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Cardiovascular Research 2007 75(3):519-529; doi:10.1016/j.cardiores.2007.04.026
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Copyright © 2007, European Society of Cardiology

Activation of PPAR{delta} inhibits cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts

Birgit E.J. Teunissena, Pascal J.H. Smeetsa, Peter H.M. Willemsena, Leon J. De Windtb, Ger J. Van der Vussea and Marc Van Bilsena,*

aDepartment of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
bHubrecht Laboratory and Interuniversity Cardiology Institute of the Netherlands, Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands

* Corresponding author. Tel.: +31 43 3881204; fax: +31 43 3884166. marc.vanbilsen{at}fys.unimaas.nl

Received 21 November 2006; revised 20 April 2007; accepted 26 April 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective The development of heart failure is invariably associated with extensive fibrosis. Treatment with Peroxisome Proliferator-Activated Receptor (PPAR) ligands has been shown to attenuate cardiac fibrosis, but the molecular mechanism underlying this protective effect has remained largely unknown. In this study the potential of each PPAR isoform (PPAR{alpha}, {delta}, and {gamma}) to attenuate cardiac fibroblast proliferation, fibroblast (CF) to myofibroblast (CMF) transdifferentiation, and collagen synthesis was investigated.

Methods and results PPAR{delta} was found to be the most abundant isoform in both CF and CMF. Only the PPAR{delta} ligand GW501516, but not PPAR{alpha} ligand Wy-14,643 or PPAR{gamma} ligand rosiglitazone, significantly increased PPAR-dependent promoter activity and expression of the PPAR-responsive gene UCP2 (~5-fold). GW501516 reduced the proliferation rate of CF (–38%) and CMF (–26%), which was associated with increased expression of the cell cycle inhibitor gene G0/G1 switch gene 2 (G0S2). Exposure of CF to the PPAR{delta} ligand or adenoviral overexpression of PPAR{delta} significantly decreased {alpha}-smooth muscle actin ({alpha}-SMA) levels, indicating a reduced CF to CMF transition. The inhibition of transdifferentiation by PPAR{delta} correlated with an increase in PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome ten) expression. 3H-Proline incorporation assays demonstrated a GW501516 induced decline in collagen synthesis (–36%) in CF.

Conclusion Cardiac fibroblast proliferation, fibroblast to myofibroblast differentiation and collagen synthesis were reduced after activation of PPAR{delta}, suggesting that PPAR{delta} represents an attractive molecular target for attenuating cardiac fibrosis.

KEYWORDS Cardiac fibroblast; Cardiac myofibroblast; Peroxisome Proliferator-Activated Receptor (PPAR); PPAR{delta}; Proliferation; Transdifferentiation; Collagen


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The development of heart failure is invariably associated with extensive fibrosis, which aggravates diastolic dysfunction and predisposes to arrhythmias [1,2]. A critical event in the initiation of fibrosis is the proliferation and differentiation of cardiac fibroblasts. Upon activation by either cytokines, growth factors or stretch, fibroblasts start proliferating and ultimately differentiate towards myofibroblasts [3,4]. When fibroblasts acquire smooth muscle-like properties, including contractile stress fibers containing {alpha}-smooth muscle actin, they are referred to as myofibroblasts [4,5]. Although fibroblasts take care of normal collagen turnover [6], myofibroblasts are held responsible for the disproportionate accumulation of extracellular matrix components, often leading to impaired organ function [3]. Accordingly, the attenuation of cardiac fibrosis, either through inhibition of fibroblast proliferation or through inhibition of their differentiation into myofibroblasts, will be beneficial for the heart.

The Peroxisome Proliferator-Activated Receptors (PPAR{alpha}, -{delta} and -{gamma}) are ligand-activated transcription factors that were first discovered because of their role in lipid metabolism [7,8]. More recently, PPARs were shown to possess anti-inflammatory and anti-proliferative properties as well [9–13]. Intriguingly, PPAR{gamma} and PPAR{alpha} ligands have been shown to attenuate cardiac fibrosis in various in situ models of cardiac hypertrophy and failure [14–18]. Also in vitro, in angiotensin II-stimulated cardiac fibroblasts collagen synthesis was reduced by the PPAR{gamma} ligand pioglitazone [19]. Up to now, however, the role of PPAR{delta} in fibrosis has not been addressed. Furthermore, the molecular mechanism underlying the anti-fibrotic effect of PPAR activation has remained elusive.

