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Departments of 1Biomedical Engineering, 2Surgery, and 3Cellular and Integrative Physiology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana; and 4Department of Community and Environmental Medicine, University of California, Irvine, California
Submitted 8 April 2006 ; accepted in final form 14 June 2006
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ABSTRACT |
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nitrite; strain; stress; elastic modulus; stiffness
Nitric oxide (NO) is an endothelium-derived relaxing factor synthesized in arterial endothelium from the amino acid L-arginine by the enzyme NO synthase (NOS), which is expressed constitutively in endothelial cells (eNOS or type III NOS) (24). Endothelium-derived NO is a potent endogenous vasodilator that contributes to resting arterial tone and affects both platelet function and smooth muscle cell proliferation (28). The eNOS is essential in maintaining basal vascular NO production that regulates blood flow, particularly coronary blood flow. Reduction in basal NO release may cause a predisposition to hypertension, thrombosis, vasospasm, and atherosclerosis (25). In vivo, CS (34) and nicotine infusion (4) impair the endothelium-dependent relaxation mediated by NO in human arteries and veins.
Arterial mechanical properties can be influenced by several factors, such as heart rate, atherosclerotic plaque, blood pressure, and age. Houdi et al. (13) found that the exposure of conscious rats to CS resulted in a decrease in heart rate and cardiac output and an increase in mean arterial pressure and total peripheral resistance. Previous studies (18) have shown that acute CS decreased arterial compliance in both large elastic and medium-sized muscular arteries. An invasive study (29) in men with coronary artery disease has shown that acute exposure to passive smoking impairs elastic properties of the aorta. Although these past studies have established that CS causes changes in the coronary blood vessels and circulation, no systematic data on the remodeling of the mouse coronary arteries exist nor have the changes been correlated with the NO bioavailability.
The present study was designed to determine the effects of short-term (6 wk) and long-term (16 wk) CS exposure on the structural and biomechanical properties of the coronary arteries. Furthermore, the reversibility of the deleterious remodeling after CS cessation was also addressed. We hypothesized that NO derived from endothelium is intimately connected with arterial wall structure and mechanical properties. In conjunction with the changes in the mechanical properties of coronary arteries, we assessed the effect of CS on basal NO production (nitrite and nitrate) and eNOS protein expression in the vessel wall. Our findings confirm a correlation between NO and the change of structural and mechanical status of arterial wall in response to CS.
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METHODS |
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Forty-two homozygous, inbred male mice (C57BL/6 strain) were used in this study. The Walton Smoke Exposure Machine (Process & Instruments, Brooklyn, NY) was used to puff 1R3 cigarettes according to the Federal Trade Commission puffing regimen (35 ml over 2 s; 1 puff/min) to generate the mainstream smoke used for exposure of mice in this study. The machine was operated to deliver a 1-day exposure equivalent to that of 1 pack per day for a human smoker. Adjusting for the difference in ventilation per unit body mass of the mouse and human, a 4-h exposure to 24 cigarettes was selected. Mice were exposed to mainstream cigarette smoke via nose-only exposure manifold (Intox Products, Moriaty, NM) at age 8 or 9 wk. This exposure was performed daily (5 days/wk). The mice were divided into two groups according to the duration of CS exposure that included controls (filter-air, nose-only exposure), CS exposure (CS), and CS exposure + recovery (CS + RE). In the first group, 12 mice were exposed to the smoke of the 1R3 commercial cigarettes (University of Kentucky) 4 h per day for 6 wk, and six of these mice were euthanized at the day of cessation of smoke exposure. The remaining six mice were then returned to the air exposure for 3 wk (CS6 + RE3). Five mice and six mice were used as sham controls for the CS and CS6 + RE3 subgroups, respectively. In the second group, 10 mice were exposed to the CS for 16 wk, and five of these mice were euthanized at the day of cessation of smoke exposure. The remaining five mice were then returned to the air exposure for 8 wk after 16 wk of smoke exposure (CS16 + RE8). Five and four mice were used as sham controls for the CS and CS16 + RE8 subgroups, respectively. The control mice were restrained daily (5 days/wk) in the smoking machine (filter-air, nose-only exposure) for the identical duration of time with the experimental groups to mimic the restraint and environmental stress of the smoking machine. The mean body weight and age of each group are listed in Table 1.
