b-induced a-SMA mRNA and protein levels were measured by qRT-PCR andand western blotting, respectively. Pirfenidone treatment reducedexpression a-SMA mRNA expression in a dose-dependent mannerin both the presence and absence of TGF-b stimulation (Fig. 3A).Consistent with this result, 0.3 mg/mL (1.6 mM) pirfenidone re-duced a-SMA protein expression in both the presence and absenceof TGF-b stimulation (Fig. 3B).

sis of COL-1A1 gene (encoding pro-collagen type I) expression, after 48 h treatmentence of TGF-b (5 ng/ml) stimulation, demonstrating a pirfenidone dose-dependentrelative fold inductions observed in three separate experiments with three differention levels of Col-I, showing a significant inhibition of TGF-b-induced protein over-

for each lane means ± SD of target/control gene ratios obtained by densitometric

E. Conte et al. / European Journal of Pharmaceutical Sciences 58 (2014) 13–19 17

3.4. Effects of pirfenidone on collagen type I expression

Aberrant deposition of type I collagen-rich ECM is the typical phe-notype of activated fibroblast/myofibroblasts in IPF lesions. We per-formed qRT-PCR and western blotting analysis to investigatepirfenidone effects on the expression of COL-1A1 gene (encodingthe pro-collagen type-I) as well as collagen type-I protein levels,respectively.TGF-bsignificantlyinducedCOL-1A1mRNAandproteinexpression and pirfenidone inhibited this induction in a dose-depen-dent manner(Fig. 4A and B). However, pirfenidone did not impact ba-sal collagen type-I mRNA or protein levels (Fig. 4A and B).

3.5. Effects of pirfenidone on Smad3 p38 and Akt activity

To elucidate molecular mechanisms underlying the observedphenotypic pirfenidone effects we investigated the long-lastingconsequences of pirfenidone treatment (24 h) on Smad3, p38 andAkt activity. Since phosphorylation is the key event for activatingthese proteins we evaluated their phosphorylation status in bothunstimulated and TGF-b-stimulated cells. In Fig. 5, we show thatpirfenidone, at least at the highest dose, significantly impairedphosphorylation of Smad3, p38 and Akt in TGF-b-induced HLFs.

4. Discussion

Pirfenidone is the only drug approved for the treatment of IPF,in Europe and Japan. However, the exact mechanisms through

Fig. 5. Long lasting Inhibitory effects of pirfenidone on Smad3 p38 and Akt activation.concentrations of pirfenidone for 24 h, phosphorylation status of Smad3, p38 and Akt windicated phosphorated proteins. Expression of GAPDH was used to normalize sample loameans ± SD of target/control gene ratios obtained by densitometric analysis of each expetreated cells.

which pirfenidone protects against lung fibrosis remain unclear.On the other hand, the pathogenesis of IPF remains poorly under-stood and controversial. Nevertheless, activated lung fibroblastsand myofibroblasts are recognized as key effector cells in IPF (Fer-nandez and Eickelberg, 2012; Sivakumar et al., 2012). In the pres-ent study we addressed the effects of pirfenidone on primary HLFsunstimulated or stimulated with TGF-b. By evaluating the cellnumbers and performing the BrdU incorporation assay, we foundthat pirfenidone at 0.3 mg/mL (1.6 mM) significantly inhibitedthe proliferation of HLFs. Bearing in mind that mesenchymalexpansion is an essential process underlying IPF, our finding in hu-man lung cells could constitute one explanation of the pirfenidoneeffectiveness in the treatment of IPF. Our results are in line withprevious observations in rat renal and cardiac fibroblasts (Hewit-son et al., 2001; Shi et al., 2011), as well as human Tenon’s fibro-blasts (Lin et al., 2009).

