Arterial proliferation contributes to the increase of blood pressure in the established phase of genetic and experimental hypertension (1). In spontaneously hypertensive rats (SHRs), an increase in both the number and the size of vascular smooth muscle cells (VSMCs) occurs during vascular growth (2). Thus treatment for hypertension should be focused on achieving the regression of excessive vascular growth.
Distinct growth phenotypes exist in cultured VSMCs from normotensive Wistar-Kyoto (WKY) rats and SHRs. SHR-derived VSMCs exhibit a higher specific growth rate, abnormal contact inhibition, and accelerated entry into S phase of the cell cycle (3). In addition, VSMCs from SHRs display nonspecific hyperproliferation in response to several growth factors such as calf serum, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), when compared with VSMCs from WKY rats (4). Because there is no blood pressure in the VSMC culture system, this hyperproliferation can be considered to be associated with intrinsic abnormalities. It has been suggested that VSMC hyperplasia is due to mitogens such as PDGF, whereas the hypertrophy of polyploid cells arises from stimulation by angiotensin (Ang) II released from within the vessel wall (5). It has been demonstrated that expression of the PDGF A-chain (6) and transforming growth factor-β1 (TGFβ1) is increased in VSMCs from SHRs compared with cells from WKY rats (7). Contributions of the PDGF A-chain and TGFβ1 to the exaggerated growth of VSMCs from SHRs have been confirmed by the growth inhibition of VSMCs with antisense oligomers to these growth factors (8-10). Furthermore, we recently demonstrated that an Ang II type 1 receptor antagonist considerably inhibits the basal growth of VSMCs from SHRs (11). More recently we found that VSMCs from SHRs show enhanced Ang II generation in homogeneous culture (12).
Catecholamines have been shown to initiate not only the immediate VSMC response of vasoconstriction, but also to promote hypertrophy of VSMCs through the α1B-adrenoreceptor (13). Catecholamines activate the progression of primary cultured VSMCs and stimulate the proliferation of rat VSMCs (14). Okazaki et al. (15) reported that α1-adrenoreceptor stimulation induced transient expression of c-fos protooncogene messenger RNA (mRNA) in cultured rat VSMCs that was completely blocked by the α1B-adrenoreceptor blocker chloroethylclonidine.
The antihypertensive agent bunazosin hydrochloride (HCl) is a quinazoline derivative α1-receptor blocker, and it decreases cultured VSMC levels of cytosolic Ca2+, which mediates vasodilatory and hypotensive effects (16,17). In addition to its potent antihypertensive effect, bunazosin was reported to inhibit arterial hyperproliferation in hypertension (18). However, the mechanism of the antiproliferative effect of bunazosin remains unclear. Our study was undertaken to investigate the mechanism of the antiproliferative effects of bunazosin by using cultured VSMCs from SHRs.
MATERIALS AND METHODS
Cultured VSMCs were grown from explants of aortic media of SHR/Izm and WKY/Izm rats and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum (Gibco Laboratories, Tokyo, Japan), 100 U/ml penicillin, and 100 mg/ml streptomycin. When the cells reached confluence in 7-10 days, they exhibited a hill-and-valley pattern typical of cultured smooth-muscle cells. They were passaged by trypsinization with 0.05% trypsin (Gibco) in Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline (PBS) and incubated in 75-cm2 tissue-culture flasks at a density of 105 cells/ml.
Establishment of quiescence
Trypsinized cells were plated into 24-well culture dishes at a density of 105 cells/cm2. They were allowed to grow in DMEM containing 10% calf serum for 24 h, and the culture medium was then changed to DMEM with 0.2% calf serum. The cells were incubated in this medium for 48-72 h to establish quiescence.
Evaluation of cytotoxicity by trypan blue staining
Bunazosin HCl-treated and untreated cells were harvested from the culture dishes by trypsinization. After centrifugation, the supernatant was discarded, and cells were suspended in 0.3% trypan blue diluted 1:1 in DMEM medium. Trypan blue-stained cells were counted in a hemocytometer with a phase microscope for 2-5 min. Bunazosin HCl-treated VSMC viability was evaluated from the percentage of unstained cells compared with untreated cells.
