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Cardiovascular Research 1998 38(2):508-515; doi:10.1016/S0008-6363(98)00027-3
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Supersensitivity of atherosclerotic artery to constrictor effect of cigarette smoke extract

Seigo Sugiyama, Kiyotaka Kugiyama*, Masamichi Ohgushi, Toshiyuki Matsumura, Yasutaka Ota, Hideki Doi, Nobuhiko Ogata, Hideki Oka and Hirofumi Yasue

Division of Cardiology, Department of Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto, Kumamoto 860, Japan

* Corresponding author. Tel.: +81 96 3735175; fax: +81 96 3623256; e-mail: kiyo@gpo.kumamoto-u.ac.jp

Received 28 July 1997; accepted 23 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to assess whether contractile response of arteries to aqueous component of cigarette smoke extract (CSE) may be modulated in atherosclerotic arteries. Methods: Thoracic aortas were isolated from control rabbits and from 1.5% cholesterol-fed rabbits, all of which had visible advanced atheromatous surface changes on the aortas. CSE was prepared by bubbling main stream of smoke from one cigarette with filter into 2 ml of phosphate-buffered saline. The thoracic aortic rings were suspended in organ chambers and tested with CSE (0.01–3.0 µl/ml buffer in the organ chamber) after precontraction with 0.1 µmol/l of phenylephrine (PE). Results: The contractile response to CSE was significantly greater in atherosclerotic aortas than in control aortas (the maximal contraction expressed as % of the precontraction; control aortas 10.8±2.8%, atherosclerotic aortas 42.6±4.7%; P<0.01). The magnitude of the precontractions by PE was not different between control and atherosclerotic aortas. Exogenous addition of superoxide dismutase (SOD) significantly attenuated the CSE-induced contraction in both control and atherosclerotic aortas and pretreatment of aortic rings with diethyldithiocarbamate to deplete of endogenous vascular CuZn-SOD activity potentiated the CSE-induced contraction in control aortas, while it had no significant effect in atherosclerotic aortas. The vascular SOD activity was significantly lower in atherosclerotic aortas than in control aortas ((U/mg protein): control aortas 38.2±3.3, atherosclerotic aortas 18.5±2.4; P<0.01). Conclusion: These results indicate that atherosclerotic arteries may be supersensitive to the constrictor effect of superoxide anion derived from CSE. The decrease in endogenous vascular SOD activity may partly contribute to the increased susceptibility to oxidative stress in atherosclerotic arteries.

KEYWORDS Oxidative stress; Nitric oxide; Superoxide anion; Superoxide dismutase; Rabbit; Atherosclerosis; Free radical; Vascular tone


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Cigarette smoking is strongly associated with cardiovascular diseases and acute coronary syndrome [1], and smoking is the major risk factor for coronary artery spasm [2]. It has been well known that cigarette smoke contains a large number of oxidants [3], and we and others have demonstrated that an aqueous extract of cigarette smoke, namely cigarette smoke extract (CSE), contains some oxygen free radicals such as superoxide anion or hydroxyl radicals [4–6]. There is increasing evidence that oxidative stress may profoundly contribute to pathogenesis of atherosclerosis and cardiovascular diseases [7], and several risk factors for atherosclerosis, such as hypercholesterolemia [8]and diabetes [9], could promote oxidative stress, and different responses to oxidative stress may be involved in susceptibility to atherosclerosis [10]. Recently, cigarette smoking has been shown to introduce reactive oxygen intermediates in human circulation [11]and decrease plasma concentrations of antioxidants, such as vitamin C and β-carotene [12], and the deteriorative actions of smoking are prevented or reversed by supplement of antioxidants [13]. Thus, there is a possibility that oxidative stress may be increased in smokers in vivo.

