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Cardiovascular Research 2000 46(3):463-475; doi:10.1016/S0008-6363(00)00037-7
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Copyright © 2000, European Society of Cardiology

Angiotensin II directly increases transforming growth factor β1 and osteopontin and indirectly affects collagen mRNA expression in the human heart

Claudio Kupfahla, Daniel Pinka, Katharina Friedricha, Heinz R. Zurbrüggb, Michael Neussa, Christina Warneckea, Jens Fielitza, Kristof Grafa, Eckart Flecka and Vera Regitz-Zagroseka,*

aInnere Medizin/Kardiologie, Virchow-Klinikum, Charité Humboldt University, and Deutsches Herzzentrum Berlin, Berlin, Germany
bHerz- Thorax- und Gefässchirurgie, Deutsches Herzzentrum Berlin, Berlin, Germany

* Corresponding author. Tel.: +49-30-4593-1000; fax: +49-30-4593-2500 zagrosek{at}dhzb.de

Received 22 June 1999; accepted 18 January 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objectives: Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFβ-1 and osteopontin are involved in this process. Methods and results I: In a first set of experiments, the direct effect of Ang II on collagen I, TGFβ-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFβ-1 (183±21% of control, p<0.05) and osteopontin mRNA (275±58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95±8%). Stimulation with TGFβ-1 led to an increase in collagen I and III mRNA (127±10%, p<0.05; 140±15%, p<0.02). Methods and results II: In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFβ-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, III or fibronectin mRNA content after 24 h. In contrast, TGFβ-1 stimulation significantly increased collagen I and III mRNA expression (124±5% and 128±5%, p<0.002). Conclusions: In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFβ-1 and osteopontin mRNA expression. Since TGFβ-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart.

KEYWORDS Ang II, Angiotensin II; AT1, AT2, angiotensin II receptor types 1 and 2; ADP, adenosine 5'-diphosphate; AMP, adenosine 5'-monophosphate; AP-1, activator protein 1; ATP, adenosine 5'-triphosphate; ATR, Ang II receptor; FN, fibronectin; HPLC, high-performance liquid chromatography; MAP, mitogen activated protein; mRNA, messenger RNA; OPN, osteopontin; PEA3, polyoma virus enhancer activator 3; PCR, polymerase chain reaction; PDH, pyruvic dehydrogenase; RT, reverse transcription; TGFβ-1, transforming growth factor β1


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
In animal experiments, cardiac angiotensin II (Ang II) plays a crucial role in remodeling after myocardial infarction and in models of hypertension (for review, see [1]). Ang II induces fibroblast proliferation, and collagen and fibronectin synthesis in rodent cell culture models [2,3]. In adult rat cardiac fibroblasts, a direct effect of Ang II on collagen gene expression was found [4,5]. The effect of Ang II on extracellular matrix synthesis is at least partially mediated by the induction of para/autocrine factors like TGFβ-1 [6–9]. Ang II upregulates TGFβ-1 in rat cardiac fibroblasts [7], and TGFβ receptors type I and II in vascular smooth muscle cells [10]. Binding of TGFβ-1 to its receptors activates a phosphorylation cascade that involves different mitogen activated protein (MAP) kinases. These kinases phosphorylate transcription factors like activator protein 1 (AP-1), SMAD (a transcription factor family related to the Drosophila gene mad) and nuclear factor 1 (NF1) that activate the promotors of the target genes, two of which are the collagen I {alpha}1 and {alpha}2 genes [11,12]. Increased collagen after stimulation with TGFβ-1 has been reported for primary fibroblasts of various origins as well as for fibroblast cell lines. TGFβ-1 interferes with the synthesis and degradation of extracellular matrix in cardiac remodeling after myocardial infarction [13], myocyte hypertrophy [14] and other fibrotic diseases (for review, see [15]).

