Angiotensin II (Ang II) and transforming growth factor-β1 (TGF-β1) are important growth-regulating factors, both in normal heart development and in hypertrophy resulting from systemic hypertension.1 Pressure-overloaded hypertrophy is characterized by increases in the accumulation of collagen and other extracellular matrix proteins and by increases in myocyte size and ventricular wall thickness.2 It is also well known that hypertension may lead to myocardial fibrosis, even before the development of myocardial hypertrophy, and that this process will impair left ventricular function, especially during diastole. Myocardial fibrosis is also an important contributor to ventricle dysfunction in chronic heart failure.
Ang II stimulates the contraction of adult rat cardiac fibroblasts plated in a 3-dimensional collagen gel lattice.3 The effects of Ang II on the collagen gel contraction in cardiac fibroblasts are Ang II type 1 (AT1)-receptor mediated because they are abolished by the AT1-receptor antagonist telmisartan and not by the AT2-receptor antagonist PD 123,319.3,4 During contraction, floating collagen matrices develop into a resting or mechanically relaxed tissue whose cells have morphologic and proliferative features resembling scar.5
In cultures of adult rat cardiac fibroblasts we have also shown that TGF-β1 is able to induce the appearance of angiotensin-converting enzyme (ACE),6 to differentiate fibroblasts into myofibroblasts,6,7 and to stimulate the contraction of cardiac fibroblasts.8 Cultures of adult rat cardiac fibroblasts contain a lisinopril-dependent activity that is obviously ACE activity and a lisinopril-independent activity or ACE-like activity that was performed by peptidase(s) different from ACE.6 ACE constitutes only 30% of the total activity.6 Recently, Petrov et al9,10 have also reported an induction of various aminopeptidases in cardiac fibroblasts by TGF-β1. The collagen production in basal and TGF-β1-treated cardiac fibroblasts is also reduced by bestatin, an aminopeptidase inhibitor with broad specificity,11 and by the ACE inhibitor lisinopril.11
The aim of the present study was to determine whether aminopeptidase inhibition could affect the Ang II-stimulated collagen contraction in control cardiac fibroblasts and in TGF-β1-treated cardiac fibroblasts or myofibroblasts.
MATERIALS AND METHODS
Collagen type I from rat tail was purchased from Collaborative Biomedical Products (Bedford, MA). Angiotensin II was obtained from Peninsula Laboratories (Belmont, CA). Tritiated water (37 MBq/mL) was purchased from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). Dulbecco Modified Eagle Medium (DMEM), ×10 concentrated MEM, streptomycin/penicillin, fetal bovine serum (FBS), ethylenediaminetetraacetic acid, and cellware were purchased from Gibco BRL (Life Technologies Ltd, Paisley, UK); Joklik medium, bestatin, leuhistin, and arphamenine A were obtained from Sigma Chemical Co (St Louis, MO), and collagenase A was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Transforming growth factor-β1 was obtained from Becton Dickinson Immunocytochemistry (San José, CA).
Isolation of Cardiac Fibroblasts
All animal procedures were in accordance with the laws, regulations, and administrative provisions of the Member States of the European Community (Council Directive 86/609/EEC of November 24, 1986) regarding the protection of animals for experimental and other scientific purposes. This research protocol was also approved by the Ethical Committee for Animal Experiments of the Katholieke Universiteit Leuven (Leuven, Belgium).
Cardiac ventricular fibroblasts were obtained from male Wistar rats, 7 to 8 weeks old, and weighing about 200 g, by a slightly modified isolation procedure as described by Brilla et al.12 In brief, rats were heparinized (625 units/100 g body weight) and anesthetized intraperitoneally with Nembutal, 50 mg/100 g body weight. The heart was removed and immediately placed in sterile Joklik medium. The extirpated hearts were allowed to eject the residual blood, and the aorta was connected with a cannula. A syringe was filled with a few milliliters of basic salt solution containing (mmol/L) NaCl 130, KCl 3, KH2PO4 1.2, MgSO4 1, CaCl2 1.25, glucose 10, N-(2-hydroxyethyl)piperazine-2′-(2-ethanesulfonic acid) (HEPES) 10, pH 7.2, and placed in the cannula to rinse the blood out of the heart. The hearts were then perfused via the ascending aorta according to the Langendorff method13 with Joklik medium for 5 minutes, then by recirculating Joklik medium containing 0.02% collagenase A and 2% bovine serum albumin (BSA) for 35 minutes with a flow of 5 mL/min. Thereafter the atria and vessels were cut off, and the ventricular tissue was placed in Joklik medium containing 1% BSA and 0.01% collagenase A for an additional 10 minutes (37°C). The tissue was then minced and filtered through a 200-μm mesh net.
