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(Circulation. 1997;96:3954-3962.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin Type 2 Receptors Are Reexpressed by Cardiac Fibroblasts From Failing Myopathic Hamster Hearts and Inhibit Cell Growth and Fibrillar Collagen Metabolism

Naohiko Ohkubo, MD; Hiroaki Matsubara, MD, PhD; Yoshihisa Nozawa, PhD; Yasukiyo Mori, MD, PhD; Satoshi Murasawa, MD, PhD; Kazuhisa Kijima, MD; Katsuya Maruyama, MD; Hiroya Masaki, MD, PhD; Yoshiaki Tsutumi, MD; Yoshinobu Shibazaki, MD; Toshiji Iwasaka, MD, PhD; ; Mitsuo Inada, MD, PhD

From the Department of Medicine II, Kansai Medical University, Osaka, and the Pharmacological Laboratory, Taiho Pharmaceutical Co, Ltd, Tokushima (Y.N.), Japan.

Correspondence to Hiroaki Matsubara, MD, Department of Medicine II, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570, Japan. E-mail matsubah{at}takii.kmu.ac.jp

	Abstract

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Background Angiotensin (Ang) II type 1 receptor (AT₁-R) induces cardiomyocyte hypertrophy and fibroblast proliferation, whereas the physiological role of AT₂-R in cardiac remodeling remains poorly defined.

Methods and Results Using Bio14.6 cardiomyopathic (CM) hamsters, we found that AT₂-R sites were increased by 153% during heart failure compared with F1B controls. AT₁-R numbers were increased by 72% in the hypertrophy stage and then decreased to the control level during heart failure. Such differential regulation of AT₂-R and AT₁-R during heart failure was consistent with changes in the respective mRNA levels. Autoradiography and immunocytochemistry revealed that both AT₂-R and AT₁-R are localized at higher densities in fibroblasts present in fibrous regions. Surrounding myocardium predominantly expressed AT₁-R, but the level of expression was less than that in fibrous regions. Cardiac fibroblasts isolated from CM hearts during heart failure but not from control hamsters expressed AT₂-R (30 fmol/mg protein). Using the cardiac fibroblasts expressing AT₂-R, we found that Ang II stimulated net collagenous protein production by 48% and pretreatment with an AT₂-R antagonist, PD123319, evoked a further elevation (83%). Ang II–induced synthesis of fibronectin and collagen type I were enhanced by 40% and 53%, respectively, by pretreatment with PD123319. Ang II–induced DNA synthesis (assessed by [³H]thymidine uptake) was significantly increased by PD123319, and the AT₂-R agonist CGP42112A reduced the serum-stimulated increase in cell numbers by 23%. Treatment with an AT₁-R antagonist, TCV116, for 20 weeks inhibited progression of interstitial fibrosis by 28%, whereas with 44-week PD123319 treatment but not 20-week treatment, the extent of the fibrous region was increased significantly, by 29%.

Conclusions These findings demonstrate that AT₂-R is reexpressed by cardiac fibroblasts present in fibrous regions in failing CM hearts and that the increased AT₂-R exerts an anti–AT₁-R action on the progression of interstitial fibrosis during cardiac remodeling by inhibiting both fibrillar collagen metabolism and growth of cardiac fibroblasts.

Key Words: angiotensin • receptors • remodeling • cardiomyopathy • cells

	Introduction

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Evidence for the presence of a renin-angiotensin system in the heart includes the expression of mRNAs encoding angiotensinogen and renin,¹ ACE,² Ang II receptors,^{3 4 5} and the detection of Ang I and II immunoreactivities.⁶ At least two main Ang II receptor subtypes, AT₁-R and AT₂-R, have been identified by use of receptor subtype–specific antagonists.⁷ Most Ang II functions in the cardiovascular system are mediated by AT₁-R, whereas there is little information regarding the physiological roles of AT₂-R.^{8 9 10} We and others have demonstrated that cardiac AT₁-R is increased in hypertrophy models^{3 11 12} and CM hamsters¹³ or after myocardial infarction.^{5 14 15} Because AT₁-R–mediated signals induce myocyte hypertrophy and proliferation of fibroblasts,^{16 17} it is suggested that increased AT₁-R in such pathological states enhances the action of Ang II. Ichiki et al¹⁹ and Hein et al,²⁰ using mice lacking the AT₂-R, found that this receptor is involved in the responsiveness of the cardiovascular system to Ang II. The AT₂-R has an antiproliferative effect on neointima formation²¹ and on coronary endothelial cells.²² The AT₂-R is abundantly present in fetal tissues^{23 24} and is reexpressed in neointima after vascular injury.²¹ These findings suggest a role of AT₂-R in the cardiovascular system as well as in cellular growth.

The CM Syrian hamster spontaneously develops muscle cell necrosis in the myocardium.²⁵ Cell necrosis later heals with interstitial fibrosis, but recurrent myocytolysis leads to hypertrophy of the remaining myocytes and eventually to failure of the function of these cells. Accumulated evidence analyzing human hearts has reported that both AT₂-R and AT₁-R are expressed in human atria and ventricles and that the distribution ratio of AT₂-R relative to AT₁-R is increased in failing human hearts as a result of downregulation of AT₁-R.^{26 27 28 29} The main aim of this study was to examine the expression pattern, cellular distribution, and physiological actions of Ang II receptors, especially ventricular AT₂-R, on failing hearts in an animal model.