As another mediator of Ang II effects on fibroblast function, osteopontin has recently been identified. Osteopontin is an acidic secreted phosphoprotein with both extracellular matrix and cytokine-like properties. Through its arginine–glycine–aspartate (RGD) domain, osteopontin binds to cell surface integrins, thereby stimulating integrin-mediated adhesion and proliferation in rat cardiac fibroblasts [16]. Osteopontin expression is upregulated in many remodeling tissues, amongst others, in heart failure in rats [17] and in human cardiac hypertrophy [18]. Controversy still exists over whether macrophages, cardiomyocytes (man) or fibroblasts (rat) represent the major source of osteopontin in the heart. Species differences may account in part for the seemingly contradictive results. Ang II is a potent stimulator of osteopontin production in cardiac fibroblasts and thereby indirectly regulates fibroblast functions like proliferation, adhesion and migration [16].

Due to the limited availability of human material, very few studies on the mechanisms that control collagen synthesis have been performed in the adult human heart. Ang II-mediated synthesis of extracellular matrix proteins in the human heart also contributes to the impairment of cardiac function in remodeling after myocardial infarction, in hypertensive heart disease and in cardiomyopathies [1]. However, significant differences have been described with regard to the expression of angiotensin receptors in the myocardium and on fibroblasts from different species, i.e., between rat, rabbit and human [19]. Comparison between neonatal, adult and senescent rat cells, and between young human cells and developmentally stage-matched rabbits showed differences in growth rate and response to growth factors [19,20]. These data suggest that human fibroblasts may behave differently from rodent cells. Nevertheless, Ang II also induces fibroblast proliferation and extracellular matrix protein synthesis in adult human cardiac fibroblasts [21,22].

The angiotensin II type 1 (AT1) receptor is the only Ang II receptor expressed in cultured adult rat cardiac fibroblasts [4]. It mediates proliferation, TGFβ-1, collagen and fibronectin synthesis [2,4,5,7]. In contrast, in fibrotic regions of the cardiomyophatic and remodeling human heart, high expression of the angiotensin II type 2 receptor (AT2) was detected, whereas the AT1 receptor was more evenly distributed in the myocardium [23,24]. The function of the AT2 is not yet completely understood, but several studies suggest that AT1 has antagonistic, antiproliferative and antifibrotic properties [23,25]. Both Ang II receptor subtypes seem to be regulated in cardiomyopathies, with a downregulation at human end-stage heart failure, but an upregulation in early stages of hypertrophy in animal models and after myocardial infarction. Some of the apparently contradictive results may be due to species-differences in receptor regulation.

In this paper, we investigated whether Ang II alters collagen mRNA expression in the adult human heart in a direct manner or whether Ang II mainly acts through the induction of intermediate factors such as TGFβ-1 and osteopontin. For this purpose, two different protocols were used: the first was designed to assess the direct effects of Ang II in a multicellular system, i.e., in fresh samples of atrial myocardium. In order to detect the effects of longer stimulation periods, which include potential indirect effects, in a second protocol we examined the effects of Ang II and TGFβ-1 in isolated human cardiac fibroblasts. In both protocols, AT1 and AT2 expression was determined to assure that Ang II was capable of inducing biological effects.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Patient characteristics
In the first protocol, we analyzed atrial myocardium from ten patients who underwent routine coronary artery bypass surgery (n=9) or aortic valve replacement (n=1) and had normal or slightly impaired global left ventricular function. The clinical characteristics and the medical therapy of the patients are summarized in Tables 1 and 2Go.


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  		Table 1 Patients for protocol 1a

 

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  		Table 2 Patients for protocol 2 (fibroblast preparation)a

 
In the second protocol, we analyzed isolated fibroblasts from seven explanted human hearts from patients that underwent heart transplantation for end-stage heart failure. The clinical characteristics are summarized in Table 2. Both experimental protocols were accepted by the ethical committee of the Charité, Campus Virchow-Klinikum, at the Humboldt University, Berlin. The investigation conforms with the principles outlined in the declaration of Helsinki.

