elevated expression of pdgf-c in coxsackievirus b3-induced

Preclinical research

Elevated expression of PDGF-C in coxsackievirusB3-induced chronic myocarditis

Katja Grun1, Boyka Markova2, Frank-D. Bohmer2, Alexander Berndt3,Hartwig Kosmehl4, and Carola Leipner1*1 Institute of Virology and Antiviral Therapy, Klinikum, Friedrich Schiller University Jena, Hans-Knoll-Strasse 2,D-07745 Jena, Germany2 Institute of Molecular Cell Biology, Klinikum, Friedrich Schiller University Jena, Drackendorfer Strasse 1,D-07747 Jena, Germany3 Institute of Pathology, Klinikum, Friedrich Schiller University Jena, Ziegelmuhlenweg 1, D-07743 Jena, Germany4HELIOS Klinikum Erfurt GmbH, Institute of Pathology, Nordhauser Strasse 74, D-099089 Erfurt, Germany

Received 5 February 2004; revised 14 December 2004; accepted 13 January 2005; online publish-ahead-of-print 9 March 2005

See page 642 for the editorial comment on this article (doi:10.1093/eurheartj/ehi201)

Aims Coxsackievirus B3 (CVB3) is a frequent cause of human chronic myocarditis andsubsequent fibrosis, leading to dilated cardiomyopathy. The molecular processesunderlying the development of fibrosis are poorly understood. Enhanced levels ofplatelet-derived growth factors (PDGFs), especially PDGF-C, have recently beenlinked with the development of different forms of fibrosis. Therefore, the expressionof PDGF was analysed in hearts of CVB3-infected major histocompatability complexclass II knockout mice. The latter were recently established as mouse model mimick-ing the chronic inflammation and fibrosis characteristic for this disease.Methods and results Expression of PDGF was analysed by reverse transcription–polymerase chain reaction, in situ hybridization, and immunohistochemistry. Heartsof C57BL/6 mice served as controls because infection of these animals leads toacute cardiac inflammation, but the hearts heal without signs of chronic inflam-mation. In uninfected hearts, basal expression of PDGF, notably PDGF-C, was detect-able throughout the heart. The chronic inflammatory process was associated withelevated and sustained expression of all tested PDGF isoforms. Immunostaining andin situ hybridization analysis localized enhanced PDGF levels to areas with highestvirus load and inflammatory infiltrations, adjacent to fibrotic areas.Conclusion PDGF may participate in fibrosis development in CVB3-induced myocar-ditis. Therefore, PDGF signalling may be considered a target for therapeuticinterference.

KEYWORDSChronic myocarditis;

Coxsackievirus B3 (CVB3);

Platelet derived growth

factor (PDGF) isoforms;

PDGF-C;

Major histocompatibility

complex (MHC) class II

knockout mouse

Introduction

Human pathogenic coxsackievirus B3 (CVB3) is consideredas the most frequent viral cause of myocarditis.1 Acutemyocarditis as a consequence of CVB3 infection is

followed by either complete healing or transition to aprogressive disease. This chronic stage is, according tothe Dallas criteria,2 characterized by chronic inflam-mation and sometimes by accumulation of connectivetissue in heart muscle, i.e. fibrosis. In these cases, fibro-sis may contribute to a loss of cardiac function, which isalso characteristic for dilated cardiomyopathy (DCM), afrequent consequence of chronic myocarditis in men.