	Methods

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Animals and Treatment With AT₁-R and AT₂-R Antagonists
CM hamsters (Bio 14.6 strain; Bio Breeders, Fitchburg, Mass) were examined at three different developmental stages (8, 24, and 48 weeks). Hamsters at 8, 24, and 48 weeks of age were classified into prehypertrophy, hypertrophy, and heart failure groups, respectively. CM hamsters were compared with age-matched normal hamsters (F1B strain, Bio Breeders). The effects of AT₁-R and AT₂-R antagonists were examined with an AT₁-R antagonist, TCV116, and an AT₂-R antagonist, PD123319. TCV116 was given in 0.5% methyl cellulose by oral gavage (6 mg/kg twice daily: 9 AM and 8 PM); in addition, the drug (10 mg · kg^-1 · d^-1) was included in the drinking water of 4-week-old CM hamsters for 20 weeks. For treatment with PD123319, miniosmotic pumps (2ML4, Alza Corp) were implanted subcutaneously in the back of the neck of 4-week-old CM hamsters, and PD123319 saline solution (20 mg · kg^-1 · d^-1) or vehicle (sterile saline) was delivered through the pumps for 20 or 44 weeks. Miniosmotic pumps were changed to new pumps every 4 weeks.

Quantification of AT₁-R and AT₂-R mRNA Levels
The mRNA levels of AT₁-R and AT₂-R were quantified by RT-PCR as described.^{3 4 5 18} Because the cDNA sequences for hamster AT₁-R and AT₂-R had not been reported, we used PCR primers designed from rat cDNAs. For the AT₁-R, the PCR product was a single band, and sequencing of this product showed a very high similarity with rat AT₁-R. For the AT₂-R, we designed PCR primers from different areas of rat AT₂-R cDNA coding region: sense (5'-CTGACCCTGAACATGTTTGCA-3') and antisense (5'-GGTGTCCATTTCTCTAAGAG-3'). The PCR product was subcloned into the pGEM-T vector (Promega). Sequencing of this product showed a very high similarity with sequences for the rat AT₂-R. To obtain deletion-mutated cRNA (AT₂-R), the subcloned PCR product was cut with Tth 111 I and self-ligated. The cRNA was made by T7 RNA polymerase after it was linearized with Sac I. Total RNA (1 µg for AT₁-R and 4 µg for AT₂-R) and cRNA (0.02 to 0.1 pg) were simultaneously mixed and assayed by RT-PCR in the presence of [³²P]dCTP.¹⁸ [³²P]dCTP counts were determined and expressed as an absolute value on the basis of the added cRNA amounts. As an internal control, GAPDH mRNA levels were also examined by Northern blotting.³⁰ Native AT₁-R and AT₁-R should give 607- and 419-bp DNA fragments, respectively. Native AT₂-R and AT₂-R should give 710- and 430-bp DNA, respectively.

Membrane Preparation and Binding Assay
The left ventricles, including septum and free wall, were dissected and minced with scissors, and membranes were prepared from pooled left ventricles (n=4).^{3 31} Membranes (60 µg of protein) were incubated with ¹²⁵I-[Sar¹, Ile⁸] Ang II in the assay volume of 300 µL for 24 hours at 4°C. K_d, B_max, and K_i values and AT₁-R and AT₂-R densities were calculated by Scatchard analyses and nonlinear least-squares regression analysis on the basis of inhibition by CGP42112A.³¹

Emulsion Autoradiography
Emulsion autoradiography was performed according to the method described by Sechi et al³² and Allen et al.³³ Sections were cut on a cryostat at -20°C, thaw-mounted onto poly-L-lysine–coated slides, and stored at -80°C. Before the assay, the slides were brought to 22°C, and endogenous Ang II bound to Ang II receptors was removed by preincubating the sections for 15 minutes at room temperature in buffer containing 10 mmol/L sodium phosphate (pH 7.4), 150 mmol/L NaCl, 1 mmol/L disodium EDTA, 0.3 mmol/L bacitracin, and 0.2% BSA. This procedure was sufficient to remove endogenous Ang II bound to Ang II receptors when Ang II binding in autoradiography was compared with the results in sections preincubated for 6 minutes at 4°C with acidic buffer (50 mmol/L glycine, 150 mmol/L NaCl, 0.2 mol/L acetic acid, pH 3.0) known to remove ligand binding to membrane receptors.³⁴ Thereafter, the buffer was replaced with fresh buffer containing ¹²⁵I-[Sar¹, Ile⁸] Ang II (0.25 nmol/L, NEN), and the sections were incubated at 16°C for 90 minutes. Preliminary experiments using membranes from left ventricles indicated that the specific binding of ¹²⁵I-[Sar¹, Ile⁸] Ang II under these incubation conditions reached >80% of saturation. Slides were rinsed and dipped in photographic emulsion (Kodak NTB3), exposed at 4°C, developed with Kodak D-19, fixed, and stained with Kernechtrot.

