Endothelial dysfunction is known to be associated with various risk factors for atherosclerosis such as hypercholesterolemia, diabetes mellitus, and hypertension (1,2). The vascular endothelium becomes more dysfunctional in the early stages of atherosclerosis, and the grade of dysfunction becomes more severe depending on the course of the disease. Such dysfunction has been shown to lead to abnormal synthesis or release of endothelial-derived nitric oxide (EDNO) (2,3). Conversely, nitric oxide is known to have an anti-atherosclerotic effect by inhibition of monocyte adhesion to endothelial cells; inhibition of smooth muscle cell chemotaxis, proliferation, and relaxation; and inhibition of platelet aggregation (3–5). Based on these findings, nitric oxide is speculated to play a role in protection against atherosclerosis. Atherosclerosis demonstrates many features, such as inflammation, associated with a reduction in nitric oxide synthesis in the endothelium (6). Chronic inhibition of EDNO was reported to cause high-cholesterol diet (HCD)-induced atherosclerosis to progress and to show inflammatory atherosclerotic lesions similar to atherosclerosis in humans (7,8).
Nitric oxide donors such as nitroglycerin are used to relieve angina pectoris by dilating stenotic coronary arteries. Nitric oxide donors in the form of a nitroglycerin patch or isosorbide dinitrate and β-adrenergic antagonists are used to protect against angina attack in patients who have had angina pectoris.
Despite their widespread and popular prescription all over the world, it is not completely understood whether nitric oxide donors and β-adrenoceptor antagonists have anti-atherosclerotic effects. Nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran) is designed to produce two complementary pharmacologic effects: β-adrenoceptor antagonist and nitric oxide–releasing actions (Fig. 1) (9). The combination of these two actions results in a highly effective and well-tolerated treatment for hypertension and coronary artery disease (10). Thus, we decided to investigate the anti-atherosclerotic effect of nitric oxide donors, including nipradilol and nitric oxide donors such as isosorbide dinitrate and nitroglycerin, in severe atherosclerosis induced by HCD and chronic nitric oxide inhibition.
MATERIALS AND METHODS
Chemicals and solutions
l -arginine hydrochloride, acetylcholine chloride, prostaglandin F2α, the calcium ionophore A23187, indomethacin, NG -nitro- l -arginine methyl ester ( l -NAME), NG -monomethyl- l -arginine acetate ( l -NMA), isosorbide dinitrate, and atenolol were all purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Nitroglycerin patch was bought from Nihon Kayaku Co., Ltd. (Tokyo, Japan). Nipradilol was obtained from Kowa Pharmaceutical Co. (Nagoya, Japan). Monoclonal anti–inducible nitric oxide synthase (anti-iNOS) and anti-nitrotyrosine antibodies were purchased from Transduction Laboratory (Lexington, KY, U.S.A.). Krebs-Henseleit solution (118 m M NaCl, 4.7 m M KCl, 1.5 m M CaCl 2, 1.2 m M MgSO 4, 1.2 m M KH 2 PO 4, 25 m M NaHCO 3, 11 m M glucose, and 0.002 m M ethylenediamine tetra-acetic acid, pH7.4) was saturated with 95% O 2 /5% CO 2.
A total of 48 male New Zealand white rabbits, 12 or 13 weeks old, weighing 2.0–2.4 kg were obtained from Kitayama Rabbis (Ina, Nagano, Japan). The rabbits were housed individually in stainless steel cages at 20 ± 3°C. The rabbits were divided into the following six groups (n = 8). Group I received HCD (standard diet plus 0.5% cholesterol) plus solvent (for isosorbide dinitrate, nipradilol, and atenolol) by oral gavage. Group II received HCD plus l -NAME. Group III received HCD plus l -NAME and isosorbide dinitrate (40 mg/kg/d). Group IV received HCD plus l -NAME and nitroglycerin patch (5 mg/d). Group V received HCD plus l -NAME and nipradilol (β-adrenergic blocker with nitric oxide–releasing action (10 mg/kg/d). Group VI received HCD plus l -NAME and atenolol (β-adrenergic blocker (50 mg/kg/d) (11). As a control group, six male rabbits were fed a regular diet for 10 weeks. l -NAME was mixed in drinking water at 80 mg/dl (7). All animals receiving l -NAME treatment drunk at least 350 ml of water, in which l -NAME was mixed, and were given water with or without l -NAME ad libitum throughout the experiment. Feeding was restricted to 100 g/d. Isosorbide dinitrate and nipradilol were administered daily at 9 AM and 6 PM by oral gavage. Atenolol was administered daily at 9 AM and solvent was administered at 6 PM by oral gavage. All experiments were conducted in accordance with institutional guidelines for animal studies. After 10 weeks of treatment, the rabbits were killed by exsanguination after being anesthetized with pentobarbital (50 mg/kg i.v.). The investigators were blinded with regards to diet regimen and medications. To decide the doses of each drug, we determined the effect of various concentrations using rabbits fed a regular diet for 2 weeks. In rabbits fed a regular chow for 2 weeks, we established that tolerance of the vascular response to nitroglycerin occurred at more than two patches (10 mg/d), and no tolerance occurred at one patch (5 mg). We selected the dose of isosorbide dinitrate to achieve an effect comparable to that of nitroglycerin. We selected the dose of atenolol to achieve β-blocking action similar to that of nipradilol.
