Biochemistry and Molecular Biology

A PPAR-Gamma Agonist Rosiglitazone Suppresses FibroticResponse in Human Pterygium Fibroblasts by Modulatingthe p38 MAPK Pathway

Selikem Abla Nuwormegbe,1 Joon Hyung Sohn,2 and Sun Woong Kim3

1Department of Global Medical Science, Yonsei University, Wonju College of Medicine, Gangwon-do, Republic of Korea2Institute of Lifestyle Medicine, Yonsei University, Wonju College of Medicine, Gangwon-do, Republic of Korea3Department of Ophthalmology, Yonsei University, Wonju College of Medicine, Gangwon-do, Republic of Korea

Correspondence: Sun Woong Kim,Yonsei University, Wonju College ofMedicine, 20 Ilsan-ro, Wonju, Gang-won-do, 26426, Republic of Korea;eyedockim@yonsei.ac.kr.

Submitted: May 11, 2017Accepted: September 18, 2017

Citation: Nuwormegbe SA, Sohn JH,Kim SW. A PPAR-gamma agonist rosi-glitazone suppresses fibrotic responsein human pterygium fibroblasts bymodulating the p38 MAPK pathway.Invest Ophthalmol Vis Sci.2017;58:5217–5226. DOI:10.1167/iovs.17-22203

PURPOSE. Fibroblast activation may play an important role in pterygium progression. Syntheticperoxisome proliferator-activated receptor c (PPAR-c) ligands have been shown to beeffective antifibrotic agents against transforming growth factor b1 (TGF-b1) induced fibrosisin several tissues. We aimed to investigate the antifibrotic effects of the PPAR-c ligandrosiglitazone in pterygium fibroblasts and the underlying mechanisms.

METHODS. Profibrotic activation was induced by TGF-b1 in primary cultured human pterygiumfibroblasts and the effect of rosiglitazone treatment on a-smooth muscle actin (a-SMA), andextra cellular matrix proteins synthesis was detected by western blotting, real-time PCR,immunostaining, and flow cytometry. Pharmaceutical inhibition of PPAR-c receptor was usedto determine the dependency or otherwise of rosiglitazone’s action on PPAR-c signaling.Major signaling pathways downstream of TGF-b1 were investigated by western blotting toassess their possible association with rosiglitazone’s effect. Cell viability and apoptosis wereinvestigated to assess drug-induced cytotoxicity, and the effect of rosiglitazone treatment oncell migration was further determined.

RESULTS. a-SMA and fibronectin synthesis induced by TGF-b1 were suppressed by rosiglitazonetreatment in a dose-dependent manner. Rosiglitazone also inhibited intrinsic TGF-b1expression. Smad2/3, ERK1/2, and P38 pathways were activated in response to TGF-b1.Rosiglitazone suppressed TGF-b1-induced P38 MAPK activation, while ERK1/2 and Smad2/3signaling remained unaffected. The observed antifibrotic effect of rosiglitazone was notaffected by the PPAR-c antagonist GW9662, indicating it is not PPAR-c dependent.Rosiglitazone also inhibited the proliferation and migration of pterygium fibroblasts.

CONCLUSIONS. Rosiglitazone suppresses TGF-b1-induced myofibroblast activation and extracellular matrix synthesis in pterygium fibroblasts at least partly through the modulation of thep38 MAPK pathway.

Keywords: pterygium, cornea, fibrosis, PPAR-c, rosiglitazone

Pterygium is an ocular disorder that clinically manifests as abenign growth of conjunctival epithelial tissues over the

cornea. The characteristic origin and location of pterygium isthe nasal limbus, from where it grows toward the cornea.Pterygium is characterized by extensive cell proliferation,connective tissue remodeling, angiogenesis and chronic inflam-mation resulting in redness and irritation in the area.1,2 Thecondition can significantly affect vision in advanced casesowing to a distortion of the shape in the cornea3 and canprogress to ocular surface squamous neoplasia.4 Surgicalexcision is the standard treatment for pterygium. Woundhealing after pterygium excision often leads to fibrosischaracterized by recurrent pterygium. The bare sclera tech-nique for instance is associated with high postsurgicalrecurrence rates reported to range from 38% to 88%.5–8

Because fibrovascular growth in recurrent pterygia is moreextensive than that in primary disease,9 recurrence is of majorconcern as it worsens prognosis. To reduce postsurgicalrecurrences, mitomycin C application10 has been widely

attempted, but numerous adverse effects such as scleralstromalysis11 and corneal melting12 have been reported. Thereis therefore the need for a safer and more targeted curative andpreventive therapy.

