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1Division of Pulmonary Medicine, Department of Medicine, Keio University and 2Department of Medicine, Tokyo Electric Power Company Hospital, Tokyo; and 3Research and Development Division, Mitsubishi Pharma Corporation, Yokohama, Japan
Submitted 20 June 2007 ; accepted in final form 24 February 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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vascular endothelial growth factor; endothelial nitric oxide synthase
3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have pleiotropic pharmacological effects, separate from cholesterol lowering, including an anti-inflammatory effect, scavenging of reactive oxygen species, improved endothelial function (17, 36, 40), inhibition of smooth muscle neointimal proliferation (28, 29), and antithrombogenesis (8, 25). The anti-inflammatory effects include suppression of the release of proinflammatory cytokines, chemokines, adhesion molecules, and matrix metalloproteinases by inflammatory cells (2, 4, 7, 12, 19, 26, 31, 37). Statins increase the production of vasodilators, suppress vasoconstrictors, and promote reendocanalization by mobilizing endothelial progenitor cells (EPCs) from the bone marrow (1, 11). They also increase the secretion of vascular endothelial growth factor (VEGF) and the expression and activity of endothelial nitric oxide synthase (eNOS) (33, 38), which improves endothelial cell function and promotes angiogenesis. Most of these effects are due to the inhibition of the prenylation of signaling proteins, for example, the GTP-binding proteins Rho and Rac (34), and can counteract the development of pulmonary emphysema (18).
Several growth factors, such as granulocyte colony-stimulating factor (G-CSF) and hepatocyte growth factor (HGF), have healing effects on emphysematous lungs in animals (16, 23). Ishizawa et al. (16) found that HGF regenerated emphysematous changes by inducing angiogenesis in injured murine lungs by mobilizing EPCs. Statins, like VEGF, G-CSF, and granulocyte-macrophage colony-stimulating factor (GM-CSF), mobilize EPCs from the bone marrow to peripheral blood (3, 30, 36). Lee et al. (18) have shown that statins also attenuate the emphysema induced by smoking in rats, mainly by their anti-inflammatory effects. In the present study, we intended to treat elastase-induced emphysema in mice with simvastatin (SS), since it is hypothesized that statins may reverse emphysema through promotion of alveolar cell proliferation in addition to their anti-inflammatory properties.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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SS was purchased from Calbiochem (La Jolla, CA). Porcine pancreas elastase (PPE) was obtained from Sigma (St. Louis, MO). The mice used in all experiments were 8-wk-old male C57BL/6 mice weighing 
20 g (Oriental Yeast, Tokyo, Japan). Human recombinant HGF was provided by Mitsubishi Pharma (Yokohama, Japan). All animals were humanely cared for, and the study was approved by the Keio University Panel on Laboratory Animal Care.
Mouse Model of Elastase-Induced Pulmonary Emphysema
Acute-phase protocol. Mice anesthetized with methoxyflurane received 20 µg of PPE in 50 µl of saline by intratracheal instillation (SS+ and SS– groups) or 50 µl of saline alone (Ctl group) at time 0. A single elastase injection causes the development of experimental emphysema over 3 wk. The mice received daily intraperitoneal injections of 20 µg of SS in 200 µl of PBS (SS+ group), or 200 µl of PBS alone (SS– and Ctl groups), beginning 1 day before the instillation of PPE. The treatment was continued until the day before death.
Morphometric measurements of air space size. After an intraperitoneal injection of pentobarbital sodium, subgroups of mice were killed 3, 7, or 14 days after the instillation of PPE. The lung tissues were inflated with 4% paraformaldehyde at a pressure of 25 cmH2O, fixed for 24 h in formalin, embedded with paraffin, sectioned in the sagittal plane, and stained with hematoxylin and eosin (H & E). The linear intercepts of 100 alveoli were measured, and the mean value (Lm) was used as a morphometric parameter of emphysema as previously reported (15, 16, 18, 23).
Bronchoalveolar lavage.