It has been shown that smoking exerts acute effects on both systemic and coronary hemodynamics [14, 15]and induces coronary spasm in some patients [16]. Previously, we have shown that CSE causes contraction of normal porcine coronary arteries through degradation of basally released nitric oxide (NO) by superoxide anion derived from CSE [4, 5]. Cigarette smoking has been reported to increase coronary vascular resistance and reduce coronary blood flow in patients with atherosclerotic coronary artery disease [14, 17]. Increased vascular tone of atherosclerotic coronary arteries has been shown to play an important role in pathogenesis of acute myocardial infarction and acute coronary syndrome [18]. The augmentation of vasoconstrictor responses to exercise [19]and vasoactive agents [20]have been well documented in atherosclerotic arteries. However, the effect of cigarette smoking on atherosclerotic arterial tone and its mechanism(s) were not fully examined. Therefore, the aim of this study was to examine effects of CSE on vascular tone of atherosclerotic arteries.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals
Male New Zealand White rabbits (2.4–2.8 kg) were fed a standard rabbit chow (CLERA, Japan) (control rabbits: n=25) or an identical chow enriched with 1.5% cholesterol (atherosclerotic rabbits) for 4 months (n=30) in an air-conditioned room at 20°C and 50% humidity with a 12 h light/12 h dark cycle at Kumamoto University Medical Animal Center. All experiments were in accordance with the guidelines on the treatment of experimental animals and were approved by the Center for Laboratory Animals of our institution and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health (NIH Publication No. 85-23, revised 1985). Serum total cholesterol level was increased in high cholesterol diet-fed rabbits but it remained normal level in control rabbits after the 4-month diet period (total serum cholesterol (mmol/l): 46.7±6.8 in high cholesterol diet, 1.2±0.2 in standard diet). Thoracic aortas were excised from rabbits under anesthesia with sodium pentobarbital (60 mg/kg i.v.) after anti-coagulation by heparin (1500 units i.v.) and the thoracic aorta was dissected free from adherent connective tissue and prepared for organ chamber experiments and assay of vascular SOD activity. The aortic rings were prepared with special care being taken not to touch the luminal surface of the aortas.

2.2 Preparation of cigarette smoke extract (CSE)
CSE was freshly prepared in every experiment as previously described [4, 5]. Briefly, the mainstream of cigarette smoke with filter was bubbled into 2 ml of phosphate-buffered saline (PBS) by constant vacuum (–3 cm H2O). The solution of cigarette smoke was filtered through a 0.22-µm filter to remove particulates. CSE was used within 2 min after the preparation. Superoxide anion generation in CSE was measured in every CSE preparation by ferricytochrome c reduction expressed as an increase in the absorbance at 550 nm monitored by spectrophotometer [4, 5]. The CSE solution (10 µl/ml PBS) had superoxide anion at the levels of 1.51±0.02, ranged from 1.64 to 1.46 (increase in absorbance at 550 nm/30 min).

2.3 Organ chamber experiment
Aortic rings of 3-mm width were prepared from the isolated thoracic aortas in a similar manner as shown in our previous reports [4, 5, 21, 22]and suspended by stainless-steel hooks in the organ chambers filled with Krebs–Henseleit bicarbonate solution (KHS, pH 7.4) composed of (mmol/l) Na+, 144.2; K+, 4.0; Ca2+, 1.5; Mg2+, 1.2; Cl–, 123.0; SO2–4, 1.2; phosphate, 1.2; HCO–3, 25.0; glucose, 5.0. The solution was aerated with 95% O2/5% CO2, maintained at 37°C and replaced every 20 min. The aortic rings were then stretched to an optimum basal tension of 3 g, and isometric tension was monitored. After equilibration for 60 min, the rings were exposed to 40 mmol/l of potassium chloride (KCl) to examine contractile response of vascular smooth muscle. After repeated washing and another equilibration for 40 min, the rings were pre-contracted with phenylephrine (PE, 0.1 µmol/l), and then tested with CSE (3.0 or 0.01–9.0 µl of CSE/ml of KHS in the organ chamber), pyrogallol (100 µmol/l), a potent generator of superoxide anion, or 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide derivatives (PTIO, 1 mmol/l), a potent scavenger of the released NO [23]. CSE, pyrogallol or PTIO was added into the organ chamber 5 min after stable plateau contraction by PE was obtained. PE at the concentration of 0.1 µmol/l used in the present study developed a tension of approximately 50% of that induced by PE at the concentration of 10 µmol/l (maximal dose tested) according to the dose–contraction relationship curve. In another set of experiments, the rings were pretreated with SOD (200 U/ml) to scavenge superoxide anion, diethyldithiocarbamate (DETCA, 10 mmol/l) to deplete CuZn-SOD, NG-monomethyl-L-arginine (L-NMMA; 500 µmol/l), a nitric oxide synthase blocker, or vehicle (PBS), and 5 min later, the pretreated rings were contracted by PE (0.1 µmol/l). Thereafter, the rings were tested with CSE. Two rings from each rabbit were examined for each treatment and the responses to each treatment in each rabbit were averaged and used for statistic analysis. The extent of contractions elicited by CSE or other chemicals was expressed as percentage of the precontraction evoked by PE (0.1 µmol/l). For contractions, effective volume of CSE causing 50% contraction of maximum contraction (EC50) was calculated from each concentration–response curve. After the organ chamber experiment, the rings were cut and opened longitudinally and their intimal surface was investigated for occurrence of atherosclerotic changes by staining with Sudan IV-lipid staining after fixing with 10% buffered formalin overnight. None of the control aortas showed any fatty streaks, but approximately 60–70% of intimal surface area on the aortas of each cholesterol-fed rabbit was covered with fatty streaks and showed advanced atheromatous changes. The endothelial cell line was preserved in all of aortic rings tested by light microscopy from both control and cholesterol-fed rabbits.

