Chronic cigarette smoking causes hypertension, increased oxidative stress, impaired NO bioavailability, endothelial dysfunction, and cardiac remodeling in mice

BP measurements in conscious mice.

BP was measured in conscious animals using the tail-cuff method (CODA-2, Kent Scientific, Torrington, CT). Briefly, mice were trained for 7 days by measuring BP daily, after which BP recordings were made 3 days/wk. Each session consisted of 5 acclimatization cycles followed by 15 BP measurements cycles. On the data collection day, 2 sessions of 15 BP measurements were obtained; a set was accepted if the computer identified >50% successful readings. The average from one session was used for systolic BP (SBP), diastolic BP (DBP), and mean BP in each individual mouse.

Mouse cardiac cine MRI.

To measure in vivo left ventricular (LV) function and cardiac morphology, cine MRI was performed in mice under light isoflurane anesthesia. Cine MRI was performed on a Bruker 11.7-T 500-MHz NMR system using an external ECG-triggered fast gradient echo. Six to eight contiguous ventricular short-axis slices (1-mm thickness) were acquired to cover the entire heart for LV end-systolic volume, LV end-diastolic volume, heart rate (HR), LV mass, wall thickening, stroke volume (SV), and ejection fraction (EF). Images were converted to DICOM format and then analyzed using ARGUS image-analysis software (Siemens Medical Solutions).

Langendorff-perfused heart preparation for cardiac function.

After scheduled smoking exposures, hearts were isolated from CS-exposed mice as previously described (46) in parallel with hearts from air-exposed mice. Briefly, mice were anesthetized with pentobarbital (50 mg/kg ip), and hearts were excised, aorta cannulated, and perfused in the Langendorff mode at a constant pressure of 80 mmHg with modified Krebs-Henseleit buffer (KHB) equilibrated with 95% O₂-5% CO₂ at 37°C. The constituents of KHB were (in mM) 120 NaCl, 5.9 KCl, 25 NaHCO₃, 1.2 MgCl₂, 2.5 CaCl₂, 0.5 EDTA, and 16.7 glucose. A fluid-filled balloon was inserted into the LV across the mitral valve and connected to a pressure transducer permitting the continuous measurement of LV pressure (LVP). Hearts were immersed in a water-jacketed bath maintained at 37°C, and the LV balloon was filled with water to yield a LV diastolic pressure of 3–6 mmHg. Coronary flow was continuously monitored via a Doppler flow probe (T206, Transonic Systems, Ithaca, NY) placed in the aortic perfusion line. Aortic and LVP were recorded on a PowerLab/400 multichannel data-acquisition system (AD Instruments, Castle Hill, Australia) using ADI Chart software (version 4.2). The LVP signal was digitally processed to yield diastolic and systolic pressures, LV developed pressure (LVDP), HR, and the rate-pressure product (RPP = LVDP × HR). After 20 min of equilibration, a pressure-volume (P-V) curve was constructed for each heart by an incremental increase in the LV balloon volume (2 μl) at 30-s intervals until the maximal developed pressure.

Isolated mouse aortic ring preparation for vascular function.

Preparation of the isolated mouse aorta was similar to that previously described (47). After the heart was collected, the thoracic aorta was gently dissected out from the same mouse. After careful removal of fat and connective tissues, the aorta was cut transversely into two to three rings of 2–3 mm in length. The rings were mounted on a wire myograph (Multi Myograph System-610M, Danish Myo, Aarhus, Denmark) with extreme care taken not to damage the endothelium and then suspended in 5-ml organ baths containing modified KHB [containing (in mM) 118 NaCl, 24 NaHCO₃, 4.6 KCl, 1.2 NaH₂PO₄, 1.2 CaCl₂, 4.6 HEPES, and 18 glucose] and continuously gassed with 95% O₂-5% CO₂ (37°C, pH 7.4). Aortic rings were allowed to equilibrate for 90 min with an initial resting tension of 1 g (the length-tension relationship was determined separately), and the bathing solution was changed at 15-min intervals. Changes in isometric tension were recorded on a PowerLab/8sp multichannel data-acquisition system (AD Instruments) using ADI Chart software (version 5.3) for digital processing and data analysis.