Determination of DNA synthesis
[3H]Thymidine incorporation into newly synthesized DNA was determined as described previously (19). Quiescent VSMCs were incubated for 24 h in 24-well plates with DMEM containing penicillin (100 U/ml), streptomycin (100 μg/ml), and various concentrations of bunazosin HCl (Eisai Pharmaceutical Company, Tokyo, Japan), Ang II (Peptide Institute, Osaka, Japan), PDGF-AA (Biochemical Technologies Inc., Stoughton, MA, U.S.A.), or EGF (Sigma Chemical, St. Louis, MO, U.S.A.). The medium was then changed to DMEM containing [3H]thymidine (0.5 μCi/ml; New England Nuclear, Irvine, CA, U.S.A.), and incubated for 2 h. Next, each well was washed with 1 ml of 150 mM NaCl to eliminate excess [3H]thymidine, and the cells were fixed in 1 ml ethanol:acetic acid (3:1) solution for 10 min. After washing with 1 ml H2O, acid-insoluble material was precipitated with 1 ml ice-cold perchloric acid, and DNA was extracted into 1.5 ml of perchloric acid by heating at 90°C for 20 min. The perchloric acid containing solubilized DNA was transferred to a scintillation vial, and the radioactivity was measured with a liquid scintillation spectrometer.
Determination of cell numbers
VSMCs were inoculated into DMEM containing 10% calf serum in 24-well culture dishes at a density of 105 cells/cm2 with or without 100 μM bunazosin HCl. Cells were trypsinized with 0.05% trypsin at 24, 48, and 72 h after inoculation, and cell numbers were counted in a Coulter Counter (Coulter Electronics Ltd., Luton, Bedfordshire, England).
Reverse-transcription and polymerase chain reaction (RT-PCR) assay for growth factor mRNAs
Quiescent VSMCs (5 × 104 cells/cm2) were incubated for 20 h in 24-well dishes with bunazosin HCl, washed with PBS, and lysed in 800 μl of RNAzol B (Biotecx Laboratories, Inc. Houston, TX, U.S.A.). Each sample was mixed with 80 μl of chloroform by vortexing for 15 s, kept on ice for 15 min, and centrifuged at 12,000 g for 15 min to extract total RNA. The colorless upper aqueous phase was mixed with an equal volume of isopropanol, let stand at −20°C for 45 min, and centrifuged at 12,000 g for 15 min at 4°C to precipitate the RNA. The RNA pellet was washed twice with 500 μl of 75% ethanol and centrifuged at 12,000 g for 8 min at 4°C, dried, and dissolved in 10 μl of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). After denaturing at 65°C for 15 min, the RNA sample was treated with 0.5 U of DNase (Gibco Life Technologies, Gaithersburg, MD, U.S.A.) in 0.5 μl of DNase buffer (20 mM Tris-HCl, pH 8.3, 50 mM KCl, and 2.5 mM MgCl2) at room temperature for 45 min. Then the DNase was inactivated by adding 0.5 μl of 20 mM EDTA and heating at 98°C for 10 min.
RT-PCR was performed as described previously (20). In brief, aliquots of RNA (1 μg/20 μl) were reverse-transcribed into single-stranded cDNA by using 0.25 U/μl avian myeloblastoma virus reverse transcriptase (Life Sciences, St. Petersburg, FL, U.S.A.) in 10 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 50 mM KCl, 1 mM deoxy-nucleoside triphosphate (NTPs), and 2.5 μM random hexamer. Five microliters of the diluted cDNA product was mixed with 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, 0.025 U/μl Taq DNA polymerase (Takara Biochemicals, Osaka, Japan), and 0.2 μM upstream sense primer and downstream antisense primer in a total volume of 25 μl. The primers used for PCR of rat bFGF, TGFβ1, PDGF-A chain, and ribosomal protein L19 are shown in Table 1. PCR was performed in an automatic thermocontroller (Perkin Elmer, Foster, CA, U.S.A.). After initial denaturation at 96°C for 5 min, PCR amplification was performed by using 30 cycles of denaturation for 45 s at 96°C for bFGF or 60 s at 94°C for TGFβ1 and PDGF-A chain, annealing for 45 s at 55°C for bFGF or for 60 s at 60°C for TGFβ1 or for 2 min at 58°C for PDGF-A chain, primer extension for 1 min at 72°C, and final extension for 10 min at 72°C. PCR with primers for L19 was performed with each reaction as an internal control. The PCR products were electrophoresed on 1.5% agarose gel.