Plasma biochemical assays
Total and free cholesterol levels were determined by using cholesterol oxidase (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) (11). A triglyceride level was measured by enzymatic techniques as described previously (12). High-density lipoprotein cholesterol was initially measured after precipitation with phosphotungstate-MgCl 2(13). Total protein level was determined by Biuret's method (14).
Blood pressure and heart rate
Blood pressure and heart rate were monitored by computer-assisted sphygmomanometer, using the indirect cuff technique developed by Cayetta et al. (8) (BP 98E: Softron Co., Ltd, Tokyo). Measurements were taken at 9 AM just before drug administration. The average of three consecutive measurements was used (15).
Histologic evaluation of atherosclerosis
Morphometric analysis was performed as described by Weiner et al. (16) with slight modifications. Six blocks were taken from the descending thoracic aorta. Each block adjacent to each segment taken for evaluation of endothelium-dependent responses was stained with hematoxylin-eosin and van Gieson elastic stain. The contours of the lumen and the internal elastic lamina were traced, and the tracings were digitized (PC-9801 ES, NEC, Tokyo, Japan) using a graphics tablet. The mean surface involvement of atherosclerotic lesion per vessel per animal was calculated. The area occupied by atherosclerotic lesions was defined as the percent area bounded by the lumen and the internal elastic lamina.
Determination of aortic cholesterol content
The segment of the aortic arch down to the bifurcation of the left subclavian arteries was removed, weighed, minced, and homogenized in 10 volumes of sucrose-tromethamine buffer with a motor-driven, glass homogenizer at 0°C–2°C. The extracted lipids were used for the examination of total cholesterol, free cholesterol (11), and esterified cholesterol by the method of Badimon et al. (17).
Isometric tension measurement
After sacrifice, the thoracic aortae were removed carefully and cut into 2-mm-wide transverse rings. Organ chamber experiments were done as described previously to determine the responsiveness of endothelium-dependent relaxation (EDR) to acetylcholine or A23187 and to determine the responsiveness of endothelium-independent vasorelaxation to nitroglycerin (18). In brief, the optimal passive loads for both control and atherosclerotic aortae were determined as the respective contractile responses to 122 m M KCl. Before starting the experiment, the rings were stretched to their predetermined optimal force, mounted on stainless steel hooks in 20-ml-capacity muscle chambers, and bathed in Krebs-Henseleit solution, pH 7.4, at 37°C, for 1 h. Force was measured isometrically using a force displacement transducer (model DSA-603 Minebea Co., Ltd., Tokyo, Japan) and displayed on a multipen recorder (model R-60, Rika Denki Co., Ltd., Tokyo, Japan). Concentration-related contractile responses to l -NMA (10 −6 –10 −4M) and concentration-related relaxation responses to superoxide dismutase (SOD) (1–100 u/ml) were also assessed to investigate tone-related basal nitric oxide release (19). In some experiments, indomethacin (5 μM) was added for 60 min before the start of the experiment to rule out the contribution of prostanoids (20).
Measurement of endothelial nitric oxide synthase mRNA
Rabbit aorta total RNA was extracted using Trizol (guanidine isocyanate; Gibco, Grand Island, NY, U.S.A.). We quantified endothelial nitric oxide synthase (eNOS) mRNA of arterial wall as copies using competitive reverse-transcription polymerase chain reaction (RT-PCR) methods (21). Briefly, to make a DNA competitor, we designed and synthesized two primers (sense and anti-sense primer) based on the sequences of human eNOS cDNA and λDNA. We synthesized an RNA competitor using the DNA competitor and a competitive RNA transcription Kit (Takara Shuzo, Kyoto, Japan). Competitive RT-PCR was done using the RNA competitor and an RNA PCR Kit (Takara Shuzo).