The coarse cell/disintegrated tissue suspension was settled for 15 minutes, and its supernatant was then centrifuged at 350 g for 10 minutes. The pellet was resuspended in DMEM supplemented with 10% FBS and 1% of a penicillin/streptomycin solution and seeded in an 80 cm2 tissue culture flask. The cell cultures were then preincubated at 37°C in humidified air (humidity 95%) with 5% CO2 for 4 hours. The medium with unattached cells was aspirated, and fresh DMEM supplemented with 10% FBS and penicillin/streptomycin added. The cultures were examined daily using phase-contrast microscopy. The medium was replaced every 2-3 days, and the cells were grown to confluence and then passaged with trypsin-EDTA. Under the above-mentioned conditions of isolation, coronary smooth muscle and endothelial cells were already rarely seen in the primary cultures. Myocytes did not survive the isolation method because oxygenation is not provided. In the present study only cardiac fibroblasts from passage 2 were used.
For the second passage, cells were transferred to 6-well dishes in DMEM with 10% FBS at a density of 2600 cells/dish for 3 days until confluency. The cells (control cardiac fibroblasts) were then harvested and washed, and 100,000 cells were added to the hydrated collagen. Another aliquot of cells was transferred to 6-well dishes in DMEM with 10% FBS at a density of 2600 cells/dish. After 24 hours, the medium was replaced with fresh DMEM with 10% FBS containing TGF-β1 (400 pmol/L) and incubated for 6 days. These cells (TGF-β1-treated cardiac fibroblasts or myofibroblasts) were also harvested and washed, and 100,000 cells were added to the collagen gel lattice in 24-well plates.
Collagen Gel Contraction Assay
The contraction of a hydrated collagen lattice by cardiac fibroblasts in serum-free conditions was determined by measuring the gel volume.14,15
BSA Treatment of Wells
An aliquot of 500 μL of 1% BSA in 10 mmol/L phosphate-buffered saline (138 mmol/l NaCl and 2.7 mmol/L KCl), pH 7.4 (PBS), was added to each well, incubated at 37°C for at least 2 hours, and twice washed with PBS.
Preparation of Collagen Solution
Collagen gels were prepared from rat tail collagen type I. A 12.55-mL collagen solution was prepared by mixing 4.25 mL collagen in 0.02 mol/L acetic acid, 1.65 mL 100 mmol/L NaOH, and 1.25 mL × 10 MEM, and 5.4 mL of bidistilled water was added to obtain a final concentration of 1.5 mg/mL collagen. The solution was kept on ice until addition of cells.
Preparation of Fibroblast-Induced Collagen Suspension
Five hundred microliters of this collagen solution was added to each BSA-treated well. Cardiac fibroblasts in DMEM (100,000 cells) were then added to this collagen solution in the wells and incubated for 1 hour for polymerization. Subsequently, 1 mL of DMEM containing no FBS but 1 μCi of tritiated water was added to each well, together with or without the test compounds angiotensin II, bestatin, leuhistin, and arphamenine A. These media were not replenished with these products during the 3 days of incubation.