Studies have shown the overexpression of transforminggrowth factor b1 (TGF-b1), a profibrotic agent, in pterygiumtissues compared to normal conjunctiva cultures and inrecurrent pterygium fibroblast cultures compared to primarypterygium fibroblast cultures.13,14 TGF-b1 signaling stimulatesfibroblast migration, proliferation, and myofibroblast differenti-ation.15 The presence of myofibroblasts in pterygium issuggestive of the involvement of TGF-b1 in the pathogenesisand progression of the disorder.16 Other research findings alsosupport the presence of myofibroblasts in the fibrovasculartissue of primary and recurrent pterygia.17,18

Previous research works have shown several synthetic ligandsof peroxisome proliferator-activated receptor c (PPAR-c) that areeffective in preventing fibrosis in various organs including theskin, kidney, and cornea.19–21 Notably, corneal fibrosis following

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laser refractive ablation was suppressed by the topicalapplication of a PPAR-c ligand.22 The topical application ofthese synthetic PPAR-c ligands and the possibility of preventingcorneal haze are advantageous to their potential use inophthalmology. We thus aimed to determine the antifibroticproperties of the PPAR-c ligand rosiglitazone in cultured primaryhuman pterygium fibroblasts and the possible signalingpathways mediating any observed effect.

METHODS

Ethical Statement

Pterygium tissues were obtained from patients who underwentpterygium excision surgery. This study protocol was approvedby the Institutional Review Board of our institute (YWMR-15-0-053), and all subjects were treated in accordance with theDeclaration of Helsinki. Written informed consent to partici-pate in this research was obtained from each patient.

Isolation and Culture of Primary HumanPterygium Fibroblasts

The pterygium tissue sample was placed in a culture plate andallowed to attach. Dulbecco’s modified Eagle’s medium F12(DMEM/F12; Welgene, Gyeongsan, Korea) with 10% fetalbovine serum (FBS) and 1% antibiotic/antimycotic (Gibco LifeTechnologies, Foster City, CA, USA) was added and the tissueincubated at 378C in a humidified atmosphere of 5% CO2

during which cells migrated from the explant. Migrated cellswere passaged with 0.25% trypsin (Gibco Life Technologies)and subcultured in DMEM (Welgene) with 5% FBS. Passage 3-5cells were used for all experiments.

Western Blotting

The pterygium fibroblasts were seeded into culture plates inDMEM with 5% FBS. After attachment, serum-free media wasadded 8 hours prior to treatment to promote cellularquiescence. A 10-mM stock solution of rosiglitazone (CaymanChemical, Ann Arbor, MI, USA) in dimethyl sulfoxide (DMSO)was added to cells at two concentrations (25 and 75 lM) for a30-minute pretreatment. TGF-b1 (PeproTech, Seoul, Korea)was then added to a 2.5-ng/mL concentration. The cells wereincubated for 48 hours after which total protein was extractedwith the Pro-prep protein extraction kit (Intron Biotechnology,Korea). Proteins were resolved with 8% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) andtransferred onto polyvinylidene fluoride (PVDF) membrane(Millipore, Milan, Italy). The membrane was blocked in 5%skimmed milk solution and incubated overnight at 48C withmonoclonal antibodies against a-smooth muscle actin (a-SMA;Sigma-Aldrich Corp., St. Louis, MO, USA), fibronectin (SantaCruz Biotechnology, Santa Cruz, CA, USA), vimentin, andtubulin (Cell Signaling Technology, Beverly, MA, USA). Themembranes were washed and then incubated with horseradishperoxidase-conjugated secondary antibodies (Cell SignalingTechnology) for 1 hour at room temperature. Proteins weredetected with enhanced chemiluminescence (ECL). Imageswere captured by UVP Bio-Spectrum 600 imaging system(Ultra-Violet Products Ltd. Cambridge, UK). The expression oftarget proteins relative to tubulin was evaluated by densitom-etry using ImageJ software (http://imagej.nih.gov/ij/index.html; provided in the public domain by the National Institutesof Health, Bethesda, MD, USA). To determine if the observedeffects of rosiglitazone were PPAR-c dependent, the cells werepretreated with 1 lM of a synthetic PPAR-c antagonist GW9662