PPAR{gamma} ligands inhibited proliferation of tumor cell lines [20,21], and PPAR{delta} has been associated with inhibition of keratinocyte proliferation [12]. More importantly, PPAR{gamma} agonists were effective in inhibiting human lung and dermal fibroblast to myofibroblast differentiation [22,23]. Together, these observations raise the possibility that anti-fibrotic effects of PPARs in the heart are mediated by mitigating both cardiac fibroblast proliferation and differentiation.

In the present study we aimed at defining the functional significance of each of the PPAR isoforms in cardiac fibroblast proliferation and differentiation, using an in vitro model of cardiac fibroblast to myofibroblast differentiation. Interestingly, the collective findings indicate that PPAR{delta} is biologically the most important isoform in cardiac (myo)fibroblasts. Activation of PPAR{delta} inhibited both cardiac fibroblast proliferation and the differentiation towards myofibroblasts, which coincided with increased levels of G0S2 (G0/ G1 switch gene 2) and PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome ten). Moreover, PPAR{delta} activation attenuated collagen synthesis.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
Rat neonatal cardiac fibroblasts (CF) were isolated from the ventricles of 1–2 day old Lewis rats. Ventricles were minced and digested in 0.05% collagenase (Gibco, Invitrogen, Carlsbad, CA, USA) and 0.05% pancreatin (Sigma, St. Louis, MO, USA) containing solution at 37 °C [24]. Adult rat CF (provided by W. Wijnen, Dept. of Molecular Genetics, Maastricht University) were collected from isolated Lewis rat hearts after perfusion with a collagenase containing buffer [25]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Neonatal and adult cells were pre-plated for 1 h on uncoated culture discs (Corning Inc., NY, USA), during which CF rapidly adhered to the dishes. After pre-plating the medium containing cardiomyocytes was removed and the attached CF were washed and further cultured in DMEM (Gibco) containing 10% fetal calf serum (FCS, Gibco) and gentamycin (0.005%, Gibco) at 37 °C and 5% CO2. These cells are referred to as passage 0 (p0) CF. Next day CF were subcultured (1:3 dilution) to passage 1 (p1). Subsequently, cells were split every 4 days and cultured up to passage 3 (p3) after which the vast majority of cells acquired a myofibroblast-like (CMF) phenotype.

P1, p2 and p3 cells were plated in a density of ~0.3.105 cells/cm2 on culture dishes and grown for 24 h, followed by serum starvation (DMEM plus 0.1% FCS) for another 24 h. Thereafter, cells were incubated with either 10 µM of the PPAR{alpha}-ligand Wy-14,643 (Biomol, Plymouth Meeting, PA, USA), the PPAR{delta}-ligand GW501516 (Calbiochem, San Diego, CA, USA), or the PPAR{gamma}-ligand rosiglitazone (Rosiglitazone-maleate, LKT Laboratories, St. Paul, MN, USA) for 24 h. The ligands were dissolved in DMSO (0.1% v/v, Sigma), which was also added as vehicle to control cells.

2.2 RNA isolation and real-time quantitative PCR
Total RNA was extracted with TRIzol Reagent (Sigma) according to the manufacturer's protocol. RNA concentration was determined at OD260/280 nm measurements. 500 ng total RNA was used for DNaseI treatment (Sigma) and subsequent cDNA synthesis (Iscript cDNA synthesis kit, Biorad Inc., Hercules, CA, USA). Gene expression analysis was performed by quantitative PCR (qPCR) on an iCycler Real-Time PCR detection system (Biorad) using the iQ SYBR-Green supermix (Biorad). For all primer sets used (Table 1), temperature and dilution curves were assessed to check for linearity. Results were normalized using the house-keeping genes cyclophilinA and HPRT. Relative changes in expression levels were calculated using GeneX (Biorad) and qBase analyzer (Hellemans et al.; http://medgen.ugent.be/qbase).