2.2 Experimental protocol 1 – Stimulation of atrial myocardial samples with Ang II and TGFβ-1
Atrial myocardial samples weighing about 50–80 mg from patients who underwent routine cardiac surgery were obtained in the operating room. Samples were dissected into pieces of about 5–20 mg. Two pieces from each patient were shock frozen immediately (time zero control), four to six pieces were immediately incubated in 50 ml Falcon tubes in 10 ml of continuously oxygenated Krebs–Henseleit buffer (pH controlled at 7.4) in a 37°C pre-warmed waterbath and were stimulated by the addition of a single dose of Ang II (Bachem, Switzerland) to a final concentration of 1 µM, to increasing Ang II concentrations ranging from 1 nM to 1 µM and used as controls, respectively. In addition, in three experiments, human recombinant TGFβ-1 (10 ng/ml; Calbiochem, Germany) was used for stimulation. Since we were interested in the effects of Ang II and TGFβ-1 independently of mechanical loading, samples were not mounted in an organ bath and stretched. A stimulation period of 4 h was selected to detect direct, short-term effects of Ang II. Pilot experiments indicated that the energy charge of the samples was not significantly altered at this time point, but decreased after 8 h. At least two samples were used in parallel for each stimulation protocol. After 4 h, samples were shock frozen and stored at –70°C until the analysis of adenine nucleotide concentrations or the mRNA preparation was performed.

Total RNA was prepared by the use of RNAzol B (Biozol, Germany). Yields of RNA were comparable after explantation, after 4 h control and after 4 h Ang II stimulation (time zero, 0.41±0.05 µg RNA/mg wet weight; 4 h control, 0.37±0.086 µg RNA/mg wet weight; 4 h Ang II, 0.37±0.07 µg RNA/mg wet weight, p=ns). From each sample, 250 ng of RNA were reverse transcribed by the use of 300 ng random hexamers and 100 U Superscript IITM(Gibco BRL, Germany). The determination of pyruvic dehydrogenase (PDH) mRNA was used as an internal standard to normalize all samples for potential variations in the mRNA content. In addition, AT1 cRNA standards in optimized concentrations were added to control for reverse transcription efficiency (see Section 2.4).

2.3 Experimental protocol 2 — Stimulation of isolated human cardiac fibroblasts with Ang II and TGFβ-1
All cell culture reagents were purchased from Gibco BRL Life Technologies (Germany). Human cardiac fibroblasts were isolated from explanted, end-stage failing adult human hearts, as described [21]. The resulting cell cultures consisted almost exclusively of fibroblasts (96–98%). Cells from passage one were plated in 10 cm tissue culture dishes for the mRNA assays. The fibroblasts were starved for 24 h with fibroblast medium (DMEM containing 4000 mg glucose/l, 2 mM L-glutamine, 100 U/0.1 mg/ml penicillin/streptomycin; 0.1% BSA) without fetal calf serum, followed by the addition of Ang II to a final concentration of 1 µM or TGFβ-1 to 10 ng/ml (triplicates from each fibroblast preparation). After 24 h, cells were harvested for RNA extraction (RNAzol B, Biozol, Germany). Yields of RNA in controls, TGFβ-1 and Ang II-stimulated samples were comparable: controls, 9.7±0.9 µg RNA/dish; Ang II, 9.7±1.0 µg RNA/dish and TGFβ-1, 11.3±1.2 µg RNA/dish (p=ns). From each sample, 250 ng of RNA was reverse transcribed and internal cRNA standards (see Section 2.4) were added prior to reverse transcription as required by the specific protocol.

Ang II-stimulated fibroblast proliferation was determined as described previously [21,26]. Briefly, 5x103 cells/well of passage one were seeded in 96-well microtiter plates and starved for 24 h followed by Ang II stimulation (1 nM to 1 µM) for 72 h. The addition of Ang II was repeated every 24 h. Proliferation was determined using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Boehringer, Mannheim, Germany) according to the manufacturer's instructions. Formazan-dye formation was detected at 550 nm using an automated microplate reader (Dynatech, USA).