Tissue sections were deparaffinized with xylene and dehydrated with graded alcohol. Immunohistochemical study was performed as previously reported (22). Briefly, the specimens were preincubated with methanol containing 0.3% hydrogen peroxidase, washed with phosphate-buffered saline (PBS), permeabilized with 0.1% triton (Sigma Aldrich, St. Louis, MO, U.S.A.) X100 in PBS, and then washed with PBS. They were incubated for 60 min with primary monoclonal antibody (for anti-macrophage iNOS, anti-rabbit T cells [P8022], anti-rabbit macrophages [RAM11], anti-smooth-muscle cells [HHF35], or anti-nitrotyrosine) diluted in PBS with horse serum, and then washed again with PBS. A biotinated rabbit anti-mouse IgG (1:500 dilution) was applied for 30 min, followed by an avidin-biotin peroxidase complex (ABC Kit, Vector Laboratories, Burlingame, CA, U.S.A.). Negative controls included substitution of primary antiserum/antibody with either PBS or irrelevant antibodies. As a control experiment, treatment with sodium dithionate (100 μM in 100 m M Na 2 CO 3 buffer, pH 9, for 5 min) prior to the antibody incubation abolished the staining for nitrotyrosine. Each field was scored for the number of target antibodies such as monocyte/macrophage–positive cells or nitrotyrosine-positive cells, and the each ratio per all cells of subintimal atherosclerotic plaque was calculated. Five samples were prepared from each rabbit.
Data are expressed as means ± SEM. Statistical analyses were performed using Student's paired and unpaired t tests. Multiple comparisons were performed by one-way analysis of variance with repeated measurements. Then, individual differences were tested by the Scheffe method after the demonstration of significant inner-group differences by analysis of variance. A value of p < 0.05 was considered statistically significant.
The structure of nipradilol is shown in Figure 1.
Plasma biochemical profile
All of the rabbits appeared to be healthy throughout the study. No significant differences in total serum protein, total cholesterol, triglyceride, high-density lipoprotein cholesterol level, or total protein existed among the six groups over the course of the study (Table 1). The addition of l -NAME to the diet did not affect plasma lipid levels (Table 1).
Blood pressure and heart rate
Systolic and diastolic blood pressures of the rabbits in group I were 112 ± 11 mm Hg and 65 ± 12 mm Hg, respectively. Heart rates of rabbits in group I were 230 ± 34 beats/min. Blood pressure tended to increase by l -NAME treatment (i.e., in group II compared with group I). However, blood pressure and heart rate tended to decrease in the nipradilol and atenolol treatment groups (groups V and VI) in comparison with group II (data not shown).
Histologic evaluation of atherosclerosis and assays for tissue cholesterol content
Atherosclerotic lesion, expressed as the mean percentage of lesional encroachment on the luminal surface and the mean area occupied by the lesion in the luminal area, showed more severe atherosclerosis in HCD plus l -NAME treatment groups; however, atherosclerosis in group V was not as severe as atherosclerosis in HCD plus l -NAME treatment groups (groups II, III, IV, and VI) (Figs. 2 and 3). The same tendency was observed in overall tissue total and esterified cholesterol concentration (Fig. 4). The atherosclerosis and tissue cholesterol concentrations in the nipradilol group (group V) were decreased in comparison with those of other l -NAME-treated groups and were comparable to that in the HCD group (group I) (Figs. 2, 3, and 4).
Endothelium-dependent and -independent relaxation
The endothelium-dependent vasodilator acetylcholine and A23187 (data not shown) produced concentration-dependent relaxation with intact endothelium (Fig. 5, left). Relaxation was impaired in groups II, III, IV, and VI in comparison with group I. However, relaxation from the nipradilol treatment animals (group V) was restored. Nitroglycerin produced a concentration-dependent relaxation in vessels without endothelium (Fig. 5, right). The magnitude of the relaxation in aortic rings from the isosorbide dinitrate treatment animals (group III) was slightly impaired, and that from the nitroglycerin-treated animals (group IV) was significantly impaired, in comparison with those from l -NAME control animals (group II). The inhibition of NOS by l -NMA led to a contractile and concentration-dependent response in aortic rings, and it is speculated to demonstrate tone-related basal nitric oxide release (Fig. 6). The magnitude of this response decreased in aortic rings from l -NAME-treated rabbits (groups II, III, IV, and VI) except in l -NAME-plus-nipradilol-treated rabbits (group V) in comparison with rings from the control (group I). The augmentation of NOS activity by SOD (1–100 u/ml) showed the same tendency (data not shown). This means that tone-related basal nitric oxide release was impaired in l -NAME treatment except in l -NAME-plus-nipradilol treatment. Preincubation with indomethacin did not affect EDR.