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  		Table 1 Sequences of oligo's used for quantitative PCR

 
2.3 Transient transfection
Neonatal CF were seeded on 6-well plates (Costar, Corning) and transfected ~16 h before serum starvation using FuGENE 6 (Roche, Indianapolis, IN, USA). Cells were transfected with 0.7 µg of promoter/reporter vector, i.e. 1.3 kb fragment of the intact or mutated Muscle-type Carnitine Palmitoyl Transferase-1 (MCPT-1) promoter cloned into the pGL3 luciferase vector [26]. In the mutated MCPT-1 promoter a point mutation (C->G) at position 879 of the human CPT1B sequence (AB003286) was introduced by Quickchange Site-Directed Mutagenesis (Stratagene, La Jolla, CA, USA), resulting in a non-functional PPAR-responsive element (PPRE). As a control for transfection efficiency 0.2 µg of the CMV-β-galactosidase pON249 was co-transfected [27]. After 4 h of serum starvation CF were incubated with the PPAR specific ligands for 24 h. Next, cell lysates were prepared and assayed for luciferase activity on the FluorS imager (BioRad) using the SteadyGo firefly luciferase assay (Promega, Madison, WI, USA). β-Galactosidase activity was determined spectrophotometrically (Titertek Multiskan Plus MKII, Thermo LabSystems, Helsinki, Finland) [27].

2.4 Recombinant adenoviral infection
Recombinant adenovirus encoding human PPAR{delta} (AdPPAR{delta}), human PPAR{alpha} (AdPPAR{alpha}) and the corresponding empty control viruses (respectively AdCMV-control and AdGFP-control) were kindly provided by Dr. B. Staels (Institut Pasteur de Lille, France). Viruses were multiplied in 293 cells (kindly provided by Dr. M. Bierhuizen, UMC Utrecht, the Netherlands) and purified using the BD Adeno-X virus purification kit (BD Biosciences Clontech, Franklin Lakes, NJ, USA). Adenoviral titers (infectious units [ifu] per ml) were determined using the AdEasy Viral Titer kit (Stratagene). Cells were infected with ~5000 ifu/per cell for 2–3 h at 37 °C and 5% CO2. After infection cells were serum starved for 4 h and subsequently incubated with PPAR{delta} ligand GW501516 (10 µM), PPAR{alpha} ligand Wy-14,643 (10 µM) or vehicle for 24 h.

2.5 Immunocytochemistry
CF cultured on LabTek II chamber slides (Nalge Nunc, Naperville, IL, USA) were fixed with 3% paraformaldehyde for 20 min, permeabilized with 0.5% Triton-X100 for 5 min and blocked with 2% BSA for 30 min. Cells were incubated overnight with an antibody against {alpha}-smooth muscle actin ({alpha}-SMA) (DakoCytomation, Glostrup, Denmark) dissolved in PBS with normal goat serum (NGS, Dako). Next, cells were washed with PBS and incubated with Texas Red (TR)-conjugated secondary anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). Finally, cells were washed again, mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA) containing the nuclear counterstain DAPI and examined using a light microscope equipped for epifluorescence.

2.6 Protein extraction and Western blotting
Protein extracts were prepared using radioimmunoprecipitation assay (RIPA) buffer [28], supplemented with protease inhibitor cocktail (Sigma). Proteins were separated by 10% SDS-polyacrylamide gels, blotted to a nitrocellulose membrane, blocked in 5% milk and incubated overnight at 4 °C with an {alpha}-SMA antibody (DakoCytomation). Subsequently, blots were incubated with horseradish peroxidase (HRP) conjugated antibody for 1 h at 4 °C. To correct for possible differences in loading and transfer, the membranes were washed and subsequently incubated with a GAPDH antibody (RDI, Flanders, NJ, USA), followed by incubation with HRP conjugated antibody. Signals were visualized using Enhanced Chemo Luminescence reagent (ECL, Amersham Biosciences Europe, Freiburg, Germany) and exposure to hyperfilm (Amersham). Signals were quantified using the FluorS Imager (Biorad).

2.7 BrdU cell proliferation assay
Cells were plated on 96 well flat clear bottom black TC-treated microplates (Corning) at a density of 4500 (p1 cells) or 7000 (p3 cells) cells per well and cultured for 24 h. After 4 h of serum starvation, cells were incubated for 40 h with BrdU labeling solution (100 µM stock) diluted 10 times in DMEM (0.1% FCS) containing either PPAR ligands or vehicle. Incorporated BrdU was detected by an anti-BrdU ELISA (Roche, Mannheim, Germany).

