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Cardiovascular Research 2003 60(3):538-546; doi:10.1016/j.cardiores.2003.08.009
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Copyright © 2003, European Society of Cardiology

Angiotensin II AT1-receptor induces biglycan in neonatal cardiac fibroblasts via autocrine release of TGFβ in vitro

Karen Tiedea, Katrin Stötera, Christian Petrikb, Wen-Bin Chenc, Hendrik Ungefrorenc, Marie-Luise Krused, Monika Stolld, Thomas Ungerb and Jens W Fischer*,e

aInstitute of Pharmacology, Christian-Albrechts-University, Kiel, Germany
bInstitute of Pharmacology and Toxicology, Charité, Humbold University, Berlin, Germany
cResearch Unit Molecular Oncology, Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany
d1st Department of Medicine, Christian-Albrechts-University, Kiel, Germany
eMolecular Pharmacology, Institute of Pharmacology and Clinical Pharmacology, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf, Germany

*Corresponding author. Tel.: +49-211-81-12513/12500; fax: +49-211-81-14781. Email address: jens.fischer{at}uni-duesseldorf.de

Received 8 May 2003; revised 3 August 2003; accepted 19 August 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
 References
 
Objective: After myocardial infarction, angiotensin II (AngII) promotes ventricular remodeling and deposition of extracellular matrix (ECM), e.g., collagen type 1 and 3. Whether AngII regulates the expression of small leucine-rich proteoglycans (SLRP) which are important modulators of collagen fibrillogenesis and are induced after experimental myocardial infarction in rats is not known. The aim of the present study was therefore to analyse in cultured cardiac fibroblasts the expression and secretion of the SLRP biglycan in response to AngII. Methods: Cardiac fibroblasts were isolated from neonatal Wistar Kyoto rats and used in the first passage. Expression of AT1- and AT2-receptors was verified by RT-PCR. Expression of protoeglycans was analyzed after metabolic labeling with [35S]-sulfate, by SDS-PAGE and Western analysis. In addition, mRNA expression was examined by means of RT-PCR and Northern analysis. The activity of the biglycan promoter was analyzed using three biglycan promoter–luciferase fusion constructs. Results: Biglycan was found to be the predominant proteoglycan produced by neonatal cardiac fibroblasts in vitro. In response to AngII (10–7 M), secretion of total [35S]-labeled proteoglycans and mRNA of biglycan were increased to 116±1.8% and 121±11% (n = 5, mean±S.E.M.) of unstimulated controls, respectively. Biglycan induction in response to AngII was sensitive to Losartan (10–5 M) and unaffected by PD123177 (10–6 M), suggesting that the AT1-receptor mediates the induction of biglycan. Direct activation of the biglycan promoter downstream of the AT1-receptor was excluded by promoter activity assays. Instead, increased release of transforming growth factor beta 1 (TGFβ1) was detected by ELISA in response to AT1-receptor stimulation. Furthermore, neutralising antibodies to TGFβ1 inhibited biglycan induction in response to AngII. Conclusion: The results indicate that in cardiac fibroblasts AngII via the AT1-receptor causes autocrine release of TGFβ1, which in turn induces biglycan expression and secretion.

KEYWORDS Small leucine-rich proteoglycan; Fibrosis; Cardiac remodeling; Collagen; Decorin; Myocardial infarction

Abbreviations: AngII, angiotensin II • ECM, extracellular matrix • SLRP, small leucine-rich proteoglycan • GAG, glycosaminoglycan • TGFβ1, transforming growth factor beta1 • ACE, angiotensin-converting enzyme • bFGF, basic fibroblast growth factor • PDGF-BB, platelet-derived growth factor BB • CPC, cetylpyridinium chloride


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods and materials
 3. Results
 4. Discussion
 References
 