Immunocytochemistry and Histological Examination
Tissue sections were cut on a cryostat at -20°C and fixed in acetone. The following primary antibodies (Sigma) were tested: monoclonal antibodies against vimentin for the detection of fibroblasts, smooth muscle -actin for vascular smooth muscle cells, desmin for cardiomyocytes, and a polyclonal antibody against von Willebrand factor for detection of endothelial cells. Immunocytochemistry was performed with the biotin/avidin system (Elite ABC kit, Vector Laboratories) with diaminobenzidine tetrahydrochloride as a substrate. The hearts were sliced transversely at the midportion, fixed in 10% buffered formalin for 2 days, and embedded in paraffin. Sliced sections were stained with hematoxylin-eosin and von Kossa stain for calcium deposition or with Azan for fibrous regions. The damaged areas of the sections were measured with an image analyzer (model SP-500, Olympus) and expressed as a percentage of total area as described elsewhere.³⁵ The mean value of damaged areas determined from three different parts of each section was used as the degree of myopathic lesions of each hamster.

Preparation and Culture of Cardiac Fibroblasts
Fibroblasts were prepared from pooled ventricles (n=5 to 8) of 48-week-old CM hamsters and age-matched controls according to a modified procedure by Brilla et al.³⁶ Briefly, hearts were perfused via the ascending aorta with Joklik's medium (Gibco) in a Langendorff apparatus. After 5 minutes of perfusion, the perfusate was changed by recirculating Joklik's medium containing 0.1% collagenase and 2% BSA for 30 minutes at a flow of 5 mL/min. Ventricles were removed, minced, and incubated in Joklik's medium with 0.1% trypsin and 0.1% collagenase for 8 minutes and pipetted several times. Dissociated cells were pelleted and seeded onto plates for 2 hours, and attached cells were cultured in 10% FCS DMEM for an additional 48 hours.

Determination of DNA, Protein, and Net Collagenous Protein Production and Cell Growth Assay
DNA and protein synthesis and collagenous protein metabolism were determined according to the method of Crabos et al.³⁷ Fibroblasts were incubated in serum-free medium for 24 hours and cultured for a further 24 hours with 5 µCi/mL of [³H]thymidine (NEN) in the presence or absence of Ang II (0.1 µmol/L). Losartan (1 µmol/L) or PD123319 (1 µmol/L) was added 30 minutes before the addition of Ang II. For protein synthesis, cells were cultured in the presence of Ang II for 48 hours and with inclusion of 1 µCi/mL of [³H]leucine (NEN) for the last 24 hours. At the end of labeling period, the medium was aspirated, and cells were washed with PBS and incubated (30 minutes at 4°C) with 10% perchloric acid. Precipitates were solubilized in 0.3N NaOH/1% SDS for 2 hours and determined by liquid scintillation counter.

For collagenous protein metabolism, cells were serum-depleted for 24 hours and then cultured for a further 24 hours with either 5 µCi/mL of [³H]proline or 25 µg/mL ascorbic acid and without or with inclusion of Ang II (0.1 µmol/L). Two aliquots of supernatant were taken, to which 3 mmol/L CaCl₂, 1 mmol/L PMSF, 5 mmol/L N-ethylmaleimide, and 100 U/mL collagenase (type VII, Sigma) were added to one aliquot and enzyme vehicle (PBS) was added to the second. Samples were incubated for 4 hours at 37°C, and then proteins were precipitated with 10% trichloroacetic acid for 30 minutes at 4°C. After centrifugation, pellets were washed with 10% trichloroacetic acid and solubilized in 0.3N NaOH/1% SDS. Radioactivities in protein pellets from PBS- and collagenase-treated supernatants represent total and noncollagenous protein, respectively. [³H]proline uptake into collagenous proteins (collagenase-sensitive [³H]proline) was calculated by subtracting collagenase-resistant [³H]proline from total uptake. For determining cell number, cells (1x10⁵ cells/well) were serum-deprived for 24 hours, then incubated in DMEM with 1% FCS in the presence and absence of CGP42112A with or without Ang II receptor antagonists for 72 hours (compounds were added every 24 hours). Thereafter, cells were counted with a hemocytometer.

[³⁵S]Methionine Labeling of Cells and Immunoprecipitation
Fibroblasts were made quiescent as described above. After 36 hours of incubation with or without Ang II (0.1 µmol/L), 100 µCi/mL of [³⁵S]methionine was added for another 12 hours. For immunoprecipitation, aliquots (100 µL) of the medium were diluted with 900 µL of RIPA buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS) and treated with normal rabbit serum (10 µL) to remove nonspecific binding, rabbit antibodies to fibronectin (Telios Pharmaceuticals), and collagen type 1 (Chemicon International) for 16 hours at 4°C. Immune complexes were removed by protein A-Sepharose beads (Pharmacia LKB). Beads were washed four times with RIPA buffer, and immunoprecipitates were analyzed by SDS-PAGE.¹⁸

Reagents and Statistical Methods
All reagents were purchased from Sigma Chemical Co unless otherwise indicated below. TCV116 and its active form CV11974 and losartan were provided by Takeda Chemical Industries and by DuPont Merck Pharmaceutical, respectively. PD123319 and CGP42112A were provided by Parke-Davis Warner-Lambert Co and Neosystem, respectively. Results are expressed as mean±SEM. ANOVA and Fisher's protected least significant difference were used for comparisons, with P<.05 considered significant.

