INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: endothelium; endothelin; nitric oxide synthase; pulmonary circulation; smoking

Cigarette smoking has important consequences on lung health, the most common of which are chronic obstructive pulmonary disease (COPD) and lung carcinoma, and has been strongly implicated in the pathogenesis of cardiovascular disorders. Even though abnormalities of pulmonary vascular bed are often present in smoking-associated respiratory disorders, the effect of cigarette smoking on pulmonary vessels has received little attention.

Pulmonary hypertension is a common feature in patients with advanced COPD, and its presence is associated with shorter life expectancy (1). Hypoxemia, which is present in advanced COPD, plays an important pathogenic role in pulmonary hypertension since hypoxia is a strong stimulus for cell proliferation in pulmonary arteries (2). However, structural abnormalities of pulmonary arteries have been shown in patients with mild COPD who are not hypoxemic (3, 4) and even in smokers with normal lung function (4, 5), suggesting that cigarette smoking may induce structural changes in pulmonary vessels.

Pulmonary endothelium plays an important role in regulating the vascular tone of pulmonary circulation through the release of potent vasoactive mediators. Nitric oxide (NO) and endothelin-1 are two of the most important endothelium-derived vasoactive agents (6, 7). Nitric oxide is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS), which is expressed constitutively in endothelial cells (eNOS or type III NOS) (6). Endothelium-derived NO is a potent endogenous vasodilator that contributes to the low pulmonary vascular tone (8) and exerts antiproliferative effects on smooth muscle cells (2, 9). By contrast, the peptide endothelin-1 is a powerful vasoconstrictor with mitogenic activity on vascular smooth muscle and fibroblasts (7). In patients with severe forms of pulmonary hypertension the expression of eNOS is reduced (10), whereas that of endothelin-1 is increased (11), suggesting that the disregulation of these endothelium-derived agents may have a pathogenic role in the development of pulmonary hypertension.

Active and passive exposure to tobacco smoke is associated with alterations in the endothelium-dependent vasodilation of coronary (12) and peripheral arteries (13, 14). Since changes in vessel structure and endothelial function are also present in pulmonary arteries of smokers with mild airway disease (4), we asked whether cigarette smoking could induce changes in the expression of eNOS and endothelin-1 in pulmonary arteries hence accounting for the functional and structural abnormalities shown in these subjects. We therefore evaluated the expression of both eNOS and endothelin-1 in lung tissue specimens from a group of smokers who underwent resective lung surgery and compared them with that in nonsmokers.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Lung tissue specimens of 32 patients (22 men/10 women) who underwent lung resection for lung carcinoma were studied. Patients were divided according to their smoking habit: 23 were current or ex-smokers and nine were lifetime nonsmokers. To rule out chronic lung disease or hypoxemia as potential causes of pulmonary vascular changes, we excluded patients with Pa_O2 < 75 mm Hg, those who had moderate to severe airflow obstruction (FEV₁ < 65% of predicted), and those with a previous diagnosis of COPD. Characteristics of the patients are shown in Table 1. The study was approved by the Committee on Human Research of our institution.

In vitro evaluation of endothelium-dependent relaxation of pulmonary arteries was performed as previously described (4), using cumulative concentrations of adenosine diphosphate (ADP) (Boehringer, Mannheim, Germany).

Morphometric characteristics of pulmonary muscular arteries with an external diameter < 1 mm and with complete elastic laminas were analyzed in H&E stains using a computerized image analyzer (Microm, Barcelona, Spain) as previously reported (15). The areas occupied by the muscular layer, the intima and the lumen, were expressed as a percentage of the total area.

Formalin-fixed paraffin-embedded tissue sections were immunostained with monoclonal antibodies antihuman eNOS (Transduction Laboratories, Lexington, KY) and antihuman endothelin-1 (Biodesign, Kennebunk, ME) using the avidin-biotin complex/horseradish peroxidase (ABC/HRP) method (Vector Laboratories, Burlingame, CA). A serial section was incubated with antihuman Factor-VIII-related antigen (Dako, Glostrup, Denmark) for control purposes. Negative control experiments were also conducted. The number of arteries showing positive immunoreactivity was counted and expressed as the percentage of the total number of arteries. The intensity of staining was additionally graded as absent, 0; mild, 1; moderate, 2; or intense, 3; and an average grading score was assigned to each patient. Slides were evaluated without knowledge of the patient's functional status.