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The mice were anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (8 mg/kg). Arterial blood pressure was measured by inserting a catheter into the common carotid artery connected to a pressure transducer. Heparin (200 U/ml) was used to prevent blood clots in the vessels via the carotid artery catheter. All animal experiments were performed in accordance with national and local ethical guidelines, including the Institute of Laboratory Animal Research Guide, Public Health Service policy, Animal Welfare Act, and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.
Nitrite/Nitrate Measurements
The mouse was euthanized with an overdose of ketamine and xylazine via the jugular vein. The left femoral and carotid arteries were isolated immediately and were coarsely grounded in methanol in an ice bath. Homogenate (
50 µl in total volume) was centrifuged at 800 g at 4°C for 10 min, and the supernatant was then assayed for nitrite (NO2–) and nitrate (NO3–). The nitrite and nitrate ion concentrations in solution were measured by the combination of a diazo-coupling method and high-performance liquid chromatography (ENO-20 NOx Analyser; EiCom, Kyoto, Japan). The method for NOx analysis has been previously described in detail (23). Briefly, the peaks of detected voltage for nitrite and nitrate were converted into nitrite and nitrate concentrations by the use of calibration solutions. The signals were corrected for contamination by subtraction of the control nitrite concentration. The endogeneous NO production was evaluated as the concentration of nitrite or nitrate per unit volume of arterial wall (in nmol/mm3).
In five control mice, the carotid, femoral, left coronary artery (LCA), and right coronary artery (RCA) were harvested, and the NOx concentrations were determined. This was done to establish a correlation between the NOx in the coronary and femoral and carotid arteries, because it is not possible to make both mechanical and biochemical measurements on the same vessels.
Western Blot Analysis
The right femoral and carotid artery were removed and homogenized in a lysis buffer containing 50 mmol/l 
-glycerophosphate, 100 µmol/l sodium orthovanadate, 2 mmol/l magnesium chloride, 1 mmol/l EGTA, 0.5% Triton X-100, 1 mmol/l dl-dithiothreitol, 20 µmol/l pepstatin, 20 µmol/l leupeptin, 0.1 U/ml aprotinin, and 1 mmol/l phenylmethylsulfonyl fluoride and then incubated on ice for 1 h. The sample was centrifuged at 1,000 g for 15 min at 1°C, and the supernatant was collected. The total protein was measured by BCA kit (Bio-Rad). Equal amounts of protein (25 µg) were loaded and electrophoresed in a 10% SDS-PAGE gel and transferred onto a ployvinylidene difluoride membrane. After being blocked for 2 h in 8% dried milk in Tris-buffered saline-Tween buffer, the membrane was incubated overnight at 4°C with specific primary antibody (1:1,000 dilution in blocking buffer, BD Transduction). The membrane was then rinsed and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h (1:3,000 dilution in blocking buffer, Bio-Rad). Specific eNOS protein was detected by enhanced chemiluminescence (Amersham Biosciences) and evaluated by densitometry (SigmaScan, Systat Software). All samples from each group were simultaneously probed with anti--actin, a mouse monoclonal antibody (primary antibody 1:1,000 dilution in blocking buffer, Santa Cruz Biotechnololgy) to correct for sample loading. Positive control protein was obtained from cultured human vascular endothelial cells provided by BD Transduction.
Mechanical Testing: Pressure-Diameter-Length Relation
The heart was immediately excised and placed in a Krebs solution after the mouse was euthanized. The anterior portion of the main trunk of the LCA and RCA close to the opening of the aorta was then exposed carefully. Water-insoluble carbon particles were used to mark the segment of the main trunk of the LCA and RCA (0.5–0.6 mm in length) to measure axial-length changes. The ascending aorta was then cannulated by a 23-gauge needle and perfused with 6% dextran solution at the various perfusion pressures. Cab-O-Sil (0.35% by weight), a colloidal silica, was mixed into the dextran solution to prevent flow through the microvessels and, hence, to attain zero-flow distensions. The LCA and RCA were preconditioned with five cyclic changes in pressure from 0 to 150 mmHg in a triangular waveform. The pressure was increased in 30-mmHg step increments from 30 to 150 mmHg in a staircase manner. The external geometry of the LCA and RCA segment, at the pressurized state, was photographed at x100 magnification to obtain the loaded outer diameter and the in vivo axial length.