	Results

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Changes in Ventricular Weight of CM Hamsters
CM hamsters in the heart failure stage had significantly greater ventricular weight than the controls (Table 1). The ratio of heart weight to body weight was increased by 17% and 31% in the hypertrophic and heart failure stages, respectively, whereas there was no significant difference in the prehypertrophy stage.

View this table: [in this window] [in a new window]   Table 1. Changes in Ventricular Weight of Cardiomyopathic and F1B Control Hamsters

AT₂-R mRNA Level Showed a Greater Enhancement During Heart Failure Than in the Hypertrophy Stage
Calculated absolute values of AT₁-R and AT₂-R mRNAs in control F1B hamsters during prehypertrophy and hypertrophy stages were 60±9.6 and 7.8±0.9 fg/µg total RNA, respectively (Fig 1). Whereas the AT₁-R mRNA level was significantly increased (60%) in the hypertrophic stage, a lesser increase (23%) was observed during heart failure (Fig 1A). AT₂-R mRNA levels were significantly increased in hypertrophy (36%) and heart failure (112%) stages compared with those in controls (Fig 1B).

View larger version (85K): [in this window] [in a new window]   Figure 1. Competitive RT-PCR analyses of changes in ventricular AT1-R (A) and AT2-R (B) mRNA levels during progression of cardiomyopathy. Total RNA (1 µg for AT1-R and 4 µg for AT2-R) and deletion-mutant cRNA were simultaneously mixed and assayed by competitive RT-PCR in presence of [32P]dCTP. PCR products were loaded onto 2% agarose gel and exposed to film for 1 day for AT1-R and 4 days for AT2-R. [32P]dCTP counts incorporated in AT1-R and AT2-R signals and in respective cRNA signals were determined and expressed as absolute values on the basis of added amount of standard cRNA. Northern blot signals of GAPDH mRNA as an internal control (7 µg of total RNA) revealed that comparable amounts of total RNA were analyzed in RT-PCR assays. Values given are mean±SEM. F1B indicates F1B control; Bio, Bio 14.6 CM hamsters; and W, weeks. *P<.05, **P<.01 vs age-matched F1B controls.

Changes of AT₁-R and AT₂-R at the Protein Level Correspond to Regulation at the mRNA Levels
Ang II binding to ventricular membranes appeared to be saturable, and Scatchard plots revealed the presence of a single class of high-affinity Ang II binding sites, as observed in rats.^{3 5} K_d values (nmol/L, n=4) of control or CM hamsters were 1.0±0.14 and 0.95±0.26 at 8 weeks, 1.1±0.26 and 0.96±0.53 at 24 weeks, and 0.98±0.26 and 0.98±0.44 at 48 weeks, respectively. Ang II binding to membranes from 48-week-old CM hamsters was saturable in the presence of 0.1 µmol/L of PD123319 (K_d=0.97±0.25 nmol/L, n=4) or losartan (K_d=1.0±0.23 nmol/L, n=4), suggesting that either binding to AT₁-R and AT₂-R was saturable. Total Ang II receptor densities were increased in hypertrophic (66%) and heart failure (38%) stages compared with those in controls (Fig 2). Competition experiments indicated the presence of two classes of binding sites with K_i values of 11.6±4.1 nmol/L and 4.0±0.72 µmol/L for losartan and 0.14±0.03 nmol/L and 0.60±0.28 µmol/L for CGP42112A in membranes from 48-week-old CM hamsters. AT₂-R sites in 8-week-old CM hamsters did not differ from those of controls, whereas at 24 and 48 weeks, the AT₂-R sites increased by 57% and 153%, respectively (Fig 2). AT₁-R numbers also increased (72%) in 24-week-old CM hamsters, whereas they decreased to the control level in 48-week-old CM hamsters.

View larger version (40K): [in this window] [in a new window]   Figure 2. Changes in ventricular Ang II receptor densities during progression of cardiomyopathy. Total Ang II receptor densities were determined by Scatchard plots, and AT1-R and AT2-R densities were calculated by nonlinear least-squares regression analysis on the basis of inhibition by CGP42112A. Membrane fractions from pooled ventricular samples (n=4) were used for assays. Specific 125I-[Sar1 Ile8] Ang II binding was determined experimentally as the difference between total and nonspecific binding in parallel assays in absence and presence of 3 µmol/L Ang II. The values are mean±SEM of experiments using four groups of separate pooled ventricular samples from myopathic or F1B control. *P<.05, **P<.01 vs age-matched F1B controls. Abbreviations as in Fig 1.