Preparation of Floating Gels
The gels detached spontaneously from the sides and bottom of the wells and were placed in a tissue culture incubator for 3 days. The medium was then removed, and the radioactivity (dpmmedium) was measured in a liquid scintillation counter (Tri Carb Model, Packard Bioscience Benelux NV, Belgium). The gels were dissolved in 0.5 mL of 1 M NaOH, the samples neutralized with 0.5 mL of 1 mol/L HCl, mixed with 5 mL of scintillation fluid, and counted in a liquid scintillation counter (dpmgel). Gel contraction was determined by calculating the gel volume (expressed in microliters) as dpmgel/dpmmedium × 100.
A decrease in gel volume induced by cardiac fibroblasts corresponds to a stimulation of the contraction of the collagen gel lattice.
Assay of Aminopeptidase Activity
Homogenates of cultured cardiac fibroblasts were prepared by dissolving them in Triton X-100. Therefore, the medium was aspirated, and then 220,000 cells were dissolved in 100 μL of PBS, pH 7.2, containing (mmol/L) 9.3 Na2HPO4, 2.9 KH2PO4, 3 KCl, and 136 NaCl. Ten microliters of Triton X-100 (20%) was added. After centrifugation (10,000 g for 15 minutes) of this suspension, the supernatant was frozen in liquid nitrogen and stored at −80°C before assay. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline at 37°C in a mixture containing 15 μL of 1 mmol/L alanine-p-nitroanilide (AlapNA) or arginine-p-nitroanilide (Arg-pNA) and 60 μL of cell homogenates in PBS buffer with 2% Triton X-100. After 30 and 60 minutes of incubation, the tubes were immediately placed in ice water, and 75 μL of 1 mol/L sodium acetate, pH 4.2, was added to the mixture to terminate the reaction. After centrifugation (12,000 g for 3 minutes at 4°C) 100 μL supernatant was pipetted in a 96-well microplate, and the absorbance at 405 nm read off against a calibration curve of standards of p-nitroaniline (range 0.01-0.5 mmol/L) in a Biorad Microplate Reader Model 550 (Bio-Rad Laboratories, Hercules, CA). The aminopeptidase activity was expressed as nanomoles p-nitroaniline liberated per minute and per 106 cells.
Measurement of DNA Content in the Cardiac Fibroblasts Inoculated in the Gels
After the collagen gels had been cultured with cardiac fibroblasts for 3 days, the medium was aspirated. The gels were solubilized by the addition of 200 μL collagenase A (1 mg/mL in 20 mmol/L HEPES buffer, pH 7.2, containing 150 mmol/L NaCl and 10 mmol/L Na-acetate). After incubation for 1 hour, the cell suspension was pipetted in an Eppendorff tube and centrifuged at 340 g for 5 minutes. The supernatant was discarded, and 50 μL EDTA (2 mmol/L) was added to the pellet. DNA was measured fluorometrically with Hoechst dye 33,258 (bisbenzimide) using an excitation wavelength of 360 nm and an emission wavelength of 460 nm,16 according to the manufacturer's instructions (Bio-Rad Labs.).
[3H]Thymidine Incorporation in the Cardiac Fibroblasts Populated Within the Gels
After the collagen gels had been cultured with cardiac fibroblasts for 2 days, 1 mL [3H]thymidine (1 mCi/mL) per 1 mL of medium was added and further incubated for 18 hours. The medium was then aspirated, and the gels were washed three times with 1 mL of 0.01 M phosphate-buffered saline, NaCl (138 mmol/L), KCl (2.7 mmol/L), pH 7.4, plus 50 μmol/L of thymidine. The gels were then solubilized by the addition of 200 μL collagenase A. After incubation for 90 minutes, the cell suspension was pipetted in an Eppendorff tube and centrifuged at 2000 g for 10 minutes. The supernatant was discarded, 500 μL NaOH (0.25 mol/L) was added to the pellet, and 500 μL of the resuspended material was added to 5 mL of scintillation fluid. The Eppendorff tube was rinsed with 250 μL of NaOH, and the rinsate was also added to the scintillation fluid. The radioactivity was measured in a liquid scintillation counter, and the [3H]thymidine incorporation was expressed as dpm/106 cells.
Values are expressed as mean ± SEM. The statistical methods used were repeated-measures analysis of variance (Tukey) and Student two-tailed test for (un)paired data when appropriate. A probability value of less than 0.05 was considered statistically significant.