Myocardial infarction is followed by extensive remodeling of the infarct site and the remaining healthy myocardium. The acute phase of this process is important to preserve cardiac function and ventricular integrity. In the long term, however, the development of ventricular hypertrophy and extracellular matrix (ECM) deposition leads to ventricular dysfunction, fibrosis, and heart failure [1,2]. Cardiac fibroblasts that are embedded between cardiac myocytes and become activated after myocardial infarction are the source of most of the cardiac ECM [3]. Angiotensin II (AngII) is known to stimulate cardiac fibroblasts to produce ECM thereby promoting fibrotic remodeling. Inhibition of AngII generation by use of angiotensin-converting enzyme (ACE) inhibitors and blockade of AT1-receptors by competitive receptor antagonists is used clinically to improve hemodynamic parameters as well as to inhibit adverse cardiac remodeling in patients after myocardial infarction [4,5]. A hallmark of cardiac fibrosis is the synthesis and deposition of fibrilar collagen type 1, which is known to be induced by AngII [6,7]. In addition to collagen synthesis, the regulation of extracellular collagen fibrillogenesis is likely to be very important with respect to the mechanical properties of the cardiac collagen network. Collagen fibrillogenesis can be modified by the interaction of collagen type 1 with other ECM molecules. The family of small leucine-rich proteoglycans (SLRPs) is known to interact with collagen type 1 and to critically control collagen fiber formation in bone, skin, and tendon [8]. Mice deficient in SLRPs show strong collagen-related phenotypes [9]. Biglycan is one member of this proteoglycan family consisting mostly of extracellular proteoglycans that are widely expressed in a variety of tissues and cell types [10]. Another member of the SLRP family is decorin which is closely related to biglycan. Recently, biglycan and decorin have been shown to be upregulated after experimental myocardial infarction [11–13].

However, the pathways that regulate biglycan and decorin expression during myocardial remodeling are not known yet.


   2. Methods and materials
Top
 Abstract
 1. Introduction
 2. Methods and materials
3. Results
 4. Discussion
 References
 
Losartan was kindly provided by Dr. R. Smith (DuPont Merck Pharmaceutical, Wilmington, USA), PD123177 by Dr. D. Tayler (Parke Davis Pharmaceutical Research, Ann Arbor, USA) and rabbit polyclonal antisera to mouse biglycan (LF-106) from Dr. L. Fisher (NIH, Bethesda, USA). The supplier of materials and reagents were the following: fetal calf serum, Boehringer Mannheim (Mannheim, Germany); neutralising antibodies to transforming growth factor β (TGFβ), platelet derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF), normal goat IgG, and normal mouse IgG1, R&D Systems (Wiesbaden, Germany); antibodies to sarcomeric actin, alpha smooth muscle actin (anti-{alpha}SMAcy3), and donkey anti-mouse 488 secondary antibody, Sigma (Deisenhof, Germany); collagenase type CL SII, Biochrom (Berlin, Germany); pancreatin, Sigma; chondroitinase ABC, ICN Biochemicals (Eschwege, Germany); angiotensin II, Bachem Biochemica (Heidelberg, Germany); DEAE Trisacryl M, Sepracor (Villeneuve la Garenne, France); H2[35S]-sulfate, Hartmann Analytic (Braunschweig, Germany); oligonucleotides, Invitrogen (Groningen, Netherlands); Trizol reagent, Life-Technologies (Karlsruhe, Germany); ELISA kit for TGFβ-quantitation (Quantikine, human TGFβ-Immunoassay), R&D Systems, and the RT-PCR OneStep Kit from Qiagen (Hilden, Germany). All other chemicals were purchased from Sigma. Wistar Kyoto breeder pairs were obtained from Charles River (Sulzfeld, Germany).

2.1. Cell culture
Cardiac fibroblasts were isolated from neonatal (2–3 days old) Wistar Kyoto rats as described previously [14]. The fibroblasts were cultured in DMEM plus 20% fetal calf serum, passaged by use of trypsin, and used for experiments after the first passage. Fibroblasts from adult rat hearts (see below) were isolated and cultured following the same protocol and used for experiments between the second and fourth passage.