Bunazosin was purchased from Eisai Pharmaceutical (Tokyo, Japan), Ang II was purchased from the Peptide Institute (Osaka, Japan), EGF was from Sigma, and PDGF-AA was from Biochemical Technologies.
Results are given as the mean ± SEM. The significance of differences between mean values was evaluated by Student's t test for unpaired data and by two-way analysis of variance (ANOVA) followed by Duncan's multiple-range test.
Effect of bunazosin HCl on DNA synthesis by VSMCs
The effect of increasing doses (1-100 μM) of bunazosin HCl on basal and serum-stimulated DNA synthesis by VSMCs from WKY rats and SHRs is illustrated in Fig. 1. In the absence of calf serum, bunazosin HCl did not affect DNA synthesis by VSMCs from WKY rats, but concentrations of 5 × 10−5 and 10−4M significantly (p < 0.05) inhibited DNA synthesis by VSMCs from SHRs. At 10−4M, bunazosin HCl significantly (p < 0.05) inhibited DNA synthesis by VSMCs from both rat strains in the presence of 10% calf serum.
Effect of bunazosin HCl on proliferation of VSMCs
In the presence of 10% calf serum, bunazosin HCl significantly (p < 0.05) inhibited an increase in the number of VSMCs from WKY rats and SHRs (Fig. 2), indicating that the drug inhibited VSMC hyperplasia in the presence of serum.
Effect of bunazosin HCl on DNA synthesis by VSMCs in response to Ang II, PDGF-AA, or EGF
Ang II dose-dependently increased DNA synthesis by VSMCs from SHRs, but not by cells from WKY rats. Bunazosin HCl did not affect the response to Ang II of DNA synthesis by VSMCs from SHRs (Fig. 3). PDGF-AA dose-dependently increased DNA synthesis by VSMCs from SHRs, but not by cells from WKY rats. Bunazosin HCl significantly (p < 0.05) suppressed the response to PDGF-AA of DNA synthesis by VSMCs from SHRs (Fig. 4). EGF dose-dependently increased DNA synthesis by VSMCs from SHRs, but not by cells from WKY rats, and bunazosin HCl also significantly suppressed the DNA response to EGF of VSMCs from SHRs (Fig. 5).
Effect of bunazosin HCl on expression of growth factor mRNAs
The effect of bunazosin HCl on expression of bFGF, TGFβ1, and PDGF A-chain mRNAs by VSMCs is illustrated in Figs. 6-8. Expression of the three growth factor mRNAs was markedly higher in VSMCs from SHRs than in cells from WKY rats. At 10−4M, bunazosin HCl significantly (p < 0.01) decreased bFGF mRNA expression in VSMCs from SHRs but did not affect it in cells from WKY rats. Bunazosin HCl significantly decreased TGFβ1 mRNA expression at both concentrations of 10−6M (p < 0.01) and 10−4M (p < 0.05) in VSMCs from SHRs but not in cells from WKY rats. Bunazosin HCl did not alter the expression of PDGF A-chain mRNA by VSMCs.
Rat VSMCs express three α1-adrenoreceptors (α1A, α1B, and α1D), which exert a trophic influence on VSMCs during normal development and also contribute to the pathogenesis of vascular hypertrophy (21). Stimulation of α1-adrenoreceptors enhances the proliferation of cultured rat VSMCs (22). It was reported that α1-adrenoreceptor blockade inhibits VSMC proliferation induced by the infusion of angiotensin in normal rats in vivo (23). Whereas noradrenaline is known to induce hyperproliferation of cultured VSMCs, addition of bunazosin significantly reduces such proliferation (18). The α1-blockers, including bunazosin, have been reported to have an antiproliferative effect on cardiovascular organ growth in vivo (24), but the mechanisms involved are still unclear.
It was demonstrated that long-term administration of bunazosin decreases the binding of calcium to Ca2+-binding sites and the norepinephrine concentration in the myocardium of SHRs (25). It was proposed that both the intercellular Ca2+-inhibitory effect and the α1-blocking effect of bunazosin are associated with its antiproliferative effect on VSMC growth (16). We previously showed that subantihypertensive doses of bunazosin inhibited the development of cardiac hypertrophy in vivo (24). In this experiment, bunazosin significantly inhibited DNA synthesis by VSMCs from both WKY rats and SHRs in the presence of 10% calf serum, whereas it inhibited basal DNA synthesis by quiescent VSMCs from SHRs, but not from WKY rats, in the absence of serum. There was no α1-adrenoceptor agonist in the culture medium without serum. Thus catecholamines may be generated by VSMCs from SHRs in a homogeneous culture system, but further investigations are needed to clarify this point.