Western blot analysis of eNOS protein was performed in frozen samples from 21 patients (seven nonsmokers and 14 smokers). After tissue homogenization, the protein concentration of the homogenates was determined. To ensure equal protein loading of all gels, 150 µg of protein was loaded in each lane of a 7.5% SDS-polyacrylamide gel and submitted to electrophoresis. Subsequently, proteins were transferred to PVDF membranes and incubated with anti-eNOS antibody (Transduction Laboratories) at a 1:1500 dilution. After incubation with the secondary antibody, specific immunoreactivity was detected with the ABC/HRP method. Protein bands were visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, UK) and their intensity quantitated by densitometry (Vilber-Lourmat, Marne-la-Vallée, France). The protein samples were additionally assayed for the expression of 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, two mitochondrial biotin-containing housekeeping gene products. Both proteins were detected with the avidin-biotin horseradish peroxidase conjugate at 76 and 72 kD, respectively. The expression of eNOS was normalized for the expression of both houskeeping proteins. Lysate derived from a human aortic endothelium cell line (Transduction) was used as positive control. All procedures were performed by duplicate and results expressed as the mean value.

Pulmonary tissue concentration of immunoreactive endothelin was measured by using radioimmunoassay (Nichils Institute, Wijchen, Netherlands) after endothelin extraction from lung homogenates on Sep-Pack C18 cartridges (Waters, Milford, MA) as described by Leivas and colleagues (16).

Data are expressed as mean ± SD. Comparisons between group means were performed with Student's unpaired t test. Correlations between variables were analyzed with Pearson's coefficient. Probability values lower than 0.05 were considered as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Smokers had a similar age than did nonsmokers, and they were heavy cigarette consumers with a mean consumption of 49 pack-years (range, 10 to 120 pack-years), which is equivalent to one pack of cigarettes per day for 49 yr. Their lung function was within the normal range or only mildly altered. Compared with the nonsmokers, there was a moderate reduction in midexpiratory flow rate (Table 1), which is consistent with dysfunction of peripheral airways, a condition that can be viewed as a preclinical stage of COPD. Arterial oxygen tension was also lower in the smokers, although it was greater than 75 mm Hg in all cases. The two groups were not matched by sex (eight women in the nonsmokers' group and two in the smokers' group) because of the general characteristics of patients who undergo resective lung surgery.

In the organ-bath study the contraction of pulmonary artery rings to L-phenylephrine was similar in the two groups. However, the endothelium-dependent relaxation was reduced in smokers, as shown by lower maximal relaxation in response to ADP (Table 2). The maximal relaxation induced by ADP was inversely correlated with the amount of cigarettes consumed, as assessed by the pack-years index (r = 0.38, p < 0.05).

The morphometric analysis of pulmonary muscular arteries revealed that the intimal coat was thicker and the arterial lumen narrower in the smokers than in the nonsmokers (Table 2).

Immunohistochemical Analyses

Immunostaining for eNOS was observed in endothelial cells of all types of pulmonary vessels (arteries, veins, and capillaries) and in the airway epithelium (Figure 1). Intense immunoreactivity to eNOS, similar to that of factor VIII, was observed in endothelial cells of pulmonary arteries of the nonsmokers (Figure 1B). By contrast, in the smokers immunostaining for eNOs was weak (Figures 1C and 1D). Semiquantitative analysis of the immunohistochemical data revealed that the proportion of pulmonary arteries showing positive immunoreaction to eNOS was lower, and the intensity of the immunoreaction lesser, in the smokers than in the nonsmokers (Table 3). No relationship could be established between the amount of cigarettes consumed and the immunoreactivity to eNOS.