2.4 PCR quantitation of collagen I, collagen III, fibronectin, AT1, AT2, TGFβ-1 and osteopontin mRNA
For quantitation of collagen I, collagen III, fibronectin and AT1 mRNA in the fibroblasts, internally standardized quantitative RT-PCR procedures were used. cRNA standards for AT1 (819 bp), collagen I (1096 bp), collagen III (1072 bp) and fibronectin (1011 bp) were constructed by the use of PCR-based mutagenesis as described (Table 3) [27]. The standards for AT1, collagen I, collagen III and fibronectin were identical to the corresponding wild-type sequences with the exception of a 6-bp deletion in the centre. The standards were added to the RNA samples prior to reverse transcription, and short fragments of the wild-type cDNA and the respective standard cDNA that spanned the deletion were co-amplified with the digoxigenin-labelled quantitation primers in the same tube (Table 4). The six-base-pair deletion was used for sequence-specific hybridization of the wild-type and standard PCR products to separate wells of a 96-well microtiter plate, with sequence specific capture probes as described in detail for AT1 [27,28]. The digoxigenin label in the primers was quantitated with an anti-digoxigenin alkaline phosphatase conjugate in a 96-channel enzyme-linked immunosorbent assay (ELISA) reader [29]. As an example, a standard curve for fibronectin is shown in Fig. 1. The total variance of the procedure including reverse transcription, PCR and ELISA was 15%. The reproducibility and accuracy of the PCR–ELISA has been described [27–29] and has recently been certified by the Deutsche Akkreditierungsgesellschaft Chemie.


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  		Table 3 Primers for the generation of cRNA standards

 

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  		Table 4 Primers for the quantitative RT-PCRs

 

Figure 1
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  		Fig. 1 Quantitative RT-PCR for fibronectin mRNA. A. To obtain a standard curve, increasing amounts of fibronectin mRNA (2x107–48x107 copies/RT tube) were reverse transcribed and amplified with the same amount of standard. The PCR products were quantitated based on the optical density (OD) after the digoxigenin/alkaline phosphatase reaction. As expected, the amplification of increasing amounts of wild-type inhibits the amplification of the standard. B. From the known amounts of fibronectin wild-type copies and the extinction of wild type (WT) and standard (STD) in each sample in A, a standard curve was constructed that attributed WT/STD optical density to fibronectin mRNA content (in the presence of a known amount of STD). This curve was used to determine fibronectin copy numbers in unknown samples to which the same number of standard copies was added. OD, optical density; Fn, fibronectin.

 
The TGFβ-1, osteopontin, collagen I, AT1 and AT2 mRNA determinations in the atrial strips were normalized in reference to PDH mRNA levels in the same tubes. Short fragments of comparable length (Table 4) were amplified from all genes and all PCRs had similar efficiencies. Duplicate samples were obtained at every time point, PCRs were run in duplicate and the PCR products were quantitated by reversed-phase high-performance liquid chromatography (HPLC) [30]. All PCR procedures were validated with respect of reproducibility and linearity within the measuring range. The variance of the PCR/HPLC procedure could be limited to less than 10% (mean from triplicates in seven samples). AT1 mRNA was quantitated in relation to the internal AT1 standard and to PDH mRNA. The mean PDH mRNA content did not change significantly during the first 4 h after tissue explantation (93±8% to 100±1%) or during 4 h stimulation with Ang II (116±6%). The respective mean mRNA level after 4 h control stimulation was set as 100% and the mRNA content after stimulation was related to this value.

2.5 Adenine nucleotide determination in myocardial samples
Adenine nucleotide concentrations were determined as described previously in endomyocardial biopsies [31]. In brief, 10 mg samples were homogenized in 4% perchloric acid and centrifuged. The supernatant was adjusted to pH 5.5, an aliquot was removed for adenine nucleotide quantitation by HPLC, and the protein concentration was determined according to the method of Bradford. For reversed-phase HPLC, a Waters system (M510, M 481, Maxima 820) with a C18 column (ODS Hypersil, 5 µm) with the detector set at 254 nm was used [32]. The ATP, ADP and AMP concentrations were measured and the energy charge ([ATP +1/2ADP]/[ATP+ADP+AMP]) was calculated according to the method of Atkinson et al. [33]. It did not differ significantly between time zero control (0.78±0.06) and the 4 h control (0.72±0.02, p=ns). This revealed that the oxygenation of the tissue was maintained during the incubation period.