At 2 and 8 h and 1, 3, 7 and 14 days after the instillation of PPE, all groups of mice were anesthetized with pentobarbital sodium (50 mg/kg ip). The trachea was exposed and intubated. The whole lungs were washed three times with 1 ml of sterile saline. Bronchoalveolar lavage fluid (BALF) was collected with a 1-ml syringe and placed on ice. The cells were counted with a hemocytometer. Cell differentials were counted on smears prepared by cytospin and stained with Diff-Quik (Kokusaishiyaku, Kobe, Japan). After centrifugation at 400 g for 5 min at 4°C, the concentrations of tumor necrosis factor (TNF)-
, macrophage inflammatory protein (MIP)-2, keratinocyte-derived chemokine (KC), and VEGF in the supernatants were measured with ELISA kits (R&D Systems, Minneapolis, MN). The lavage fluid was lyophilized and hydrolyzed in 2 ml of 6 M hydrocholic acid for 48 h at 110°C, and parts of the hydrolysate were used for measuring concentrations of desmosine and hydroxyproline. Plasma VEGF concentrations were also measured by ELISA. The sensitivity of the ELISA kits was 5.1 pg/ml for TNF-, 1.5 pg/ml for MIP-2, 2.0 pg/ml for KC, and 3.0 pg/ml for VEGF.
Immunohistochemical staining for proliferating cell nuclear antigen. The lung sections were incubated with a proliferating cell nuclear antigen (PCNA) antibody (mouse monoclonal clone PC-10 antibody, Abcam, Cambridge, MA) followed by incubation with goat anti-mouse antibody conjugated with horseradish peroxidase. The percentage of PCNA-positive alveolar epithelial cells was calculated as (number of PCNA-positive cells/all epithelial cells in each section) x 100.
Late-phase protocol. The mice received 20 µg of PPE in 50 µl of saline or 50 µl of saline alone by intratracheal instillation at time 0 with the procedures described above. Beginning on day 21, the mice received daily intraperitoneal injections of 4, 20, or 100 µg of SS in 200 µl of PBS (SS+ group), 5 µg of human HGF in 200 µl of PBS (HGF group), or 200 µl of PBS alone (SS– and Ctl groups) for 12 days, at which time we measured Lm and percentages of PCNA-positive stained cells by the methods described above. We also collected BALF and plasma to measure VEGF concentrations. The activity of HGF in C57BL/6 mice has been verified previously (39).
Immunohistochemical staining and real-time RT-PCR for eNOS. Vascular expression of eNOS in pulmonary vessels of 50- to 200-µm diameter was estimated with anti-eNOS monoclonal antibody (BD Transduction Laboratories, Lexington, KY) and biotin-labeled goat anti-mouse secondary antibody (Dako, Carpinteria, CA). Immunoreactivity was determined in semiquantified scales from 0 (no staining) to 3 (very intense staining) as previously reported (18).
Total lung RNA was isolated with an RNeasy Mini kit (Qiagen, Valencia, CA) and reverse transcribed with an Omniscript RT kit (Qiagen). TaqMan gene expression assays detecting mRNA encoding mouse eNOS (Mm00435204_m1) were performed according to the supplier's recommendations by ABI PRISM 7000 real-time cycler (Applied Biosystems, Foster City, CA). Fluorescence values for eNOS were normalized to those of a housekeeping gene, GAPDH, and expressed as fold change over the Ctl group.
Statistical Analysis
Data are presented as means ± SD in Tables 1 and 2 and Figs. 1–3. Comparisons between groups (SS+ vs. SS–) were performed with Mann-Whitney U-test in the acute phase protocol. Kruskal-Wallis test and Mann-Whitney U-test were used for comparisons among the groups in the late-phase protocol, including dose-response experiments. Dose dependence was evaluated by Jonckheere test. All statistical tests were two sided, and significance was defined as P < 0.05.
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RESULTS |
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There were significant differences in the values of each experiment in the late-phase protocol by Kruskal-Wallis test except eNOS protein expression (P = 0.08) and BALF VEGF levels (P = 0.25).