(Cayman Chemical) to block PPAR-c receptor function.23 The effect of rosiglitazone on intrinsic TGF-b1 production was also investigated by western blotting with anti-TGF-b1 antibodies (Cell Signaling Technology). Major signaling pathways down-stream of TGF-b1 were investigated to see their relation to the observed effect of rosiglitazone. Primary antibodies to p38 MAPK, p44/42MAPK (Erk1/2), and Smad2/3 (Cell Signaling Technology) were used. The p38 kinase inhibitor SB203580 (InvivoGen, San Diego, CA, USA) was used to further investigate the effect of blocking p38 MAPK signaling on myofibroblast differentiation and extracellular matrix (ECM) synthesis. The effect of rosiglitazone on the expression of matrix metal-loproteinases (MMPs)-1, 3, and 9 were also investigated.

Quantitative Real-Time PCR

The pterygium fibroblasts were seeded and treated with GW9662, rosiglitazone, and TGF-b1 as described above. After 48 hours of incubation, the cells were harvested and total RNA extracted using an RNA extraction kit (Qiagen, Germany) according to the manufacturer’s protocol. The quality and quantity of the RNA extract were assessed by spectrophotom-etry. The qualified RNA was reverse-transcribed into cDNA with LaboPass cDNA synthesis kit (Cosmo Genetech, Seoul, Korea) according to the manufacturer’s protocol and used for real-time PCR. Primer pairs of target genes used are given in Supplementary Table S1. The reaction was performed in a 10-lL reaction volume containing SYBR green PCR mix (Applied Biosystems Life Technologies, Foster City, CA, USA), cDNA, primers, and nuclease free water.

Immunofluorescence Staining and Indirect FlowCytometry

The pterygium fibroblasts were cultured on glass chamber slides and pretreated with rosiglitazone (75 lM) in serum-free DMEM, followed by TGF-b1 (2.5 ng/mL). After 48 hours of incubation, the cells were fixed with 4% paraformaldehyde (PFA) followed by peroxidase and protein blocking. The slides were then incubated with primary antibodies against a-SMA and fibronectin overnight at 48C and then with Alexa Fluor labelled secondary antibodies (Thermo Fisher Scientific, Rock-ford, IL, USA) and incubated at room temperature in the dark for 2 hours. The nuclei were counterstained with 4 0,6-diamidino-2-phenylindole (DAPI), and immunofluorescence was visualized and digitally captured using a confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany). Internal control was performed using PBS and secondary antibodies without the primary antibodies. The proportion of a-SMA positive cells was quantified using indirect flow cytometry. Briefly, cell suspensions (approximately 1 3 105

cells/100 lL) prepared in stain buffer (ice cold PBS, 2% FBS, 5%sodium azide) were fixed for 10 minutes with 1% PFA. After washing, the cells were suspended in 500 lL permeability buffer (stain buffer þ 0.2% saponin), incubated in the dark for 5 minutes, and washed again. The cell pellets obtained after centrifugation at 1500 rpm for 5 minutes were resuspended in permeability buffer and incubated with primary antibodies against a-SMA for 30 minutes on ice in the dark. After washing, the cells were then incubated with an Alexa Fluor 488-conjugated secondary antibody for another 30 minutes, washed, and analyzed by flow cytometry.