AT₂-R Expression is Localized in Regions With Interstitial Fibrosis
Staining with hematoxylin-eosin distinguished regions with interstitial fibrosis (indicated by F in Fig 3, panel A1) from surrounding myocardium. Binding sites for Ang II indicated by the presence of silver grains were localized at higher densities in the fibrous regions (Fig 3, panel B1), and the degree of Ang II receptor expression in this region was comparable to that in vessels (indicated by V in Fig 3, panel A2). Experiments using selective antagonists revealed that binding throughout myocardium was uniformly inhibited by saralasin (Fig 3, panels C1 and C2) and an AT₁-R antagonist CV11974 (Fig 3, panels D1 and D2). In contrast, PD123319 preferentially inhibited binding in fibrous regions rather than in myocardium (Fig 3, panel E1), and binding in fibrous regions was also suppressed to a similar extent by CV11974 (Fig 3, panel D1). Binding in the vessels penetrating the myocardium was inhibited by both saralasin (Fig 3, panel C2) and CV11974 (Fig 3, panel D2) but not by PD123319 (Fig 3, panel E2). These findings indicated that AT₂-R is highly concentrated in fibrous regions in which AT₁-R is also expressed, whereas myocardium expresses mainly AT₁-R, but to a lesser extent. The fibrous regions contained collagen fibers, shown in blue by Azan staining (indicated by arrows in Fig 4, panels 1 and 2). To identify the cell types in this region, we stained the sections with antibodies against vimentin, desmin, smooth muscle -actin, and von Willebrand factor (for endothelial cells). Cells were positive for vimentin (Fig 4, panel 3) but were negative for other cell markers (data not shown), whereas cells surrounding this region were positive for desmin (Fig 4, panel 4), suggesting that the majority of the cells present in fibrous regions are fibroblasts.

View larger version (102K): [in this window] [in a new window]   Figure 3. Cellular localization by emulsion autoradiography of Ang II receptor subtypes in myopathic ventricle during heart failure. Adjacent sections were stained with hematoxylin-eosin or were incubated with 125I-[Sar1, Ile8] Ang II (0.25 nmol/L), dipped in photographic emulsion, developed, fixed, and then stained with Kernechtrot. A1 and A2, Hematoxylin-eosin staining showing presence of fibrous regions (F) or vessels penetrating myocardium (V). B1 and B2, Sections were incubated with 125I-[Sar1, Ile8] Ang II alone without antagonist pretreatment. Ang II binding sites are represented by distribution of silver grains (black dots). Amounts of silver grains in (F) and (V) were greater than those in surrounding myocardium. C1 and C2, Sections pretreated with saralasin (50 nmol/L) were incubated with 125I-[Sar1, Ile8] Ang II. Deposition of silver grains was completely inhibited. D1 and D2, Sections pretreated with AT1-R antagonist CV11974 (50 nmol/L) were incubated with 125I-[Sar1, Ile8] Ang II. Deposition of silver grains in surrounding myocardium and (V) was strongly inhibited, whereas binding in (F) was blocked to a lesser extent. E1 and E2, Sections pretreated with AT2-R antagonist PD123319 (50 nmol/L) were incubated with 125I-[Sar1, Ile8] Ang II. Deposition of silver grains in (F) was inhibited compared with that in Fig B1 but not in surrounding myocardium or (V). Exposure time was 7 days.

View larger version (111K): [in this window] [in a new window]   Figure 4. Immunocytochemical analysis of myopathic hearts during heart failure (A) and detection of AT2-R in cardiac fibroblasts by emulsion autoradiography (B). A, Tissue sections including regions with interstitial fibrosis were stained with Azan, and adjacent sections were analyzed by fluorescence immunocytochemistry with biotin/avidin-immunoperoxidase system. In Azan-stained sections, collagen fibers were blue (1 and 2), as indicated by arrows. Adjacent sections were stained with monoclonal antibodies against vimentin (3) and desmin (4). Cells present in fibrous regions were positive for vimentin, and surrounding myocardium was positive for desmin. B, Primary cultures of cardiac fibroblasts isolated from CM hamsters in heart failure state were incubated for 2 hours at 24°C in serum-free DMEM with 125I-CGP42112A alone (0.2 nmol/L) (2200 Ci/mmol, NEN) (1) or after pretreatment for 30 minutes with PD123319 (1 µmol/L) (2). Cells were dipped in photographic emulsion, developed, fixed, and then stained with Kernechtrot. Presence of AT2-R was shown by black dots, which were blocked by pretreatment with PD123319. Exposure time was 10 days.

Cardiac Fibroblasts Isolated From CM Hamsters Express Both AT₂-R and AT₁-R
To confirm the presence of AT₂-R in cardiac fibroblasts and its potential function, we prepared primary cultures of fibroblasts from CM hamsters in the heart failure state. Saturation binding experiments using ¹²⁵I-[Sar¹, Ile⁸]–Ang II revealed that B_max (fmol/mg protein) and K_d (nmol/L) values (n=6) for Ang II receptors were 125.5±8.1 and 0.63±0.14, respectively, in cells from CM hamsters and 42.7±4.8 and 0.64±0.13 in cells from control hamsters, indicating that densities of Ang II receptors present in fibroblasts are higher (P<.01) in CM than in normal hamsters.