2.2. Myocardial infarction
Myocardial infarction was induced in 3 months old Wistar Kyoto rats (Charles River) by permanent ligation of the left coronary artery as previously described [15]. One week after myocardial infarction, rats were sacrificed, the hearts removed and cut transversally into four slices. From the two middle slices, the infarct zone was obtained and used to isolate cardiac fibroblasts as described above for neonatal fibroblasts. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3. Experimental design
Unless stated otherwise, the following experimental design was used: Neonatal fibroblasts of the first passage were seeded at 10,000 cells/cm2, grown to near confluence and starved for 48 h by complete serum withdrawal. Neutralizing antibodies to TGFβ, PDGF-BB, bFGF, and control IgG-antibodies were added simultaneously with AngII, whereas antagonists of AT1- and AT2-receptors were added 30 min prior to AngII. AngII was generally used at 10–7 M, Losartan at 10–5 M, and PD123177 at 10–6 M. Proteoglycan secretion into the culture medium and mRNA expression was analyzed 24 h after stimulation with AngII.

2.4. Immunohistochemistry
Methanol-fixed cells plated at 3000 cells/cm2 were stained with mouse monoclonal antibodies to sarcomeric actin (1:100; secondary antibody: 1:1000, donkey anti-mouse Alexa Fluor 488) and alpha smooth muscle actin (anti-{alpha}SMAcy3, 1:500).

2.5. RT-PCR and Northern analysis
Total mRNA was isolated by the single-step method using TRIzol reagent. The RT-PCR for AT1- and AT2-receptors was performed as described previously [16]. The following primers and conditions were used for detection of proteoglycan mRNA expression using the RT-PCR OneStep kit: rat biglycan: forward 5'-gatgacttcaaaggcctcca-3', reverse 5'-tcaggctcccattctcaatc-3', cycles 30, expected size, 499 bp; rat versican V0/1: forward 5'-cgagactggagctactgatgg-3', reverse 5'-gcttcctcagttggagacagg-3', cycles 30, expected size, 480 bp; rat decorin, forward 5'-atctccgagtggtgcagtg-3', reverse 5'-tgtcgtggagtcgaagctc-3', cycles, 30, expected size, 297 bp; GAPDH, forward 5'-tgatgacatcaagaaggtggtgaa-3', reverse 5'-tccttggaggccatgtaggccat-3', cycles 27, expected size, 238 bp. In addition, mRNA expression of biglycan, decorin, and versican-V0/1 was analyzed by Northern analysis as described before [17]; cDNA probes: 562-bp fragment of rat decorin (Dr. R. Forough, Texas A&M University System Health Science Center, Temple, USA); beta-glycosaminoglycan (GAG) domain of rat versican (V0/1, Dr. T. N. Wight, Hope Heart Institute, Seattle, USA) and complete cDNA of human biglycan (Dr. L. Fisher, NIH).

2.6. Proteoglycan analysis
For metabolic labeling of proteoglycans, 10 µCi/ml carrier-free H2[35S]-sulfate was added to the culture medium. Incorporation of radiosulfate into GAG chains was used to quantitate proteoglycan secretion. For this purpose, duplicate aliquots of the culture medium were analyzed by cetylpyridinium chloride (CPC) precipitation [18]. Partial purification of proteoglycans by DEAE chromatography, digestion of GAG chains by chondroitinase ABC, and analytical separation of metabolically labeled proteoglycans by SDS–polyacrylamide electrophoresis (SDS-PAGE) were performed as described [19]. Radiolabeled samples were run on a 4–12% gradient SDS-PAGE gel and detected by autoradiography, and nonradioactive samples were run on a 10% SDS-PAGE and analyzed by immunoblotting using the rabbit polyclonal antisera to mouse biglycan (LF-106, 1:1000) as described before [19].