Detection of mRNA for endothelial nitric oxide synthase in the rabbit aorta
The signal for eNOS mRNA was increased by about 80% in reverse-transcribed RNA samples from aorta of hypercholesterolemic rabbits (group I) as compared with those from normal rabbits (data not shown). In contrast, the yield of PCR products of the predicted size for eNOS was decreased in aortas from the HCD-plus- l -NAME-treated groups excepting the nipradilol group (group V) as compared with the HCD group (group I) (Fig. 7). The signal for eNOS mRNA increased in aortas from the nipradilol group (group V) as compared with other l -NAME-treated groups (Fig. 7).
Histologically, not only macrophages and T cells but also smooth muscle cells were distributed in atherosclerotic lesion in this HCD- and l -NAME-induced atherosclerosis (groups II, III, IV, V, and VI). iNOS was apparent in T cells and some macrophages of the advanced atherosclerotic lesions, and peroxynitrite shown by staining with nitrotyrosine was distributed not only in necrotic cores of fibrous plaques but also in subintimal areas of fibrous plaques (groups II, III, IV, V, and VI). The numbers of monocytes/macrophages in the atheroma of each group were 20.4 ± 4.1, 27.9 ± 5.6, 26.8 ± 3.9, 23.5 ± 4.6, 16.9* ± 3.2, and 23.8 ± 5.4% (*p < 0.05 versus group II) cells in the subintimal atherosclerotic plaque areas of groups I, II, III, IV, V, and VI (Fig. 3, lower). These numbers were decreased in group V in comparison with other l -NAME-treated groups. Further, l -NAME treatment, but not in group V, increased the adhesion of monocytes on endothelium and area of necrotic core also increased in atherosclerotic plaque compared with those from group I. Immunohistochemical staining for nitrotyrosine demonstrated that 28.2 ± 3.1, 17.9 ± 3.6, 20.8 ± 3.4, 18.5 ± 2.6, 13.9 ± 3.2, and 20.8 ± 3.4% cells were nitrotyrosine positive among the cells in the subintimal atherosclerotic plaque areas of aorta from groups I, II, III, IV, V, and VI. No significant differences were observed among l -NAME-treated groups, although values were decreased in comparison with values in group I.
Hypercholesterolemia is a principal cause of the development of atherosclerosis, and the action of EDNO is known to be impaired during this process (1–4). The action of EDNO was also reported to be impaired directly relative to the number of coronary risk factors including hypercholesterolemia as well as hypertension, smoking, diabetes mellitus, and aging, and it was almost abolished in the presence of many complicated risk factors (1–5,23). It is widely accepted that inhibition of nitric oxide production accelerates atherosclerosis (7,8). Conversely, it is not yet known clearly whether nitric oxide donors such as nitroglycerin have anti-atherosclerotic effects. The current study was designed to investigate the effect of nitric oxide donors on hypercholesterolemia and chronic inhibition of nitric oxide synthesis–induced severe atherosclerosis in rabbits. Smooth muscle cells as well as macrophages and T cells appeared in atherosclerotic lesion in this HCD- and l -NAME-induced atherosclerosis, a feature that is relevant to human coronary atherosclerosis and is different from features of the lesion in simple HCD-induced atherosclerosis (24,25).