No-load and Zero-Stress State
After the mechanical testing was completed, the marked blood vessel segment of the LCA and RCA were carefully dissected and placed into a Ca2+-free Krebs solution, aerated with 95% O2-5% CO2. The vessel segment was then cut transversely into five or six rings. Each ring was photographed at x100 magnification in no-load state (zero transmural pressure) and then cut radially by a scissor to reveal the zero-stress state. The ring opened into a sector and gradually approached a constant opening angle. The cross section of each sector was photographed 1 h after the radial cut. The morphological measurements of the in vitro axial length, inner and outer circumference, wall thickness (WT), and area in the no-load and zero-stress state were made from the images using a morphometric analysis system (SigmaScan).
Biomechanical Analysis
Incompressibility condition.
The loaded inner radius and WT of vessel were determined from the incompressibility assumption. The incompressibility condition for a cylindrical vessel can be expressed as follows:
 | (1a) |
z is l/lo as the stretch ratio in the axial direction where l and lo are the vessel length in the loaded and zero-stress state, respectively; and Ao is the wall area in the no-load state. WT, at the loaded state, was computed as the difference between the outer and inner radius of the vessel at various pressures as follows:
 | (1b) |
z were measured quantities.
Strain and stress.
The circumferential deformation of the artery may be described by Green strain, 
, which is defined as follows:
 | (2) |
is the midwall circumferential stretch ratio ( = c/czs), c refers to the midwall circumference of the vessel in the loaded or no-load state, and czs refers to the midwall circumference in the zero-stress state.
At equilibrium, the average circumferential Cauchy and second Piola-Kirchoff stress in a cylinder can be computed as follows:
 | (3a) |
 | (3b) |
are the inner radius and the circumferential stretch ratio of the vessel, respectively. Equations 2 and 3 allow the determination of the circumferential stresses and strains, respectively, for different pressure distensions.
Elastic moduli.
The computation of elastic modulus has been previously described in detail (10). In analogy to an isotropic tube, the circumferential elastic modulus (E) can be defined as:
 | (4a) |
 | (4b) |
and Ez are constant and independent of pressure. Statistical Analysis
All values for mechanical analysis and quantitative analysis for Western blot analysis were expressed as means ± SD, expect for NO by-product (nitrite) measurements that were expressed as means ± SE. Significance of the differences between the different groups were evaluated by two-way ANOVA or t-test. The results were considered statistically significant when P < 0.05 (2-tailed).
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RESULTS |
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Figure 3 shows Western blot and quantitative analysis of eNOS protein expression in the femoral and carotid artery for control, CS, and CS + RE mice after 6 and 16 wk of smoke exposure. A band at 140 kDa was detected, which was similar in size with the band for human endothelium cell protein standard. The anti--actin antibody reacted with a 42-kDa protein corresponding to the size of -actin. The final value for eNOS densitometry was computed as the ratio of eNOS to -actin. The CS and CS + RE group did not differ from the control in eNOS protein expression after 6 wk of CS exposure. The eNOS protein expression of femoral and carotid artery was significantly lower in CS than in control mice (P < 0.05) after 16 wk of smoking exposure. There was no significant difference in eNOS protein expression between CS16 + RE8 and control mice.
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DISCUSSION |
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There are several interacting homeostatic regulators of blood pressure, including the rennin-angiotensin system, the autonomic nervous system, and local mediators. The myogenic tone and flow-dependent vasodilation are of major importance for the regulation of blood pressure under various physiological circumstances. Some studies (8) have shown that CS is associated with an acute and marked increase in blood pressure and heart rate. This study shows that CS increases the blood pressure of mouse, as shown in Fig. 1. It is well known that NO is a vasodilator released by the endothelium in response to flow or shear stress. NO has been associated with the regulation of blood pressure and regional blood flow (14). Rees et al. (27) found that the pharmacological blockage of NO synthesis induced a dose-dependent, long-lasting increase in mean systemic arterial blood pressure. Our data confirmed that the NO bioavailability was significantly decreased by CS (Fig. 2A). Furthermore, a negative linear relation between NO production (nitrite) of femoral and carotid artery and blood pressure was observed (Fig. 2B). Although regulation of blood pressure occurs primarily by resistance-size arteries, these data on epicardial (150–200 µm in diameter) arteries show that the increase of the mean arterial pressure of mouse in response to CS correlates with vascular NO.