Cell Viability Test

The pterygium fibroblasts were seeded into a 96-well plate at a density of 5000 per well and incubated for 24 hours. The cells were then treated with rosiglitazone (25, 50, 75, and 100 lM).

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Control cells were vehicle treated. After 48 hours ofincubation, the cell counting kit 8 (CCK-8) assay wasperformed according to kit manufacturer’s protocol (DojindoLaboratories, Kumamoto, Japan). After a 2-hour incubation, theoptical absorbance was read at 450 nm on a microplate reader.The experiment was done in triplicate for each concentrationof rosiglitazone.

Apoptosis Test

The pterygium fibroblasts were pretreated with rosiglitazone(25 and 75 lM) in serum-free DMEM after which TGF-b1 (2.5ng/mL) was added. Cells with only TGF-b1 treatment wereused as control. After 48 hours of incubation, the cells werewashed with cold PBS and resuspended in 1X binding buffer ata concentration of 1 3 106 cells/mL. One hundred microlitersof the solution with approximately 1 3 105 cells wastransferred to a 5-mL culture tube and 5 lL each of FITCannexin V and propidium iodide (PI) (BD Pharmingen, SanJose, CA, USA) added. After 15 minutes of incubation at roomtemperature in the dark, 400 lL 1X binding buffer was addedto each tube and analyzed by flow cytometry within 1 hour.

Cell Migration Test

The pterygium fibroblasts were seeded into culture plates andincubated for 24 hours to allow the cells to attach to the platesurface. Further, scratch wounds were induced with a pipettetip (1000 lL), and the suspended cells were washed off. Thecells were then pretreated with rosiglitazone (75 lM), followedby treatment with TGF-b1 (2.5 ng/mL) in serum-free media.The wounds were monitored using phase-contrast microscopyand photographed at 12, 24, and 48 hours. The ImageJsoftware was used to determine the wound area. Vehicle-treated cells were used as controls.

Statistical Analysis

The data were analyzed using Prism 5.0 software. Statisticalcomparisons were done using Student’s t-test or 1-way analysisof variance (ANOVA), as appropriate. All results are expressedas mean 6 SD. Statistical significance was defined as P < 0.05in all cases.

RESULTS

Rosiglitazone Decreased TGF-b1-Induced Synthesisof ECM Proteins and Myofibroblast Differentiationin Pterygium Fibroblasts Independent of PPAR-c

Basal levels of a-SMA, a key marker for myofibroblastdifferentiation, and fibronectin, an ECM protein, in thepterygium fibroblasts were detectable (Fig. 1). There was amarked increase of approximately threefold in the expressiona-SMA (P ¼ 0.0001) and approximately sixfold for fibronectin(P < 0.0001) relative to vehicle-treated control cultures after48 hours of TGF-b1 stimulation. Rosiglitazone treatment for 48hours decreased a-SMA and fibronectin expression in a dose-dependent manner. The expression of vimentin, a marker formesenchymal fibroblasts, was unaffected by both TGF-b1 androsiglitazone treatments. The observed antifibrotic effect ofrosiglitazone was seen to be independent of PPAR-c receptor astreatment with rosiglitazone together with GW9662 did notaffect its antifibrotic effect. These western blot results wereconfirmed by the relative mRNA expression of these proteins,as well as collagen 1 and serum response factor (SRF), which isa transcription factor for actin cytoskeletal reorganization andmyofibroblast transformation.24 The mRNA expression levelsof a-SMA, collagen 1, fibronectin, and SRF also tended to showa dose-dependent antifibrotic effect of rosiglitazone in pteryg-ium fibroblasts, whereas no significant change was observed in

FIGURE 1. Effect of rosiglitazone on TGF-b1-induced ECM protein synthesis. (A) Representative western blot images of a-SMA, fibronectin, andvimentin protein expression. Quantitative analysis of immunoblots with summary data from three independent experiments from (B) a-SMA (P <0.0001, F[5, 12]¼ 31.75), (C) fibronectin (P < 0.0001, F[5, 12]¼ 145.1), and (D) vimentin (P¼ 0.1989, F[5, 12]¼ 1.745). #Represents P < 0.05compared to vehicle-treated control cells; *represents P < 0.05 compared to only TGF-b-treated cells.