& The European Society of Cardiology 2005. All rights reserved. For Permissions, please e-mail: [email protected]

*Corresponding author. Tel: þ49 3641 657358; fax: þ49 3641 657202.E-mail address: [email protected]

European Heart Journal (2005) 26, 728–739doi:10.1093/eurheartj/ehi168

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The molecular mechanism of fibrosis development inchronic myocarditis and DCM is currently not wellunderstood.In general, fibrosis is characterized by substantial

proliferation of resident fibroblasts with subsequentdeposition of extracellular matrix components like col-lagen, fibronectin,3,4 or tenascin.5 Both proliferation aswell as matrix protein synthesis are stimulated by a setof cytokines and growth factors, including transforminggrowth factor-b, tumour necrosis factor-a (TNF-a),interleukin-1 (IL-1), and platelet-derived growth factors(PDGF).3,6 Immune cells invading tissue upon inflam-mation are a substantial source of these growth factors.Isoforms of the PDGF family are potent mitogens

for fibroblasts and other mesenchymal cells. The PDGFfamily comprises several disulfide-linked dimeric growthfactors including PDGF-AA, -BB, -AB, and the recently dis-covered molecules PDGF-C (also designated Fallotein)and PDGF-D.7–10 Their activity is exerted by activationof PDGF receptor (R)-a or PDGFR-b, two structurallyrelated receptor tyrosine kinases. Investigations in diffe-rent tissues have suggested an important role for thePDGF system in fibrotic processes, including fibrosis oflung and kidney.6,11–15 So far, little is known about therole of PDGF in cardiac fibrosis. PDGF-C overexpressionin the myocardium of transgenic mice leads to substantialfibrosis7,16 suggesting that PDGF-C may be an importantplayer in this disease. Similar studies with PDGF-A and-B have not yet been reported.In the present study, the possible involvement of PDGF

in the development of cardiac fibrosis was investigated ina previously established mouse model of CVB3-inducedchronic myocarditis.17 Immunodeficient C57BL/6 majorhistocompatability complex (MHC) class II knockoutmice develop a chronic myocarditis upon CVB3 infection.In contrast, immunocompetent wild-type mice of identi-cal genetic background exhibit acute myocarditis, whichheals completely, accompanied by virus elimination.Elevated expression of PDGF isoforms, notably PDGF-C,correlated with the inflammatory process in both mouse

strains. Thus, we show here for the first time a connec-tion of PDGF-C up-regulation with the development offibrosis in virus-infected heart tissue, leading to chronicmyocarditis.

Methods

Virus

The CVB3 Nancy strain (CVB3-Mu/J) was kindly provided byProfessor Kandolf (Department of Molecular Pathology,University of Tubingen, Germany) and propagated four times inHeLa cells.

Mice

B6-Aa0/Aa0 MHC class II knockout mice (genetic backgroundH-2b) were kindly provided by Professor Bluthmann (RocheCenter for Medical Genomics, Basel, Switzerland). C57BL/6mice were purchased from the Laboratory Animal Centre,Friedrich Schiller University Jena, Jena, Germany. Experimentswere performed in accordance with the animal protection law(registration no. 02-30/99).

Five experiments were performed using identical protocols: Ineach experiment, we started with groups of five 8–13-week-oldmale C57BL/6 and B6-Aa0/Aa0 MHC class II knockout mice.18 Themice were inoculated intraperitoneally with 104 pfu of CVB3-Mu/J and sacrificed under ether narcosis at days 7, 21, and 56post-infectionem (p.i.), respectively. A clearly visible necrosisof the exocrine pancreas was taken as indication of successfulvirus infection because this organ is the first target of CVB3infection.19 Those animals (‘responders’) (usually two to fiveout of five) were included into the further studies. Non-respon-ders were excluded.

Table 1 describes the number of totally analysed animals pergroup, 9–12 mice were used as non-infected controls.

Histology

Samples of murine hearts were treated as reported previously.17

Sections were evaluated with the Axioskop 2 (Carl Zeiss, Jena,Germany). An AxioCamHR electronic camera (Carl Zeiss Vision

Table 1 Number of animals per experimental group

Total number ofinfected micea

Histologyb RT–PCRb In situhybridizationb

Immunohistochemistryb

C57BL/6 miceNaive control mice 12 6 6 4 37d p.i. 16 9 6 10 521d p.i. 16 10 9 9 356d p.i. 14 10 8 3 4

MHC class II knockout miceNaive control mice 9 9 7 3 37d p.i. 17 9 8 7 321d p.i. 18 11 5 6 756d p.i. 24 11 9 8 4

It has to be mentioned that not all four analyses from one heart could be performed because the fixation of tissue is diffe-rent for different analyses (see Methods). The choice which animal was used for a certain analysis was done at random at thetime of sacrificing.

aSum of all animals studied in five experiments.bNumber of animal samples analysed with the given method.

