Introduction

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Abnormal accumulation of vascular smooth muscle cells (VSMCs) likely participates in smooth muscle hypertrophy, which is often associated with many vascular diseases including atherosclerosis, restenosis after balloon angioplasty, and primary hypertension (see Ross, 1993 for review). This may result from an uncontrolled proliferation of VSMCs in response to growth factors. Within the vasculature the renin-angiotensin system plays an important physiological role, not only through the regulation of blood pressure (Vallotton, 1987), but also through the modulation by its components, of the so-called vascular remodeling (Griffin et al., 1991). In this respect, the modulation in vitro by angiotensin II (AII) of VSMC growth, i.e., the modulation of mitogenic and trophic actions of AII, has been documented (Campbell-Boswell and Robertson, 1981; Geisterfer et al., 1988; Powell et al., 1989). In vivo, the local AII production has been recently shown to directly affect vascular hypertrophy (Morishita et al., 1994). Although the mechanisms whereby AII stimulates VSMC growth are not fully understood and remain controversial (Geisterfer et al., 1988; Paquet et al., 1990), it is widely accepted that these growth-related events are mediated through AT₁ receptors (Timmermans et al., 1993; Sung et al., 1994).

Recently, a novel class of AT₁ receptor antagonists that are nonpeptidic and devoid of agonistic properties has been developed (Timmermans et al., 1993). In VSMC, losartan, the prototype of AT₁ receptor antagonists, has been demonstrated to be a potent antihypertensive agent that inhibits most of AII-induced intracellular responses including the growth-related cellular events (Ko et al., 1992; Lyall et al., 1992; Catalioto et al., 1995; Duff et al., 1995; Leduc et al., 1995). BAY 10-6734 (Embusartan; 6-butyl-1-[(3-fluoro-2'-1H-tetrazol-5-yl-biphenyl-4-yl)-methyl]-2-oxo-1,2-dihydropyridine-4-carboxylic) acid methylester is another newly developed orally active antihypertensive compound. Embusartan is a nonpeptide-specific AT₁ receptor antagonist that has proven to be efficacious in various animal models of hypertension (Stasch et al., 1997). The influence of BAY 10-6734 upon AII-induced vascular hypertrophy has not been investigated. This study was therefore undertaken to determine whether BAY 10-6734 could inhibit AII-stimulated growth of cultured VSMCs isolated from spontaneously hypertensive (SHR) and control normotensive Wistar Kyoto (WKY) rats. The effects obtained with BAY 10-6734 were compared to those obtained with losartan, used as a positive control. In addition, the effects of BAY 10-6735, the predominant therapeutically active moiety of BAY 10-6734, and those of EXP 3174, the active metabolite of losartan, were also investigated.

	Experimental Procedures

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Materials. Ten-week-old male WKYs and SHRs were purchased from Iffa-Credo (les Oncins, France). Plasticware for culture was obtained from Costar Corp. (Cambridge, MA). Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, trypsin, and HEPES buffer were purchased from Life Technologies (Cergy Pontoise, France). Fetal calf serum (FCS) was obtained from Boehringer Mannheim GmBH (Mannheim, Germany). [¹²⁵I]AII (81.4 TBq/mmol) and [³H]L-leucine (5.18 TBq/mmol) were purchased from New England Nuclear (Boston, MA). Unlabeled AII and all other chemicals were obtained from Sigma (St. Louis, MO). BAY 10-6734, BAY 10-6735 (the carboxylic acid derivative of BAY 10-6734), EXP 3174, and losartan were synthesized by Bayer AG (Leverkusen, Germany) for pharmacological research.

Cell Culture. VSMCs were cultured according to Ross (1971) as previously described (Stepien et al., 1998). Briefly, the media of thoracic aortae were isolated and cut into pieces of ~1 mm². After removal of adventitia and endothelium using fine forceps, explants of the media were then cultured in DMEM containing 15% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 8 mM HEPES. Cells grown to confluence were detached with 0.125% trypsin and subcultured every week in a similar culture medium containing 10% FCS. For each experiment, the cells were allowed to grow for 4 to 5 days in 5% CO₂, 95% air at 37°C, until they were subconfluent. Then VSMCs were made quiescent, i.e., synchronized to the G₀/G₁ phase, by serum deprivation for 48 h before stimulation. Unless specified, subconfluent VSMCs between passages 4 and 13 were used for experiments. Cell viability was not affected by the various compounds tested, as assessed by the measurement of lactate dehydrogenase activity released from damaged cells, using the cytotoxicity detection kit (Boehringer Mannheim).