Decrease in Elastase-Induced Enlargement of Lm by Simvastatin
In the acute-phase protocol, median Lm values increased over time in the SS– group, from 37 µm on day 3 to 45 µm on day 7 to 50 µm on day 14 after a single instillation of PPE. In contrast, in the Ctl group, Lm size was 27 µm on days 3, 7, and 14. Lm was significantly smaller in the SS+ group than in the SS– group on day 3 (34 vs. 37 µm; P < 0.05) and on day 7 (35 vs. 45 µm; P < 0.01, 21% reduction) (Fig. 1A). Representative light microscopic photographs of lung sections stained with H & E are shown in Fig. 2, A (SS–, day 7) and B (SS+, day 7). In the late-phase protocol, histological analysis showed a 52% reduction of median Lm in the SS+ group compared with the SS– group (29 vs. 60 µm; P < 0.05) when administered 3 wk after the PPE instillation (Fig. 1B). The regenerative effects of SS were significantly more prominent than those of HGF (P < 0.05) in this experimental protocol. Lm in the SS–, SS+, and HGF groups significantly increased compared with that in the Ctl group (P < 0.01). Representative light microscopic photographs of lung sections in the late-phase protocol are shown in Fig. 2, C (SS–), D (SS+), E (HGF), and F (Ctl).
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The percentage of PCNA-positively stained alveolar epithelial cells was significantly higher in the SS+ group than in the SS– group (Fig. 3A) on day 3, day 7, and day 14 (P < 0.05) in the acute-phase protocol. Representative light microscopic photographs of lung sections stained with the PCNA antibody are shown in Fig. 4, A (SS–, day 7) and B (SS+, day 7). In the late-phase protocol, the percentage of PCNA-positively stained alveolar epithelial cells, which tended to be lower than those in the acute phase, was also significantly higher in the SS+ group than in the SS– (P < 0.01) or the HGF (P < 0.05) group (Fig. 3B). The percentage of positive cells was higher in the SS+ and HGF groups than in the Ctl group (P < 0.01). Representative light microscopic photographs of lung sections are shown in Fig. 4, C (SS–), D (SS+), E (HGF), and F (Ctl).
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Jonckheere test demonstrated that Lm was dose-dependently decreased by SS in the late-phase protocol (P < 0.0001). Lm in the SS+ 4, 20, and 100 µg groups was significantly smaller than that in the SS– group (P < 0.01, Fig. 5A). Lm in the SS+ 100 µg group was significantly smaller than that in the SS+ 4 and 20 µg groups (P < 0.01 and 0.05, respectively). Jonckheere test suggested that the percentage of PCNA-positive alveolar epithelial cells was dose-dependently increased by SS (P < 0.0001). The percentage of the positively stained cells in the SS+ 4, 20, and 100 µg groups was significantly higher than that in the SS– group (P < 0.01, Fig. 5B). The percentage in the SS+ 20 µg group was higher than that in the SS+ 4 µg group (P < 0.05).
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The percentage of neutrophils in BALF was significantly increased during the acute inflammatory phase, i.e., 2 h to 3 days after the instillation of PPE, and tended to be lower in the SS+ group than in the SS– group (Table 1). However, this tendency was not observed in later time points during days 7–14 when the neutrophil percentages were <3% (Table 1). The total cell count was similar in both groups at each time point (data not shown).
The number of mice, median value, and range (%) in each experiment for 2 h (SS– and SS+); 8 h (SS– and SS+); day 1 (SS– and SS+); day 3 (SS– and SS+); day 7 (SS– and SS+); and day 14 (SS– and SS+) were [8, 27.2 (11.8–33.0)] and [8, 16.3 (0.0–24.4)]; [8, 33.8 (25.1–60.4)] and [9, 23.8 (5.5–43.1)]; [8, 45.9 (30.8–63.9)] and [7, 27.4 (22.4–40.0)]; [8, 24.0 (3.8–55.3)] and [10, 8.4 (1.3–31.9)]; [10, 1.3 (0.0–4.6)] and [8, 0.7 (0.0–10.7)]; and [4, 0.0 (0.0–0.7)] and [5, 1.0 (0.3–5.1)], respectively.
