American Journal of Respiratory and Critical Care Medicine

Rationale: In cigarette smoking–induced chronic obstructive pulmonary disease, structural and functional derangements are characterized by parenchymal destruction and pulmonary hypertension. Statins are 3-hydroxy-3-methyl-glutaryl–coenzyme-A reductase inhibitors that have been used as lipid-lowering agents. These drugs also have additional pharmacologic properties, including antiinflammation, scavenging reactive oxygen species, restoring endothelial function, and antithrombogenesis, all of which can counteract the harmful effects of cigarette smoking.

Objective: We performed assays to determine whether simvastatin could attenuate lung damage induced by chronic cigarette smoking in rats.

Methods: In Sprague-Dawley rats exposed to cigarette smoke for 16 weeks, morphologic changes in the lungs and pulmonary arterial pressure were examined.

Main Results: Simvastatin inhibited lung parenchymal destruction and development of pulmonary hypertension, and also inhibited peribronchial and perivascular infiltration of inflammatory cells and induction of matrix metalloproteinase-9 activity in lung tissue. Simvastatin additionally prevented pulmonary vascular remodeling and the changes in endothelial nitric oxide synthase expression induced by smoking. In human lung microvascular endothelial cells, simvastatin increased expression of endothelial nitric oxide synthase mRNA.

Conclusions: Simvastatin ameliorated the structural and functional derangements of the lungs caused by cigarette smoking, partly by suppressing inflammation and matrix metalloproteinase-9 induction and preventing pulmonary vascular abnormality. These findings indicate that statins may play a role in the treatment of cigarette smoking–induced chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is defined as a disease associated with an abnormal inflammatory response of the lungs to noxious particles or gas (1). Cigarette smoking is the most important risk factor for the development of COPD. Chronic smoke exposure causes airway and lung parenchymal inflammation, which is characterized by increased numbers of macrophages, lymphocytes, neutrophils, and/or eosinophils (2). Several kinds of proteases, including neutrophil elastase, macrophage elastase, matrix metalloproteinases (MMPs), and cathepsins, from these inflammatory cells may contribute to alveolar destruction and result in pulmonary emphysema in chronic cigarette smokers (37). In addition, reactive oxygen species produced by cigarette smoke (8) or inflammatory cells, including activated lung macrophages and infiltrating neutrophils (9), may contribute to emphysematous changes, in that massive and continuous oxidative stress may overwhelm the antioxidant capacity of lung tissue, thereby causing damage.

In addition to causing structural damage of lung tissue, cigarette smoking has been shown to induce prominent pulmonary vascular changes characterized by endothelial dysfunction and vascular remodeling, which lead to pulmonary hypertension (1013). Suppression of endothelial nitric oxide (NO) production is suggested as a potential cause of the endothelial dysfunction and impaired vasodilation in humans (11), although chronic exposure to cigarette smoke oppositely induced endothelial NO synthase (eNOS) expression in guinea pig pulmonary arteries (14). In addition, pulmonary vascular remodeling, characterized by intimal and medial thickening with proliferation of smooth muscle cells and deposition of elastic and collagen fibers in pulmonary arteries, may be an underlying mechanism for pulmonary hypertension in COPD (12). In smoking-induced structural and functional derangements of pulmonary circulation, potential pathologic causative factors may include direct oxidative damage by cigarette smoke products and subsequent inflammation, as well as exposure to chronic hypoxia (13). Furthermore, a tendency toward hypercoagulation induced by cigarette smoking may be an additional factor contributing to pulmonary hypertension (15).

Statins are 3-hydroxy-3-methyl-glutaryl–coenzyme-A (HMG-CoA) reductase inhibitors that have been used clinically as lipid-lowering agents. Statins, however, have additional pleiotropic pharmacologic properties, including antiinflammatory, antioxidant, antithrombogenic, and vascular function–restoring actions (16). Interestingly, all of these additional actions may counteract the harmful effects of cigarette smoking and chronic inflammation. We therefore performed assays to determine the beneficial effects of simvastatin on chronic cigarette smoking–induced lung damage, using a rat model. To our knowledge, this study is the first to show that simvastatin ameliorates cigarette smoking–induced structural and functional derangement of the lungs, possibly by reducing inflammatory infiltration, inhibiting MMP-9 induction and preventing pathologic changes in pulmonary vasculature. These findings indicate that statins may be used in the treatment of cigarette smoking–induced COPD.