2.7. Transient transfections of cardiac fibroblasts with biglycan promoter–reporter fusion genes and reporter gene assays
Three (human) biglycan promoter–reporter fusion constructs were used as described before [20]: Sac–luc (–1218, +42); Bam–luc (–686, +42) and 78-luc (–78, +42). Negative numbers indicate the 5' end of the promoter fragment relative to the major transcription start site (+1) and refer to the restriction sites used (SacI, –1218; BamHI, –686 and –78). The empty vector (pGL2-E) served as a control. For transient transfections, primary neonatal cardiac fibroblasts were seeded in 12-well plates at a density of 5 x 104 cells/well on day 1. On the next day when cells were 70–80% confluent, they were transfected by serum-free lipofection using Lipofectin (Invitrogen) according to the manufacturer's instructions (5 µl Lipofectin+1 µg DNA/well for 5 h). Following removal of the transfection mixture, cells were allowed to recover in normal growth medium (containing 20% FCS) for 20–24 h. Subsequently, cells were treated with AngII (10–7 M), or TNF-{alpha} (1000 U/ml) as control, for another 24 h. At the end of the stimulation period, cells were lysed in "Glo Lysis Buffer" (150 µl), and luciferase activities were determined following addition of 150 µl of Bright-Glo Assay Reagent (Bright GloTM Luciferase Assay System, Promega) with a MicroBeta TriLux 1450 system (Wallac, Gaithersburg, MD) for 2 s. Measurements for each condition were performed using three to six wells processed in parallel.

2.8. Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA). Subsequently, Bonferroni's multiple comparison post hoc test was performed where appropriate. A p-value of <0.05 was considered significant. Data from individual experiments were normalized to the values of unstimulated controls (100%) in order to pool these data.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
4. Discussion
 References
 
3.1. Cardiac fibroblasts
The employed isolation procedure was optimized to yield pure cultures of neonatal cardiac fibroblasts. However, cultured fibroblasts were monitored for contamination with cardiac myocytes by immunostaining of sarcomeric alpha-actin, which is expressed only in myocytes derived from skeletal and cardiac muscle. Fig. 1A and B shows a representative culture of cardiac fibroblasts that was double-stained with antibodies against alpha-sarcomeric actin (green) [21] and alpha-SM actin (red), which is expressed in cardiac fibroblasts but not in cardiac myocytes [22]. As shown in Fig. 1A, cardiac myocytes are absent from this culture. In comparison, Fig. 1C shows the detection of a contaminating cardiac myocyte in another culture dish. The expression of AT1- and AT2-receptors was verified by RT-PCR (not shown).


Figure 1
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  		Fig. 1 Characterization of neonatal cardiac fibroblasts. Immunofluorescent staining of cultured cardiac fibroblasts of the first passage using sarcomeric actin to detect contamination with cardiac myocytes (A) and alpha-smooth muscle actin to identify cardiac fibroblasts (B). (A) and (B) are images of the same cells incubated with both antibodies; excitation wavelength in (A) was 488 nm and in (B) 543 nm; in (C), images taken at both excitation wavelengths were superimposed. (C) shows a contaminating myocyte next to a fibroblast in a different culture dish; original magnifications: 100 x in (A) and (B); 200 x in (C).

 
3.2. Characterization of proteoglycan expression
The majority of labeled proteoglycans run as a single band above 220 kDa on a SDS-PAGE gradient gel (4–12%), which is typical for biglycan [23] (Fig. 2A). An additional proportion of high-molecular-weight PGs hardly entered the separating gel, which is characteristic for versican [24]. Using RT-PCR (Fig. 2B) and Northern blot analysis (not shown), biglycan and versican transcripts were detected easily, whereas decorin mRNA expression could hardly be detected (Fig. 2B). The expression of the predominant proteoglycan, biglycan, was verified by Western blot analysis using a polyclonal antiserum to biglycan (LF-106 [25]) (Fig. 2C). The data indicate that biglycan is the major chondroitin/dermatan sulphate proteoglycan secreted by neonatal cardiac fibroblasts in culture. In addition, the profile of proteoglycans secreted by cardiac fibroblasts isolated from the infarcted myocardium of adult rats was analyzed. By RT-PCR expression of decorin mRNA in addition to the expression of biglycan and versican V0/1 mRNA was detected in these cells (not shown).