Nonlinear least-squares regression analysis indicated two classes of binding sites, with K_i values of 0.14±0.04 nmol/L and 0.57±0.16 µmol/L for CGP42112A (n=6) and 11.1±3.8 nmol/L and 4.6±0.61 µmol/L for losartan (n=6), and the relative ratio of AT₁-R/AT₂-R was calculated to be 75±4.7/24±3.2%. AT₁-R and AT₂-R numbers were 94±5.1 and 30±3.7 fmol/mg protein, respectively. Fibroblasts from control hamsters had a single class of binding sites, with K_i values of 16.4±1.3 nmol/L for losartan and >1000 nmol/L for CGP42112A (n=6). The expression of AT₂-R was further confirmed by autoradiography using ¹²⁵I-CGP42112A. Most of cells had binding sites for the ligand (Fig 4A) that were blocked by pretreatment with PD123319 (Fig 4B), indicating that the majority of cells in primary culture substantially express AT₂-R.

AT₂-R Exerts Inhibitory Effects not Only on DNA Synthesis and Cell Growth but Also on Protein Synthesis and Fibrillar Collagen Metabolism
Effects of Ang II mediated through both AT₁-R and AT₂-R caused increases in [³H]thymidine (19%) (Fig 5A) and [³H]leucine (27%) (Fig 5B) uptake. Blockade of AT₂-R by PD123319 (1 µmol/L) further elevated these uptake rates, which were increased by 33% and 49% over the control values. When cells were pretreated with losartan, addition of Ang II elicited a decrease (16%) in [³H]thymidine uptake, whereas the rate of [³H]leucine uptake was significantly reduced (20%) to below the basal level. We further examined AT₂-R–mediated effects on cell growth by counting the cell numbers. Cells (1x10⁵ cells/well) were serum-deprived for 24 hours and then incubated in medium with 1% FCS in the presence and absence of CGP42112A. Cell numbers in the absence of CGP42112A were increased by 433±19% over a period of 72 hours with 1% FCS. After cells were incubated with 1% FCS for 72 hours in the presence of the AT₂-R agonist CGP42112A at 0.1 µmol/L, the cell number was 73% to 78% of that in the absence of CGP42112A. This change was blocked by PD123319 but not losartan (each 5 µmol/L) (Fig 5C). The effects of AT₂-R on net collagenous protein production were examined by measurement of [³H]proline uptake (Fig 5D). Ang II stimulated uptake of collagenase-sensitive [³H]proline by 48% relative to the basal level, and pretreatment with PD123319 evoked a further elevation (by 83%), whereas exposure to losartan reduced (by 21%) the uptake to below the basal level.

View larger version (52K): [in this window] [in a new window]   Figure 5. Effects of Ang II on DNA (A), protein (B), cell growth (C), and net production of collagenous protein (D) in cardiac fibroblasts. Levels of incorporation of [3H]thymidine into DNA, [3H]leucine into protein, and [3H]proline into collagenous protein were examined after stimulation of cardiac fibroblasts with Ang II (0.1 µmol/L) in presence or absence of losartan (1 µmol/L) and PD123319 (1 µmol/L). All experimental details are described in "Methods." Increases in collagenase-sensitive [3H]proline incorporation are expressed relative to uptake rates of controls without Ang II. For determination of cell number, cells (1x105 cells/well) were cultured in serum-free DMEM for 24 hours, then incubated in DMEM with 1% FCS for 72 hours with CGP42112A (0.1 µmol/L) in presence or absence of losartan (1 µmol/L) and PD123319 (1 µmol/L). Values represent mean±SEM of six (A, B, D) and four (C) separate experiments, with duplicate determinations in each experiment. CTL indicates control without Ang II. *P<.05, **P<.01 vs controls without addition of Ang II.

AT₂-R Inhibits Synthesis of Matrix Components in Cardiac Fibroblasts
Because it might be possible that collagenase and trichloroacetic acid used in [³H]proline uptake rates contains nonspecific proteases and precipitates small fragments, affecting the evaluation of fibrillar collagen metabolism, we further examined the effect of Ang II on major fibrillar collagenous protein.^{36 37} Immunoprecipitated fibronectin and collagen type 1 secreted into medium were increased by 0.1 µmol/L Ang II to 44% and 60% over the control levels, and pretreatment with PD123319 induced further increases, while losartan reduced the levels by 23% and 28% to below the control levels (Fig 6).

View larger version (48K): [in this window] [in a new window]   Figure 6. Effect of Ang II on cardiac fibroblast synthesis of extracellular matrix components. Cardiac fibroblast cultures incubated in presence of Ang II with or without pretreatment by PD123319 or losartan were radiolabeled with [35S]methionine. Culture supernatants were used for immunoprecipitation of (A) fibronectin and (B) collagen type I. Signals for 220-kD fibronectin and for 190-kD of collagen type I in SDS-PAGE were measured by densitometry, and a value in control was arbitrarily expressed as 1 unit. Values represent mean±SEM of three separate experiments. Cont. indicates control. *P<.05, **P<.01 vs controls without addition of Ang II.