2.8 3H-Proline incorporation assay
Serum-starved (0.1% FCS; 24 h) CF and CMF were incubated with 1 µCi/ml 3H-Proline (Amersham) along with the PPAR ligands. After 24 h cells were washed with ice cold PBS and proteins were precipitated with 15% trichloroacetic acid (TCA) for 30–60 min at 4 °C. Protein pellets were washed three times with ice cold ethanol and air-dried for 30 min. Next, proteins were solubilized in lysis buffer (40 mM Tris, 2% SDS, 1 mM EDTA, pH 7.4) for liquid scintillation counting or determination of protein content (BCA kit, Pierce, Rockford, IL, USA). 3H-Proline incorporation, measured as radioactive counts per minute (CPM), was normalized to total protein content.

2.9 Statistics
Data are presented as mean±SEM (standard error of the mean) and reflect duplicate or triplicate measurements from at least two separate cell isolations. Comparison between groups was performed by one-way analysis of variance (ANOVA), using either Bonferonni or Dunnet's post hoc testing (SPSS 12) where appropriate. Differences were considered significant at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Cardiac fibroblast to myofibroblast differentiation
As reported earlier [29], neonatal rat cardiac fibroblasts cultured on rigid substrate show progressive fibroblast to myofibroblast differentiation. Indeed, from passage 1 (p1) to passage 3 (p3) expression of the myofibroblast marker {alpha}-SMA markedly increased at the mRNA level (Fig. 1A) and, to a lesser extent, at the protein level (Fig. 1B). Moreover, relative to p1, in p3 cellular proliferation rate was reduced (data not shown) and a marked increase in {alpha}-SMA stress fibers and cell size was observed (Fig. 1C). Adult cardiac fibroblasts behaved similarly when propagated on rigid culture discs (data not shown). As in p3 the far majority of cells (>95%) had acquired a myofibroblast-like phenotype, while in p1 most cells still had a fibroblast-like appearance, we categorize p1-cells as cardiac fibroblasts (CF) and p3-cells as cardiac myofibroblasts (CMF) throughout the remainder of this paper.


Figure 1
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  		Fig. 1 {alpha}-SMA expression in neonatal rat cardiac fibroblasts propagated on rigid culture discs. {alpha}-SMA content increased from p1 to p3 both at the RNA (A) and protein level (B). The level of expression in p1 cells was arbitrarily set 1.0; *indicates p<0.05 versus corresponding p1 cells. Immunocytochemically (C) a clear increase in {alpha}-SMA positive stress fibers accompanied by an increase in cell size was observed in p3 versus p1 cells.

 
3.2 PPAR isoforms in CF and CM
QPCR demonstrated the presence of the three PPAR isoforms in p1-CF and p3-CMF of both neonatal and adult rats, albeit at different levels (Fig. 2A and B, respectively). In neonatal CF the relative abundance for PPAR{delta}, PPAR{alpha} and PPAR{gamma} mRNA amounts to 1.00: 0.29: 0.08, which is very similar to adult CF (1.00: 0.14: 0.13). From p1 to p3 the mRNA level of PPAR{alpha} significantly decreased in neonatal, but not in adult fibroblasts. Changes in PPAR{delta} and PPAR{gamma} mRNA were not observed during passaging. Unless indicated otherwise, further experiments were conducted with neonatal CF and CMF.


Figure 2
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  		Fig. 2 Expression pattern and activity of the PPAR isoforms in rat cardiac fibroblasts. PPAR mRNA levels in neonatal (A) and adult (B) CF (p1) and CMF (p3). PPAR{delta} mRNA level in p1 cells was set 1.0. Other mRNA levels were expressed relative to this value. * indicates p<0.05 versus corresponding p1 cells. (C) On top a schematic presentation of the intact and mutated MCPT-1 constructs, in which the MCPT-1 promoter directs luciferase (LUC). TIS indicates the transcription initiation site. After transfection neonatal CF (p1) were exposed to either the PPAR{alpha} ligand Wy-14,643 (Wy, 10 µmol/L), the PPAR{delta} ligand GW501516 (GW, 10 µmol/L) or the PPAR{gamma} ligand rosiglitazone (Rosi, 10 µmol/L) for 24 h. Luciferase activity of the intact MCPT-1 construct after incubation with vehicle (veh) was arbitrarily set at 1.0 and all other luciferase activities were expressed relative to this value. (D) mRNA levels of Uncoupling Protein 2 (UCP2) in neonatal CF (p1) and CMF (p3) after 24 h treatment with PPAR ligands. The UCP2 level in vehicle-treated p1-CF was arbitrarily set at 1.0. *Indicates p<0.05 versus vehicle-treated cells.