Figure 2
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  		Fig. 2 Characterization of proteoglycans expressed by cultured neonatal fibroblasts. (A) SDS-PAGE (3.5% stacking gel; 4–12% resolving gel) of [35S]O42–-labeled proteoglycans secreted into the culture medium of neonatal fibroblasts (first passage) cultured in normal growth medium (20% FCS). Proteoglycans were purified by DEAE-anion exchange chromatography and 15,000 dpm of the ethanol-precipitated eluate was loaded on the gel. The pattern of separated proteoglycans was subsequently visualized by autoradiography. (B) Semiquantitative RT-PCR of biglycan (BG), versican (Vc), and decorin (Dec) using mRNA isolated from cultured cardiac fibroblasts of the first passage. GAPDH was amplified in the same reaction as internal control. Lanes 2 and 3, mRNA of cardiac fibroblasts grown in normal growth medium, lane 4, positive controls: mRNA of cultured rat smooth muscle cells was used as positive control for biglycan and versican, mRNA isolated from the myocardium of male Wistar Kyoto rats was used as positive control for decorin. (C) Western blot analysis of biglycan; the same material as in (A) was digested with chondroitinase ABC to remove GAG-chains and run on a 10% SDS-PAGE gel. Biglycan was subsequently detected by Western analysis using the polyclonal rabbit antibody LF106.

 
3.3. Angiotensin II induces secretion of proteoglycans by neonatal cardiac fibroblasts
In order to characterize the influence of AngII on the expression of proteoglycans in cardiac fibroblasts, total secreted proteoglycans were quantitated by CPC-precipitation after metabolic labeling of sulphated GAG chains by [35S]O42– (10 µCi/ml). Treatment of neonatal cardiac fibroblasts with different concentrations of AngII (10–8, 10–7, and 10–6 M) revealed a maximum induction of proteoglycan secretion at 10–7 M (Fig. 3A). Furthermore, the measurement of the proteoglycan secretion in response to AngII (10–7 M) after 24, 48, and 72 h revealed (not shown) that the stimulatory effect was maximal already 24 h after addition of AngII. Therefore, in all subsequent experiments, proteoglycan secretion and expression was studied 24 h after addition of 10–7 M AngII. The induction of total proteoglycan secretion, although moderate, was very consistent in all experiments which were performed in quadruplicates. In average, in response to AngII (10–7 M), secretion of [35S]O42–-labeled proteoglycans was increased to 116±1.8% (n = 5, mean±S.E.M.) over unstimulated controls in a 24-h time interval (Fig. 3B). In the same time interval, the cell number and thymidine incorporation as indicators of cell proliferation were not altered by AngII compared to unstimulated controls (data not shown). The AT1-receptor antagonist Losartan (10–5 M) completely abolished the induction of proteoglycan secretion by AngII, whereas the AT2-receptor antagonist PD123177 (10–6 M) had no effect (Fig. 3B). Furthermore, Losartan (10–5 M) and PD123177 (10–6 M), in the absence of AngII, had no effect (not shown). Based on the data presented in Fig. 2, neonatal cardiac fibroblasts express almost exclusively biglycan. These cells appear therefore to be particular well suited to investigate the effects of AngII on the regulation of biglycan. Thus, subsequently experiments addressing the mechanism of AngII-induced biglycan expression were undertaken. As shown by Northern analysis (Fig. 4), biglycan mRNA levels were increased 24 h after addition of AngII. The densitometric quantitation of mRNA levels of five independent experiments revealed an increase in biglycan mRNA of 121±11% (n = 5, mean±S.E.M.). This induction of biglycan mRNA expression by AngII was mediated by the AT1-receptor since Losartan was completely inhibitory, and PD123177 had no effect. AngII did not induce the expression of versican V0/V1, the main splice variants of versican, and decorin as analyzed by RT-PCR (not shown).