Elevated expression of PDGF-C in CVB3 729

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GmbH, Munchen-Halbergmoos, Germany) was used for imagecapturing and pictures were processed with the Axiovisionimage processing system (Carl Zeiss, Munchen-Halbergmoos,Germany). For each myocardial sample, histological indicatorsof myocarditis were graded semi-quantitatively according tothe extent of cellular infiltration/fibrosis on a four-point scaleranging from 0 (no infiltration, no fibrosis) to 3 (strong infiltra-tion, strong fibrosis).17

Virus titre

The CVB3 titre of the heart tissue was determined on HeLacells17 by the tissue culture infectious dose 50 (TCID50) assaymethod according to Reed and Muench20 (see Supplementarymaterial online).

Semi-quantitative PCR

Total RNA was isolated according to Chomczynski and Sacchi.21

Reverse transcription was performed with 10 mg total RNA witholigo (dT) and 500 U MMLV reverse transcriptase (Superscript II,Invitrogen) corresponding to the manufacturers protocol. HeartcDNA was adjusted to equal concentrations by competitive poly-merase chain reaction (PCR) between b-actin cDNA and a corre-sponding control fragment.22 A constant amount of experimentalcDNA (4 � 106 molecules) was used as template for any amplifi-cation. The 50 mL PCR reactions contained 0.2 mM of each dNTP(Promega), 20 nM of each primer, 1.25 U HotStar Taq polymer-ase, 5 mL 10� concentrated PCR buffer, and 10 mL solution Qand MgCl2 (all reagents from Qiagen). The final MgCl2 con-centration was 2.5 mM for amplification of b-actin, VP1,TNF-a, PDGF-A, -B, and -C cDNAs, and 1.5 mM for all othercDNAs. Amplification was performed for 30 cycles according tothe manufacturer’s protocol. Primer sequences are mentionedin the Supplementary material online. The primers always corres-pond to different exons. Thereby, amplification of genomic DNAis either precluded or would lead to formation of clearly largerproducts. Fifteen microlitres of the PCR products were analysedon 1.5% agarose gels.

In situ hybridization

Detection of viral RNA in the myocardium by in situ hybridizationusing a digoxigenin (DIG)-labelled DNA probe detecting the CVB3capsid protein gene VP1 was performed according to Fleminget al.23 (see Supplementary material online).

A probe for the detection of PDGF-C mRNA was generatedby PCR amplification with PfU polymerase (Stratagene) usingPDGF-C cDNA (‘Fallotein’ cDNA, kindly provided by Dr Yuan-JangTsai, Division of Reproduction and Endocrinolgy, Department ofMedical Research, Mackay Memorial Hospital, Tamshui, Taiwan)as template and the primers 50-TCC AGC GAC AAG GAA CAG AACG-30 (forward) and 50-GGA GAT TCA GAT TCA CCA CTT TGC-30

(reverse). The resulting 625 bp blunt-end product was clonedinto EcoRV-digested pBluescript vector II KS (þ). For synthesis ofantisense and sense RNA probes, the plasmid was linearized andin vitro transcription was performed with the DIG-RNA LabellingKit (Roche) according to the instructions of the manufacturerusing T3 and T7 polymerase, respectively. The linearized probeswere precipitated with 2.5 mM LiCl–ethanol, aliquoted, andstored at 2808C until use for in situ hybridization according toDijkman et al.24 For further details see Supplementary materialonline. Immunological detection of the bound probes was per-formed as described for VP1 (see Supplementary materialonline), except that the anti-DIG-antibody was used in a dilutionof 1:200.