View larger version (130K): [in this window] [in a new window]   View larger version (130K): [in this window] [in a new window]   View larger version (137K): [in this window] [in a new window]   View larger version (135K): [in this window] [in a new window]   View larger version (140K): [in this window] [in a new window]   Figure 1.   Immunohistochemical expression of eNOS and endothelin-1 in lung sections of smokers and nonsmokers. (A) Control section stained for factor-VIII-related antigen, a marker of endothelial cells. Staining (brown) is apparent in the endothelium of a pulmonary artery and bronchial vessels (original magnification: ×200). (B ) Immunoreactivity to eNOS in a nonsmoker's lung. Intense staining of endothelial cells (similar to that shown with factor-VIII-related antigen) is present in a small-sized pulmonary artery and in capillaries (original magnification: ×400). (C ) Expression of eNOS in a smoker's lung. Note the decreased signal in the pulmonary artery endothelium, whereas positive staining is apparent in the bronchial epithelium (original magnification: ×100). (D) Endothelial NOS immunoreactivity in a smoker showing marked staining in bronchial epithelium and bronchial vessels, contrasting with the faint signal in the endothelium of the pulmonary muscular artery (original magnification: ×200). (E ) Endothelin-1 immunoreactivity in a smoker's lung. Note the expression of endothelin-1 in the bronchial epithelium and in the medial layer of the accompanying pulmonary artery, but not in the arterial endothelium (original magnification: ×200). Panels A, C, and E are consecutive sections of the same lung tissue specimen.

Immunostaining for endothelin-1 was observed in endothelial cells, smooth muscle cells, and bronchial epithelium (Figure 1E). Overall, immunostaining for endothelin-1 in pulmonary endothelium was weaker than for eNOS. The number of arteries showing positive immunoreaction to endothelin-1 was low and the intensity of the immunoreaction was reduced, as compared with the immunostaining for factor VIII or eNOS (Table 3). No differences were shown between the smokers and the nonsmokers in the semiquantitative analysis of the immunostaining for endothelin-1 (Table 3).

Western Blot Analysis of eNOS Protein Levels

Protein extracts from lung tissue samples were examined for eNOS protein content by Western blot analysis (Figure 2). The monoclonal antibody to eNOS detected a protein band at a molecular mass of 140 kD. Lysates from an endothelial cell line served as positive controls. Densitometric analysis of the protein band, normalized for the expression of housekeeping gene products, revealed a higher level of eNOS protein in lung tissue extracts from the group of nonsmokers than from the group of smokers (Table 3), in agreement with the findings of the immunohistochemical analysis. The eNOS protein level did not correlate with the pack-years index.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Western blot analysis for eNOS in protein extracts from lung tissue of two nonsmokers and four smokers, and from a human aortic endothelium cell line (CTL). Band at 140 kD is consistent with the size of eNOS (M denotes the molecular weight marker). The intensity of the band is reduced in smokers as compared with nonsmokers.

Endothelin-1 Content in Lung Tissue

Endothelin concentration in lung tissue homogenates was measured by using radioimmunoassay. Endothelin concentration in lung tissue was high, as compared with the concentration usually seen in other organs (16). Endothelin levels were slightly higher in the nonsmokers than in the smokers, although the difference was not statistically significant (Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of the present study demonstrate a reduced expression of eNOS protein in pulmonary artery endothelium of heavy smokers. Smokers also had diminished endothelium-dependent relaxation and intimal thickening in pulmonary arteries as compared with nonsmokers. No differences between smokers and nonsmokers were shown in the immunostaining for endothelin-1 in pulmonary endothelium or in the endothelin concentration in lung tissue.

Endothelial cells play a crucial role in vascular homeostasis (8, 17). Dysfunction of pulmonary endothelium has been shown in patients with end-stage COPD (21). Because the degree of endothelial function impairment correlated with the severity of vascular remodeling, it was suggested that the altered endothelial function might predispose to pulmonary hypertension (21), which is common at this disease stage. Nitric oxide is one of the major endothelium-derived relaxing factors. Giaid and Saleh (10) showed significant reduction of eNOS expression in severe forms of both primary and secondary pulmonary hypertension, suggesting that downregulation of NO might contribute to the development of pulmonary hypertension. We have previously shown structural abnormalities (5, 15) and impairment of endothelial function (4) in pulmonary arteries of patients with mild COPD. Because these patients were not hypoxemic we suggested that additional factors might promote the structural and functional abnormalities of the pulmonary circulation at an early disease stage. Based on the fact that cigarette smoking is the principal etiologic factor for COPD and that circulating smoking products have the capability to damage vascular endothelium (22, 23), we hypothesized that this could also occur in the pulmonary circulation. The present study has shown that the expression of eNOS in pulmonary endothelial cells was reduced in smokers who did not have chronic airway obstruction. Smokers also had lower relaxation of pulmonary artery rings in response to ADP, a vasodilator the action of which depends on the endothelial synthesis of NO. Accordingly, in smokers, the reduction of eNOS expression in endothelial cells may result in a lower bioavailability of NO and thus explain the altered endothelium-dependent relaxation of pulmonary arteries.