EDR was worsened in l -NAME-plus-HCD-supplemented rabbits (Fig. 5). Experimental evidence suggests that the inhibition of NOS induces the expression of various genes including those encoding adhesion molecules and inflammatory cytokines (5,26). The l -NAME- and HCD-mediated decrease of EDR was improved significantly only in nipradilol-treated rabbits and was not improved by other nitric oxide donors or β-adrenergic blocker treatment. Intimal thickening was more severe in HCD plus l -NAME, with or without nitric oxide donors or β-adrenergic-blocker-treated rabbits (groups II, III, IV, and VI) as compared with that in HCD rabbits (group I) or with that in HCD-plus- l -NAME and nipradilol-treated rabbits (group V). EDNO has a short half-life, which is reduced further by superoxide anion. O 2− reacts with nitric oxide and peroxynitrite was produced. The reaction is faster than the reaction that O 2− is scavenged by SOD (27). Preliminary measurement of the amount of O 2− release from the vessel by modified the chemiluminescence method did not show the increase in response to l -NAME treatment in this study (data not shown) (30). This finding might indicate that the early scavenge of O 2−, however, does not occur. The immunohistochemical staining area for nitrotyrosine, the footprint of peroxynitrite, was decreased by HCD-plus- l -NAME treatment as compared with HCD treatment and tended to be further decreased in nipradilol groups. We speculate that inhibition of eNOS and iNOS by l -NAME induces depletion of nitric oxide as substrate for peroxynitrite. However, the mechanism should be elucidated further. Although we recently reported that ONOO − impaired EDR in the artery, its contribution may not be principal in this study (28). We speculate that the decrease of EDR in groups II, III, IV, and VI was caused by severe atherosclerosis, especially lipids in atheroma, in these groups of rabbits. A correlation between intimal thickening and impairment of EDR has been reported in an atherosclerotic porcine model, and atherogenic lipoproteins such as oxidized low-density lipoprotein and β-very-low-density lipoprotein were known to impair EDR (29–32).
For this study, we selected nitroglycerin and isosorbide dinitrate, which are widely used in the treatment of angina pectoris and congestive heart failure, and are prescribed for long-term use (i.e. years) for the prevention of angina. Vascular responses to these nitrovasodilators are considered to be mediated by nitric oxide (33,34). Given that the purpose of our study is based on the clinical demand, we selected two kinds of drugs used widely. We did not investigate more ideal nitric oxide donors such as S-nitroso-glutathione, S-nitroso-penicillamine, SIN-1, or sodium nitroprusside, because these donors are usually applied i.v., and absorption from the digestive tract and side effects are not well known (35). SIN-1 is also reported to release nitric oxide as well as O 2− and ONOO −(35). The endothelium-independent vasodilator nitroglycerin produced a concentration-dependent relaxation. The relaxation of aortic rings from nitroglycerin-treated animals (group III) was impaired as compared with that in rings from other l -NAME-treated animals. It is possible that tolerance to nitric oxide donors such as nitroglycerin occurred in these vessels. EDRF and other nitrovasodilators produce relaxations predominantly by increase of cGMP in smooth muscle cells.
The mechanism of the anti-atherosclerotic effect of nipradilol is discussed as follows. Nipradilol is a β-adrenoceptor blocker with nitric oxide–releasing action (9); i.e., nipradilol is a nonselective β-blocker with nitroglycerin-like activity (36). It is clear that the anti-atherosclerotic effect of nipradilol in this study was not caused by the effect of plasma lipid levels nor was it caused by the effect of blood pressure, because these parameters were not notably changed by nipradilol treatment. The β-blocking activity of nipradilol in vitro is reported to be approximately twice that of propranolol (36). Given that atenolol, a pure β1-blocker that shows β-blocking action three times stronger than that of propranolol, did not show an anti-atherosclerotic effect, the effect of nipradilol may not be caused by β1-blocking action. The number of inflammatory cells such as macrophages and T lymphocytes decreased in the atherosclerotic area of nipradilol-treated rabbits. Therefore, the anti-atherosclerotic effect of nipradilol may be caused by the increased action of nitric oxide. Nipradilol treatment showed a remarkable anti-atherosclerotic effect on severe atherosclerosis induced by inhibition of intrinsic nitric oxide activity with HCD. We demonstrated the possibility that nipradilol released nitric oxide after binding with β-adrenoceptor existing only in the caveolae of cultured endothelial or smooth muscle cells (37). Caveolae, which occupied a small percentage of the cell surface, is known to the site of eNOS activation (38). We found that eNOS mRNA, protein, and activity were not inhibited by nipradilol but they were augmented up to 1.6 times by nipradilol (37). Nipradilol-induced nitric oxide release might be derived from denitration of itself and enhancement of eNOS, although eNOS activity was decreased by treatment with l -NAME, with or without nitric oxide donors except nipradilol. The beneficial effect on atherosclerosis by nipradilol is partially caused by activation of intrinsic eNOS activity.
Conclusively, nipradilol, a β-adrenergic blocker with nitric oxide–releasing action, retarded the progression of atherosclerosis produced by chronic inhibition of intrinsic nitric oxide release and HCD-induced atherosclerosis. However, neither nitric oxide donor (nitroglycerin and isosorbide dinitrate) nor another β-adrenergic blocker (atenolol) had this beneficial effect.
This study was supported in part by grant-in-aid No. 09470166 and 11670672 of the Japanese ministry of Education. The authors thank Yuriko Kato for excellent technical assistance.
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