 
To determine whether the PPAR isoforms are functionally important, CF were transfected with the intact or mutated (mutant PPRE) MCPT-1 promoter (Fig. 2C, upper panel) and subsequently chased with PPAR ligands. The PPAR{delta} ligand GW501516 increased MCPT-1 promoter activity more than 5-fold, an effect requiring an intact PPRE (Fig. 2C, lower panel). In contrast, Wy-14,643 (PPAR{alpha} ligand) and rosiglitazone (PPAR{gamma} ligand) increased MCPT-1 promoter activity only modestly and non-significantly. Furthermore, the mRNA level of the established PPAR target gene UCP2 (Uncoupling Protein 2) increased significantly in both CF (~5-fold) and CMF (~3-fold) after exposure to the PPAR{delta} ligand GW501516, but not after exposure to rosiglitazone or Wy-14,643 (Fig. 2D). The collective findings indicate that functionally PPAR{delta} is the most important isoform in CF and CMF.

3.3 Fibroblast proliferation
Given their reported anti-proliferative properties [11–13], the effect of PPAR ligands on CF and CMF proliferation was explored. In CF BrdU incorporation was significantly reduced by ~38% following GW501516 treatment (Fig. 3A). CF proliferation was not affected by Wy-14,643 or rosiglitazone. Also in CMF proliferation rate (~26%) was decreased after GW501516 treatment (Fig. 3B). Exposure of CMF to rosiglitazone resulted in a small, but significant decrease in cell proliferation as well (~11%), whereas Wy-14,643 was ineffective. None of the PPAR ligands induced apoptosis in either CF or CMF as determined by AnnexinA5-FITC staining (data not shown).


Figure 3
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  		Fig. 3 Effect of isoform-specific PPAR ligands on CF and CMF proliferation and the cell cycle inhibitor genes G0S2 and p16INK4a. (A) Neonatal CF (p1) and (B) CMF (p3) were exposed to either Wy-14,643 (Wy, 10 µmol/L), GW501516 (GW, 10 µmol/L) or rosiglitazone (Rosi, 10 µmol/L) or vehicle (veh) along with BrdU for 40 h. BrdU incorporation of vehicle-treated cells was arbitrarily set at 100%. *Denotes p<0.05 versus vehicle-treated cells. (C) mRNA levels of G0S2 and (D) p16INK4a were determined in CF (p1) and CMF (p3) 24 h after treatment with 10 µmol/L of isoform-specific PPAR ligands. G0S2 and p16INK4a levels in vehicle-treated p1-CF were arbitrarily set at 1.0. *Denotes p<0.05 versus vehicle-treated cells; #denotes p<0.05 between p3 and p1 cells.

 
To explore potential mechanisms by which PPARs influence cardiac fibroblast proliferation, mRNA levels of the cell cycle inhibitory genes G0S2 (G0/G1 switch gene 2) and p16INK4a were determined. Both G0S2 and p16INK4a are associated with growth arrest and behave like PPAR target genes [11,30]. In CF the mRNA level of G0S2 significantly increased (~2.6 fold) after GW501516 treatment (Fig. 3C). Treating CF with the other PPAR ligands did not affect G0S2 expression. In CMF none of the PPAR ligands increased G0S2 mRNA content to a significant extent. While p16INK4a mRNA levels were higher (~7 fold) in CMF compared to CF (Fig. 3D), this gene did not respond to PPAR ligands.

3.4 Myofibroblast differentiation
Treatment of CF with the PPAR{delta} ligand GW501516 led to a significant decline in {alpha}-SMA mRNA (–60%) and protein (–25%) expression, indicative of a reduction in CF to CMF differentiation (Fig. 4A and B). Also immunocytochemically a clear decrease in {alpha}-SMA-containing stress fibers was observed after GW501516 treatment (Fig. 4C). In contrast, differentiation was not attenuated by Wy-14,643 or rosiglitazone (Fig. 4A, B and C).