Changes in BALF Concentrations of TNF-, MIP-2, and KC by Simvastatin
Despite its inhibitory effects on the accumulation of neutrophils, treatment with SS was associated with a significant increase in concentrations of TNF- and MIP-2 in BALF 2 h after the instillation of PPE (Table 1). However, BALF concentrations of TNF- and MIP-2 were both lower on day 1 in the SS+ group than in the SS– group. In contrast, BALF concentrations of KC, another potent neutrophil chemoattractant in mice, tended to decrease in the SS+ group compared with the SS–group throughout the acute phase (2 h to day 7).
The number, median value, and range (pg/ml) for TNF- at 2 h (SS–, SS+); 8 h (SS–, SS+); day 1 (SS–, SS+); day 3 (SS–, SS+); day 7 (SS–, SS+); and day 14 (SS–, SS+) were [12, 99 (69–129)] and [11, 125 (89–246)]; [12, 75 (65–112)] and [12, 86 (62–202)]; [9, 67 (59–88)] and [12, 58 (0–69)]; [8, 52 (48–78)] and [10, 57 (49–65)]; [9, 44 (39–46)] and [8, 47 (42–51)]; and [8, 39 (35–44)] and [12, 39 (0–43)], respectively. The number, median value, and range (pg/ml) for MIP-2 at 2 h (SS–, SS+); 8 h (SS–, SS+); day 1 (SS–, SS+); day 3 (SS–, SS+); day 7 (SS–, SS+); and day 14 (SS–, SS+) were [12, 68 (3–119)] and [9, 130 (48–318)]; [12, 80 (15–339)] and [13, 192 (3–324)]; [12, 28 (18–105)] and [10, 13 (8–17)]; [9, 12 (8–31)] and [12, 14 (8–32)]; [8, 11 (10–15)] and [10, 11 (9–13)]; and [12, 10 (9–14)] and [10, 10 (9–11)], respectively. The number, median value, and range (pg/ml) for KC at 2 h (SS–, SS+); 8 h (SS–, SS+); day 1 (SS–, SS+); day 3 (SS–, SS+); day 7 (SS–, SS+); and day 14 (SS–, SS+) were [6, 120 (101–143)] and [6, 104 (88–116)]; [6, 107 (89–119)] and [6, 96 (91–109)]; [7, 60 (55–88)] and [7, 46 (43–80)]; [7, 40 (38–45)] and [7, 39 (36–45)]; [5, 46 (40–50)] and [7, 38 (34–44)]; and [3, 46 (38–47)] and [4, 47 (44–50)], respectively.
Mitigation of Elastase-Induced Collagen Breakdown by Simvastatin
Hydroxyproline and desmosine are markers of collagen and elastin breakdown, respectively. SS significantly decreased the concentrations of hydroxyproline in BALF on day 3 (Table 1). However, there was no significant difference in BALF desmosine levels between the groups.
The number, median value, and range (ng/ml) for hydroxyproline at day 1 (SS–, SS+); day 3 (SS–, SS+); day 7 (SS–, SS+); and day 16 (SS–, SS+) were [6, 1,910 (760–4,120)] and [6, 2,500 (960–3,220)]; [6, 1,930 (970–3,890)] and [6, 910 (440–1,330)]; [6, 870 (670–1,120)] and [6, 920 (640–1,570)]; and [6, 1,360 (540–2,820)] and [4, 700 (400–1,410)], respectively. The number, median value, and range (ng/ml) for desmosine at 2 h (SS–, SS+); day 1 (SS–, SS+); day 3 (SS–, SS+); day 7 (SS–, SS+); and day 16 (SS–, SS+) were [5, 5,720 (0–11,300)] and [5, 160 (0–8,100)]; [5, 540 (0–1,320)] and [5, 0 (0–920)]; [5, 120 (60–410)] and [5, 230 (0–610)]; [4, 50 (0–150)] and [5, 110 (0–140)]; and [5, 70 (0–90)] and [4, 70 (50–80)], respectively.