Myofibroblasts, exhibit increased proliferative, migratory andsecretory properties, playing a major role in the aberrantwound-healing and remodeling processes active in IPF. Therefore,phenotypic transformation of fibroblasts into myofibroblasts,characterized by the expression of a-SMA, is perceived to be a keyevent in the IPF pathogenesis. We demonstrate here that a-SMAmRNA expression was significantly inhibited by pirfenidone inboth unstimulated and TGF-b-stimulated HLFs, in a dose-dependentmanner. Moreover, we show that pirfenidone at 0.3 mg/mL(1.6 mM) significantly affected basal and TGF-b-stimulated levelsof a-SMA protein. This finding is in agreement with a previous

Following treatments with TGF-b (5 ng/ml) in the absence or presence of differentas assessed by western blot analysis with antibodies that specifically recognize theding. (A) Representative blots of one out three separate experiments. (B) Graph withriment results. The asterisks indicate a P value <0.05 in all comparisons with TGF-b-

18 E. Conte et al. / European Journal of Pharmaceutical Sciences 58 (2014) 13–19

report on rat cardiac fibroblasts (Shi et al., 2011) and is noteworthyconsidering that prevention of myofibroblast differentiation mayrepresent a crucial target for therapies aimed at limiting fibrosisin the IPF lung. Furthermore, by using qRT-PCR and western blotanalysis, we clearly demonstrate that pirfenidone inhibited TGF-b-induced synthesis of collagen type I at both mRNA and proteinlevels These results complement a previously published study onHLFs utilizing Northern blot analysis and immunocytochemistry(Nakayama et al., 2008), and supporting observations in hepaticstellate cells (Di et al., 2002), leiomyoma cells (Lee et al., 1998), cul-tured orbital fibroblasts (Kim et al., 2010), and alveolar epithelialcells (Hisatomi et al., 2012).

The major signaling pathway of TGF-b1 is through its trans-membrane receptor serine/threonine kinases activating the cyto-plasmic Smad proteins (Shi and Massague, 2003). Among those,Smad3 has been shown to be the major contributor in regulatingmyofibroblast differentiation (Gu et al., 2007) and thus we askedif pirfenidone were able to inhibit the Smad 3 activation. In this pa-per, we demonstrate for the first time that pirfenidone treatmentsignificantly reduced TGF-b-induced Smad3 phosphorylation inHLFs. In contrast, in human retinal pigment epithelial cell lineARPE-19 it was shown that pirfenidone inhibited TGF-b signalingby preventing nuclear accumulation of active Smad2/3 complexesrather than phosphorylation of Smad2/3 (Choi et al., 2012). In addi-tion to Smad, other signaling pathways have been shown to con-tribute at inducing a-SMA gene expression and fibrogenicactivity of myofibroblasts (Wilkes et al., 2005; Martin et al.,2007; Conte et al., 2011). In this study we demonstrate that TGF-b-induced phosphorylation of mitogen-activated protein kinasep38 was inhibited by pirfenidone treatment. Accordingly, in bothrat and human hepatic stellate cells PDGF-BB-induced p38activation was inhibited by pirfenidone as well as by the novelwater-soluble pyridone agent fluorofenidone (Peng et al., 2013),whereas in a rat model of experimental liver fibrosis it wasobserved that pirfenidone reduced p38 activation in the liverhomogenate (Navarro-Partida et al., 2012). Moreover, pirfenidoneinhibited lipopolysaccharide-dependent p38 phosphorylation inmouse dendritic cells (Bizargity et al., 2012). Differently, no pirfe-nidone effects on p38 activation were reported in human retinalpigment epithelial cells (Choi et al., 2012). Finally, since in a previouspaper we provided evidence that the PI3K pathway plays a majorrole in both lung fibroblast proliferation and TGF-b-induced differ-entiation into myofibroblasts (Conte et al., 2011), in this study weinvestigated putative effects of pirfenidone on the Akt activity(downstream of PI3K). We show that pirfenidone significantlyimpaired Akt phosphorylation in TGF-b-induced HLFs. In agree-ment with our data, in human hepatocytes pirfenidone was shownto inhibit Akt activation as a consequence of IL-1b stimulation andup-regulation of IL-1b receptor (Nakanishi et al., 2004).

In conclusion, demonstrating that pirfenidone directly inhibitedproliferation as well as a-SMA and Col-I expression in HLFs wesuggest that lung fibroblasts represent a major target of pirfeni-done in IPF treatment. Moreover, in this study we show for the firsttime that pirfenidone inhibited TGF-b-induced Smad3, p38 and Aktphosphorylation in HLFs, gaining insight into molecular mecha-nisms of pirfenidone behavior in IPF fibroblasts.

Acknowledgements

The authors are indebted to InterMune, Inc. that supported thiswork by providing pirfenidone and research funding.

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