2.6 Statistical tests
Data were calculated as the mean±S.E.M. Comparison of groups was performed by paired t-tests and correction for multi-group comparison was obtained by the use of an ANOVA procedure with Bonferroni correction. Indicated levels of significance for differences between groups are based on the ANOVA procedure.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Protocol 1: Direct induction of gene expression in strips of human atrial myocardium after short-term stimulation with Ang II and TGFβ-1
The expression of TGFβ-1/PDH mRNA increased significantly after 4 h stimulation with Ang II in comparison to 4 h control incubation (183±21% versus 100±8%, p<0.05; Figs. 2 and 3Go). Ang II-stimulated samples also expressed a significantly higher osteopontin/PDH mRNA ratio than controls (275±58% versus 100±10%, p<0.02; Fig. 3). In contrast, the collagen I/PDH mRNA expression was not altered in Ang II-stimulated (95±8%) compared with the control samples (100±7%, p=ns; Figs. 2 and 3Go). Both AT1 and AT2 mRNA was expressed in the human atrial myocardial samples. AT1/PDH expression increased significantly under stimulation with Ang II, to 259±42% of control (Ang II versus control, p<0.0005). No change was found during the 4 h control incubation (107±27% versus 100±10%). The increase in AT1 mRNA expression by Ang II was dose-dependent with a maximum at 1 µM (Fig. 4A) and reached its plateau after 1 h (Fig. 4B). The AT2/PDH mRNA was also increased in Ang II-stimulated samples (194±34 versus 100±10%, p<0.02) (Fig. 4C). Stimulation of human atrial samples with recombinant human TGFβ-1 led to a significant increase of collagen I (127±10%, p<0.05) and collagen III (140±15%, p<0.02) but not fibronectin mRNA expression (115±8%, p=ns; Fig. 5).


Figure 2
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  		Fig. 2 Representative PCR products of atrial samples after 4 h stimulation with Ang II. Products of A, TGFβ-1; B, osteopontin; C, collagen I and D, PDH RT-PCRs after 4 h stimulation with 1 µM Ang II. A and B, co, 4 h control stimulation of atrial tissue samples of two different patients; AII, Ang II-stimulated atrial samples of the two patients, duplicate RT-PCR; C, co, 4 h control of atrial samples of the two patients, duplicate RT-PCR; AII, Ang II stimulation of the two atrial samples; D, 0 h co, time zero control; co, 4 h control, AII, Ang II-stimulated samples of two patients. M, 100 bp ladder, thick band=600 bp.

 

Figure 3
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  		Fig. 3 TGFβ-1, osteopontin and collagen I mRNA expression in atrial samples after 4 h of Ang II stimulation. Significant increases were observed for TGFβ-1/PDH and osteopontin/PDH (OPN) but not for collagen I/PDH mRNA. Mean values from ten hearts, with duplicate samples. Levels of significance for differences between groups are based on the ANOVA procedure.

 

Figure 4
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  		Fig. 4 Increase in AT1 mRNA expression in atrial samples after 4 h of Ang II stimulation. A. Time course of the increase of AT1/PDH mRNA expression during 4 h Ang II stimulation (1 µM). The plateau was reached after 1 h. Data are expressed as percentages of the 4 h control incubation, with mean values from two experiments being given. B. Dose-dependent increase in AT1 mRNA after 4 h of Ang II stimulation. Data are expressed as percentages of the 4 h control incubation, with mean values from two experiments being given. C. Time course of the increase of AT2/PDH mRNA expression during 4 h of Ang II stimulation. Data are expressed as percentages of the 4 h control incubation, with the means from two experiments being given.

 

Figure 5
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  		Fig. 5 Induction by TGFβ-1 of collagen I and III, but not fibronectin mRNA in human atrial myocardium. Significant increases were observed for collagen I and III/PDH mRNA but not for fibronectin/PDH mRNA levels. Mean values from three hearts, using duplicate samples are given. Levels of significance for differences between groups are based on the ANOVA procedure.