2.4 Preparation of tissue extract of thoracic aorta
The remaining thoracic aortic segments were cleared off the perivascular connective tissue and washed with ice-cold 0.1 mol/l phosphate buffer (pH 7.8), and the aortas were homogenized in 1 ml of 0.1 mol/l phosphate buffer contained protease inhibitors by using a glass-to-glass homogenizer, and they were then sonicated by Bronson sonicator at 4°C. The homogenates were centrifuged (100,000xg, 30 min, 4°C) and the resultant supernatant was collected as the tissue extract of aortas and stored at –80°C until using for SOD-activity assay. Protein concentration was measured with method of Bradford using bovine serum albumin as a standard.

2.5 SOD activity
SOD activity was determined by the modified method of Spitz and Oberley [24]. The reaction mixture contained: xanthine, 0.4 mmol/l; defatted bovine serum albumin, 0.13 mg/ml; nitroblue tetrazolium (NBT), 220 µmol/l; disodium salt of bathocuproine disulfonic acid, 0.1 mmol/l; catalase, 1 U/ml; 2 ml of 0.1 mol/l phosphate buffer (pH 7.8). Xanthine oxidase was added to the reaction tube containing sample or standard SOD, and the mixtures were then incubated at 37°C for 20 min. Manganese SOD (Mn-SOD) activity was measured after incubation with 5 mmol/l NaCN for 30 min in the assay mixture. After the incubation, the reaction was terminated by addition of 2% sodium dodecyl sulfate, and then produced formazan was spectrophotometrically measured at 560 nm. The vascular CuZn-SOD activity was calculated by subtracting the Mn-SOD activity value from the total SOD value.

2.6 Reagents
PTIO was obtained from DOJINDO, Kumamoto Japan. SOD (S5395, bovine erythrocytes), catalase (C40), DETCA and other chemicals were obtained from Sigma Chemical, St. Louis, MO, USA.

2.7 Data analysis
Results are expressed as mean±s.e.m. Statistical evaluation of the data was performed by Student's t-test for unpaired observation. When more than two groups were compared, analysis of variance was used. Values were considered to be statistically different at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 CSE-induced contraction in control and atherosclerotic rabbit aortas
When single dose of CSE (3.0 µl of CSE/ml) was added into the organ chambers with aortic rings precontracted by PE (0.1 µmol/l), CSE elicited transient contraction in both control and atherosclerotic aortas (Fig. 1). As shown in Fig. 1, the CSE-induced contraction after the PE-induced precontraction was significantly greater in atherosclerotic aortas than in control (% of the precontraction by PE: control aortas 10.8±2.8%, atherosclerotic aortas 42.6±4.7%; P<0.01). In contrast, maximum response to PE (0.1 µmol/l) or KCl (40 mmol/l) was not significantly different in the two groups (Table 1). The addition of CSE caused smaller contraction in the quiescent aortas before the precontraction by PE (addition of CSE (3.0 µl/ml) before the precontraction by PE (% of the contraction by PE): control aortas 3.5±0.32%* vs. atherosclerotic aortas 8.8±0.74%*, P<0.05 between control and atherosclerotic aortas, n=6, respectively; *P<0.01 vs. the respective contractions in control and atherosclerotic aortas by CSE after precontraction by PE).


