Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig

Patients with chronic obstructive pulmonary disease (COPD) show intimal hyperplasia in pulmonary muscular arteries, which results from the proliferation of smooth muscle cells (SMCs), and an increased proportion of muscularized arterioles. In addition, pulmonary arteries of COPD patients show abnormal endothelium-dependent vascular reactivity [1, 2]. Studies conducted in healthy smokers have also revealed intimal hyperplasia in pulmonary muscular arteries, which does not differ from that in patients with mild COPD [3]. Furthermore, endothelial function of pulmonary arteries in healthy smokers lies between that in non-smokers and COPD patients, thereby indicating that endothelial dysfunction is present at the origin of the disease [2]. The impairment of endothelial function results from changes in the expression and release of vasoactive mediators that also regulate cell growth [4]. Overall, these initial alterations may lead to persistent changes in the vascular structure and function that underlie the development of pulmonary hypertension in COPD [5].

Studies performed in animal models have attempted to reproduce some of the pulmonary alterations that occur in COPD [6, 7]. Among these, the model of airflow obstruction resulting from exposure to cigarette smoke (CS) is probably the most satisfactory approach. Chronic exposure of the guinea pig to CS is a widely recognized model of COPD [6, 8]. In this model, Wright et al. [9–12] have shown muscularization of small pulmonary vessels, which precedes the development of emphysema, as well as changes in the expression of vascular mediators. In a recent study performed in guinea pig precision-cut lung slices, short-term exposure to CS induced a delayed response to vasoactive agents in intracinar arteries [13]. Whether long-term exposure to CS in this animal model produces endothelial dysfunction in pulmonary arteries has not been addressed using the organ-bath methodology, which is the conventional method to assess endothelial function of pulmonary arteries in humans [2, 14] and in animal models [15–18]. Furthermore, the extent to which changes in endothelial function are related to vessel remodeling and/or expression of endothelium-derived mediators remains to be established.

We hypothesized that in the guinea pig, long-term exposure to CS alters endothelial function, induces the proliferation of poorly differentiated SMCs in pulmonary vessels, and reduces the expression of endothelium-derived vasodilators, in a similar way as in humans [2–4]. Accordingly, the present study was addressed to investigate in the guinea pig the effects of chronic exposure to CS on the endothelial function of pulmonary arteries and the lung expression of endothelial nitric oxide synthase (eNOS), and to determine whether CS induces muscularization in small pulmonary vessels. We also studied the chronological sequence of the functional and morphological changes induced by CS on pulmonary vessels.

Five of the 24 guinea pigs exposed to CS died during the study while no deaths occurred in the control group. The animals that died during the experiment were excluded from the analysis.

At the end of the study, animals showed normal behavior and activity. CS-exposed guinea pigs were smaller and had lower body weights than the controls (data not shown). The anesthesia was deep in all cases and anesthetic dosages were identical between controls and CS-exposed animals. The anatomical examination revealed no signs of respiratory infection or other major abnormalities in lung tissue.

Endothelial function

Guinea pigs exposed to CS showed a shift to the right in the dose-response curve of pulmonary arteries to ADP, as shown by a greater AUC and higher (less diluted) EC₅₀, compared with control guinea pigs (Table 1, Figure 1). The ANOVA failed to show any interaction between CS exposure and time (Table 1), thereby indicating that reduced reactivity of pulmonary arteries in CS-exposed animals was independent of the length of exposure.

The ANOVA also revealed a marked effect of time on endothelium-dependent relaxation. Irrespective of whether the animals were exposed to CS, the endothelium-dependent vasodilatation of pulmonary arteries was lower at 6 months than at 3 months.

The maximal relaxation induced by ADP was close to 100% in all groups. When pulmonary artery rings were exposed to the competitive eNOS inhibitor, L-NAME, the relaxation induced by ADP was almost completely abolished (Table 1), indicating that ADP operated through the L-arginine-NO pathway. In all cases, pulmonary arteries reached maximal relaxation when they were assessed with SNP, an exogenous NO donor.

In rings of the aorta, no effect of CS exposure was found in any of the relaxation responses to the pharmacological agents that were tested (Table 2). Yet, the ANOVA revealed a time effect in both the endothelium-dependent and – independent relaxation responses.