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the expression of vimentin (Fig. 2). Immunostaining resultsshowed decreased expression of a-SMA and fibronectin inrosiglitazone-treated cells (Fig. 3A). Indirect flow cytometryresults revealed that rosiglitazone treatment decreased theproportion of a-SMA-positive cells, which further confirmed itsantifibrotic effect in pterygium fibroblasts (Fig. 3B).

Rosiglitazone Decreased Intrinsic TGF-b1Production in Pterygium Fibroblasts Independentof PPAR-c

Our results showed a dose-dependent decrease in the intrinsicexpression of TGF-b1 when the cells were treated withrosiglitazone, indicating a modulatory effect of rosiglitazoneon TGF-b1 protein synthesis. In addition, this effect wasindependent of PPAR-c receptor, as cotreatment with rosigli-tazone and GW9662 did not reverse the decreased synthesis ofTGF-b1. The basal levels of a-SMA and fibronectin proteins alsotended to decrease in a dose-dependent manner subsequent tothe decreased TGF-b1 expression (Fig. 4).

Rosiglitazone Inhibited TGF-b1-Induced Activationof p38 MAPK Signaling Pathway in PterygiumFibroblasts

To determine the possible mechanisms underlying theantifibrotic effect of rosiglitazone, the major signal transduc-tion pathways downstream of TGF-b1 were examined. Asshown, TGF-b1 increased the activation of p38 MAPK, ERK1/

2, and Smad2/3 from the basal level indicating increasedsignaling. However, the p38 MAPK signaling was decreasedby rosiglitazone treatment as seen by the decreasedphosphorylated p38 MAPK protein levels. This effect ofrosiglitazone was not observed with ERK1/2 and Smad2/3signaling as the phosphorylated protein levels remainunaltered (Fig. 5).

TGF-b1 Induces Synthesis of ECM Proteins and

Differentiation of Myofibroblasts via p38 MAPK

Signaling in Pterygium Fibroblasts

To test whether p38 was involved in the TGF-b1-dependentphenotypic transformation of myofibroblasts in pterygiumfibroblasts, we first investigated the time course of p38 MAPKactivation. As shown in Figure 6A, the TGF-b1-inducedactivation of p38 MAPK signaling peaked at 45 minutes andlasted for at least 24 hours. This indicated that rosiglitazonedownregulated the p38 MAPK pathway. Maintenance of thisdownregulation by rosiglitazone on p38 activation for at least24 hours (Fig. 6B) confirmed the role of p38 signaling in theantifibrotic effect of rosiglitazone. Further, the kinase inhibitor,SB203580, blocked the p38 signaling pathway and led to adose-dependent decrease in the expression of a-SMA andfibronectin proteins (Figs. 6C, 6D, 6E). This indicated that TGF-b1-induced myofibroblast transformation and ECM synthesisoccurs at least partly through p38 MAPK signaling inpterygium fibroblasts.

FIGURE 2. Real-time PCR analysis. Quantitative analysis of mRNA expression levels of (A) a-SMA (P < 0.0001, F[5, 12]¼ 18.78), (B) collagen 1 (P¼0.0396, F[5, 12]¼ 3.471), (C) fibronectin (P¼ 0.0011, F[5, 12]¼ 9.447), (D) SRF (P¼ 0.0398, F[5, 12]¼ 9.447), and (E) vimentin (P¼ 0.2412, F[5,12]¼ 1.593). #Represents P < 0.05 compared to vehicle-treated control cells; *represents P < 0.05 compared to only TGF-b1-treated cells.