 
3.2 Protocol 2: Induction of gene expresssion by Ang II and TGFβ-1 in cultured human cardiac fibroblasts
Since Ang II has been reported to induce collagen synthesis in cardiac fibroblasts, and since Ang II induced TGFβ-1, but not collagen mRNA in the first protocol, we determined in a second protocol whether Ang II or TGFβ-1 induced collagen mRNA expression in adult human cardiac fibroblasts. After 24 h, collagen I and III mRNA expression was significantly increased by TGFβ-1 to 124±5 and 128±5% of control (both p<0.002; Fig. 6) whereas Ang II had no effect on collagen I (102±8%) or collagen III (96±6% of control) mRNA. However, Ang II significantly increased TGFβ-1/PDH mRNA to 190 and 317% of control in three independent stimulations (Table 5). Fibronectin mRNA was not changed by Ang II or TGFβ-1 (99±3 and 99±5%).


Figure 6
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  		Fig. 6 Induction by TGFβ-1 of Col I, Col III and FN mRNA in cultured human cardiac fibroblasts. Regulation of Col I, III and FN mRNA expresssion by Ang II and TGFβ-1. After 24 h, Col I and III mRNA expression was significantly increased by TGFβ-1, whereas Ang II had no effect. The results given are the mean values from seven hearts. All stimulation data were related to the respective negative controls after 24 h and expressed as percentages of the controls. Col I, collagen I; Col III, collagen III; FN, fibronectin.

 

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  		Table 5 Stimulation of human cardiac fibroblasts with Ang II — Effect on TGFβ-1 mRNAa

 
AT1, but not AT2 mRNA was expressed in the isolated cardiac fibroblasts. AT1 mRNA was detected after 22 cycles, whereas AT2 mRNA could not even be detected after 40 cycles. Stimulation of the isolated cardiac fibroblasts for 24 h led to a downregulation of the AT1 receptor mRNA by Ang II to 53±22% and by TGFβ-1 to 28±4% of controls.

No differences were found between fibroblasts prepared from the unused donor heart, from the patients in whom the ventricle was unloaded with a biventricular assist device and from the explanted end-stage failing hearts.

3.3 Stimulation of cell proliferation by Ang II in human cardiac fibroblasts
As in previous studies, Ang II caused a dose-dependent stimulation of human cardiac fibroblast proliferation over a dose range of 10 to 1000 nM (Table 6). The fibroblasts were from the same preparations and first passages as the cells used in parallel for the determination of collagen I, III and fibronectin mRNA levels.


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  		Table 6 Dose-dependent stimulation of fibroblast proliferation by Ang IIa

 
3.4 Effects of medical therapy in protocol 1
There were no significant differences between the six patients treated with ACE inhibitors in protocol 1 and the four non-treated patients. However, patients treated with ACE inhibitors exhibited a trend towards higher basal AT1, AT2 and osteopontin and lower TGFβ-1 and collagen I/PDH mRNA expression (Table 7).


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  		Table 7 Effects of ACE inhibitor therapy on basal RNA expression in the human atriuma

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
The main finding of this study is that, in human atrial myocardium, Ang II leads to an increase in TGFβ-1 and osteopontin mRNA levels but does not increase the collagen mRNA content directly. Secondly, Ang II itself does not increase collagen I mRNA in isolated human cardiac fibroblasts, although it does induce fibroblast proliferation. Thirdly, TGFβ-1, which is induced by Ang II in the atrium, increases the collagen I and III but not the fibronectin mRNA content in the atrial myocardium and in isolated fibroblasts. These data strongly suggest that Ang II induces collagen mRNA synthesis in the adult human heart by indirect pathways that may involve TGFβ-1 and osteopontin. Since TGFβ-1 increases collagen I and III, but not fibronectin mRNA expression, other factors may contribute to the induction of fibronectin.