Morphological evaluation

The thickness of the walls of the right and left main pulmonary arteries was enlarged in CS-exposed animals (Figure 2A and 2B). Wall thickening of pulmonary arteries was due to both smooth muscle cell proliferation and elastic fiber deposition (data not shown). In contrast, the thickness of the aorta was not affected at either 3 or 6 months (Figure 2C and 2D, Table 3).

		3 months		6 months
		Control (n = 8)	CS-Exposed (n = 5)	Control (n = 8)	CS-Exposed (n = 13)
Pulmonary artery	Diameter (mm)	2.26 (2.10–2.49)	2.37 (2.11–2.45)	2.26 (2.13–2.39)	2.19 (2.15–2.40)
	Wall area (mm 2 )	0.26 (0.24–0.33)	0.38 (0.30–0.42)	0.27 (0.24–0.31)	0.35* (0.32–0.41)
	Wall thickness (μm)	38 (36–43)	50 (43–57)	35 (33–40)	51‡ (45–53)
Aorta	Diameter (mm)	2.21 (2.14–2.30)	2.34 (2.17–3.80)	2.22 (2.07–2.32)	2.28 (2.24–2.36)
	Wall area (mm 2 )	0.58 (0.51–0.65)	0.77 (0.59–0.92)	0.50 (0.46–0.65)	0.63 (0.57–0.74)
	Wall thickness (μm)	82 (74–92)	96 (86–117)	77 (70–87)	88 (80–97)

Wall thickness was calculated as total area/external perimeter

Values are median and interquartile range

* p < 0.05

^‡ p ≤ 0.01

The percentage of intra-pulmonary vessels with double elastic laminas increased 2-fold after 6 months of CS exposure compared to controls (12.3 ± 4.8 vs. 6.3 ± 5.7 respectively). This effect was not observed at 3 months (6.1 ± 3.9 vs. 7.4 ± 8.1 respectively) (Figure 3). There were no differences in the percentage of muscularized arterioles at 3 months of exposure.

Immunohistochemical evaluation

Immunohistochemical evaluation was performed in vessels with a diameter <50 μm. The number of vessels that were positive to smooth muscle α-actin was significantly greater in CS-exposed animals, at both 3 and 6 months of exposure (Figures 4A, B and 4C). In contrast, no changes were observed in the number of desmin-positive vessels at 3 or 6 months (Figure 4D), nor in the intensity or extension of protein expression (data not shown).

Gene expression of eNOS

Gene expression of eNOS was evaluated by Real-Time PCR in whole lung homogenates and normalized by the expression of β-actin. Compared with control animals, eNOS expression was decreased at 3 and 6 months of exposure to CS (Figure 5).

Our results show that guinea pigs chronically exposed to CS developed endothelial dysfunction in the pulmonary artery, which was already apparent after 3 months of exposure. In this period of time, animals exposed to CS showed reduced expression of eNOS in lung tissue and developed SMC proliferation in small intrapulmonary arteries. Longer exposure resulted in complete muscularization of small pulmonary vessels, as well as emphysematous changes in the alveolar spaces.

Endothelial dysfunction in pulmonary arteries was shown by a diminished response to the endothelium-dependent vasodilator ADP, which was abolished by the eNOS inhibitor L-NAME. Contrasting with this observation, no differences between groups were found in the endothelium-dependent relaxation of the aorta, thereby suggesting that CS exposure exerted a direct effect on pulmonary circulation. These results are in agreement with previous studies showing diminished endothelium-dependent relaxation in pulmonary arteries of smokers [2, 4] and in guinea pigs after a short period of CS exposure [13]. Moreover, in the present study endothelial dysfunction preceded the complete muscularization of small intrapulmonary vessels (vessels with double elastic lamina), thereby supporting the hypothesis that in COPD endothelial dysfunction is an early event that antecedes pulmonary vascular remodeling [22]. The mechanisms by which CS impairs pulmonary endothelium remain to be established. Our results show a decrease in eNOS gene expression in the lungs of animals exposed to CS. This finding is in agreement with those of Su and co-workers [23], who demonstrated that eNOS is down-regulated in endothelial cell cultures exposed to cumulative doses of CS extract. The expression of eNOS is also reduced in pulmonary arteries of smokers and in patients with different degrees of COPD severity [4, 24]. Accordingly, it is conceivable that CS might alter endothelium-dependent relaxation by down-regulating eNOS expression in pulmonary arteries of guinea pigs.