Figure 3
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  		Fig. 3 AT1-receptor stimulates secretion of [35S]O42–-labeled proteoglycans. Secretion of total [35S]O42–-labeled proteoglycans into the culture supernatant of neonatal fibroblasts in response to AngII. (A) Concentration dependency, raw data (dpm) from a representative experiment performed in quadruplicates; (B) effect of the AT1-receptor antagonist Losartan (10–5 M) and the AT2-receptor antagonist PD123177 (10–6 M); n = 5. Values are expressed as means±S.E.M.; *p<0.05; **p<0.01.

 

Figure 4
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  		Fig. 4 AT1-receptor induces biglycan mRNA expression. Biglycan mRNA expression (Northern blot) in neonatal fibroblasts treated with AngII (10–7 M) in the presence or absence of the AT1-receptor antagonist Losartan (10–5 M) or the AT2-receptor antagonist PD123177 (10–6 M) for 24 h. (A) Representative Northern blot; (B) quantitative analysis of independent experiments, biglycan signals were normalized to the signals of 18S RNA and expressed as percentage of control values (serum-starved fibroblasts); means±S.E.M., n = 5 for AngII; n = 4 for AngII+Losartan; *p<0.05; in case of AngII plus PD123177, the mean of two experiments is shown.

 
3.4. Angiotensin II does not activate the biglycan promoter
To investigate whether the increase in biglycan mRNA in response to AngII is mediated by activation of the biglycan gene promoter, transient transfections of neonatal fibroblasts were performed with the full-length (1218 bp) and two 5' deletions of the human biglycan promoter fused to the luciferase reporter gene [20]. All constructs containing at least 78 bp of 5'-flanking sequence showed considerable promoter activity (Fig. 5). However, none of the biglycan promoter constructs tested responded to AngII stimulation with significantly enhanced activity. Various modifications, i.e., reducing the serum in the medium to 0.5% FCS, increasing the AngII concentration to 0.5 x 10–6 or 10–6 M, or simultaneously treating the cells with the AT2-receptor antagonist PD123177 (10–6 M) did not result in reproducible and statistically significant induction of AngII-induced over basal promoter activity (data not shown). Since no factor is known to date that increases biglycan promoter activity, the sensitivity of the promoter assay was demonstrated by use of TNF{alpha}, which is known to repress the biglycan promoter in a variety of cell types [20]. Under the same experimental conditions, TNF{alpha} (1000 U/ml) decreased the transcription from the full-length biglycan promoter by 38% (not shown). These results indicate that AngII does not increase biglycan mRNA levels via stimulation of transcription from the endogenous biglycan gene.


Figure 5
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  		Fig. 5 AngII does not activate the biglycan promoter. Neonatal, cardiac fibroblasts were transiently transfected by lipofection with three biglycan promoter–luciferase fusion genes as indicated, or the empty pGL2-E vector as control. After 5 h the transfection solution was removed and replaced by normal growth medium for 20–24 h. Subsequently, cells were treated with AngII (10–7 M) for additional 24 h. Relative luciferase activities of four independent experiments are shown (mean±S.D.).