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Rosiglitazone Reduced Pterygium Fibroblasts

Proliferation and Cell Migration, But Did Not

Induce Cell Death

Results of fluorescence-activated cell sorting (FACS) analysis ofcells treated with rosiglitazone compared with vehicle-treatedcontrols and cells treated with only TGF-b1 showed approx-imately the same percentage of distribution of live cells (80%),cells at early apoptosis (6%), cells at late apoptosis (11%), andnecrotic cells (3%), indicating that rosiglitazone treatment didnot induce apoptosis. However, CCK-8 assay results showedthat rosiglitazone treatment significantly caused a reduction incell number dose dependently (Fig. 7) indicative of aninhibitory effect of rosiglitazone on pterygium fibroblastproliferation. Rosiglitazone also suppressed the TGF-b1-in-duced cell migration in pterygium fibroblasts (Fig. 8).

Rosiglitazone Reduced the TGF-b1-induced

Increase in MMP-9 in Pterygium Fibroblasts

We investigated the effect of rosiglitazone on the expression ofMMP-1, -3, and -9, which are known to be highly expressed inpterygium fibroblasts.16 Results showed that TGF-b1 increasedthe expression of MMP-3 and -9, but not MMP-1. In addition,rosiglitazone significantly suppressed the TGF-b1-induced

expression of MMP-9. However, it had no effect on the expression of MMP-1 and -3 (Supplementary Fig. S1).

DISCUSSION

Recent studies identifying PPAR-c as an important cellular antifibrotic mechanism and its importance in the negative regulation of connective tissue biosynthesis during both physiologic and pathological matrix remodeling,25,26 and the subsequent consistent findings in different tissue types with different PPAR-c ligands27–31 have increased research focus in this area in the quest to finding potential therapeutic agents for the myriad chronic debilitating fibrotic disorders. In this regard, PPAR-c ligands have also been reported to be effective corneal antifibrotics in vitro and in vivo in a PPAR-c-independent manner.21,22 However, their effects on other ocular fibrotic disorders, such as pterygium, which is known to overexpress TGF-b1, is not known. Our results showed that the PPAR-c ligand rosiglitazone is an effective antifibrotic agent in pterygium fibroblasts as well in vitro.

Notably, our results also showed that rosiglitazone can reduce the basal levels of a-SMA and fibronectin, thereby making it a very suitable candidate for therapy. This assertion is further consolidated by the additive downregulation of the intrinsic expression of TGF-b1 in pterygium fibroblasts, which

FIGURE 3. Immunofluorescence and indirect flow cytometry analysis of the antifibrotic effect of rosiglitazone in pterygium fibroblasts. (A) Confocalimages of immunostains with a-SMA (green) and fibronectin (red) antibodies of vehicle-treated control cells, cells treated with only TGF-b1, andcells treated with TGF-b1 and 75 lM rosiglitazone (3100 magnification). Nuclei were counterstained with DAPI (blue). (B) Proportions of a-SMA-positive cells in pterygium fibroblasts treated with TGF-b1 (2.5 ng/mL), rosiglitazone (75 lM), and GW9662 (1 lM) as determined by indirect flowcytometry. The shadow graph indicates isotype IgG, and the white graph indicates a-SMA-positive cells.

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FIGURE 4. Effect of rosiglitazone on intrinsic TGF-b1 synthesis in pterygium fibroblast. (A) Representative western blot images of a-SMA andfibronectin protein expression. Quantitative analysis of immunoblots with summary data from three independent experiments from (B) a-SMA (P¼0.0004, F[4, 10] ¼ 14.05) and (C) fibronectin (P ¼ 0.0007, F[4, 10] ¼ 12.18). (D) Representative western blot images of TGF-b1 expression. (E)Quantitative analysis of immunoblots with summary data from three independent experiments (P¼ 0.0068, F[4, 10]¼6.714). *Represents P < 0.05compared to control cells.

FIGURE 5. Effects of rosiglitazone on signaling pathways following exposure to TGF-b1 for 2 hours. (A) Representative western blot images.Quantitative analysis of immunoblots for (B) P-P38/T-P38 (P¼ 0.0008, F[5, 12]¼ 9.378), (C) P-ERK/T-ERK (P¼ 0.1599, F[5, 12]¼ 1.946), and (D) P-Smad/T-Smad (P¼0.0638, F[5, 12]¼2.848), with summary data from three independent experiments. *Represents P < 0.05 compared to only TGF-b1-treated cells.