The mechanisms by which cigarette smoke may reduce the expression of eNOS are uncertain. Tobacco smoke contains a high concentration of NO and NO itself exerts a negative feedback on its synthesis (24, 25). Downregulation of NOS in cells of the respiratory tract by this mechanism has been suggested to explain the reduced concentration of exhaled NO in smokers (26). It is conceivable that chronic inhalation of NO contained in cigarette smoke could also regulate NOS expression in pulmonary endothelial cells. Su and colleagues (27) showed that cigarette smoke extract decreases eNOS activity and protein content in cultured pulmonary artery endothelial cells because of an inhibitory action at the level of gene transcription (27). This inhibition was not due to inactivation by protein kinase C or the effect of oxidants contained in cigarette smoke (27). Despite that the factors involved on eNOS inhibition by tobacco smoke remain unclear, it is likely to be produced by a smoke constituent that diffuses across the pulmonary capillaries and enters into the bloodstream, since it has been consistently shown that the endothelial function of the systemic circulation is impaired in both active and passive smokers (12- 14). Therefore, it seems reasonable to think that the cigarette smoke constituent responsible for this systemic effect might also exert a direct effect on endothelial cells of pulmonary arteries, that are exposed to a higher smoke concentration.

Smokers and nonsmokers in our study were not matched by sex. Two reasons accounted for such a difference. First, the current incidence of lung carcinoma in women is very low (28), thus the number of women who undergo lung resection is reduced (29). Second, epidemiologic data indicate that the proportion of current or ex-smokers among those with the same age range of our series is much higher among men than among women (30). Hypothetically, such a difference in sex distribution might influence our findings since estrogens exert a protective effect on the cardiovascular system (31), caused in part by the enhancement of endothelium-dependent relaxation through the release of NO (32). However, the beneficial effects of estrogens disappear in postmenopausal women, when estrogen levels are similar or even lower than in men (31). Based on the fact that in our series all but one woman were postmenopausal and older than 55 yr of age, we consider that the difference in eNOS expression we have found was not due to the greater proportion of women in the group of nonsmokers.

In our series, both endothelin-1 expression in pulmonary arteries and endothelin content in lung tissue extracts were similar in smokers and nonsmokers. This suggests that cigarette smoking does not augment the production of endothelin-1 in pulmonary endothelial cells and, according to the immunoassay results, presumably not in other lung cells either. Previous studies have demonstrated an increased production of endothelin-1 in the pulmonary circulation of patients with pulmonary hypertension (35, 36). Furthermore, Giaid and colleague (11) showed an increased expression of endothelin-1 in pulmonary vessels of patients with severe pulmonary hypertension, suggesting that endothelin-1 might play a role in its pathogenesis. Our findings indicate that tobacco smoke components seem to exert no effect on the expression of endothelin-1, and they suggest that this molecule does not contribute to the initial changes of the pulmonary circulation shown in smokers. However, since chronic hypoxia and elevated vascular pressures are potent stimuli for the production of endothelin-1 by pulmonary vessels (7, 37), we do not exclude that in advanced COPD endothelin-1 might play a role in the pathogenesis of pulmonary vessel remodeling and promote the development of pulmonary hypertension.

In summary, the present study has shown that smokers have reduced expression of eNOS in pulmonary endothelium, which is associated with endothelial dysfunction and enlargement of the intimal coat of pulmonary arteries. These findings suggest that inhibition of eNOS by tobacco smoke components in pulmonary vessels is an early event that may antecede the full development of cigarette-smoke-induced respiratory disease (COPD). The resultant endothelial dysfunction may serve as a base where additional factors such as arterial hypoxemia may add on and produce further changes in vascular structure and function, ultimately leading to pulmonary hypertension.