After equilibration, the responsiveness and stability of each individual ring were checked by the successive administration of a submaximally effective concentration of l-phenylephrine hydrochloride (phenylephrine; 1 μM). The integrity of the vascular endothelium was assessed pharmacologically by ACh-induced relaxation of phenylephrine-precontracted rings. Preparations were then washed three times with drug-free buffer and allowed to relax fully for 30 min before the experimental protocol began. To determine the vasodilator responses to ACh, the aortic rings were precontracted with phenylephrine, and dose-response curves for aortic relaxation were obtained by the cumulative addition of ACh in the organ bath. The concentration of agonist in the organ bath was increased in steps of 1-log units. ACh was added to yield the next higher concentration only when the response to the lower dose reached a steady state. One dose-response curve for ACh was constructed for each ring. The vasodilator (relaxant) responses were expressed as percent decreases of phenylephrine-induced precontraction, where the contraction produced by 1 μM phenylephrine in each ring from its initial resting tension (1 g) was considered as 100%. The terms “vasodilation” and “vasorelaxation” are used interchangeably throughout this report.

Preparation of white blood cells and measurement of ROS.

Whole blood was obtained from the mice (CS or air exposed) through a cardiac puncture. Blood cells were separated from the plasma by centrifugation at 1,000 rpm for 2 min. The buffy coat was carefully aspirated from the remaining red blood cells (RBCs), and the sample was resuspended in HBSS (pH 7.4). ROS generation in white blood cells (WBCs) was determined using the redox-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA; for H₂O₂) or dihydroethidium (DHE; for superoxide) along with nuclear stain (Hoechst-33342 dye) as previously reported (22). H₂DCF-DA can readily pass through cells and is deacetylated by cellular esterases to the nonfluorescent probe 2′,7′-dichlorohydrofluorescein (DCFH), which is then trapped inside the cell by its polar nature. DCFH is converted by intracellular oxidants, such as H₂O₂, to the green fluorescent molecule 2′,7′-dichlorofluorescein (DCF). Similarly, DHE enters the cell freely and is dehydrogenated to ethidium. H₂DCF-DA and DHE at a concentration of 50 μM were added simultaneously with Hoechst-33342 dye (1 μM) to the cell suspensions and incubated for 30 min at 37°C. To investigate the effects of a known potent activator of leukocyte ROS generation (48), additional sets of WBCs were incubated with 50 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 30 min. After an incubation, cells were washed three times and then mounted on a glass slide using mounting media. The fluorescence emissions for DCF and DHE were detected at excitation/emission wavelengths of 490 and 555 nm, respectively, and images were acquired by a Nikon Fluorescent microscope using a ×40 objective. Averaged individual intensities of WBCs were analyzed by Metamorph software (version 7.0).

Electrochemical measurements of the NO decay rate in blood.

Blood was collected into a heparinized tube from control and CS-exposed mice 24 h after the last exposure. Blood samples were centrifuged at 2,300 g for 10 min. The supernatant containing the plasma was removed, and the RBC and WBC pellet was washed three times with PBS [15 mM phosphate (potassium) plus 0.9% NaCl; pH 7.4]. The packed RBCs/WBCs were then resuspended in the same buffer and stored on ice for use. Measurements of NO concentration were performed in a four-port water-jacketed electrochemical chamber (NOCHM-4, WPI, Sarasota, FL) at 37°C with a carbon fiber NO electrode (ISO-NOPF200, WPI). The carbon fiber NO electrode was inserted (through the electrode port on the cap of the chamber) into the chamber. NO (1 μM) was added into the solution (through a hole on the cap of the chamber) with a Hamilton syringe by a bolus injection in the presence or absence of diluted (1:3,000) blood samples to measure the NO decay rate after hemoglobin of the RBCs was completely oxidized by previously added NO. The NO decay rate was determined in a manner similar to that previously described (29).

Chronic smoking and BP.

Hypertension is a major risk factor for CVD, and the risk for cardiovascular events doubles with each 20-mmHg increase in SBP (∼10-mmHg DBP) (28). Several epidemiological studies including healthy subjects, hypertensive subjects, diabetic, and renal patients have clearly documented that smokers have higher BPs than nonsmokers (for a review, see Ref. 37). Importantly, the prevalence of hypertension was higher in former smokers than in never smokers (37), and the risk of hypertension was associated with the number of cigarettes smoked daily and the duration of smoking (37). Despite the strong association between smoking and BP in humans, there is a significant lack of animal studies for the cardiovascular effects of chronic CS exposure. In a rat model, CS exposure for 4 days mildly increased mean arterial BP in CS-smoked rats compared with sham control rats (24). Recently, in a mouse model, CS exposure for 6 or 16 wk has been shown to mildly increase mean arterial BP to a similar extent in both groups. Interestingly, switching to air exposure decreased BP in the 6-wk group but not in the 16-wk group (21), indicating an irreversible state in BP with long-term CS exposure.