Figure 1
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  		Fig. 1 Representative recordings and bar graphs showing contractile effects of cigarette smoke extract (CSE) on control and atherosclerotic aortic rings. Upper panels: CSE (3.0 µl/ml buffer in organ chamber) was added into the organ chambers with rabbit thoracic aortic rings during stable contraction evoked by phenylephrine (0.1 µmol/l). CSE elicited transient contraction in both control and atherosclerotic aortic rings. CSE, cigarette smoke extract; PE, phenylephrine; W, washout. Lower panels: the CSE (3.0 µl/ml)-induced contraction was significantly greater in atherosclerotic aortas (42.6±4.7%) than control aortas (10.8±2.8%). Data are expressed as percent increase from the precontraction by phenylephrine. Values are mean±s.e.m. *P<0.01 vs. control; n=6 in both.

 

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  		Table 1 Contraction in response to KCl and phenylephrine in isolated rabbit aortic rings

 
To determine concentration–response relations in control and atherosclerotic aortas, cumulative doses of CSE (0.01–9.0 µl of CSE/ml) were delivered into the organ chambers with aortic rings precontracted by PE (0.1 µmol/l). As shown in Fig. 2, the contractions elicited by CSE were in a dose-dependent manner from 0.01 µl/ml to 3.0 µl/ml and the maximum contraction was significantly greater in atherosclerotic aortas than in control aortas (maximum contraction (g): control aortas 0.42±0.06, atherosclerotic aortas 0.81±0.08; P<0.01, n=8 in both). EC50 was significantly lower in atherosclerotic aortas than in control aortas (EC50 (µl/ml): control aortas 0.28±0.03, atherosclerotic aortas 0.18±0.02; P<0.05, n=8 in both). These results indicate that the contractile response to CSE was hypersensitive and hyperreactive in atherosclerotic aortas.


Figure 2
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  		Fig. 2 Graph showing concentration–response relation of CSE-induced contraction in control and atherosclerotic aortas. Cumulative doses of CSE (0.01–9.0 µl/ml buffer in organ chamber) were delivered into the organ chambers with aortic rings precontracted by phenylephrine (0.1 µmol/l). Data are expressed as percent increase from precontraction by phenylephrine. CSE-induced contractions were in a dose-dependent manner from 0.01 µl/ml to 3.0 µl/ml of CSE in both control and atherosclerotic groups. The maximum contraction was significantly greater in atherosclerotic aortas than control aortas. Values are mean±s.e.m. *P<0.01 vs. control; n=8 in both.

 
3.2 Role of nitric oxide released from arteries in vasocontraction by CSE
The addition of L-NMMA (500 µmol/l), which is a blocker of NO synthase, caused smaller contraction of the quiescent aortas before the precontraction with PE ((g): control aortas 0.11±0.11 vs. atherosclerotic aortas 0.14±0.12; P=not significant (n.s.), n=6 in both). The contraction by the subsequent addition of PE (0.1 µmol/l) tended to be greater but was not significantly increased in the aortas pretreated by L-NMMA as compared with those not pretreated by L-NMMA (PE-induced contraction (g). Control aortas: vehicle 2.8±0.3 vs. L-NMMA 3.0±0.3; P=n.s. Atherosclerotic aortas: vehicle 2.6±0.3 vs. L-NMMA 2.9±0.3; P=n.s.; n=10 in both). After pretreatment of aortic rings with L-NMMA (500 µmol/l), the CSE-induced contraction was almost abolished in both control and atherosclerotic aortas (Fig. 3). Furthermore, we investigated effects of PTIO which is a potent scavenger of released NO [23]. As shown in Fig. 4, PTIO (1 mmol/l) elicited contractions in both control and atherosclerotic aortas. The PTIO-induced contractions in both aortas were also abolished by pretreatment of aortic rings with L-NMMA (PTIO-induced contractions after L-NMMA treatment (% contraction by PE): control aortas 1.2±0.1%, atherosclerotic aortas 2.8±0.1%; n=6). The PTIO-induced contraction was significantly greater than the CSE-induced contraction in control aortas, while it was comparable magnitude with the CSE-induced contraction in atherosclerotic aortas. The magnitude of the PTIO-induced contraction in atherosclerotic aortas was tended to be greater but it was not significantly different as compared with that in control aortas (% maximum contraction: control 28.6±3.9, atherosclerosis 37.2±4.2; P=n.s., n=9 in both) (Fig. 4).