Radioligand Binding. Binding of [¹²⁵I]-labeled AII was performed as previously described (Sachinidis et al., 1993). Quiescent and confluent cells cultured in 24-well plates were washed with 0.5 ml of binding buffer (Tris 50 mmol/l, NaCl 100 mmol/l, and BSA 0.25%, pH 7.2) and incubated in the same buffer for 1 h at room temperature to allow dissociation of endogenous AII. Then cells were washed and incubated for 1 h in 0.5 ml of binding buffer containing 200 pmol [¹²⁵I]AII in the presence or absence of antagonists and/or unlabeled AII. Nonspecific binding, determined in the presence of 1 µM unlabeled AII, was <20%. Five-tenths nanomolar AII was used for studying the inhibition of AII binding by AT₁ antagonists. Reaction was stopped by removing the incubation medium and washing the cells twice. The attached cells were dissolved in 0.5 ml of 0.1 N NaOH. The amount of AII bound to cells was quantified by radioactivity counting in a gamma spectrometer.

Determination of DNA Synthesis. In preliminary experiments, we determined DNA synthesis by measuring the incorporation of [³H]thymidine into VSMCs, as already detailed (Zhu et al., 1991) and the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into VSMCs by using the BrdU proliferation kit (Boehringer Mannheim) according to the manufacturer's instructions. These two methods gave essentially similar results. For practical reasons, the BrdU incorporation method was chosen for the present investigations. Quiescent cells cultured in 96-well plates were stimulated for 24 h with activators (or vehicle) in the presence or absence of antagonists and then incubated with BrdU (10 µM) for 2 h. Cells were fixed and BrdU was labeled with a peroxidase-conjugated anti-BrdU antibody followed by addition of a peroxidase substrate. Reaction was stopped by adding 30 µl of 1 M H₂SO₄ solution and optical density at 450 nm was measured in each well with the microplate reader mentioned above.

Protein Synthesis. Protein synthesis was measured as the [³H]L-leucine into the trichloroacetic acid (TCA)-insoluble precipitate as previously described (Takahashi et al., 1996). Briefly, quiescent VSMCs were washed and then stimulated with activators in the presence or absence of antagonists and incubated for 24 h in a medium containing 0.5 µCi/ml of [³H]L-leucine and 0.45 mM L-leucine. Then VSMCs were washed 3 times with ice-cold PBS and incubated with 5% TCA for 30 min at 4°C. The cells were washed 2 times with 5% TCA. The TCA precipitate was dissolved in 0.1 N NaOH and the radioactivity incorporated was determined by scintillation counting.

Measurement of Cytosolic Calcium. Cytosolic calcium concentration ([Ca²⁺]_i) was measured using the fluorescent Ca²⁺ indicator Fura-2 (Grynkiewicz et al., 1985). VSMCs were incubated with serum-free DMEM for 48 h and washed with buffered solution containing (in mM): 135 NaCl, 5.4 KCl, 44 NaHCO₃, 5 glucose, 0.8 MgSO₄, 0.9 NaH₂PO₄, and 10 HEPES, pH 7,4 at 37°C. VSMCs were then incubated in the same medium in the presence of 2 µM Fura-2/AM for 30 min at 37°C. The cells were washed twice to remove the external dye, and placed in the quartz cuvette for [Ca²⁺]_i measurement at 37°C. Fluorescence was recorded with excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm on a spectrofluorometer SPEX CMIII (ISA-Jobin-Yvon, Longjumeau, France). [Ca²⁺]_i was calculated as described (Grynkiewicz et al., 1985).

Data Analysis. All experiments were performed in triplicate and values are expressed as means ± S.E.M. of n distinct experiments. When AT₁ antagonists were tested, the experiments included the four antagonists, irrespective of the passage used. The IC₅₀ values were calculated by linear regression. Multiple comparisons and dose-response and time-dependent effects were examined by one-way ANOVA and posthoc Fisher's test. Comparisons of dose-response effects between two different groups were assessed by two-way ANOVA.