Figure 4
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  		Fig. 4 PPAR{delta} activation and overexpression reduces {alpha}-SMA expression. CF (p1) were exposed to 10 µmol/L of either Wy-14,643, GW501516 or rosiglitazone for 24 h and assayed for {alpha}-SMA mRNA (A) and protein (B) expression. The {alpha}-SMA level in vehicle-treated CF was arbitrarily set at 1.0. *Indicates p<0.05 versus vehicle-treated cells. (C) immunocytochemically a clear decrease in {alpha}-SMA-containing stress fibers was observed 24 h after exposure to GW501516, but not Wy-14,643 or rosiglitazone. Bar=100 µm. (D) CF (p1) were infected with adenovirus encoding for PPAR{delta} (AdPPAR{delta}), PPAR{alpha} (AdPPAR{alpha}), or control adenovirus (Ad-control). 24 h after infection cells were incubated with 10 µmol/L of PPAR ligands or vehicle for another 24 h. The {alpha}-SMA level of Ad-control infected, vehicle-treated CF, was arbitrarily set at 1.0. *Denotes p<0.05 versus cells infected with control virus and treated with vehicle.

 
To further investigate the modulatory role of PPAR{delta} on myofibroblast differentiation, CF were infected with adenovirus encoding either PPAR{delta} (AdPPAR{delta}) or PPAR{alpha} (AdPPAR{alpha}), or with empty control adenovirus (Ad-control). After 48 h, {alpha}-SMA mRNA was significantly lower (46% of control) after infection with AdPPAR{delta} (Fig. 4D). A similar reduction of the {alpha}-SMA mRNA level was observed after treatment with Ad-control along with GW501516. Combining AdPPAR{delta} overexpression with GW501516 treatment further reduced {alpha}-SMA expression to 32% of control. Notably, infection with AdPPAR{alpha} in the absence or presence of Wy-14,643 did not affect {alpha}-SMA mRNA levels.

In contrast to CF, in CMF neither {alpha}-SMA mRNA and protein levels (Fig. 5A and B), nor the amount of {alpha}-SMA-containing stress fibers (Fig. 5C), were reduced after treatment with GW501516. The same holds when CMF were treated with PPAR{alpha} or PPAR{gamma} ligands. However, when CMF were infected with AdPPAR{delta}, in the absence or presence of GW501516, {alpha}-SMA mRNA expression was reduced to 34% and 26% relative to control virus, respectively (Fig. 5D). Overexpression of PPAR{alpha} did not alter {alpha}-SMA mRNA levels in CMF.


Figure 5
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  		Fig. 5 Effect of PPAR{delta} activation and overexpression on {alpha}-SMA. (A) {alpha}-SMA mRNA and (B) protein expression was determined in CMF (p3) incubated either with 10 µmol/L of Wy-14,643, GW501516 or rosiglitazone for 24 h. The {alpha}-SMA level in vehicle-treated CMF was arbitrarily set at 1.0. Significant differences in {alpha}-SMA levels were not observed. (C) Similarly, immunocytochemical differences in {alpha}-SMA staining 24 h after treatment with the PPAR isoform-specific ligands were not observed. Bar=100 µm. (D) CMF were infected with adenovirus encoding for PPAR{delta} (AdPPAR{delta}), PPAR{alpha} (AdPPAR{alpha}), or control adenovirus (Ad-control). 24 h after infection, CMF were incubated with either GW501516, Wy-14,643 or vehicle for another 24 h. The {alpha}-SMA level of Ad-control infected, vehicle-treated CMF was arbitrarily set at 1.0. *Denotes p<0.05 versus cells infected with control virus and exposed to vehicle.

 
3.5 PTEN expression
PTEN is known to attenuate lung fibroblast to myofibroblast differentiation [31] and to have a functional PPRE in its promoter [32]. Indeed, treatment of CF with the PPAR{delta} ligand GW501516 significantly (p=0.01) increased PTEN expression by approximately 3.5 fold (Fig. 6). Wy-14,643 and rosiglitazone also tended to increase PTEN mRNA expression in CF, however, these differences did not reach the level of significance (p=0.06 and p=0.15, respectively). PTEN mRNA levels in CMF were not affected by exposure to PPAR ligands.


Figure 6
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  		Fig. 6 Effect of isoform-specific PPAR ligands on PTEN expression. mRNA levels of PTEN were determined 24 h after exposure of neonatal CF (p1) or CMF (p3) to 10 µmol/L of Wy-14,643, GW501516 or rosiglitazone. PTEN level in vehicle-treated p1-CF was arbitrarily set at 1.0. *Denotes p<0.05 versus vehicle-treated cells.