Immunohistochemistry

Heart tissue was mounted in Cryomatrix (Thermo Shandon,Pittsburgh, PA, USA), snap-frozen, and stored at 2808C untiluse. Immunohistochemistry was performed as describedearlier.17 Incubation with primary antibodies was performedat 48C over night using goat anti-mPDGF-A (I-20; Santa CruzBiotechnology), 1:100; rabbit anti-mPDGF-B (H-55; Santa CruzBiotechnology), 1:100; rabbit anti-mPDGF-C10 (kindly provi-ded by Dr Y.J. Tsai; 1:300 in the absence or presence of blockingpeptide CDCVCRGNAGG-amide), rabbit anti-PDGF-Ra (951;Santa Cruz Biotechnology) 1:200; rabbit anti-phospho Akt(Ser473) (Cell Signaling Technology, Inc., Cummings CentreBeverly, USA), 1:100. Rabbit IgG (4 mg/mL) (SC-2027; SantaCruz Biotechnology) was used as negative control. Sectionswere counter-stained with Hemalaun.

Results

CVB3 infection leads to chronic inflammationand progressive cardiac fibrosis in MHC class IIknockout mice

Immunocompetent C57BL/6 mice or immunodeficientMHC class II knockout mice were infected with CVB3-Mu/J (Nancy strain) and hearts were analysed until day56 p.i. As described earlier,17 CVB3 induced a severeacute myocarditis characterized by marked cell infiltra-tion in the myocardium of both mouse strains.

Figure 1 Chronic inflammation and fibrosis development in immunodefi-cient MHC class II knockout mice. Histopathology of paraffin-embeddedheart sections from immunodeficient MHC class II knockout mice (A andB ) and immunocompetent C57BL/6 mice (C and D ) 56 days after infectionwith 1 � 104 plaque-forming units (pfu) of CVB3-Mu/J. Tissue sectionswere stained with Haematoxylin and Eosin (A and C ) or Sirius Red (Band D ). Inflammatory infiltrates (arrows in A ) accompanied by the deve-lopment of fibrosis (B ) are only visible in MHC class II knock out animals.Original magnification �200.

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The inflammatory infiltrates in hearts of acutely CVB3-infected C57BI/6 mice contain CD8þ, CD4þ, andCD11bþ cells. In the MHC class II knockout mice, CD8þand CD11bþ cells, but no CD4þ cells, can be detected.Here, we show that in MHC class II knockout mice, themyocarditis was accompanied by a necrosis of cardiomyo-cytes indicated by a loss of nuclear staining, a focalincreased eosinophilia of cardiomyocyte cytoplasm, andcardiomyocyte fragmentation resulting in a reduction offunctional heart tissue (Figure 1A ). The necrosis ofgrouped cardiomyocytes and the presence of infiltratingcells persisting until day 56 was accompanied by severefibrosis as shown by staining with Sirius Red(Figure 1B ). In contrast, the CVB3-induced myocarditis

healed completely in immunocompetent C57BL/6 micewithout any further interstitial inflammatory cells orsigns of fibrosis (Figure 1C and D ).

Elevated expression of PDGF isoforms correlateswith CVB3-induced myocarditis

To evaluate the possible participation of PDGF in thefibrotic process, mRNA expression of different PDGF-isoforms was monitored by semi-quantitative PCR. Forcomparison, mRNA expression of TNF-a, IL-1a, andIL-1b was also detected. Representative experimentsare shown in Figure 2. The histological score for

Figure 2 Expression of PDGF isoforms and cytokines during the CVB3 infection. The mRNA expression of PDGF-A, -B, and -C as well as TNF-a, IL-1a, andIL-1b in the myocardium of C57BL/6 and MHC class II knockout mice was examined at days 7, 21, and 56 after CVB3-infection. Each column represents thePCR products synthesized with identical cDNA templates isolated from heart tissue of one animal. Only two examples are shown, for the number ofanimals per group see Methods. A constant amount of 4 � 106 cDNA molecules were used as template for PCR amplification. Basic expression in non-infected control (co) mice is indicated for comparison. PCR conditions were modified in order to make the background level of PDGF isoforms expressionin control mice nearly non-visible. VP1 indicates the expression of CVB3 capsid protein gene VP1. The score of histological indicators of myocarditis, signsof fibrosis, and the amount of infectious virus determined by TCID50, are shown in the upper part. Left lane: MW ladder.