Effect of Ang II on Collagen Gel Contraction by Cardiac Fibroblasts
The original gel volume of the hydrated collagen gel in the absence of cardiac fibroblasts averaged 233.5 ± 2.9 μL and decreased to 161.8 ± 16.9 μL in control fibroblasts and to 70.4 ± 3.9 μL in TGF-β1-treated cardiac fibroblasts. As shown in Figure 1, Ang II (100 nmol/L) reduced the gel volume in control and TGF-β1-treated fibroblasts, respectively, by 24.6 ± 2.4% and 17.9 ± 4.1%.
DNA Content and [3H]Thymidine Incorporation in the Collagen Gel Matrix Inoculated by Cardiac Fibroblasts
The DNA content and [3H]thymidine incorporation in the collagen gel lattice populated by cardiac fibroblasts for 3 days averaged 574 ± 33 ng/well and 8360 ± 1320 dpm/106 cells (n = 4), respectively, and was not affected by Ang II.
Stimulation of Aminopeptidase Activity in Cardiac Fibroblasts by TGF-β1
Homogenates of cardiac fibroblasts hydrolyzed Ala- and Arg-pNA and TGF-β1 stimulated, by approximately two- to three-fold, the hydrolytic activity of Ala-pNA and Arg-pNa performed by aminopeptidases (Table 1).
Inhibition of Ala-pNA Hydrolysis
Bestatin, a broad-range aminopeptidase inhibitor, and leuhistin, a specific inhibitor of Ala-aminopeptidase, dose-dependently inhibited the Ala-aminopeptidase activity in control and TGF-β1-treated cardiac fibroblasts (Fig. 2A,B). Complete inhibition of Ala-aminopeptidase activity was obtained at a concentration of 100 μmol/L of leuhistin, whereas bestatin (100 μmol/L) inhibited the hydrolysis of Ala-pNA by 90%.
Arphamenine A, a specific inhibitor of Arg-aminopeptidase, particularly of aminopeptidase B, also dose-dependently inhibited the hydrolysis of Ala-pNa in control and TGF-β1-treated cardiac fibroblasts; arphamenine A at the highest studied concentration of 10 mmol/L inhibited Ala-aminopeptidase up to 15% of the basal level (Fig. 2C). At 100 μmol/L, arphamenine A blocked Ala-aminopeptidase by only 25% in control and TGF-β1-treated fibroblasts.
Inhibition of Arg-pNA Hydrolysis
Bestatin at a concentration of 300 μmol/L inhibited Arg-aminopeptidase up to 10% of the basal level, and TGF-β1 decreased this inhibition (Fig. 3A). Leuhistin inhibited the Arg-pNa hydrolysis up to 40% at a concentration of 300 μM, independently of the treatment with TGF-β1(Fig. 3B). Arphamenine A inhibited the Arg-pNa hydrolysis dose-dependently up to a concentration of 1 μmol/L. A maximal inhibition of 40% was found at a concentration of 1 μmol/L, and further increase in the concentration of arphamenine A did not induce an additional inhibition of Arg-pNA. Arphamenine A inhibited the hydrolysis of Arg-pNA to a lesser extent in the TGF-β1-treated cells (Fig. 3C).
Effect of Aminopeptidase Inhibition on Collagen Gel Contraction
In nonstimulated, control cardiac fibroblasts, neither bestatin, leuhistin, nor arphamenine A affected the collagen gel contraction. The Ang II-induced reduction in gel volume in control cardiac fibroblasts was almost completely abolished by leuhistin (100 μmol/L) and arphamenine A (100 μmol/L) and only partly by bestatin (100 μmol/L) (Fig. 4). In TGF-β1-treated cardiac fibroblasts, leuhistin and arphamenine A also completely reversed the Ang II-induced decrease in gel volume, whereas bestatin did not affect the Ang II-stimulated collagen gel contraction.