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FIGURE 6. Role of p38 signaling in the TGF-b1-mediated fibrosis and the effect of rosiglitazone. (A) Representative western blot images of the timecourse of p38 protein activation, (B) effect of rosiglitazone on p38 signaling following exposure to TGF-b1 for 24 hours, and (C) protein expressionof a-SMA and fibronectin after blocking p38 signaling with SB203580. Quantitative analysis of the immunoblots for (D) a-SMA (P < 0.0001, F[5, 12]¼ 36.09) and (E) fibronectin (P < 0.0001, F[5, 12]¼ 97.39) with representative data from three independent experiments. #Represents P < 0.05compared to vehicle-treated control cells; *represents P < 0.05 compared to only TGF-b1-treated cells.

FIGURE 7. The effect of rosiglitazone on cell viability. FACS results of (A) vehicle-treated control cells, (B) cells treated with only TGF-b1, (C) cellstreated with TGF-b1 and 25 lM rosiglitazone, (D) cells treated with TGF-b1 and 75 lM rosiglitazone, and (E) their quantitative representation. (F)Quantitative representation of CCK-8 assay results after treatment with different concentrations of rosiglitazone compared with vehicle-treatedcontrol cells (P < 0.0001, F[4, 10] ¼ 56.74). *Represents P < 0.05 compared to vehicle-treated control.

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suggests that rosiglitazone modulates TGF-b1 induced fibro-genesis. Given that pterygium fibroblasts exhibit aberrant ECMremodeling and are associated with MMPs,16 the effect ofrosiglitazone on the production of MMPs provides anotherpromising perspective. Investigation of the expression of MMP-1, -3, and -9 in pterygium fibroblasts showed that rosiglitazonesignificantly suppressed the TGF-b1-induced expression ofMMP-9. However, it had no effect on MMP-1 and -3. BecauseMMP production is mainly associated with NF-kB pathway,further studies to understand the underlying mechanism arewarranted. The noncytotoxic and antiproliferative effects ofrosiglitazone treatment as shown by the cell viability assays arealso an added advantage compared to the currently usedtherapeutic applications. Thus, rosiglitazone’s effect appears tobe mediated by limiting the proliferation of pterygiumfibroblasts and their transformation into myofibroblasts.

Our evaluation of the possible molecular mechanismsunderlying the observed antifibrotic effects of rosiglitazoneindicated that rosiglitazone inhibits TGF-b1-induced fibrosis inpterygium fibroblasts at least in part by modulating thenoncanonical p38 MAPK cellular signaling pathway. Asnoticed, TGF-b1-induced phosphorylated Smad protein levelremained unaltered with rosiglitazone treatment, while phos-phorylated P38 protein levels were downregulated with aconsequent significant reduction in ECM synthesis andmyofibroblast differentiation in a dose-dependent manner,albeit not to the baseline levels. Blocking of the p38 signalingin pterygium fibroblasts led a dose-dependent decrease inmyofibroblast differentiation and ECM synthesis similar to theeffect of rosiglitazone treatment. The MAPK signaling path-ways, which include ERK1/2, Jun kinase (JNK/SAPK), and p38pathways, are known to play a key role in the transduction ofextracellular signals to elicit cellular responses including

proliferation, differentiation, development, inflammation, andapoptosis. These pathways relay, amplify, and integrate signalsfrom different stimuli, such as growth factors by phosphory-lating proteins, which then translocate to the nucleus toactivate transcription factors to regulate the expression oftarget genes.32 Indeed, TGF-b1 stimulated p38 MAPK activationhas been reported to be involved in fibrosis and scar tissueformation,33–35 and research findings in human cornea andtenon fibroblasts indicate that rosiglitazone inhibits TGF-b1-induced activation of these fibroblasts via the p38 MAPKsignaling pathway.21,36 The involvement of this pathway in theTGF-b1 stimulated fibrotic process in pterygium fibroblasts asour results also indicate is consistent with these reports.However, both rosiglitazone and p38 inhibition failed tocompletely block myofibroblast differentiation and ECMsynthesis. This indicated that the TGF-b1-induced fibrosis isonly partially dependent on p38 MAPK signaling. Previousstudies have shown that the canonical Smad2/3 signaling andseveral other noncanonical pathways are involved in TGF-b1-induced fibrosis, thus indicating a complex regulatory linkagebetween TGF-b1 signaling and the development of fibrosis.