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myocarditis and fibrosis, the virus titre, as well as thepresence of VP1 RNA are shown to characterize thedisease state of the analysed animals.

High virus titres were detectable on day 7 after infec-tion. Acute myocarditis in both mouse strains was charac-terized by strong infiltration of the heart muscle withinflammatory cells. In correlation with inflammation,a moderate to strong expression of the inflammatory

cytokines TNF-a, IL-1a, and IL-1b could be detected inthe myocardium. Notably, also the mRNAs of all threePDGF isoforms were clearly up-regulated. At days 21and 56, the differences in the disease course betweenboth mouse strains became apparent: the cytokinesTNF-a, IL-1a, and IL-1b, as well as the virus titre, werealready below the detection limit in C57BL/6 mice.In addition, the expression of the PDGF mRNAs was

Figure 3 In situ hybridization of PDGF-C mRNA and viral RNA in CVB3-infected hearts. (A ) In situ hybridization of PDGF-C mRNA in paraffin-embeddedheart tissue sections of MHC class II knockout mice (left column) and immunocompetent C57BL/6 mice (right column) from days 7, 21, and 56 after infec-tion with CVB3. Insets show the controls with the sense probe. The sections are not counter stained. Dark blue to brown stained areas are positive forPDGF-C mRNA. It is possible to distinguish strongly positively reactive inflammatory cells (black arrows) and weakly reactive cardiomyocytes (whitearrows). Original magnification �200; co: non-infected controls. (B ) In situ hybridization for detection of viral RNA in paraffin-embedded heart tissuesections of MHC class II knockout mice (left column) and immunocompetent C57BL/6 mice (right column) from days 7, 21, and 56 after infection withCVB3. Insets show negative controls without probe. Positive regions are coloured in dark blue to brown. Sections are counterstained withHaematoxylin. VP1 RNA is strongly detected in cardiomyocytes (white arrows). Original magnification �400; co: non-infected controls.

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down-regulated to levels of non-infected controls at day56 p.i., only PDGF-A still seems to be slightly enhancedcompared with the controls (Figure 2 ). In contrastto immunocompetent mice, virus infection and inflam-mation persisted in MHC class II knockout mice. The per-sisting inflammation was associated with a sustainedexpression of PDGF-A, -B, and -C mRNAs.

Elevated PDGF-C production occurs in theinfiltrated heart tissue

To obtain information about the cellular origin of PDGF-C,we localized PDGF-C mRNA and protein by in situ

hybridization and immunohistochemistry, respectively.For comparison, and to characterize the inflammatoryprocess, virus RNA (VP1 gene) was also analysed by insitu hybridization. PDGF-C mRNA reactivity was detectedin heart sections of MHC class II knockout mice using theRNA antisense probe (Figure 3A, left). The specificity ofdetection was verified by negative staining with thesense probe (Figure 3A, see insets). The main localizationof this mRNA was found in areas of infiltrating inflamma-tory cells. However, basal expression of PDGF-C mRNAwas also detectable in cardiomyocytes. A diffuse PDGF-C mRNA expression could also be shown in cardiomyo-cytes of cured C57BL/6 mice at 21 and 56 days p.i.

Figure 3 Continued.


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(Figure 3A, right). The inflamed areas are surrounded byRNA of the VP1 gene (Figure 3B ). This distributionpattern of PDGF-C and VP1 was seen in heartsfrom MHC class II knockout mice at all time points ofinfection, as well as in hearts of C57BL/6 mice at day7 p.i.