In our study protocol, BP showed a trend to increase at 16 wk of CS exposure, and it was significantly different from that of control mice at 32 wk of CS exposure. The significantly elevated BP and reduced BW gain at 32 wk in CS-exposed mice compared with air-exposed mice indicate that a biologically active significant level of CS exposure was achieved in our model. Although the mechanism of elevated BP in our model was not investigated, it has been reported that the increase in BP with CS is mediated via direct stimulation of the sympathetic nervous system and subsequent increased plasma concentrations of norepinephrine and epinephrine (19). Of note, the main biologically active ingredients in CS that have been considered as casual constituents for CVD are nicotine, carbon monoxide, oxidant gases, and particulate matter (1, 4, 5, 11).

Chronic smoking and ED.

ED is an early event of atherosclerosis in chronic smokers as well as after acute cigarette smoking (for a review, see Ref. 41), and an important manifestation of ED is reduced endothelium-dependent vasodilation/vasorelaxation. Endothelium-dependent vasomotion is the most widely used clinical end point for the assessment of endothelial function, and it is often compared with an endothelium-independent dilator such as nitroglycerine. In vivo and in vitro studies have demonstrated that smoking impairs endothelium-dependent vascular responses in virtually every vascular bed (for a review, see Ref. 41); however, there is limited information regarding the temporal onset of vascular ED and its relationship with CS-induced alterations in physiological and biological parameters (41). Using a chronic CS exposure protocol in otherwise healthy mice, we observe that 16 wk of CS exposure did not, but 32 wk of CS exposure did, significantly impair endothelium-dependent vasorelaxation (Fig. 2). The CS exposure-related vascular ED support a causative role of smoking in hypertension and atherogenesis. Importantly, the impaired aortic endothelial function was concurrent with elevated BP (Fig. 1). ED with accompanying risk factors, such as high BP, may be a critical pathological state preceding the development of overt atherosclerosis with long-term or higher-level exposure. Of note, 8 wk of CS exposure decreased endothelium-dependent aortic relaxation in hypercholesterolemic rabbits (33). The precise mechanisms of CS-induced ED are not fully understood, but several potential mechanisms are likely to be involved. Oxidative damage seems to constitute the primary mechanism because CS contains a large number of oxidants and introduces ROS in the circulatory system (4, 11, 39). ROS generated by long-term smoking has been reported to directly damage endothelial cells and inactivate NO (11, 39). Decreased NO release, dysfunctional NO synthase, and increased consumption of NO have been reported with ED in healthy smokers (2, 11, 23, 39).

Chronic smoking and the heart.

In addition to the well-established effects of smoking on BP and endothelial function (2, 19, 23, 37, 39, 41), smoking also can impair cardiac performance independent of coronary atherosclerosis. A previous study (32) showed that smoking compromises cardiac function and leads to cardiac remodeling. Greater LV mass was reported in chronic smokers independent of any changes in their BP and LV fractional shortening (16), and a recent report (38) showed a positive association of smoking with exercise-induced LV growth in young men. Cardiac hypertrophy was determined by the crude HW-to-BW ratio, and this was complemented by in vivo cardiac MRI. We observed that 32 wk of CS exposure caused an increase in the LV wall thickness both at diastole and systole with a resultant decrease in SV and cardiac output (Table 1); however, global in vivo cardiac contractile function (EF) was unaffected. Of note, LV mass and the HW-to-BW ratio at 32 wk were significantly larger in smoking mice compared with control mice (Fig. 3), and this was concurrent with elevated BP (Fig. 1). Thus, it is possible that the adaptive cardiac hypertrophic changes might have contributed to the maintenance of cardiac contractile function under normal physiological conditions. In Wistar rats (6), CS exposure has been shown to increase BP and ventricular weight without altering BW. Moreover, in spontaneously hypertensive rats (SHRs), smoking significantly enhanced the progression of cardiac hypertrophy with an increased HW-to-BW ratio compared with sham SHRs (31). We did not observe any adverse effect on the in vitro baseline cardiac contractile function at the organ level (Table 2); however, the LV P-V relationship demonstrated a trend toward LV systolic and diastolic dysfunction at high afterload in isolated hearts of CS-exposed mice compared with control mice (Fig. 3C). This impairment in the LV P-V relationship suggests that under stress conditions, CS-exposed animals would be at a greater risk of cardiac contractile dysfunction. Importantly, in the Multi-Ethnic Study of Atherosclerosis, MRI demonstrated regional myocardial dysfunction in smokers compared with nonsmokers, despite the absence of clinical manifestation of disease (42).