	Results

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Effect on Specific AII Binding. The capacity of cultured VSMCs to bind AII was assessed in a first set of experiments. Both WKY- and SHR-derived VSMCs bound [¹²⁵I]AII with high affinity, and Scatchard analysis of results (data not shown) indicated the presence of one single class of binding sites with K_D values of 0.51 ± 0.14 nM and 0.50 ± 0.08 nM, and a binding capacity of 45 ± 7 fmol/mg protein and 27 ± 2 fmol/mg protein, for WKY- and SHR-derived VSMCs, respectively. These characteristics are in agreement with what has already been reported (Sachinidis et al., 1993; Cahill et al., 1995).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Inhibition by BAY 10-6734, losartan, and their metabolites of the specific binding of [125I]AII to WKY- and SHR-derived VSMCs. Quiescent VSMCs were incubated with [125I]AII in the presence or absence of various concentrations of the antagonist for 1 h at room temperature and experiments were performed as described in Experimental Procedures. BAY 10-6734 (open circles), BAY 10-6735 (closed circles), losartan (open triangles), EXP 3174 (closed triangles). Results are expressed as the means ± S.E.M. of at least three different experiments from different cell cultures.

Effect of Bay 10-6734 on DNA Synthesis. Exposure of VSMC cultures to AII significantly increased DNA synthesis in a concentration-dependent manner as indicated by the increase in BrdU incorporation (p < .001 for both, n = 7-21; Fig. 2A). In both SHR and WKY cultures, the maximum BrdU incorporation-3 to 5 times the incorporation in unstimulated cells-was reached with 1 µM AII (Fig. 2A). For AII concentrations above 10 nM, DNA synthesis was significantly increased in SHR compared to WKY, when analyzed by two-way ANOVA (p < .01).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   AII-induced DNA synthesis in WKY- and SHR-derived VSMCs and its inhibition by BAY 10-6734, losartan, and their metabolites. Quiescent VSMCs were stimulated by AII for 24 h in the absence (A) or presence (B and C) of the antagonist at various concentrations and then incubated for 2 h with 10 µM BrdU. For the inhibition experiments, the antagonist was added to 100 nM AII. BrdU incorporation was quantified as described in Experimental Procedures. Results are expressed as the means ± S.E.M. of at least four experiments from different cell cultures. A, SHR-derived VSMCs (open squares), WKY-derived VSMCs (closed squares); B, WKY-derived VSMCs; C, SHR-derived VSMCs. In (B) and (C), symbols are as in Fig. 1.

Effect on Protein Synthesis. In both WKY- and SHR-derived VSMCs, AII induced protein synthesis in a concentration-dependent manner, as assessed by [³H]-leucine incorporation (Fig. 3A; p < .001 for both, n = 5-7). Maximum [³H]-leucine incorporations were ~2- and 3-fold the basal level in WKY- and SHR-derived VSMCs, respectively. For AII concentrations above 0.1 µM, [³H]-leucine incorporations were significantly higher in SHR VSMCs than in WKY ones (p < .01, n = 5-7).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   AII-induced protein synthesis in WKY- and SHR-derived VSMCs and its inhibition by BAY 10-6734, losartan, and their metabolites. Quiescent VSMCs were stimulated by AII for 24 h in the absence (A) or presence (B and C) of the antagonist at various concentrations. For the inhibition experiments, the antagonist was added to 100 nM AII. [3H]-leucine incorporation was quantified as described in Experimental Procedures. Results are expressed as the means ± S.E.M. of at least four experiments from different cell cultures. A, SHR-derived VSMCs (open squares), WKY-derived VSMCs (closed squares); B, WKY-derived VSMCs; C, SHR-derived VSMCs. In (B) and (C), symbols are as in Fig. 1.