 
3.5. Autocrine release of TGFβ1 stimulates proteoglycan secretion in response to AngII
An alternative explanation for the increase in biglycan mRNA levels without activation of the biglycan promoter could be a transforming growth factor beta 1 (TGFβ1)-dependent pathway [20]. Indeed, the TGFβ1 concentration in the culture supernatant was increased twofold after stimulation of serum-starved cells by AngII for 24 h (Fig. 6A). This increase in TGFβ1 concentration was completely blocked by Losartan (10–5 M) and the combination of Losartan and PD123177 (10–6 M), whereas PD123177 had no effect. The addition of either Losartan or PD123177 in the absence of AngII had no effect on TGFβ1 levels (not shown). Adding neutralizing antibodies to TGFβ1 into the cell culture medium completely abolished the stimulation of proteoglycan secretion in response to AngII (Fig. 6B). Control IgG antibodies applied at the same concentration had no effect. Moreover, the induction of biglycan mRNA by AngII was also inhibited by neutralizing antibodies to TGFβ1 (not shown). Since it has been shown that AngII induces in some cell types also the autocrine release of bFGF and PDGF-BB [26,27], neutralizing antibodies to these growth factors were applied as well. Neutralizing antibodies to bFGF and PDGF-BB did not affect proteoglycan secretion in response to AngII (Fig. 6C). Furthermore, addition of exogenous TGFβ1 (0.1–10.0 ng/ml) dose-dependently increased secretion of total proteoglycans under identical experimental conditions (Fig. 6D). Since biglycan was found to be the predominant proteoglycan and AngII did not stimulate the expression of versican and decorin, we assume that the amount of CPC precipitable radioactivity in the cell culture supernatant correlated directly to secreted biglycan.


Figure 6
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  		Fig. 6 Stimulation of autocrine release of TGFβ1 in response to AngII. (A) TGFβ1 concentration in the culture supernatant of neonatal fibroblasts; n = 3. (B–D), total secreted [35S]O42–-labeled proteoglycans; (B) cells were stimulated with AngII (10–7 M) in the presence or absence of a neutralizing antibody to TGFβ1 (0.5 µg/ml) or a nonimmune control antibody (0.5 µg/ml); *p<0.05, n = 3; (C) effects of neutralizing antibodies to bFGF (1 µg/ml) or PDGF-BB (10 µg/ml) and the respective control IgGs on AngII (10–7 M)-stimulated proteoglycan secretion; n = 3. (D) Response to exogenous TGFβ1; n = 3; *p<0.05; means±S.E.M.; all measurements 24 h after addition of AngII.

 
3.6. Adult cardiac fibroblasts show increased proteoglycan secretion in response to AngII
In order to investigate whether the above results might be applicable to cardiac fibroblasts derived from adult rats as well, fibroblasts were isolated from the infarcted area 1 week after experimental myocardial infarction. AngII (10–7 and 10–6 M) and TGFβ (not shown) evoked a marked increase of total secreted proteoglycans (Fig. 7) in adult fibroblasts derived from the infarct zone. The stimulation of proteoglycan synthesis was mediated by the AT1-receptor, since it was sensitive to Losartan.


Figure 7
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  		Fig. 7 AT1-receptor stimulates secretion of [35S]O42–-labeled proteoglycans in adult cardiac fibroblasts. Secretion of total [35S]O42–-labeled proteoglycans in response to AngII, adult cardiac fibroblasts derived from the infarct zone; representative experiment performed in quadruplicates; *p<0.05; means±S.E.M.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
References
 
During remodeling after myocardial infarction, ECM accumulates in the non-infarcted areas as well as in the infarct scar [1]. The accumulation of ECM impairs the contractile compliance of the ventricle but, on the other hand, insures the integrity of the infarct scar. The functional properties of the cardiac ECM are determined by the balance between fibrilar components of the ECM, e.g., collagen types 1 and 3, glycoproteins and proteoglycans such as the SLRPs, decorin, and biglycan. However, little is known about the regulatory pathways that control the expression of SLRPs in cardiac cells. In the current study, the expression of biglycan was examined in cultured cardiac fibroblasts. AngII induced an increase of total proteoglycan secretion that was sensitive to preincubation with Losartan indicating that the AT1-receptor mediated the induction of proteoglycan secretion. This stimulation of total proteoglycan secretion was observed in fibroblasts from neonatal rats and from the infarcted area of adult rats. Cardiac fibroblasts from neonates represented a particular suitable model to investigate the regulation of biglycan by AngII, since biglycan is the predominant proteoglycan in these cells, and both AT1- and AT2-receptors are still expressed at the first and second passage. Neonatal fibroblasts were therefore chosen for further mechanistic studies. In cardiac fibroblasts from neonatal rats, the elevation of total proteoglycan secretion corresponded to an increase in biglycan mRNA levels that was mediated by the AT1-receptor as well. Promoter–reportor assays with three different biglycan promoter constructs revealed that AngII did not activate the biglycan promoter.