Long-term Treatment With AT₁-R Antagonist Prevents Progression of Interstitial Fibrosis, Whereas AT₂-R Antagonist Increases the Extent of Fibrous Regions
Treatment of 4-week-old CM hamsters with the AT₁-R antagonist TCV116 for 20 weeks significantly reduced the ratio of ventricle weight to body weight and progression of regions with interstitial fibrosis or calcification (Table 2 and Fig 7, panel 2). The degree of interstitial fibrosis or focal necrosis was not affected by 20-week PD123319 treatment, whereas the extent of fibrous regions and calcification was significantly increased (29% and 40%) by 44-week treatment (Table 2 and Fig 7, panel 3). The expression of AT₂-R was increased in parallel with the development of heart failure, whereas that of AT₁-R was downregulated in heart failure (Fig 2). Such a differential expression pattern of AT₁-R and AT₂-R during the progression of heart failure may account for a difference in the duration necessary to induce significant cardiac effects between TCV116 and PD123319. The dose of PD123319 used here resulted in a plasma drug level that was sufficient to selectively block AT₂-R but had no effect on AT₁-R in rats.²¹ Although we could not measure the plasma level of PD123319 in hamsters, it was likely that the drug level was in an effective range to block AT₂-R, in light of the fact that treatment with PD123319 resulted in an action opposite to that of TCV116 on the extent of fibrous regions. We also examined the influence of invasive treatment using osmotic minipumps. Ventricular weight and areas of necrosis, fibrosis, and calcification in untreated hamsters were similar to those in hamsters treated with vehicle through osmotic minipumps (Table 2), indicating that the influence of treatment by minipumps was negligible.

View this table: [in this window] [in a new window]   Table 2. Changes in Ventricular Weight and Myopathic Lesions After Treatments With AT1-R and AT2-R Antagonists

View larger version (145K): [in this window] [in a new window]   Figure 7. Microscopic analysis of effects of AT1-R and AT2-R antagonist on cardiomyopathic lesions. Four-week-old CM hamsters were treated with TCV116 for 20 weeks (2) or with PD123319 for 44 weeks (3). Ventricular sections from treated hamsters were stained with Azan and compared with those in vehicle-treated CM (1) or control hamsters (4) at 48 weeks of age. Representative results are shown, indicating that focal lesions with interstitial replacement fibrosis are inhibited by treatment with TCV116 and increased by treatment with PD123319.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we found that the expression of AT₂-R in left ventricles was increased in the heart failure stage and that the AT₂-R was mainly localized in fibroblasts present in fibrous regions rather than in myocardium. We also found that cardiac fibroblasts isolated from CM hamsters express AT₂-R at high levels. These findings suggest that fibroblasts are the major cell type expressing AT₂-R in failing CM hearts. Ichiki and Inagami³⁸ showed that the binding sites for C/EBP and NF-IL6 are located in the promoter region of the mouse AT₂-R gene and that interleukin-1ß induces AT₂-R expression in R3T3 fibroblasts,³⁹ suggesting that cytokine production due to myopathic changes is partially involved in the reexpression of AT₂-R. It was also reported that cardiac myocytes produced cytokines when exposed to hypoxic stress such as that which occurs in the myopathic heart.⁴⁰ Taken together with the results showing that AT₂-R is reexpressed during the healing process of skin wounds⁴¹ and that most cells expressing AT₂-R in fetal mesenchymal tissues are undifferentiated fibroblasts,²³ induction of AT₂-R in fibrous regions might be a general feature in pathological processes, during which fibroblasts may dedifferentiate by proliferation stimulus and become more fetus-like in nature.

Accumulated evidence analyzing human hearts has reported that both AT₂-R and AT₁-R are expressed in human hearts and that the distribution ratio of AT₂-R relative to AT₁-R is increased in failing human hearts as result of downregulation of AT₁-R.^{26 27 28 29} It was shown that AT₂-R sites in human atrium during heart failure were localized in the fibrous regions.²⁷ We also found that AT₂-R was increased in fibrous regions of left ventricles in patients with idiopathic cardiomyopathy (N. Ohkubo et al, unpublished data, 1997). These findings in human studies were in good agreement with the present observations in CM hamsters, suggesting that this particular strain of animal could be a good model for studying the physiological roles of cardiac AT₂-R on the human failing hearts. The physiological role of AT₂-R and its signal transduction pathway remain poorly defined. Previous studies showed that AT₂-R had an inhibitory action on DNA synthesis or cell growth.^{21 22} Consistent with these reports, we also confirmed that AT₂-R mediated inhibitory effects on DNA synthesis and cell growth in cardiac fibroblasts. In addition, we provided the first evidence that AT₂-R suppressed synthesis and secretion of major matrix components such as fibronectin and collagen type 1. These findings suggest that cardiac fibroblasts acquire the ability to inhibit self-growth and fibrillar collagen metabolism by reexpressing AT₂-R. Overall metabolism of fibrillar collagens is dependent on the balance between the rate of synthesis and degradation by collagenases.⁴² Brilla et al³⁶ reported that AT₂-R decreased collagenase activity in fibroblasts isolated from normal adult rat hearts, resulting in increased collagen synthesis, whereas they did not identify the existence of AT₂-R in the cells, and adult rat cardiac fibroblasts had been shown to express only AT₁-R but not AT₂-R.^{42 43} In addition, previous studies using adult rat cardiac fibroblasts reported that AT₁-R but not AT₂-R decreased collagenase activity⁴² and that AT₂-R exerted no effect on DNA and protein synthesis.³⁷ Thus, AT₂-R action on collagenase activity in cardiac fibroblasts expressing AT₂-R remains to be determined.