The observed antifibrotic effects of rosiglitazone were alsoseen to be independent of PPAR-c receptors as treatment withthe PPAR-c receptor antagonist GW9662 did not reverse theeffects. This is consistent with results obtained in corneafibroblasts treated with rosiglitazone and other PPAR-cligands.21,37 PPAR-c agonists modulate multiple cellular func-tions both in a PPAR-c-dependent and PPAR-c-independentmanner as indicated by several research findings with differentPPAR-c ligands from different tissues. The dependence orotherwise of rosiglitazone on PPAR-c signaling with regard toits antifibrotic effects seems to be tissue specific as similarresults demonstrated in autosomal dominant polycystic kidney

FIGURE 8. Effect of rosiglitazone on cell migration in pterygium fibroblasts. (A) Phase-contrast microscopic images of the scratch wound area ofcells treated with vehicle, TGF-b1 (2.5 ng/mL) only, and TGF-b1 (2.5 ng/mL) along with rosiglitazone (75 lM), taken at 12, 24, and 48 hours aftertreatment. (B) Quantitative analysis of the scratch wound area with representative data from three different sites of the vehicle-treated control cells(P¼ 0.0292, F[2, 6] ¼ 6.739), cells treated with only TGF-b1 (P < 0.0001, F[2, 6] ¼ 432.2), and cells treated with TGF-b1 and rosiglitazone (P ¼0.0101, F[2, 6] ¼ 10.89). *Represents P < 0.05 compared to the scratch wound area at 12 hours after treatment.

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disease (ADPKD) cyst-lining epithelial cells were observed tobe PPAR-c dependent.19 In human corneal fibroblasts, theantifibrotic effects of two other synthetic PPAR-c ligands,CDDO and 15d-PGJ2, were seen to be largely independent ofPPAR-c,38 also indicative of the different mechanisms by whichthese ligands function. Further research is necessary toevaluate the antifibrotic effect of other PPAR-c ligands toobtain the best alternative for therapeutic use. Although thePPAR-c-independent nongenomic effect of rosiglitazone hasbeen reported, the receptors possibly targeted and underlyingmechanisms are not fully understood. We speculate that Ca2þ

acts as a possible mediator for the cross-talk betweenrosiglitazone and TGF-b1 signaling. Previous studies haveshown that TGF-b1 evokes Ca2þ transients in human pulmo-nary fibroblasts, which when disrupted interfere with ECMproduction39; rosiglitazone is known to affect Ca2þ homeosta-sis.40 In corneal fibroblasts, activation of transient receptorpotential cation channel subfamily V member 1 (TRPV1) with asubsequent increase in intracellular Ca2þ is associated withmyofibroblast transformation,41 and it is also upregulated in thepterygium.42 Further research is necessary to fully understandthe mechanism underlying the antifibrotic effect of rosiglita-zone.

In summary, our study demonstrated that rosiglitazone iseffective in reducing ECM production and myofibroblastdifferentiation independent of PPAR-c. These findings maypresent the basis for its use as a promising candidate drug foradjuvant therapy after surgery to prevent recurrent pterygium.

Acknowledgments

Supported by Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by the Ministry ofEducation, Science and Technology (2017R1D1A3B03036549) and agrant from the Korea Health Technology R&D Project through theKorea Health Industry Development Institute (KHIDI), funded bythe Ministry of Health and Welfare, Republic of Korea (H I15C2237).

Disclosure: S.A. Nuwormegbe, None; J.H. Sohn, None; S.W.Kim, None

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