Differential Regulation of VEGF Concentrations in BALF and Plasma by Simvastatin
In the acute phase, mean concentrations of BALF VEGF in the SS+ group appeared to be higher than the SS– group, but there was no significant difference (Table 1). The plasma concentrations were unchanged by treatment with SS in this protocol. SS tended to increase VEGF concentrations in BALF in the late-phase protocol (P = 0.07, Table 2). In contrast, VEGF concentrations in plasma were decreased by instillation of PPE in this protocol (P < 0.05, SS– vs. Ctl; Table 2) and tended to be further reduced by SS (P = 0.07, SS+ vs. SS–), while they seemed to be restored by HGF (P = 0.07, HGF vs. SS–).
The number, median value, and range (pg/ml) for BALF VEGF in the acute phase at day 3 (SS–, SS+); day 7 (SS–, SS+); and day 14 (SS–, SS+) were [12, 149 (108–218)] and [12, 135 (117–302)]; [12, 121 (89–144)] and [12, 125 (96–409)]; and [8, 117 (103–171)] and [12, 117 (93–266)], respectively. The number, median value, and range (pg/ml) for plasma VEGF in the acute phase at day 1 (SS–, SS+); day 3 (SS–, SS+); day 7 (SS–, SS+); and day 14 (SS–, SS+) were [10, 147 (131–152)] and [9, 144 (124–169)]; [6, 148 (134–158)] and [8, 150 (143–161)]; [10, 155 (143–171)] and [8, 153 (132–161)]; and [4, 150 (144–155)] and [11, 146 (136–155)], respectively.
Immunohistochemical Staining and Real-Time RT-PCR for eNOS
Endothelial expression of eNOS protein in pulmonary vessels was attenuated by instillation of PPE (SS– vs. Ctl, P < 0.05) and tended to be reversed by SS (SS+ vs. SS–, P = 0.07) in the late-phase protocol (Table 2 and Fig. 6). In contrast, eNOS mRNA expression in the lungs was increased in the SS–, SS+, and HGF groups, in which PPE was instilled, compared with the Ctl group (P < 0.01, Table 2).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A growing amount of information indicates that statins modulate proinflammatory cellular signaling and function, resulting in anti-inflammatory effects via the regulation of small GTP-binding protein and Akt pathway in vitro and in vivo (8, 20). Lee et al. (18) found that SS decreased the infiltration of inflammatory cells in BALF and matrix metalloproteinase-9 in the lungs of rats with smoking-induced emphysema. In contrast, we did not observe consistent anti-inflammatory effects conferred by SS in our study. Anti-inflammatory effects were apparent as a reduction in percentage of neutrophils and hydroxyproline concentrations in BALF. TNF- and MIP-2 levels in BALF were paradoxically increased at 2 h but were decreased on day 1 in the SS+ group compared with those in the SS– group. In contrast, BALF KC levels tended to continuously decrease from 2 h to day 7 in the SS+ group. Neutrophil accumulation was conspicuous for 3 days after the elastase stimulation and appeared to be attenuated by treatment with SS. However, this trend was not observed during days 7–14, when the percentage of neutrophils was <3% but slightly increased in the SS+ group compared with the SS– group. The modest and transient decrease in hydroxyproline may be related to less neutrophil infiltration and, therefore, a lesser amount of neutrophil-derived collagenases. Previous reports have suggested that statins also exert proinflammatory effects. Matsumoto et al. (24) showed that SS increases the LPS-dependent activation of interleukin (IL)-12p40 and TNF- promoter in macrophages. Sun and Fernandes (32) reported that lovastatin enhanced the production of IL-6, IL-12, and TNF- in dendritic cells. These observations suggest that statins have both anti- and proinflammatory effects, which may account for the biphasic behaviors of BALF cytokines and neutrophils in the acute-phase protocol.