NO Production and eNOS Protein Expression
The association between CS and vascular diseases is widely recognized, and there is a general consensus that CS targets the vascular endothelial cells. Endothelial integrity is essential for homeostatic function of blood vessels and for maintaining a nonthrombotic and nonatherogenic state. NO is a potent vasodilator that inhibits extracellular matrix turnover and can thus modify the mechanical properties of the arterial wall (33). Higman et al. (11) reported that the release of NO from saphenous veins of nonsmokers was significantly higher than that from veins of heavy smokers. Using the NO antagonist NG-monomethyl-L-arginine, several investigators (19) have found indirect impairment of endothelium-dependent vasodilatation in smokers with decreased NO. In the present study, the measurement of nitrite (aqueous oxidation products of NO) from femoral and carotid artery after short-term and long-term CS exposure provides evidence that CS significantly decreases the bioavaibility of NO. Furthermore, the impaired release of endothelial NO was reversible for mice at 3 wk after cessation of short-term exposure of CS (Fig. 2).
Impaired release of endothelial NO is thought to be related to the reduced synthesis or activity of eNOS (11). Recently, both an increase and a decrease of eNOS mRNA and protein have been reported in relation to the effect of cigarette smoke in various experimental models. CS has been shown to inhibit pulmonary artery eNOS (31) and to suppress eNOS by 52% in cultured endothelial cells (35). Conversely, Barua et al. (2) found a higher eNOS expression in human umbilical vein endothelial cells treated with serum from smokers. To date, the changes in protein expression of eNOS of mouse coronary arteries have not been documented. The present data show that eNOS protein expression is markedly affected only by long-term, but not by short-term, CS exposure. Moreover, the decrease of eNOS protein expression was reversed after cessation of smoke for 8 wk after 16 wk of CS exposure (Fig. 3). It has been reported that CS contains a variety of oxidants, including nitrogen oxides, hydrogen peroxide, hydrogen cyanide, and acrolein (12), that are capable of affecting eNOS expression. In the present study, the protein expression of eNOS appears to be a less sensitive biomarker for the structural and mechanical remodeling of coronary artery than the NO concentration. Future studies should investigate the activity of eNOS as a biomarker. Furthermore, the role of increased reactive oxygen species as a cause of decreased NO bioavailability in this model warrants attention. Finally, other enzymes responsible for NO synthesis, such as inducible NOS and neuronal NOS, should also be investigated.
Structural Properties
Intimal-medial WT has emerged as an index of cardiovascular disease and is generally regarded as a biomarker of atherosclerosis. The study by Auerbach et al. (1) has shown qualitatively that smokers have an unusual hyaline thickening of the arterioles that are rare in nonsmokers. A study in mice found that after 5 wk of exposure to CS, the carotid intimal area was significantly increased in the exposure group (32). Our data showed an increase in WT (intima-media) and WTTR ratio of the coronary wall after CS exposure (Fig. 4), which may predispose the coronary arteries to atherosclerosis.
The WTTR ratio was not restored to control value in either recovery group. The effect of short-term exposure to CS on the WT, however, was reversible after the cessation of CS exposure. This is likely due to the restoration of NO bioavailability that can reduce hyperplasia. Moreover, we found that mean arterial pressure was increased by 8% to 10% and that WTTR ratio was increased by 13 to 14% after 6 and 16 wk of CS exposure. This suggests that the increase in WTTR ratio may be partly due to the blood pressure and partly due to the biochemical affect of CS (decrease in NO bioavailability).