To detect PDGF-C protein, we used an antibody recog-nizing an epitope of the biologically active domain ofPDGF-C. This antibody stained the regions of massiveimmune cell infiltration likewise (Figure 4A ), confirmingthe expression of PDGF-C in these areas. No signalswere obtained in controls either omitting the primaryantibody or upon blocking of anti-PDGF-C antibody bythe corresponding peptide antigen (data not shown).PDGF-B and -A were also expressed in areas of infiltrationas revealed by immunohistochemistry (Figure 4B and C ).

In summary, these data suggest that the inflammatorycells are the main source of elevated PDGF levels in theCVB3-infected hearts.The cognate receptor for PDGF-C, PDGF-Ra, is also

detectable by immunohistochemistry in the regions ofinflammation and fibrosis (Figure 4D ). The expressionqualitatively parallels the expression of PDGF-C.The receptor expression is prominent not in the cardio-myocytes, but in small cells within and also outside theinflammatory lesions, which represent invaded immunecells and probably also resident fibroblasts.Visualization of activated receptors in situ is not possibleuntil today. In order to obtain an indication for theactivation status of PDGF-R, we, therefore, used animportant down-stream signalling mediator of PDGFreceptors, Akt/PKB kinase. Antibodies recognizing only

Figure 4 Immunohistochemical detection of PDGF-C, -B, and -A, PDGF-Ra, and activated Akt/PKB. Frozen myocardial sections of CVB3-infected MHCclass II knockout mice (left column) or immunocompetent C57BL/6 mice (right column), respectively, from days 7, 21, or 56 after infection were reactedwith antibodies to PDGF-C (A ), -B (B ), or -A (C ), PDGF-Ra (D ), and phospho-Akt (Akt/PKB) (E ). The respective PDGF immunoreactive cells in inflamma-tory regions and outside are indicated by black arrows. Insets show isotype controls (non-immune IgG as primary antibody). Original magnification �200.

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the activated form of Akt/PKB again revealed signalsin the hearts of infected MHC class II knockoutanimals with a similar distribution as in case ofPDGF-Ra staining (Figure 4E ). This finding is consistentwith a possible PDGF-R activation in resident cardiaccells.

Discussion

The molecular mechanisms involved in the transition ofan acute myocarditis into an ongoing chronic diseaseare still not clear. Human chronic myocarditis is some-times characterized by the development of progressivefibrosis,2 whose molecular mechanisms are not wellunderstood. To analyse the underlying mechanisms, weemployed a mouse model of chronic myocarditis, whichis accompanied by fibrosis. The fibrotic process comprises

the replacement of normal tissue by mesenchymal cellsand the extracellular matrix produced by these cells,and thereby leads to distortion of normal tissuefunction.3 Different cytokines and growth factors canstimulate mesenchymal cell proliferation and matrixsynthesis and may be involved in the pathological fibrosis,which accompanies chronic myocarditis. For example,transgenic expression of TNF-a in mouse hearts leadsto severe myocarditis, fibrosis, and cardiomegaly.25,26

Persistent expression of cytokines in the chronic stageof CVB3-induced myocarditis has been described forvarious mouse models,27,28 as well as for human DCM.29

Although chronic inflammation is characteristic for thehuman disease as well as for our mouse model, fibrosisis more prevalent in the latter.Here, we explored the possibility that factors of the

PDGF family are involved in the pathology of chronic

Figure 4 Continued.


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myocarditis. A model of CVB3-induced myocarditis inmice was used for this study. This mouse model comprisesseveral hallmarks of the human disease. For example, thecourse of the disease depends strongly on the immunestatus of the recipient mice.17,30–32 We took advantageof this fact by comparing two mice strains in ourexpression analysis: immunodeficient MHC class II knock-out mice develop a chronic myocarditis upon CVB3 infec-tion, whereas immunocompetent C57BL/6 wild-typemice exhibit only an acute myocarditis, which is comple-tely reversible.