Figure 3
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  		Fig. 3 Bar graphs showing effects of L-NMMA on the CSE-induced contraction in control and atherosclerotic rabbit aortas. Rabbit aortic rings were pretreated with vehicle (PBS) or L-NMMA (500 µmol/l) and then CSE (3.0 µl/ml buffer in organ chamber) was added into the organ chambers with rabbit aortic rings after stable precontraction with phenylephrine (0.1 µmol/l) in the presence of vehicle or L-NMMA. The CSE-induced contraction was abolished in both control and atherosclerotic aortas pretreated with L-NMMA. Data are expressed as percent increase from precontraction by phenylephrine. Values are mean±s.e.m. *P<0.01 vs. vehicle; n=6 in both. Vehicle, pretreated with PBS; L-NMMA, pretreated with L-NMMA.

 

Figure 4
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  		Fig. 4 Representative recordings showing contractile effects of PTIO on control and atherosclerotic aortic rings (upper) and the bar graphs showing values of the CSE-induced contraction and the PTIO-induced contraction in control and atherosclerotic rabbit aortas. CSE (3.0 µl/ml buffer in organ chamber) or PTIO (1.0 mmol/l) was added into the organ chambers with rabbit aortic rings after stable precontraction with phenylephrine (0.1 µmol/l). PTIO (1 mmol/l) elicited contraction in both control and atherosclerotic aortas, and the PTIO-induced contraction was significantly greater than the CSE-induced contraction in control aortas, but there was no significant difference in the magnitude of the PTIO-induced contraction between control and atherosclerotic aortas. Data are expressed as percent increase from precontraction by phenylephrine. Values are mean±s.e.m. *P<0.01; NS=not significant; n=9 in both. CSE, CSE-induced contraction; PTIO, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide derivatives (PTIO)-induced contraction.

 
3.3 Role of superoxide anion in contraction by CSE in atherosclerotic aortas
As shown in Fig. 5, treatment of aortic rings with exogenous SOD significantly attenuated the CSE-induced contraction in both control and atherosclerotic aortas, but atherosclerotic aortas still had a greater contractile response to CSE after treatment with SOD than control aortas. There was no significant difference in the preincubation time with SOD between control and atherosclerotic aortas (15±1.2 min vs. 16±1.5 min; P=n.s., n=6 in both). Catalase (1500 U/ml) did not significantly affect the CSE-induced contraction in both control and atherosclerotic aortas (CSE-induced contraction after catalase treatment ((% of precontraction by PE): control aortas 9.8±3.2, atherosclerotic aortas 39.2±5.8; P=n.s., n=6, respectively). These findings suggest that superoxide anion contained in CSE may be involved in the contraction elicited by CSE in both control and atherosclerotic aortas and confirm the results in our previous reports [4, 5]. Then, we further examined effects of pyrogallol as a potent superoxide anion generator on the vascular tone. Fig. 6 shows that pyrogallol (100 µmol/l) also elicited contraction in both control and atherosclerotic aortas and the pyrogallol-induced contraction was significantly greater in atherosclerotic aortas than in control aortas, mimicking the effects of CSE. Further, pretreatment of the aortas with SOD (200 U/ml) completely abolished the pyrogallol-induced contraction.