Since hypertension and smoking are main risk factors for CVD, either of these individual risk factors can cause cardiac hypertrophy, and they may accelerate the progression of hypertrophy into overt cardiac remodeling when present together. Indeed, CS-induced cardiac remodeling has been reported in rats with potential mechanisms including neurohumoral activation, oxidative stress, and MAPK activation (32). Although the mechanisms underlying the clear association of CS exposure with cardiac hypertrophy and remodeling were not investigated in mice, the increased BP in our study provides strong evidence that exposure to CS results in the stimulation of intracellular signaling pathways of cardiac hypertrophy and remodeling.

Chronic smoking and systemic oxidative stress.

The association between CS and oxidative stress is widely recognized, and there is a general consensus that CS-induced increased oxidative stress plays a major role in the pathogenesis of different cardiovascular disorders (1, 4, 11). In the setting of cigarette smoking, free radicals could arise from the CS itself; circulating or in situ activated neutrophils, monocytes, and macrophages; and endogenous sources of ROS, such as uncoupled endothelial NO synthase, xanthine oxidase, and the mitochondrial electron transport chain (4, 11, 39, 57, 60). Blood is the first body fluid exposed to substances either inhaled or ingested, and reactive oxidants contained in CS may readily traverse into the blood and modify the function of blood cells.

We observed that chronic CS exposure increased ROS generation in both mononuclear and polymorphonuclear leukocytes compared with control mice (Figs. 4 and ​and5),5), and these findings are in agreement with increased systemic oxidative stress reported in humans (3, 40). CS has been reported to cause marked activation of leukocytes and platelets and contributes to oxidative vascular damage in smokers (1, 11). Interestingly, WBCs of the CS-exposed group exhibited markedly enhanced ROS generation with levels much above those of untreated WBCs of control mice, approaching the levels seen when control cells were treated with TPA. Thus, these data indicate that CS exposure alone caused increased oxidative stress in CS-exposed animals. While high-level ROS formation was seen in WBCs of CS-exposed mice, a further increase was seen after TPA treatment, indicating that further activation was possible. Importantly, along with ROS formation, myeloperoxidase release by activated neutrophils has been reported to contribute to CS-dependent vascular inflammation and atherosclerosis by reducing endothelial NO bioavailability (43). Lipid peroxidation and protein nitration by increased ROS/reactive nitrogen species might play a role in the inflammatory response. While the causes of CS-induced systemic oxidative stress and inflammation are multifaceted, our results provide clear evidence that free radicals and secondary oxidants are released endogenously from activated leukocytes of chronically CS-exposed mice, and this, in turn, can induce vascular dysfunction, leading to hypertension and atherogenesis (1, 3, 4, 11, 39, 40, 43).

Chronic smoking and NO bioavailability.

Several clinical and animal studies have clearly documented the critical role of NO in smoking-induced ED (for reviews, see Refs. 34, 38, and 41), and altered NO function with CS exposure may result from a break or alteration in the cascade of NO metabolism (2, 21, 23, 59). We observed that the NO decay rate was significantly accelerated with a shortened half-life in RBCs/WBCs of CS-exposed mice compared with RBCs/WBCs of control mice (Fig. 6). Of note, the increased NO decay was inhibited by the flavoprotein inhibitor diphenyleneiodonium, which is well known to inhibit leukocyte NADPH oxidase (30), suggesting that the increased NO decay rate is caused by the raised superoxide generation from leukocytes. We also observed increased oxidative stress in the blood of CS-exposed mice (Figs. 4 and ​and5).5). If ROS levels are raised, NO would be trapped by superoxide in the vascular wall to form the potent oxidant peroxynitrite (51) there, and this would decrease NO bioavailability, contributing to ED.