Interestingly, all analysed isoforms of PDGF-A, -B, and -Cwere up-regulated in close correlation with the inflamma-tory process. High levels of the growth factors persistedonly in CVB3-infected MHC class II knockout mice.

Localization of elevated PDGF expression in the CVB3-infected hearts showed that PDGF expression occurs in

the areas of inflammatory cell invasion. Thus, infiltratingimmune cells are likely to be the source of PDGF in thissetting. In this context, PDGF-C is of particular interest.Earlier, it has been found that PDGF-AA is a very potentmitogen for cardiac fibroblasts, suggesting that itscognate receptor, the PDGFa-receptor, drives their pro-liferation.33 PDGF-C is another recently discoveredligand for the PDGFa-receptor and therefore potentiallyimportant for proliferation of cardiac fibroblasts. In linewith such a presumed role, selective transgenicexpression of PDGF-C in the mouse heart induces strongproliferation of interstitial cells as cardiac fibroblasts.7,16

These data convincingly support the concept that PDGF-Ccan play a causative role for myocardial fibrosis develop-ment. Evidence for elevated PDGF receptor signalling inresident cardiac cells as consequence of PDGF over-expression is still elusive. PDGF-Ra is expressed in the

Figure 4 Continued.

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inflamed hearts, suggesting that up-regulation of PDGF-Cmay be functional. Further, we were able to detect acti-vation of Akt/PKB, a major known signalling mediator ofPDGF receptors, in the regions of inflammatorycell invasion. Although this finding is consistent withpossible resident cell activation by PDGF, furtherwork is required to better characterize the cell typesinvolved in PDGF expression and perception of PDGFsignals.A role for members of the PDGF family in development

of fibrosis has previously been proposed also for lung andkidney.11–15 For example, AG1295, a selective inhibitor ofthe PDGF receptors attenuated fibrosis in rat kidney afterunilateral obstruction suggesting a causal role of thissignalling system.15 Very recently, Eitner et al.34 demon-strated up-regulation of PDGF-C in fibrotic processesassociated with tubulo-interstitial kidney injuries,further supporting an important role of this factor for

fibrosis. That activation of resident PDGF receptors maycritically contribute to fibrosis development has alsobeen concluded from recent experiments with the PDGFreceptor blocker STI571 (Glivec). In chronic myeoloidleukemia (CML) patients, treatment with this compoundreduced fibrosis in bone marrow.35 In a rat model ofliver fibrosis, STI571-treatment attenuated the disease,concomitant with an inhibition of myofibroblast conver-sion.36 Our observation that enhanced mRNA andprotein levels of PDGF-C are associated with CVB3-induced myocarditis and fibrosis is consistent with thepossible participation of PDGF-C in initiation or mainten-ance of the cardiac fibrotic process. Notwithstanding thatother growth factors and cytokines may also contributeto the disease, it seems tempting to speculate in thelight of existing literature data that PDGF-C up-regulation plays a role for disease development ratherthan being merely a secondary phenomenon.

Figure 4 Continued.


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As an obvious continuation of these experiments, it willbe necessary to directly test this hypothesis. InhibitingPDGF receptor signal transduction in the available mousemodel of CVB3-induced chronic myocarditis with selectivekinase inhibitors should be one possible approach. Thisshould help to better understand the disease and maypotentially reveal a novel therapeutic strategy for theprevention of fibrosis in chronic myocarditis.

Supplementary material

Supplementary material is available at European HeartJournal online.

Acknowledgements

The authors would like to thank Birgit Meißner, BirgitSchikowski, and Heike Urban for excellent technical

assistance and Carl Zeiss, Jena GmbH, for technicalsupport. The study was supported by a grant fromDeutsche Forschungsgemeinschaft (DFG: BO 1043/5-1)and partially by the Thuringian Ministry of Science,Research, and Art (TMWFK) and the IZKF of theUniversity Jena (FKZ: B307-04004).

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