Figure 5
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  		Fig. 5 Bar graphs showing effects of SOD on the CSE-induced contraction in control and atherosclerotic rabbit aortas. Rabbit aortic rings were pretreated with vehicle (PBS) or SOD (200 U/ml). CSE (3.0 µl/ml buffer in organ chamber) was then added into the organ chambers with rabbit aortic rings after stable contraction evoked by phenylephrine (0.1 µmol/l) in the presence of vehicle or SOD. The pretreatment with SOD significantly attenuates the CSE-induced contraction in both control and atherosclerotic aortas. Data are expressed as percent increase from precontraction by phenylephrine. Values are mean±s.e.m. *P<0.01; n=6 in both. Vehicle, pretreated with PBS; SOD, pretreated with SOD.

 

Figure 6
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  		Fig. 6 Representative recordings showing contractile effects of pyrogallol on control and atherosclerotic aortic rings (upper) and the bar graph showing values of the pyrogallol-induced contraction in control and atherosclerotic rabbit aortas (below). Pyrogallol (100 µmol/l) was added into the organ chambers with rabbit aortic rings during stable precontraction evoked by phenylephrine (0.1 µmol/l). Pyrogallol elicited transient contraction in both control and atherosclerotic aortic ring, and the contractile response to pyrogallol was greater in atherosclerotic aortas than control aortas. Data are expressed as percent increase from the precontraction by phenylephrine. Values are mean±s.e.m. *P<0.01 vs. control; n=6 in both.

 
Since it has been known that SOD activity exists in vascular walls, there is a possibility that endogenous vascular SOD could modulate effects of superoxide anion derived from CSE on the vascular tone in control and atherosclerotic aortas. To determine the possible involvement of intrinsic vascular SOD in regulation of the CSE-induced contraction, aortic rings were pretreated with DETCA (10 mmol/l) to deplete of endogenous vascular CuZn-SOD activity and CSE (3.0 µl/ml) was then delivered into the organ chamber with the DETCA-pretreated rings after the precontraction with PE (0.1 µmol/l) in the presence of DETCA. As shown in Fig. 7, pretreatment with DETCA potentiated the CSE-induced contraction only in control aortas, while it had no significant effect on atherosclerotic aortas. There was the similar magnitude of the CSE-induced contraction in control and atherosclerotic aortas after pretreatment with DETCA (Fig. 7). These results indicate that endogenous vascular CuZn-SOD could effectively scavenge and inactivate superoxide anion derived from CSE in control aortas, but it could not sufficiently inactivate the superoxide anion derived from CSE in atherosclerotic aortas and DETCA-treated control aortas.


Figure 7
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  		Fig. 7 Bar graphs showing effects of DETCA on the CSE-induced contraction in control and atherosclerotic rabbit aortas. Rabbit aortic rings were pretreated with vehicle (PBS) or DETCA (10 mmol/l). CSE (3.0 µl/ml buffer in organ chamber) was then added into the organ chambers with rabbit aortic rings after stable precontraction evoked by phenylephrine (0.1 µmol/l) in the presence of vehicle or DETCA. Pretreatment with DETCA potentiated the CSE-induced contraction in control aortas, while it had no significant effect in atherosclerotic aortas. Data are expressed as percent increase from the precontraction by phenylephrine. Values are mean±s.e.m. *P<0.01; n=6 in both. Vehicle, pretreated with PBS; DETCA, pretreated with DETCA.

 
3.4 Vascular SOD activity
Vascular SOD could modulate vascular superoxide level and regulate vascular tone, then, we examined SOD activity in tissue extract of aortas by modified NBT assay system [24]. In the present study, Mn-SOD activity was undetectable in all samples, therefore the vascular SOD activity in the tissue extract of the aortas means the CuZn-SOD activity. The vascular SOD activity of atherosclerotic aortas was significantly lower than that of control aortas ((U/mg protein): control aortas 38.2±3.3 vs. atherosclerotic aortas 18.5±2.4; P<0.01, n=10 in both).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have previously demonstrated that CSE contracts isolated normal porcine coronary arteries probably through degradation of basally released NO by superoxide anion derived from CSE [4]. The present study also showed that CSE contracted both control and atherosclerotic rabbit thoracic aortas. Furthermore, we showed that pyrogallol-induced contractions in control and atherosclerotic aortas were similar to the CSE-induced contractions, and the pretreatment of aortic rings with SOD attenuated the CSE-induced contraction in both aortas, and further the pretreatment of aortic rings with L-NMMA almost completely abolished the constrictor effect of CSE in both aortas. These present results confirm our previous data [4]. The main new finding of the present study is that the contractile response to CSE was significantly greater in atherosclerotic aortas than in control aortas.