4.1 Direct and indirect effects of Ang II and TGFβ-1
In the human myocardial strips, Ang II upregulated TGFβ-1 and osteopontin mRNA. This is comparable to rat cardiac fibroblasts where Ang II upregulated TGFβ-1 mRNA for 24–48 h [34,35] and led to an increase in TGFβ-1 activity and protein levels [7,8]. Therefore, we postulated that Ang II also increased TGFβ-1 protein in the human heart and used an ELISA to estimate the total (latent and active) TGFβ-1 protein content. In our pooled samples, we measured 250 pg TGFβ-1/mg protein (i.e. approximately 40 ng TGFβ-1/g wet weight) in control atrium and 290 pg/mg protein after stimulation with Ang II, which suggested that the upregulation observed at the mRNA level was paralleled by an increase of TGFβ-1 protein content (unpublished results). The TGFβ-1 concentrations in the human myocardium were lower than in the normal rat heart (5 ng/mg protein) [13], but, given a ratio for latent to active TGFβ-1 of 4:1, they are comparable to those that we and others used for stimulation experiments (10 ng/ml).

Both the atrial myocardium and the isolated fibroblasts respond similarly to Ang II by an upregulation of TGFβ-1 but not of collagen mRNA. Furthermore, both respond to TGFβ-1 by an upregulation of collagen I and III mRNA, which is in partial agreement with animal models [36,37]. This supports the hypothesis that TGFβ-1 is involved in Ang II-mediated collagen synthesis in the human heart. Possible signalling pathways leading to TGFβ-1 induction include the activation of the MAP kinases ERK (extracellular signal-regulated kinases) 1 and 2.

The effects of Ang II on osteopontin mRNA levels have also been shown in the rat heart, cardiac fibroblasts and arteries [16,17,38,39] and may be mediated by protein kinase C (PKC) activation. Our data demonstrate for the first time that, in the human heart, osteopontin expression is also increased by Ang II. Whether its function is restricted to the control of fibroblast proliferation and fibroblast–matrix interactions [16,40] or whether it also affects collagen or fibronectin synthesis remains to be elucidated.

A direct effect of Ang II on collagen mRNA synthesis, as has been suggested in rat heart cells [2,4,5,41], could be largely excluded in the adult human heart. This is in agreement with a recent study in a rat cell co-culture model in which a myocyte–fibroblast crosstalk seemed to be necessary for the induction of collagen synthesis by Ang II [42]. Our data fit well within the hypothesis that myocardial Ang II contributes to the phenotype switch of fibroblasts into myofibroblasts, which is followed by the secretion of TGFβ-1 and the induction of collagen and tissue metalloproteinases [43].

4.2 Ang II receptor expression as a prerequisite for the biological effects of Ang II
Ang II receptor expression was determined in both models since it is a prerequisite for the biological functions of Ang II and differs considerably between species. AT1 and AT2 mRNAs were both expressed in the atrial samples, as reported earlier [27]. Previously, we found that the Ang II receptor (ATR) mRNA content in the human myocardium parallels the ATR protein expression [30]. In the tissue samples, AT1 mRNA was upregulated after 1 h of Ang II stimulation. This may be explained by an activation of PKC and MAP kinases, which activate the AT1 promoter via AP-1 and PEA3 (polyoma virus enhancer activator 3) cis-acting DNA elements [44]. This effect is of short duration since PKC is rapidly downregulated after stimulation with Ang II. The upregulation of AT2 in parallel with AT1 may be due to a transcriptional activation of the AT2 promotor via its numerous PEA3 elements [45]. In the fibroblasts, AT1 mRNA was downregulated after 24 h of Ang II exposure as has also been reported for rat cells [46,47]. We hypothesize that the initial time window prior to AT1 downregulation was sufficient to mediate the effects of Ang II and to initiate the signalling cascades that led to TGFβ-1 synthesis. AT2 mRNA was not detected in the isolated fibroblasts although it was present in the tissue samples. Downregulation of AT2 in primary cell cultures by growth factors is a well known phenomenon, which impedes the investigation of this receptor [48].