On the other hand, VSMCs from SHRs were reported to display nonspecific enhancement of DNA synthesis in response to several growth factors, such as EGF, PDGF, and bFGF (4). In our study, Ang II, PDGF-AA, and EGF stimulated DNA synthesis by VSMCs from SHRs, whereas these mitogens did not stimulate DNA synthesis by cells from WKY rats. Bunazosin significantly suppressed the DNA response to PDGF-AA and EGF, but not to Ang II, by VSMCs from SHRs. Intracellular signaling pathways regulating VSMC growth are initiated by activation of receptor tyrosine kinases or by protein kinase C or by both. Ang II and PDGF induce VSMC growth by the activation of protein kinase C (26,27), whereas EGF induces VSMC growth by the activation of only tyrosine kinase (28). It is therefore considered that bunazosin may inhibit the growth of VSMCs from SHRs through a tyrosine kinase-dependent pathway.
Ang II was shown to induce cell hypertrophy (29), and an in vitro hyperplastic effect was described for some cells, including VSMCs from WKY rats and SHRs (30). Moreover, Ang II is known to induce expression of growth factors such as TGFβ1(26), PDGF A-chain (31), and bFGF (32). In our study, bunazosin significantly suppressed the increase of VSMCs from both rat strains in the presence of 10% calf serum, indicating that the antiproliferative effect of this drug is associated with inhibition of VSMC hyperplasia. The inhibitory effect of bunazosin on VSMC hyperplasia may be mediated through the suppression of these growth factors.
Growth factors such as PDGF, TGFβ, EGF, and FGF were reported to interact with each other. Injured rat VSMCs in culture release FGF, which activates DNA synthesis in neighboring VSMCs both by a direct mechanism and by stimulating the production of PDGF-AA (33). TGFβ was shown to stimulate VSMC growth by induction of PDGF-AA expression (34), and it can also potentiate the mitogenic effects of PDGF-AA and bFGF in VSMCs (35). Saltis et al. (36) demonstrated that TGFβ1 enhances the proliferative effect of EGF on VSMCs from SHRs. It is therefore considered that the inhibition of TGFβ1 by bunazosin is relevant to suppression of the growth response of VSMCs from SHRs to PDGF-AA and EGF in these experiments.
We recently observed that VSMCs from SHRs generate Ang II, but not cells from WKY rats, along with increased expression of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE) mRNAs (12). In our study, basal expression of bFGF, TGFβ1, and PDGF A-chain mRNAs was markedly higher in VSMCs from SHRs than in cells from WKY rats. We also found that an angiotensin II type 1 receptor antagonist, as well as an ACE inhibitor, decreased expression of mRNA for these three growth factors (unpublished data), indicating that increased growth-factor expression is induced by endogenous production of Ang II. In these experiments, bunazosin significantly inhibited expression of bFGF and TGFβ1 mRNAs in VSMCs from SHRs, but not in cells from WKY rats, whereas this agent did not affect expression of PDGF A-chain mRNA. It was reported that bFGF stimulates ACE expression in VSMCs (37) and that ACE is associated with generation of Ang II, which stimulates VSMC growth. Zhu et al. (38) demonstrated that bFGF contributes to hyperplasia and hypertrophy of VSMCs from SHRs. On the other hand, TGFβ has a dual effect on VSMCs, suppressing growth at low cell densities and stimulating growth at high densities (39). This dual effect was reported to result from an interaction of TGFβ with distinct receptor phenotypes (35). We previously reported that exogenous TGFβ inhibits DNA synthesis by VSMCs from WKY rats, whereas it stimulates DNA synthesis by VSMCs from SHRs, which show increased expression of the TGFβ type II receptor compared with cells from WKY rats, and that this difference may contribute to the exaggerated growth of SHR-derived VSMCs (40). From these findings, it is considered that bunazosin inhibits the growth of VSMCs from SHRs by suppression of the enhanced expression of bFGF and TGFβ1.
In summary, bunazosin inhibited the hyperproliferation of VSMCs from SHRs by suppression of the increased expression of bFGF and TGFβ1 mRNAs and by suppressing the increased response to PDGF-AA and EGF.
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