Effect on AII-Induced [Ca²⁺]_i Variations. In both WKY- and SHR-derived VSMCs, and in the presence of transmembrane calcium gradient, i.e., in the presence of 1 mM [Ca²⁺]_ex, AII (100 nM) induced a biphasic [Ca²⁺]_i rise comprising a transient peak and a sustained phase represented by a plateau (Fig. 4). The observed transient and sustained phases likely reflect the calcium release from internal stores and the calcium influx activation, respectively, because the absence of the transmembrane calcium gradient ([Ca²⁺]_ex = 50 nM) suppressed the latest phase without affecting the former (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of AII on intracellular cytosolic Ca2+ concentration ([Ca2+]i) in WKY- or SHR-derived VSMCs. Inhibition by BAY 10-6734 and losartan. A and B, WKY- and SHR-derived VSMCs, respectively. In a calcium-containing medium ([Ca2+]ex=1 mM), quiescent VSMCs that have been previously loaded with Fura-2 were preincubated for 30 min at 37°C with vehicle (a), 100 nM BAY 10-6734 (b), or losartan (c), and then stimulated by AII (100 nM). AII-induced [Ca2+]i variations were then measured as described in Experimental Procedures. Tracings are representative of at least three distinct experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of BAY 10-6734, losartan, and their active metabolites on AII-induced [Ca2+]i variations. Experiments were carried out as described in the legend of Fig. 5, in the presence of external calcium ([Ca2+]ex = 1 mM). [Ca2+]i at peak level (A) and at plateau level (B) were calculated as the [Ca2+]i value at peak and plateau, respectively, diminished by the basal [Ca2+]i value. Values are expressed as the means ± S.E.M. of at least four experiments from different cell cultures. Open columns: control, i.e., AII alone; slantwise hatched columns: VSMCs pretreated with BAY 16734; vertically hatched columns: VSMCs pretreated with BAY 16735; closed columns: VSMCs pretreated with losartan; horizontally hatched columns: VSMCs pretreated with EXP 3174.

	Discussion

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The present investigations were designed to determine the effects of BAY 10-6734, a newly developed orally active antihypertensive drug, which has been classified as an AT₁ receptor antagonist (Stasch et al., 1997; Bohm et al., 1998), on AII-induced hypertrophy/hyperplasia of VSMCs. The in vitro effects of BAY 10-6734 on AII-induced growth-related events were, therefore, studied in VSMCs isolated from normotensive and hypertensive rats. Using WKY- and SHR-derived cultured VSMCs, we examined the effect of BAY 10-6734, losartan, and their metabolites BAY 10-6735 and EXP 3174, respectively, on AII binding, AII-induced DNA and protein synthesis, and AII-induced variations in intracellular [Ca²⁺]. There is compelling evidence that such AII-induced hyperplasia and hypertrophy are mediated by the type I AII receptors (Geisterfer et al., 1988; Berk et al., 1989; Chiu et al., 1991; Bunkenburg et al., 1992). Moreover, in culture, VSMCs have been demonstrated to be devoid of type II AII receptors (de Gasparo et al., 1990).

In the present study, BAY 10-6734 has been shown to antagonize the specific binding of [¹²⁵I]AII to both WKY- and SHR-derived VSMCs (Fig. 1). In VSMCs isolated from WKY, both BAY 10-6734 and losartan inhibited AII binding with the same affinity, as indicated by their IC₅₀ values (Table 1). However, BAY 10-6734 was significantly (~10 times) more potent than losartan for displacing [¹²⁵I]AII binding to VSMCs isolated from SHR (Table 1). IC₅₀ values similar to those obtained here have been reported for other AT₁ receptor antagonists, including TCV-116, UP 269-6, HR 720, and SK&F 108566 (Sung et al., 1994; Flesch et al., 1995; Dunn et al., 1997; Virone-Oddos et al., 1997). Regarding the potency of each AT₁ receptor antagonist used to inhibit [¹²⁵I]AII binding, Table 1 also indicates that SHR VSMCs were more sensitive than WKY ones, as already reported with UP 269-6 (Virone-Oddos et al., 1997).