4.3 Model characteristics
In the first protocol to study the effects of Ang II and TGFβ-1 in strips of human atrial myocardium independently of mechanical load, the samples were not stretched. Isolated muscle strips may stay alive in organ baths for several hours without signs of disintegration or cell death [49], and we found no alterations in energy charge or expression of the genes of interest in our controls. With the short stimulation time of 4 h, we concentrated on the direct effects of Ang II, since this time period was considered too short for complete de novo synthesis and stimulation of their respective receptors by secondary mediators like TGFβ-1 and osteopontin. Therefore, interactions between the different cell types within this multicellular system as well as fibroblast proliferation probably did not account for the observed results. In the second protocol, the long-term effects of Ang II and TGFβ-1 were assessed in cultured cardiac fibroblasts. As in previous investigations [21], fibroblast proliferation was induced by Ang II, which indicated that the cells responded to Ang II although it had no effect on the collagen mRNA content. In previous studies, we did not observe any differences between fibroblasts from atria and ventricles or different patient groups (unpublished), although proliferation was more pronounced in fibroblasts from younger donors and an induction of collagen mRNA by Ang II was only seen in cells from a two-year-old child. However, differences between different passages from the same donor were found. Therefore, only cells from passage one were used in this study. Fibroblasts from six end-stage failing adult hearts and one unused donor heart responded comparably. The end-stage failing hearts are characterized by altered calcium handling, β-adrenergic and Ang II receptor downregulation, cytosceletal disorganization, alterations in energy metabolism and in contractile and sarcomeric proteins (for review, see [50]). The increased amount of fibrous tissue in these hearts suggests that the number and activity of fibroblasts is increased. However, in our study, all end-stage failing hearts as well as the donor heart homogeneously lacked a direct inducibility of collagen and fibronectin mRNA by Ang II. Therefore, this lack of response may not be due to end-stage heart disease but may reflect an inherent quality of the adult human cardiac fibroblasts.

4.4 Effects of medical therapy
Although the number of specimens used in protocol 1, in which different forms of medical therapy might have affected the results, was small, an interesting observation was made: patients treated with ACE inhibitors exhibited a trend towards higher basal AT1, AT2 and osteopontin mRNA expression and lower TGFβ-1 and collagen I mRNA expression. This may have been caused by chronically reduced tissue Ang II levels that led in the long term to an upregulation of Ang II receptors, but a reduction of the Ang II-stimulated TGFβ-1 and collagen expression.

4.5 Limitations of the study
We concentrated on Ang II-regulated gene expression at the level of the respective mRNAs. Large effects on the protein levels were not expected in the atrial samples after 4 h and were therefore not analyzed. We realize that a correlation between mRNA and protein expression does not necessarily exist, although animal experiments for TGFβ-1 and osteopontin [7,39] and the preliminary results of our TGFβ-1 protein determinations support a parallel regulation. In contrast, collagen protein turnover is regulated by several parameters, which include tissue metalloproteinases. Therefore, the upregulation of collagen mRNA by TGFβ-1 is not necessarily accompanied by an increase in collagen protein.

4.6 Conclusions
In atrial tissue and in isolated fibroblasts from the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFβ-1 and osteopontin mRNA levels, and, according to preliminary results, also TGFβ-1 protein. Since TGFβ-1 induces collagen I and III mRNA in the human atrium and in cultured cardiac fibroblasts, and osteopontin is known to enhance cardiac fibroblast proliferation, these factors may represent necessary mediators for the effects of Ang II in the human heart.

Time for primary review 21 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This study was partially supported by the Deutsche Forschungsgemeinschaft, DFG, Re 662/2-2. We thank Ernst Wellnhofer for the statistical calculations, Joscha Buckendahl, Britta Hannack and Bernhard Kruedewagen for expert laboratory work and Tonie Derwent for editorial assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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P. J Lijnen, V. V Petrov, and R. H Fagard
Angiotensin II-induced stimulation of collagen secretion and production in cardiac fibroblasts is mediated via angiotensin II subtype 1 receptors
Journal of Renin-Angiotensin-Aldosterone System, June 1, 2001; 2(2): 117 - 122.
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