Since AngII has been shown to induce the autocrine release of TGFβ1 in cardiac fibroblasts [28,29], it was analyzed whether the induction of biglycan might be mediated by autocrine release of TGFβ1 in response to AngII. This is very likely the case, since in response to AngII neonatal cardiac fibroblasts (i) released strongly increased amounts of TGFβ1 into the culture medium, (ii) the release of TGFβ1 in response to AngII was mediated by the AT1-receptor, (iii) neutralizing antibodies to TGFβ1 blocked the effect of AngII on proteoglycan secretion and biglycan mRNA expression, and (iiii) application of exogenous TGFβ1 resulted in a comparable increase of proteoglycan secretion. Furthermore, the present data are in line with two recent studies in vascular smooth muscle cells analyzing the effect of AngII on ECM-gene expression. One study describes that biglycan expression is induced by AT1-receptor [30] and the other that collagen type I is induced by AngII via the AT1-receptor and autocrine release of TGFβ1 [31]. The current study extends these observations to cardiac fibroblasts. In addition, it shows by the use of the selective AT2-receptor antagonist PD123177 that the AT2-receptor does not participate in the regulation of biglycan expression in neonatal cardiac fibroblasts. Identification of TGFβ as the mediator of AngII-induced biglycan expression and the unresponsiveness of the biglycan promoter are in accordance with previous studies investigating the effect of TGFβ on biglycan expression. In the osteoblastic MG-63 cell line and the pancreatic carcinoma cell line, PANC-1, TGFβ1 leads to increased biglycan mRNA levels without stimulating the biglycan promoter activity as assessed from transfection of biglycan promoter–reporter fusion genes and transcriptional run-on analysis [20,32]. Instead, available data suggest that TGFβ1 induces an accumulation of mature mRNA via enhanced nuclear processing, stability, and/or export [20], presumably via Smad-dependent stimulation of p38 MAPK [33].

The functions of biglycan and other SLRPs in the myocardium are unknown. In other cells and organs, biglycan has been shown to modulate collagen fibrillogenesis [9], TGFβ1 activity [34], and cell migration [23]. Irregular collagen fibrils are formed in bone, skin, and tendon of biglycan-deficient mice [35] that develop an osteoporosis-like phenotype, osteoarthritis, and ectopic tendon ossification [9,36]. Whether biglycan is a regulator of collagen type 1 fibrillogenesis in the myocardium as well is not known. Such modulation of cardiac collagen fibrillogenesis by biglycan could be of particular importance for the post-infarct healing, since collagen type 1 is next to collagen type 3, the main collagen type involved in myocardial fibrosis and remodeling [37].

In response to AngII, total proteoglycan secretion and biglycan expression were increased in the range of 10–20% over unstimulated controls. Although this increase is moderate, it could be of biological relevance since (i) biglycan is a secreted molecule that accumulates in the interstitium over time, and (ii) long-term activation of the renin angiotensin system occurs both systemically and locally after myocardial infarction [1]. The present finding that AngII via the AT1-receptor induces proteoglycan expression also in fibroblasts derived from the infarct zone of adult rats supports the hypothesis that AngII might be a regulator of proteoglycan synthesis in adult cardiac tissue as well. However, whether the present findings are applicable to the regulation of biglycan in the heart in vivo and whether this might have functional consequences, needs to be addressed in animal studies in the future.


   Acknowledgements
 
The polyclonal antisera to biglycan (LF106) were kindly provided by Dr. Larry Fisher, NIH, Bethesda, MD, USA. The excellent technical assistance of Peggy Mann (Institute of Pharmacology and Clinical Pharmacology, Heinrich-Heine-University, Düsseldorf) is greatly acknowledged.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
 References
 

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