Mechanical Properties
Arterial stiffness is recognized as an important cardiovascular risk factor and an independent predictor of all-cause and cardiovascular death. Hence, a number of studies have focused on the changes in mechanical properties of blood vessels in response to CS. Liu and Fung (21) studied the effect of CS (2 and 3 mo) on the mechanical properties of the pulmonary arteries of the rat. They found an increase in the stiffness of the pulmonary arteries in smoke-exposed rats. Kool et al. (18), using noninvasive methodology in smokers, showed an acute decrease in the distensibility of both elastic common carotid artery and muscular brachial artery. There are no similar data on the coronary arteries, which are very susceptible to the effects of CS. Our results indicate that CS significantly increases the circumferential elastic modulus of coronary arteries, suggesting an increase in arterial stiffness (Fig. 6). We also found the elastic modulus to be restored to the normal value after 6 wk of CS exposure, whereas it remained larger than the control value after 16 wk of CS exposure. These findings underscore the relation between reversibility of the changes in mechanical properties and the dose of CS.
The zero-stress state of a blood vessel is an open sector that is quantified by the opening angle. The remodeling of the zero-stress state is an index of the nonuniformity of growth and remodeling (7). A recent study (22) reported that flow overload induced growth of adventitia that exceeded that of intima and, hence, decreased the opening angle. The opening angle was found to be increased in hypertension (6). The present data show that the opening angle of coronary artery was decreased after long-term exposure of CS (Fig. 7). This suggests that the growth of adventitia exceeds that of intima or that the intima resorbs more than the adventitia. This is in contrast to the pulmonary arteries that are in more direct contact with the products of smoke (21).
Uniform Strain and Stress Hypothesis
It is well accepted that a homeostatic state of stress exists in the cardiovascular system. Our laboratory (9) has recently found that the strain (computed in reference to the zero-stress state) is very uniform along the cardiovascular system, i.e., from small to large vessels. We also found that in hypertension or flow overload where the homeostatic wall strain is suddenly altered, the vessel will remodel (including the zero-stress state) in such a way as to normalize the wall strain and restore mechanical homeostasis (5, 22). Here we show that CS markedly decreased the circumferential strain and stress in coronary artery. The strain was decreased by 19% to 25%, and the stress was decreased by 9% to 12% in response to CS. Moreover, this change in stress and strain was irreversible after cessation of CS exposure for the two groups (Fig. 5). These findings reflect a change of mechanical homeostasis of vessel wall and possibly function.
Correlation Between NO and Structural and Mechanical Properties
NO appears to be intimately connected with vessel wall structure and mechanical properties (16, 30). Here we report a significant relationship between the NO bioavailability (nitrite) and the structural and mechanical properties as shown in Fig. 8. The stress and strain show a significant linear relation, whereas the WT and modulus reveal an inverse relation. Although the data on mechanical properties and nitrite concentration stemmed from different arteries in this study, we have confirmed that there existed a linear relation for NO by-products between coronary and combined femoral and carotid arteries in mice (R2 = 0.79, P < 0.05). Our results demonstrated that the reduction of NO production by CS may be responsible, at least in part, for the increased intima-medial thickness and arterial stiffness of coronary arteries.
The present data provide a basis for future mechanistic investigations of the interactions between NO and the mechanical properties of the vessel. For example, is the dysfunction in NO synthesis the cause of the mechanical remodeling on the mouse arteries? This and other questions may be assessed by pharmacological blockers and transgenic models in future studies.
In summary and in conclusion, the present study demonstrates that CS has an immediate and substantial effect on structural and elastic properties of mouse coronary arteries which highly correlated with a decrease in NO bioavailability. Moreover, long-term CS exposure can cause an irreversible deterioration of structural and elastic properties of coronary arteries. The decrease in NO bioavailability in vessel wall stiffness may explain why CS is an important risk factor for coronary artery disease. The observed recovery of NO bioavailability after short-term, but not long-term, smoke cessation is an important public health message.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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; C), elastic modulus (E; D) and concentration of nitrite (N) at pressure of 120 mmHg for all mice. A linear least-square fit is used to fit data as follows: wall thickness = –6.2E – 03N + 0.01 (R2 = 0.45, P < 0.01);