We submitted abstracts and presented part of this study at the Aspen Lung Conference (Aspen, CO, 2004) and COPD 4 (Birmingham, UK, 2004). This article reports our complete study findings.

Smoking Exposure and Treatment Groups

This animal study was approved by the Panel on Laboratory Animal Care of Asan Medical Center. Male Sprague-Dawley rats (Orient, South Korea) were exposed to the smoke of 10 commercial cigarettes (Eighty Eight Lights, South Korea) per day for 16 weeks (17). Rats were divided into four groups: control (CTL), smoking-only (SM), smoking-plus-simvastatin (SMST), and statin-only (ST) groups. Simvastatin, at a dose of 5 mg/kg, was administered orally to rats in the SMST and ST groups once per day for 16 weeks.

Hemodynamic and Histologic Analysis

At the end of the 16 weeks, mean pulmonary arterial pressure (MPAP) was measured by cannulation through the right jugular vein. After the lungs were removed en bloc, the right and left lungs were used for histologic and biochemical analysis, respectively.

In hematoxylin-and-eosin–stained lung sections, average interalveolar septal wall distance (mean linear intercept [MLI]) (18), and alveolar surface-to-volume ratio (S/V) were measured (19). The degree of peribronchial and perivascular inflammation was scored on a subjective scale of 0 (no) to 4 (severe) in a blind manner (20).

After elastin–van Gieson staining, approximately 100 consecutive peripheral vessels were classified into three categories; fully muscularized, partially muscularized, and nonmuscularized (21). Also, medial thickness in 20 fully muscularized arteries was evaluated by calculating the percentage of medial wall thickness as (medial thickness × 2/external diameter) × 100 (21).

Vascular expression of eNOS and endothelin (ET)-1 proteins in pulmonary vessels of 50 to 200 μm in diameter was estimated using anti-eNOS (BDB Transduction Laboratories, Lexington, KY) and anti–ET-1 monoclonal antibody (Affinity BioReagents, Golden, CO), and immunoreactivity was determined in semiquantified scales from 0 (no staining) to 3 (very intense staining) (11).

Gelatin Zymography

MMP-2 and MMP-9 activities were determined by gelatin zymography (22). Proteins from each lung extract (20 μg) were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis containing 1 mg/ml gelatin (Invitrogen, Carlsbad, CA), and, after developing and staining the gels, gelatinolytic activities were analyzed with LAS-1000 and the ImageGuage program (Fuji, Tokyo, Japan).

Human Lung Microvascular Endothelial Cells

Human lung microvascular endothelial cells (Cambrex, Rockland, ME) of passages 6 to 8 at 70% confluence in 60-mm dishes were exposed to cigarette smoke extract (CSE). CSE solution was prepared by drawing mainstream smoke from two cigarettes through 10 ml modified Eagle medium containing 25 mM N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid (23). After 24-hour exposure to CSE, eNOS mRNA expression was determined by reverse transcriptase–polymerase chain reaction.

Statistical Analysis

Data are presented as mean ± SE. Data were analyzed by two-way analysis of variance with multiple comparisons to detect whether smoking causes functional and structural changes in lungs and whether simvastatin treatment had a protective effect during smoking.

Additional details on the methods for making the above measurements are provided in an online supplement.

General Conditions of Rats Exposed to Cigarette Smoke for 16 Weeks

All 35 rats in the four groups survived the 16 weeks of the study. The mean ± SE bodyweights of the rats in the CTL, SM, SMST, and ST groups at 16 weeks were 598 ± 24, 554 ± 20, 580 ± 17, and 575 ± 19 g, respectively. Although the mean bodyweight of the rats in the SM group was slightly lower than those in the other three groups, this difference was not significant.