 
3.6 Collagen synthesis
To investigate the effect of PPAR ligands on collagen synthesis 3H-Proline incorporation was assessed. PPAR{delta} activation led to a significant decline (–36%) in 3H-Proline incorporation in CF, reflecting a diminished collagen synthesis (Fig. 7A). Collagen synthesis by CMF was not affected by GW501516 (Fig. 7B). Strikingly, rosiglitazone increased 3H-Proline incorporation in both CF (+43%) and CMF (+67%). Collagen synthesis was insensitive to Wy-14,643.


Figure 7
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  		Fig. 7 Effect of isoform-specific PPAR ligands on 3H-Proline incorporation. (A) Neonatal CF (p1) and (B) CMF (p3) were exposed to either Wy-14,643 (Wy, 10 µmol/L), GW501516 (GW, 10 µmol/L) or rosiglitazone (Rosi, 10 µmol/L) or vehicle (veh) along with 3H-Proline (1 µCi/ml). Incorporated 3H-Proline was corrected for total protein content. 3H-Proline content of vehicle-treated cells was arbitrarily set at 1.0. *Denotes p<0.05 versus vehicle-treated cells.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present, PPAR{delta} was found to be the most abundant and biologically most active isoform in both CF and CMF. Fibroblast proliferation, transdifferentiation, and collagen synthesis were all reduced after activation of PPAR{delta}, thereby identifying this particular PPAR isoform as a potential therapeutic target for intervening with cardiac fibrosis.

4.1 Stretch-mediated cardiac fibroblast transdifferentiation
To assess the role of the PPAR isoforms in myocardial fibrosis, primary cultures of rat cardiac fibroblasts (CF) propagated on rigid culture discs were used as an in vitro model of stretch-mediated fibroblast to myofibroblast differentiation [29]. It is shown that during passaging from p1 to p3 the cells acquire a CMF-like phenotype. It should be noted, however, that although the majority of p1-cells have a fibroblast-like appearance, it is possible that rapidly after plating these cells enter a so-called "transition phenotype", characterized by expression of {alpha}-SMA protein, that is not organized in stress fibers as yet [33,34]. This may explain the observed discrepancy between the relatively mild increase in {alpha}-SMA protein content and marked increase in {alpha}-SMA-containing stress fibers from p1 to p3.

4.2 PPAR{delta} and cardiac fibroblast proliferation
This study reveals that activation of PPAR{delta} diminishes the proliferation of both CF and CMF substantially. In contrast, activation of the other PPAR isoforms only marginally affected cell proliferation. Consistent with this, PPAR{delta} was reported to inhibit the proliferation of keratinocytes and osteoblastic cells [12,13].

To explore the molecular mechanism mediating the anti-proliferative effect of the PPAR{delta} ligand GW501516, the expression of the cell cycle inhibitory genes G0S2 and p16INK4a in CF and CMF was determined. Both genes contain a functional PPRE in their promoter region [11,30]. G0S2 was up-regulated after activation of PPAR{delta}, strongly suggesting that this gene is involved in the PPAR{delta}–mediated mitigation of cardiac fibroblast proliferation. In contrast, p16INK4a expression was not affected by any of the PPAR ligands. Gizard et al. have shown that the inhibition of smooth muscle cell (SMC) proliferation by PPAR{alpha} requires p16INK4a and that p16INK4a transcription is directly regulated by PPAR{alpha} [30]. Apparently, in the cardiac fibroblast context PPAR{alpha} modulates neither p16INK4a expression nor cell proliferation. Concerning the role of PPAR{delta} in SMC, it was reported that the PPAR{delta} ligand GW501516 had no effect on porcine coronary artery SMC proliferation [35]. However, proliferation in rat aortic vascular SMCs stably overexpressing PPAR{delta} was enhanced [36], suggesting that the effects of PPAR{delta} activation on cell proliferation are cell type specific.