Statins play important roles in improving endothelial cell function and in promoting angiogenesis. Statins mobilize circulating EPC from the bone marrow (22), promote their adhesiveness by increasing the integrin subunits, enhance their proliferative and migratory activities and survival by activating intracellular Akt protein kinase, and inhibit their senescence (5). Statins also increase the production of vasodilators, such as nitric oxide, PGI2, and VEGF, and decrease the production of vasoconstrictors, such as endothelin-1, ANG II, and oxidative stress (6). In our experiments, SS tended to increase the concentrations of BALF VEGF in both protocols. In contrast, plasma VEGF concentrations were decreased by SS in the late-phase protocol, while they were unchanged during the time course in the acute-phase protocol. The presence or absence of airway inflammation induced by elastase instillation may account for the differential mobilization of VEGF between the protocols. Previous reports suggested that the VEGF concentration gradients in vivo may promote local enhancement of angiogenesis (13, 14, 21). It was interesting that VEGF concentration gradients between the lung and circulation appeared to be enhanced by SS in our murine emphysema model. The BALF consisted of an airway epithelial cell lining fluid (ELF) diluted with 1 ml of saline in each mouse, and the VEGF concentration in ELF was thought to be higher than that in plasma, since the measured BALF concentrations were similar to those in plasma (Tables 1 and 2). However, the mechanisms by which alveolar epithelial cell proliferation was enhanced after the putative improvement in endothelial cell function by SS after PPE stimulation are unclear. Recent studies have reported that VEGF also induces EPC mobilization (3) and stimulates resident alveolar epithelial and endothelial cells (10). Thus VEGF might promote the proliferation of alveolar epithelial cells, indirectly as well as directly, in a tissue-specific manner. In addition, eNOS protein expression in the endothelial cells of pulmonary vessels was preserved by SS, supporting the notion that SS contributes to the reversal of emphysema by restoring endothelial cell functions. eNOS mRNA levels were not increased in the SS+ group compared with the SS– group. However, they were significantly increased in the SS–, SS+, and HGF groups compared with the Ctl group, in which eNOS protein expression was intact without increase in its mRNA level (Table 2). We speculate that pulmonary vasculature in elastase-treated groups, i.e., the SS–, SS+, and HGF groups, was similarly damaged by PPE and SS increased eNOS mRNA levels, as previously reported (18), leading to the recovery of eNOS protein expression at the end of the late-phase protocol.
Biphasic dose-dependent effects of statins on angiogenesis, associated with alterations in endothelial apoptosis and VEGF signaling, have also been reported by Weis et al. (35). It was demonstrated that low-dose atorvastatin (0.5 mg·kg–1·day–1) enhanced inflammation-induced angiogenesis, while it was inhibited by high-dose administration (2.5 mg·kg–1·day–1) in a murine model (35). Our experiments suggested that epithelial cell proliferation and reversal of emphysema were observed dose-dependently within the range of 0–5 mg·kg–1·day–1 of SS. Although decreased plasma VEGF concentrations in the late-phase model may reflect the inhibitory effects of high-dose statins on endothelial functions as previously reported (9, 35), BALF VEGF levels tended to increase and emphysematous changes were significantly improved by treatment with the medium dose of SS (1 mg·kg–1·day–1), as a consequence, in this protocol. Since the low and medium doses of SS (0.2 and 1 mg·kg–1·day–1, respectively) correspond to those in patients with hyperlipidemia, our findings related to its dose-dependent favorable effects on emphysema may promise further clinical implication.
In conclusion, we demonstrated for the first time that SS exerts therapeutic effects in pulmonary emphysema induced by elastase in mice, mainly by promoting the proliferation of epithelial cells. Since they are widely and safely used in elderly hyperlipidemic patients, the potential of statins to improve the pathophysiology and alleviate the symptoms of COPD, as well as their therapeutic mechanisms, warrants further investigations.
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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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