Inhibition of Cigarette Smoke–induced Emphysema Development by Simvastatin

We first evaluated the effect of simvastatin on the cigarette smoking–induced destruction of alveolar architecture. Compared with the normal alveolar structure (CTL group, Figure 1A

, a), chronic exposure to cigarette smoke for 16 weeks induced lung parenchymal destruction, leading to the enlargement of air-spaces (SM group, Figure 1A, b). However, simvastatin dramatically inhibited lung destruction by cigarette smoke (SMST group, Figure 1A, c). After 16 weeks of smoking, the MLI in the SM group was 174% of that in the CTL group (116.2 ± 7.7 vs. 66.6 ± 1.8 μm, p < 0.01; Figure 1B, a), and the S/V in the SM group was half that in the CTL group (0.065 ± 0.003 vs. 0.130 ± 0.005 μm−1, p < 0.01; Figure 1B, b). Although simvastatin alone did not significantly change MLI (60.1 ± 2.0 μm) and S/V (0.128 ± 0.004 μm−1), as observed in the ST group, simvastatin treatment of rats exposed to cigarette smoke (SMST group) significantly decreased MLI (66.1 ± 1.5 μm) and increased S/V (0.125 ± 0.007 μm−1), nearly to the levels observed in the CTL group.

Reduction of Peribronchial and Perivascular Inflammation by Simvastatin

During smoke exposure, infiltrating inflammatory cells are believed to contribute to structural derangement of lung tissues in COPD via the release of reactive oxygen species and proteases (35, 8, 9). We therefore tested the effects of simvastatin on inflammatory infiltration induced by cigarette smoking. In rat lungs, cigarette smoking for 16 weeks caused infiltration of primarily mononuclear cells into the peribronchial and perivascular areas (Figure 2A

, b), whereas simvastatin inhibited inflammatory infiltration around the bronchial trees and blood vessels (Figure 2A, c). By histologic scoring, simvastatin significantly reduced peribronchial, perivascular, and total inflammation scores in the SMST group, when compared with the SM group (Figure 2B).

Effect of Simvastatin on Smoking-induced Pulmonary Hypertension

Because pulmonary hypertension is one of the most important functional derangements in chronic smoking-induced COPD (13), we tested the effects of simvastatin on pulmonary arterial pressure (Figure 3)

. After exposure to cigarette smoke for 16 weeks, MPAP in the SM group (21.9 ± 2.0 mm Hg) was significantly higher than in the CTL group (15.0 ± 1.7 mm Hg). In contrast, although simvastatin alone did not affect MPAP (ST group, 14.5 ± 0.3 mm Hg), it significantly inhibited the smoking-induced increase in MPAP (SMST group, 12.4 ± 0.5 mm Hg).

Possible Mechanisms by which Simvastatin Prevents Development of Pulmonary Hypertension

Potential mechanisms for pulmonary hypertension in COPD are those involving pulmonary vascular remodeling, which is characterized by muscularization of the small vessels (12) and alterations in expression of proteins that control pulmonary vascular tone, such as eNOS and ET-1 (11, 23, 24). We therefore tested the effects of simvastatin on pulmonary vascular remodeling and expression of eNOS and ET-1.

After 16 weeks of exposure to cigarette smoke, rats in the SM group had more muscularized pulmonary vessels showing double elastic lamina (internal elastic lamina and external elastic lamina) with medial thickening than rats in the CTL group (Figure 4A

, a, b). Simvastatin treatment, however, inhibited double lamina formation and medial thickening induced by cigarette smoke, as observed in the SMST group (Figure 4A, c). When we categorized the small pulmonary vessels, we found that the proportion of fully or partially muscularized vessels was higher, and the proportion of nonmuscularized vessels was significantly lower, in the SM group than in the CTL group (Figure 4B). Simvastatin treatment of rats exposed to cigarette smoke normalized the proportion of each type of vessel (SMST group), whereas simvastatin alone did not induce morphologic changes in pulmonary vasculature (Figures 4A and 4B). Compared with the CTL group, the percentage of medial wall thickness of fully muscularized arterioles in the SM group was increased, but there were no significant differences in percentage of medial wall thickness between the CTL and the other two groups (Figure 4C).

When we tested the effect of simvastatin on endothelial expression of ET-1 and eNOS proteins, we found that the microvascular endothelium showed eNOS immunostaining similar to that of control (Figures 5A and 5B)

, but had no effect on ET-1 protein expression (data not shown). Similarly, simvastatin dose-dependently increased eNOS expression in human lung microvascular endothelial cells treated with or without CSE (Figure 6). Together with its inhibition of pulmonary vascular remodeling, the simvastatin-induced restoration of eNOS expression may explain its beneficial effects on pulmonary hypertension.