4.3 PPAR{delta} and cardiac myofibroblast transdifferentiation
Activation of endogenous PPAR{delta} by its cognate ligand GW501516 inhibits transdifferentiation of CF to CMF, as deduced from the marked attenuation of {alpha}-SMA expression and morphological appearance. In contrast, PPAR{alpha} or PPAR{gamma}-specific ligands did not influence fibroblast transdifferentiation. Increasing PPAR{delta} levels by adenoviral infection only exerted a modest additional decline, indicating that in cardiac fibroblasts endogenous PPAR{delta} levels are sufficient to evoke a substantial response after administration of its cognate ligand. In contrast, in CMF, overexpression of PPAR{delta} was necessary to inhibit the differentiation process.

Interestingly, raising intracellular PPAR{alpha} levels by adenoviral infection, even in the presence of the PPAR{alpha} ligand Wy-14,643, did not attenuate transdifferentiation (see Figs. 4C and 5C), although it markedly enhanced the expression of the PPAR-responsive gene UCP2 (data not shown). These findings indicate that the ability of PPAR{delta} to inhibit cardiac myofibroblast transdifferentiation is not merely a reflection of its abundance, but represents a feature unique for this isoform.

It was recently shown that inhibition of PTEN allowed for increased transdifferentiation of pulmonary fibroblasts [31], implicating an important role of this phosphatase in the transdifferentiation process. In line with this, our results showed a simultaneous upregulation of PTEN and downregulation of {alpha}-SMA after treating cardiac CF with the PPAR{delta} ligand GW501516. These data are consistent with the notion that the PTEN promoter contains a functional PPRE [32]. Earlier studies, using other cell types, demonstrated increased PTEN expression upon PPAR{gamma} activation [32,37,38]. The present findings add to that by showing that in cardiac fibroblasts PTEN is induced by PPAR{delta} as well.

PTEN inhibits stretch-mediated integrin signaling by antagonizing FAK (focal adhesion kinase) or PI3K signal transduction [31,39]. Both kinases can activate the transcription factor SRF (serum response factor), which drives {alpha}-SMA expression [40]. The spontaneous differentiation of CF to CMF propagated on rigid culture discs is due to stretch activation [4,41]. Accordingly, it is tempting to speculate that PPAR{delta} ligands inhibit stretch-induced CF to CMF differentiation through upregulation of PTEN. As such, PPAR{delta} might be a target for attenuating stretch-mediated transdifferentiation of CF during pressure or volume overload. Further studies are warranted to clarify the underlying molecular mechanisms and the role of PPAR{delta} in cardiac fibrosis in vivo.

The attenuation of myofibroblast transdifferentiation by PPAR{delta} activation coincides with a diminished collagen production, underpinning the notion that myofibroblast transdifferentiation is accompanied by increased collagen production [3]. Remarkably, the PPAR{gamma} ligand rosiglitazone was found to increase collagen synthesis under these conditions. Since rosiglitazone increased collagen production without significantly increasing PPAR target genes or myofibroblast transdifferentiation, we presume this effect to be PPAR{gamma} independent.

Previous studies have shown that both PPAR{gamma} and PPAR{alpha} ligands attenuate myocardial fibrosis following hypertension or myocardial infarction in vivo [14–18], which was associated with inhibition of the production of cytokines. The latter observations suggest that the anti-fibrotic effects of the PPAR{gamma} and PPAR{alpha} ligands in vivo may be indirect and related to their anti-inflammatory properties [9,10]. The role of PPAR{delta} on in vivo cardiac fibrotic remodeling has not been addressed thus far. However, from the present in vitro observations it can be hypothesized that PPAR{delta} might have an even more potent and direct effect in attenuating cardiac fibrosis.

In conclusion, although all three PPAR isoforms are simultaneously expressed in CF and CMF, PPAR{delta} appeared to be the most relevant isoform in terms of biological function. We found that cardiac (myo)fibroblast proliferation was reduced after activation of PPAR{delta}, an effect that was associated with upregulation of the PPAR-responsive cell cycle inhibitory G0S2 gene. In addition, PPAR{delta}, but not PPAR{alpha} or PPAR{gamma}, diminished CF to CMF transdifferentiation, possibly via increased levels of PTEN. Furthermore, collagen synthesis was inhibited only after PPAR{delta} activation. Since PPAR{delta} is able to inhibit multiple aspects of the fibrotic process, it may provide an interesting new therapeutic target for cardiac fibrotic diseases such as congestive heart failure.


   Acknowledgements
 
This work was supported by the Netherlands Organization for Scientific Research NWO (912-04-017), the EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart and Medtronic Inc. (Minneapolis, Minnesota, USA).


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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