The present study demonstrated that an increase in heart weight observed in CM hamsters during heart failure was significantly inhibited with TCV116 but not with PD123319 (Table 2). Peripheral signs of heart failure, such as subcutaneous edema or respiratory distress, were also improved with TCV116 but not with PD123319. Nakamura et al⁴⁴ also reported that treatment of CM hamsters with TCV116 significantly improved cardiac contractility assessed by left ventricular dP/dt_max. These findings suggested that the progression of heart failure and deterioration in cardiac function in CM hearts were prevented by an AT₁-R antagonist, whereas AT₂-R–mediated effects on cardiac function seemed to be smaller. Treatment with the AT₁-R antagonist has been reported to cause an increase in plasma Ang II level,⁴⁵ which in turn preferentially binds to AT₂-R, indicating that inhibition of fibrosis by the AT₁-R antagonist results from the additive effects of AT₁-R blockade and AT₂-R stimulation. Although treatment with PD123319 had the effect expected from in vitro study with cardiac fibroblasts, such as enlargement of fibrous regions by blocking the inhibitory effect of AT₂-R on cell growth and collagen synthesis, the effect was much weaker than its in vitro effect on collagen synthesis. Considering that overexpression of AT₂-R causes pronounced inhibitory effects on neointimal formation and DNA synthesis in fetal aortas,²¹ the level of AT₂-R expression may play a key role in the in vivo effects of AT₂-R.

A decrease in Ang II binding sites in myocardium by PD123319 treatment, indicated by a comparison of Fig 3, panel B1 with Fig 3, panel E1, suggests that AT₂-R is present in myocardium at a low level. In contrast, Ang II bindings in myocardium were completely inhibited by CV11974 (Fig 3, panels D1 and D2). Since the K_i value is higher (approximately sevenfold) in CV11974 than in PD123319 (N. Ohkubo et al, unpublished data, 1997), this discrepancy might partially result from the difference in the binding affinities between CV11974 and PD123319. In normal hamster hearts, Ang II binding sites were uniformly distributed throughout myocardium, as observed in CM hamsters, and this binding was completely inhibited by CV11974 but not PD123319 (data not shown). Considering that myocytes isolated from normal neonatal rats express both AT₁-R and AT₂-R^{4 18} and mechanical stretch of myocytes enhances the expression of AT₂-R,¹⁸ these findings raise the possibility that AT₂-R is not expressed in normal myocytes or if it is, the expression is so small as to be undetectable with autoradiography and that during cardiac remodeling, its expression is locally increased in certain regions, such as subendomyocardium, in response to an increase in intraventricular pressure.

Recently, Lambert et al¹³ reported that AT₂-R protein was not detected in failing hearts of different strains of CM hamster (CHF146 strain). In contrast, we confirmed the presence of AT₂-R at the mRNA, protein, and cellular levels and found that the extent of fibrous region played a role in the expression level of AT₂-R. Given that the cardiac AT₁-R level of CHF146 strain was higher than the control at all developmental stages, a difference in extent of fibrous regions in CM lesions or in the genetic background might partly account for this discrepancy.

The findings in this study have important implications for the predicted actions of AT₁-R antagonists. Accumulated evidence indicated that cardiac AT₂-R was increased in cardiac remodeling^{3 5} and that the distribution ratio of AT₂-R was increased by downregulation of AT₁-R in failing human hearts.^{26 27 28 29} Because circulating Ang II levels are increased by treatment with AT₁-R antagonists⁴⁵ and Ang II preferentially binds to cardiac AT₂-R, AT₂-R–mediated actions are expected to be further activated under these pathological conditions. This study demonstrates that AT₂-R stimulation has a novel cardioprotective effect, such as inhibition of fibrillar collagen synthesis. Because blockade of the renin-angiotensin system is essential for the management of patients with cardiovascular diseases,⁴⁶ it is expected that AT₁-R antagonists are as widely used for these patients as ACE inhibitors. Although it is not clear whether the findings obtained from the particular strain of CM hamster are relevant to the general problem of cardiomyopathy, this novel cardioprotective effect of AT₂-R should be considerable and confirmed in clinical settings.

	Selected Abbreviations and Acronyms

 

Ang = angiotensin AT1-R, AT2-R = Ang II receptor subtypes CM = cardiomyopathic PCR = polymerase chain reaction RT = reverse transcription

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science, and Culture, Japan; the Study Group of Molecular Cardiology; and the Japan Medical Association and Japan Smoking Foundation.

Received May 23, 1997; revision received August 14, 1997; accepted August 27, 1997.

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