We also observed a marked effect of time on the endothelial function of pulmonary arteries. The vascular relaxation induced by ADP was lower at 6 months than at 3 months, irrespective of whether the animals were exposed to CS or not. The effect of time on vascular reactivity was also apparent in the aorta. Since the guinea pigs used in our study were in their growing period (mean weight increased by 231% at 3 months, and by 404% at 6 months), we consider that growth (or maturation) might affect vascular reactivity, in particular the endothelial function of pulmonary arteries, by mechanisms that have not been fully elucidated. Indeed, it has been observed that hormonal changes associated with sexual maturity may affect posttranscriptional and/or translational regulation of eNOS protein and result in lower plasma NO levels in adult male pigs, thereby exerting an effect on vascular function [25]. On the other hand, maturation also induces increased production of reactive oxygen species (ROS) in vessels, which, in turn, may impair vessel function as a result of decreased NO bioavailability [26–28].

We characterized the phenotype of the SMC responsible for vascular remodeling in guinea pigs exposed to CS by evaluating the expression of the intermediate filaments smooth muscle α-actin and desmin in small pulmonary vessels. Animals exposed to CS for 3 months showed an increase in the number of α-actin-positive vessels, which persisted after 6 months of exposure. In contrast, there were no differences in the number of vessels positive for the contractile filament desmin, either at 3 or at 6 months. Accordingly, CS-exposed guinea pigs showed proliferation of α-actin⁺/desmin^- SMC, which represent a subpopulation of less differentiated SMCs with synthetic capacity [5]. These results are consistent with those obtained in COPD showing that vascular remodeling is produced by the intimal proliferation of poorly differentiated SMCs [3]. The structural alterations in the pulmonary circulation of CS-exposed guinea pigs might be a consequence of changes in the synthesis and release of vasoactive and cell proliferative mediators, since endothelin and VEGF expression are increased in the arterial wall of remodeled vessels in animals exposed to CS [12]. After 6 months of exposure, we found an increased percentage of vessels with double elastic laminas. In the same experimental model, Wright et al. [9] also reported that the greater number of vessels with double elastic laminas was accompanied by an increase in pulmonary artery pressure (PAP). This observation suggests that muscularization of small vessels induced by CS exposure is associated with pulmonary hypertension. In keeping with this, we observed a wall enlargement in main pulmonary arteries after 6 months of CS exposure, which correlated with the contractility to KCl.

Smooth muscle cell proliferation in small vessels was already present at 3 months of exposure and preceded the development of emphysema. These findings confirm previous observations made by Yamato et al. [29] and are in agreement with studies performed in healthy smokers, who showed pulmonary vascular remodeling and endothelial dysfunction [2]. Although hypoxemia, which is associated with emphysema, is one of the strongest agents producing vasoconstriction and vessel remodeling, our results corroborate that CS exposure may have a similar effect on pulmonary vessels [30]. Nevertheless, the presence of hypoxemia in patients with COPD might exert a synergistic effect on the pathogenesis of pulmonary hypertension. Further studies are required to elucidate the potential synergism between cigarette smoke and hypoxia in this experimental model.

It is interesting to note that endothelial dysfunction and vessel remodeling associated with CS exposure affected selectively pulmonary arteries while the aorta remained unaltered. We consider this could be due to the fact that pulmonary vessels are exposed to greater concentrations of CS products, whereas the effects on the aorta might be eventually caused by products diffusing to the blood. We do not disregard that longer exposure to CS might exert an effect on the aorta or systemic vessels. Although, it is conceivable that longer exposure would also result in greater structural and functional damage of pulmonary vessels. Yet, it is noteworthy that in this experimental model pulmonary vessels develop changes after a short period of CS exposure that antecede changes in lung structure or systemic vascular involvement.

In conclusion, the guinea pig chronically exposed to CS develops endothelial dysfunction selectively in pulmonary arteries, without presenting changes in systemic arteries. This endothelial dysfunction is accompanied by reduced lung expression of eNOS and proliferation of poorly differentiated SMCs in small pulmonary vessels. These changes are already apparent after a relatively short period of CS exposure and precede the full muscularization of small pulmonary vessels and the development of emphysema. Results in this experimental model confirm that CS has direct and deleterious effects on the structure and function of pulmonary vessels that might contribute to the development of pulmonary hypertension in COPD.