The hyperplastic effect of AII in cultured VSMCs is still controversial (Geisterfer et al., 1988; Berk et al., 1989; Sachinidis et al., 1993). Under the present experimental conditions, AII behaved as a weak mitogen for VSMCs (EC₅₀ ~10 nM). Nevertheless, AII did stimulate DNA synthesis in a concentration-dependent manner in both WKY- and SHR-derived VSMCs, the latter being significantly more sensitive (Fig. 2A), consistent with previous reports (Hamada et al., 1990; Paquet et al., 1990; Morton et al., 1995). In both cell types examined, BAY 10-6734 and losartan, as well as their respective metabolites, inhibited AII-induced DNA synthesis in a concentration-dependent manner (Fig. 2, B and C). IC₅₀ values similar to those presented in Table 2 have been reported for other AT₁ antagonists, including SK&F 108566, TCV-116, losartan, and their respective metabolites CV 11974 and EXP 3174 (Briand et al., 1994; Sung et al., 1994; Flesch et al., 1995; Itazaki et al., 1995). In both WKY- and SHR-derived VSMCs, BAY 10-6734 was significantly more potent that losartan (Table 2). BAY 10-6734 was as potent as its metabolite BAY 10-6735 (Table 2); however, EXP 3174 was more potent than losartan, its parent compound, in agreement with a previous report (Sachinidis et al., 1993). As might be expected with a specific AT₁ antagonist, BAY 10-6734 did not influence serum-, AVP-, or bFGF-induced DNA synthesis (not shown).

As already published by others (Berk et al., 1989; Catalioto et al., 1995; Dunn et al., 1997; Virone-Oddos et al., 1997), AII also stimulated protein synthesis in VSMCs (Fig. 3A). The various compounds tested in this study inhibited AII-induced protein synthesis in a concentration-dependent manner in both WKY- and SHR-derived VSMCs (Fig. 3, B and C). Although BAY 10-6734 and losartan exhibited similar affinity for inhibiting protein synthesis in WKY-derived VSMCs, the affinity of BAY 10-6734 appeared ~10 times greater than that of losartan in SHR-derived VSMCs (Table 3). Table 3 also indicates that IC₅₀ values for BAY 10-6734 and its metabolite were in the range of those reported for other AT₁ antagonists (Dunn et al., 1997; Virone-Oddos et al., 1997) and SHR-derived VSMCs were more sensitive to BAY 10-6734 than WKY-derived cells.

In VSMC, AII-induced elevation of [Ca²⁺]_i is a primary signaling event for stimulating mitogen-activated protein kinase pathways (see Schmitz and Berk, 1997 for review). AII-induced [Ca²⁺]_i variations presented here (Fig. 4) are consistent with what has been previously reported (Neusser et al., 1993; Sachinidis et al., 1993; Koh et al., 1994). BAY 10-6734, losartan, and the metabolites tested did not affect the basal level of [Ca²⁺]_i, but they inhibited AII-induced Ca²⁺ movements (Figs. 4 and 5). Irrespective of the cells examined, BAY 10-6734 and its metabolite BAY 10-6735 tremendously reduced AII-induced transient Ca²⁺ elevation (i.e., AII-induced Ca²⁺ mobilization from internal stores) and also abolished the sustained phase (i.e., AII-induced Ca²⁺ influx). Losartan and its metabolite behave similarly, as expected from previous reports dealing with AT₁ receptor antagonists (Ko et al., 1992; Sachinidis et al., 1993; Koh et al., 1994; Itazaki et al., 1995). Both BAY 10-6735 and EXP 3174 appeared more potent than their respective parent compounds; this observation has already been reported for the losartan metabolite (Sachinidis et al., 1993). As a specific AT₁ receptor antagonist, BAY 10-6734 did not affect thrombin-, AVP-, or bFGF-induced variations of [Ca²⁺]_i (results not shown), indicating that the compound did not inhibit cellular Ca²⁺-ATPases.

Taken together, our results demonstrate that BAY 10-6734 and its active metabolite BAY 10-6735 behave as other AT₁ antagonists and are potent and specific inhibitors of AII-induced growth-related events in VSMCs. One may envisage that such a potency participates in the antihypertensive properties of BAY 10-6734 in the various animal models of hypertension (Stasch et al., 1997), particularly in the SHR, where AII has been shown to exert more marked hyperplastic and hypertrophic effects (Bunkenburg et al., 1992 and this study) and where cellular hyperactivity has been well documented (Marche et al., 1995).