Simvastatin Inhibits MMP-9 Induction by Cigarette Smoking

Because increased MMP-9 expression in the lungs has been reported to be associated with emphysema, both in clinical and experimental studies (57, 2528), we tested whether simvastatin inhibits MMP-9 induction in cigarette smoke–exposed lung tissues.

Gelatin zymography showed that MMP-9 activity was higher in rats exposed to cigarette smoke (SM group) than in control rats (CTL group), whereas MMP-2 activity was equivalent in all three groups (Figure 7A)

. In contrast, simvastatin treatment significantly reduced the smoking-induced increase in MMP-9 activity (SMST group; Figures 7A and 7B).

We have shown here that simvastatin inhibits the development of lung damage caused by chronic cigarette smoking. Simvastatin attenuated lung parenchymal destruction, inflammatory infiltration, and pulmonary hypertension induced by chronic cigarette smoking. These results indicate that simvastatin prevents smoking-induced lung damage via pleiotropic effects.

In addition to cardiovascular diseases associated with hypercholesterolemia, statins have antiinflammatory activity in several models of human diseases. For example, simvastatin inhibited the development of collagen-induced inflammatory arthritis in a mouse model (29), and decreased lung inflammation and Th2 cytokine secretion in a murine model of allergic asthma (20). Statins have also been shown to inhibit the LPS-induced expression of tumor necrosis factor α, interleukin 1β, and interleukin 6 in macrophages (30); the expression of vascular cell adhesion molecule-1 and E-selectin in endothelial cells (31); and the interaction between leukocyte function antigen-1 and intercellular adhesion molecule-1 (32). Because tumor necrosis factor α (33) and interleukin 1β (28) are believed to mediate emphysematous change in mice, and because ICAM-1 is increased in small airway epithelium of smokers (34), simvastatin may reduce the inflammatory infiltration observed in this study by inhibiting the expression of these proinflammatory molecules.

Among inflammatory cells, activated alveolar macrophages (35) and infiltrated/activated neutrophils (3, 33) are believed to play a major role in the destruction of lung parenchyma by releasing various kinds of proteinases, although the contribution of each type of inflammatory cell is not clear. We have shown here that simvastatin inhibited the induction of MMP-9, a proteinase that may be associated with emphysema (25, 28, 35). Although the exact mechanism by which simvastatin inhibits MMP-9 expression is not yet known, our results suggest that it may occur through inhibition of inflammatory cell infiltration. Another possible explanation is that simvastatin may directly inhibit MMP-9 expression in parenchymal and inflammatory cells by inhibiting the prenylation of Rho (36) or Ras (37).

Although we have shown that MMP-9 activity in the lungs is increased by exposure to cigarette smoke, further studies are needed to determine the causal relationship between MMP-9 induction and emphysema development, as well as the potential contribution of other MMPs to the destruction of lung tissue observed in this study. For example, MMP-1 (38) and MMP-12 (4) have been reported to be associated with smoke-induced lung injury. Furthermore, every type of lung parenchymal cell, as well as infiltrating cells, may be a source of MMPs. It is clinically important that CP-471,474, an MMP inhibitor, attenuated MMP-9 induction and the development of smoke-induced lung inflammation and emphysema in guinea pig lungs (5), although this MMP inhibitor did not completely block the development of emphysema, suggesting that other factors are involved in the complicated pathogenesis of COPD.

In addition to showing the effects of simvastatin on inflammatory cell infiltration and MMP-9 expression, we found that simvastatin inhibited suppression of smoking-induced eNOS expression, pulmonary vascular remodeling, and development of pulmonary hypertension. Cigarette smoking has been shown to induce physiologic and biochemical alterations, including impaired endothelium-dependent relaxation of pulmonary arteries (13) and reduced eNOS expression in pulmonary arteries of smokers (11) and in pulmonary artery endothelial cells (23). Because the stability of eNOS mRNA and the phosphorylation of eNOS for optimum activity are negatively regulated through Rho (39, 40), the effect of simvastatin on eNOS may be mediated through a reduction in Rho activity caused by inhibiting isoprenoid synthesis and subsequent prenylation of Rho.