Basal release of NO has been shown to play an important role in regulation of arterial tone [25], and it is known to be degraded by endogenous and exogenous superoxide anion in arterial walls [26], but superoxide anion itself is inactivated by intrinsic SOD in arterial walls at the same time. Thus, although superoxide anion can contract arteries, the superoxide-mediated contractile response may be modulated by balance of the basally released NO and the endogenous vascular SOD activity. The present study showed that PTIO, a potent scavenger of released NO [23], caused arterial contraction with comparable magnitude between control and atherosclerotic aortas, suggesting that the basally released NO may be equivalent between control and atherosclerotic aortas. It is generally accepted that NO bioactivity is decreased in atherosclerotic arteries. It was proposed that the increased superoxide anion in atherosclerotic arterial walls [8]caused breakdown of the released NO [26], leading to decrease in the NO bioactivity in spite of the same or increased level of NO production and release in atherosclerotic arterial walls as compared with normal arteries [27]. Recently, it has been demonstrated that NO synthase is expressed not only in endothelial cells but also in smooth muscle cells and macrophages of atherosclerotic arterial walls [28], raising a possibility that atherosclerotic arteries could basally release abundant NO.

In the present study, we showed that the CSE-induced contractions were significantly inhibited by SOD-treatment in both control and atherosclerotic arteries, indicating that the CSE-induced contractions seem to be mediated through superoxide anion derived from CSE. We and others have previously shown that cigarette smoke and cigarette smoke extract (CSE) contain a huge amount of oxygen free radicals such as superoxide anion and hydrogen peroxide [3–6], and Zang et al. demonstrated that superoxide anion is produced from autooxidation of hydroquinone radicals contained in CSE [29]. Because the quinone radicals have a long life in aqueous solution and hydrophobic environment [3], it is possible that the quinone radicals generated in CSE may be one of the sources of superoxide anion derived from CSE in the present study. Furthermore, we demonstrated that pretreatment of arteries with DETCA, which depletes of endogenous SOD activity, potentiated the CSE-induced contraction in control aortas, while the DETCA-treatment had no effect in atherosclerotic arteries, indicating that endogenous vascular SOD activity may play an important role in regulation of the CSE-induced contraction, and then we here presented that the endogenous vascular SOD activity in atherosclerotic arteries was found to be much less as compared with that in control arteries. These results suggest that decreased endogenous vascular CuZn-SOD activity may fail to inactivate superoxide anion derived from CSE, leading to the greater contraction by CSE in atherosclerotic arteries.