The present study shows that the Ang II-stimulated collagen gel contraction in control and TGF-β1-treated cardiac fibroblasts (or myofibroblasts) is reversed almost completely by leuhistin and arphamenine A. Bestatin only partially inhibited the Ang II-stimulated collagen gel contraction in control fibroblasts, but it did not affect the Ang II-induced contraction in TGF-β1-treated fibroblasts (Fig. 4).
In neonatal cardiac fibroblasts, Ang II also stimulated the ability of these fibroblasts to contract the 3-dimensional collagen gels.17-20 In adult cardiac fibroblasts, we3,4 have reported a dose-dependent stimulation of the collagen gel contraction by Ang II. Not only Ang II but also Ang I and Ang III stimulated the collagen gel contraction in control cardiac fibroblasts.21 The aminopeptidase inhibitor with broad specificity bestatin reduced the Ang I-, Ang II-, and Ang III- induced collagen gel contraction in control cardiac fibroblasts, suggesting that aminopeptidases are involved not only in the Ang II- but also in the Ang I- and Ang III-induced collagen gel contraction.21
Figure 4 shows that bestatin only partially inhibited the Ang II-stimulated collagen gel contraction, whereas arphamenine A and leuhistin almost completely blocked the Ang II-induced collagen gel contraction in control cardiac fibroblasts, suggesting that Ala- and Arg-aminopeptidases are both involved in the Ang II-stimulated contraction. Bestatin is considered an aminopeptidase inhibitor with a broad specificity; leuhistin is a specific inhibitor of Ala-aminopeptidase, and arphamenine A is a specific inhibitor of Arg-aminopeptidase, particularly of aminopeptidase B.9,10 Indeed, in control and TGF-β1-treated cardiac fibroblasts, leuhistin and arphamenine A only partially inhibited Ala-aminopeptidase activity at 100 μmol/L, whereas bestatin (100 μmol/L) completely inhibited the Ala-aminopeptidase activity. At this concentration the Ang II-stimulated collagen gel contraction is almost completely reversed by leuhistin and arphamenine A but not by bestatin.
The Arg-aminopeptidase activity is also only partially inhibited by leuhistin and arphamenine A at 100 μmol/L in control and TGF-β1-treated fibroblasts. Bestatin completely blocked the Arg-aminopeptidase activity in control fibroblasts and only partially in TGF-β1-treated fibroblasts. These data suggest that both Arg- and Ala-aminopeptidase are involved in the reversal of the Ang II-stimulated collagen gel contraction. It is also well known that aminopeptidase inhibitors such as amastatin and bestatin efficiently reduced the degradation of angiotensin II.22,23
Experiments with arphamenine A have, however, shown the presence of two Arg-aminopeptidase activities: arphamenine-sensitive, chloride-stimulated and arphamenine-insensitive, chloride-insensitive aminopeptidase.10 TGF-β1 stimulated both Arg-aminopeptidase activities by three-fold.10 Immunoblot with an antibody specific to rat aminopeptidase B has revealed that arphamenine-sensitive, chloride-stimulated AP is aminopeptidase B.10 Thus, the inhibition of Ang II-stimulated collagen gel contraction can at least partly be attributed to aminopeptidase B. We have also reported that Ang II is able to inhibit Arg-pNA hydrolysis.10 A 50% inhibition of the Arg-pNA hydrolysis by Ang II was found at 3 μmol/L, whereas the concentration of Arg-pNA was 60-fold higher. This indicates that the affinity of Arg-aminopeptidase to Ang II in cardiac fibroblasts is high.
In conclusion, our data indicate that Ala-AP and Arg-AP, probably AP-B, are involved in the reversal of the Ang II-stimulated collagen gel contraction in control and TGF-β1-treated cardiac fibroblasts (or myofibroblasts).
The authors gratefully acknowledge the technical and secretarial assistance of Ms T. Coenen, Ms L. Lommelen, and Ms Y. Piccart. This work was supported by an educational grant from AstraZeneca (Belgium). P. J. Lijnen is holder of the Boehringer Ingelheim Chair in Hypertension. G. Diaz-Araya received a fellowship from MECESUP program for Pharmacology (Ministry of Education, Chile).
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