Other mechanisms may be involved in the simvastatin-induced effect on eNOS expression in pulmonary vascular endothelial cells exposed to cigarette smoke. Because reactive oxygen species in the sera of smokers has been shown to reduce eNOS activity in human coronary endothelial cells (41), simvastatin, via its antioxidant activity (42), may restore eNOS activity by scavenging reactive oxygen species. In addition, simvastatin may reduce expression of tumor necrosis factor α, a major proinflammatory mediator in cigarette smoke–induced lung damage (33) that has been shown to inhibit eNOS expression by destabilizing its mRNA (43) or inhibiting its transcription (44).

Another beneficial effect of simvastatin demonstrated in this study was its inhibition of pulmonary vascular remodeling. Pulmonary vascular remodeling accompanies intimal thickening, muscularization of arterioles, and the loss of capillaries and precapillary arterioles (12). Several underlying factors, including chronic hypoxemia, inflammation, and endothelial dysfunction, may contribute to pulmonary vascular remodeling and subsequent pulmonary hypertension in COPD (13). Chronic hypoxemia, however, must be carefully evaluated because vascular remodeling and endothelial dysfunction have been observed in patients with mild COPD and even in smokers with normal lung function (13). The intensity of inflammatory infiltration around pulmonary vessels showed a correlation with endothelium-dependent relaxation as well as with structural abnormalities of pulmonary arteries (45). Although the role of MMPs in vascular remodeling in COPD is not known, there is some evidence that MMPs contribute to the development of pulmonary hypertension (46, 47). MMPs may be involved in the degradation of basement membrane, and in the migration and proliferation of smooth muscle cells (48). Evidence against the role of MMPs in vascular remodeling includes the finding that simvastatin inhibited the proliferation and migration of saphenous vein neointimal smooth muscle by reducing MMP-9 activity (49).

Statins have also been shown to prevent vascular remodeling by inducing apoptosis of smooth muscle cells (5053). Similar to our results, simvastatin attenuated pulmonary hypertension and pulmonary vascular remodeling induced by hypoxia (21) or by pneumonectomy/monocrotalin (52, 53). This was especially shown in the pneumonectomy/monocrotalin model, in which characteristic neointimal smooth muscle proliferation and vascular obliteration of small pulmonary arterioles were attenuated by the simvastatin-induced apoptosis of these smooth muscle cells (53). Although several mechanisms, including the upregulation of p27 (50, 53) and the downregulation of Bcl-2 expression (51), have been suggested as preventing vascular remodeling, the precise mechanism by which simvastatin prevents pulmonary arterial hypertension in each model requires further study.

In a study using knockout mice, Nrf-2, a redox-sensitive transcription factor, was recently reported to play a critical role in cigarette smoking–induced emphysema, in that Nrf2 knockout mice exposed to cigarette smoke showed earlier onset and more extensive emphysema, which was associated with pronounced inflammation, increased oxidative DNA damage, and increased apoptosis of alveolar epithelial cells (54). In addition, many Nrf2-dependent genes encoded antioxidants and cytoprotective enzymes, suggesting that overwhelming oxidative stress from smoking may cause development of COPD. Thus, the protective inhibitory effect of simvastatin on lungs exposed to cigarette smoke may be due to its antioxidant activity and its inhibitory effect on infiltration of inflammatory cells to produce reactive oxygen species.

In conclusion, we have shown here that simvastatin, an HMG-CoA reductase inhibitor, attenuated the development of cigarette smoking–induced emphysema and pulmonary hypertension, partly by inhibiting inflammatory cell infiltration into the lungs. In addition, simvastatin inhibited smoking-induced MMP-9 expression, as well as ameliorated smoking-associated structural derangements and normalized eNOS expression in the pulmonary vasculature. The detailed mechanisms by which simvastatin acts to prevent smoking-induced COPD still remain to be determined, both because of the pleiotropic effects of simvastatin and the complex pathogenesis of COPD. Taken together, these findings indicate that statins could potentially play a role in the treatment of cigarette smoking–induced COPD.

The authors thank Ju-Young Kim for technical assistance. Simvastatin was kindly provided by Merck and Co. (Rahway, NJ).

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*These authors contributed equally to this article.

Correspondence and requests for reprints should be addressed to Sang-Do Lee, M.D., Ph.D., Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, 388-1 Poongnab-dong, Songpa-gu, Seoul 138-736, South Korea. E-mail:

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American Journal of Respiratory and Critical Care Medicine
172
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