The present study clinically implies that atherosclerotic arteries may have the supersensitivity to constrictor effect of cigarette smoking. In fact, Klein et al. demonstrated that smoking significantly increased coronary resistance in patients with atherosclerotic coronary artery disease [17], Nicod et al. indicated an association between smoking-induced decrease of coronary flow and severity of atherosclerosis [14], and Maouad et al. showed that cigarette smoking induces coronary spasm in some patients [16]. Although the present study confirms our previous report [4]and shows that atherosclerotic arteries exhibited the supersensitive constrictor response to superoxide anion derived from CSE, it may be, however, difficult to directly extrapolate the present results to human smokers. It remains undetermined whether free radicals generated by cigarette smoking actually reach the systemic and coronary arteries because it is believed that the half-life of free radicals is extremely short in the circulation. However, cigarette smoking has recently been shown to decrease plasma concentrations of antioxidants, such as vitamin C, E, and β-carotene [11]. Deteriorative actions of smoking on endothelial functions were prevented or reversed by supplement of antioxidants in vivo [13]. Furthermore, the plasma indexes of lipid peroxidation and oxidized lipoprotein are shown to be increased in smokers [11, 30, 31]. Therefore, it is quite possible that cigarette smoking could induce oxidative stress and affect vascular functions in smokers in vivo. We have previously shown that oxidatively modified low-density lipoprotein impairs NO-mediated regulation of the vascular tone and causes arterial contraction [21, 22]. Because lipid peroxidation products and reactive oxygen intermediates such as quinone radicals by smoking have a long life and they could be a continuous source of oxidative stress in human circulation [3], the oxidative stress generated by smoking may directly or indirectly inhibit NO-mediated regulation of arterial tone especially in atherosclerotic arteries. Since cigarette smoking is a major risk factor for ischemic heart disease [1]and the increased tone of atherosclerotic coronary arteries has been shown to play an important role in acute coronary syndrome [18], it may be conceivable that the increase in vascular tone by cigarette smoking-induced oxidative stress could be involved in the pathogenesis of ischemic heart disease. In the meantime, we cannot completely exclude a possibility that CSE contracts atherosclerotic arteries via other mechanisms (stimulation of sympathetic nerve system) than superoxide anion because the present study shows that the CSE-induced contraction after the treatment with exogenous SOD was still greater in atherosclerotic aortas than in control aortas. The maximum contraction induced by a single dose of CSE (3.0 µl/ml) was greater than that induced by cumulative doses of CSE at 3.0 µl/ml in both control and atherosclerotic aortas. There is a possibility that it may be caused by the effect of CSE-induced late phase relaxation in the cumulative doses of CSE as shown in our previous report [4]. However, the late phase relaxation may play a minimal role in this study since CSE induced little relaxation subsequently after the initial contraction in the isolated rabbit aortas.

In the present study, we demonstrated that the vascular SOD activity was significantly lower in atherosclerotic aortas than control aortas. However, Del Boccio et al. reported an increase in arterial SOD activity in hypercholesterolemic diet-fed rabbits [30], but Mugge et al. and White et al. demonstrated no difference in the SOD activity between normal and atherosclerotic arteries [26, 32]. The reason for the discrepancy in the SOD activity between ours and their studies is uncertain but it may be caused by a difference in the stages of atherosclerotic lesions. For example, Del Boccio et al. used lower cholesterol diet for short period (0.5% of cholesterol diet for 1–2 months) and their atherosclerotic changes were only fatty streaks, but we used high cholesterol for longer period (1.5% of cholesterol diet for 4 months) and our vascular lesions then showed fatty streaks and advanced atheromatous changes (60–70% of total surface), raising the possibility that vascular SOD activity may initially increase in the fatty streaks (in their studies) and thereafter decrease in the advanced atheromatous plaque lesion (in our study). The time course of vascular SOD activity during various concentrations of cholesterol treatment and its mechanisms remain to be investigated.

The arterial tone is determined by the balance between contracting and relaxing factors and it is not under quiescent condition but may be tensed in vivo. This is the reason why we chose the precontracted aortic rings for the experiments. In the present study, the contraction by CSE was smaller in quiescent aortas but it was significantly exaggerated after precontraction with phenylephrine, a phenomenon which is in agreement with our previous report [4]. The similar difference in the contraction of the aortas between before and after precontraction was also observed in the cases of L-NMMA and oxidized low-density lipoprotein in our previous and others' reports [22, 33]. Its precise mechanism remains unknown in the present study. However, there is a possibility that basal release of endothelium-derived NO may be augmented in the precontracted aortas as compared with the quiescent aortas, and there may be a phenomenon of ‘contractile synergism’ between NO inhibition and vasocontracting agonists.

In conclusion, the present study shows that atherosclerotic arteries exhibit supersensitive contractile response to superoxide anion derived from CSE. This may be partly caused by decreased endogenous vascular SOD activity in atherosclerotic arteries. The susceptibility to oxidative stress by smoking may be increased in atherosclerotic arteries, which may lead to the genesis of coronary spasm and acute coronary syndrome in atherosclerotic arteries.

Time for primary review 25 days.


   Acknowledgements
 
This work was partly presented at the 67th American Heart Association Meeting at Atlanta. This work was supported in part by the grant-in-aid for Scientific Research on Priority Areas A08407019, C07670793 and C08670807 from the Ministry of Education, Science and Culture in Japan, and Smoking Research Foundation Grant for Biomedical Research, Tokyo, Japan.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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