The Antimalarial Drug Artesunate Inhibits Primary ... - ATS Journals

ORIGINAL RESEARCH The Antimalarial Drug Artesunate Inhibits Primary Human Cultured Airway Smooth Muscle Cell Proliferation Sheryl S. L. Tan1, Benjamin Ong1, Chang Cheng2, Wanxing Eugene Ho4, John K. C. Tam3, Alastair G. Stewart5, Trudi Harris5, W. S. Fred Wong2, and Thai Tran1 1

Department of Physiology, 2Department of Pharmacology, and 3Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 4Saw Swee Hock School of Public Health, National University Health System, Singapore; and 5Department of Pharmacology, University of Melbourne, Australia

PI3K/Akt/p70S6K pathway and suggests that artesunate may be used as combination therapy with glucocorticoids.

Abstract Airway smooth muscle (ASM) cell hyperplasia contributes to airway wall remodeling (AWR) in asthma. Glucocorticoids, which are used as first-line therapy for the treatment of inflammation in asthma, have limited impact on AWR, and protracted usage of high doses of glucocorticoids is associated with an increased risk of side effects. Moreover, patients with severe asthma often show reduced sensitivity to glucocorticoids. Artesunate, a semisynthetic artemisinin derivative used to treat malaria with minimal toxicity, attenuates allergic airway inflammation in mice, but its impact on AWR is not known. We examined the effects of artesunate on ASM proliferation in vitro and in vivo. Primary human ASM cells derived from nonasthmatic donors were treated with artesunate before mitogen stimulation. Artesunate reduced mitogen-stimulated increases in cell number and cyclin D1 protein abundance but had no significant effect on ERK1/2 phosphorylation. Artesunate, but not dexamethasone, inhibited phospho-Akt and phospho-p70S6K protein abundance. Artesunate, but not dexamethasone, inhibited mitogen-stimulated increases in cell number, cyclin D1, and phospho-Akt protein abundance on ASM cells derived from asthmatic donors. In a murine model of allergic asthma, artesunate reduced the area of a-smooth muscle actin–positive cells and decreased cyclin D1 protein abundance. Our study provides a basis for the future development of artesunate as a novel anti-AWR agent that targets ASM hyperplasia via the

An essential component of airway wall remodeling (AWR) in asthma is the thickening of the airway smooth muscle (ASM) layer, which is attributed to ASM hypertrophy and/or hyperplasia (1). Hyperplasia of ASM cells further amplifies contraction, leading to obstruction of the airways (2). ASM has been identified as an

Keywords: asthma; airway wall remodeling; PI3K; cyclin D1;

p70S6K

Clinical Relevance Our study describes, for the first time, a novel application for the antimalarial drug artesunate, which shows anti-airway wall remodeling properties that target airway smooth muscle proliferation via the PI3K/Akt/p70S6K pathway, a pathway that is not targeted by glucocorticoids. Our study may offer an additional therapeutic strategy in combination therapy for use by patients who are insensitive to glucocorticoids due to an elevated PI3K/Akt signaling pathway. Results from our study will increase our understanding of the mechanisms by which airway wall remodeling occurs, and the findings have implications for other lung-related diseases, such as chronic obstructive pulmonary disease and lymphangioleiomyomatosis, where hyperproliferation of abnormal airway smooth muscle cells in the lungs of women of child-bearing age are targets.

attractive target for the development of antiasthma agents (3, 4). However, the degree to which treatment can reverse remodeling of ASM is unknown. Glucocorticoids are antiinflammatory agents that reduce AHR (3, 5) but show low efficacy regarding features of AWR (6). Glucocorticoid therapy is accompanied by

many side effects (7), such as infection that accompanies attained immunodeficiency due to chronic steroid use (8). Therefore, there is a need for alternative and supplementary anti-AWR drugs with high efficacy and minimal side effects. Artesunate is a partially synthetic artemisinin derivative extracted from the

( Received in original form June 18, 2013; accepted in final form September 10, 2013 ) Correspondence and requests for reprints should be addressed to Thai Tran, Ph.D., Department of Physiology, MD9, 2 Medical Drive, National University of Singapore, Singapore 117597. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 50, Iss 2, pp 451–458, Feb 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0273OC on September 25, 2013 Internet address: www.atsjournals.org

Tan, Ong, Cheng, et al.: Artesunate Inhibits ASM Cell Proliferation

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ORIGINAL RESEARCH Chinese herb sweet wormwood (Artemisia annua) (9). With minimal toxicity and the absence of apparent side effects, artesunate has been approved for the treatment of malaria (10). Artesunate exerts antiproliferative effects in human osteosarcoma cells and myeloma cells via unknown mechanisms (11, 12). In asthma, artesunate attenuates allergic airway inflammation in mice (13). However, no studies to date have assessed the role of artesunate against features of AWR. We hypothesize that treatment of human ASM cells with artesunate may inhibit hyperplasia to influence AWR. We demonstrate for the first time that artesunate exerts inhibitory effects on the PI3K/Akt/p70S6K pathway to regulate cyclin

D1, resulting in antiproliferative effects in primary human cultured ASM cells derived from nonasthmatic and asthmatic donors and in a murine model of allergic asthma.

by the Institutional Review Board of the National University of Singapore and the University of Melbourne. Experimental System

Materials and Methods Cell Culture

Primary human ASM cell cultures were obtained from macroscopically healthy segments of the second to fourth generation main bronchus from patients with asthma (n = 4; 50% male; age, 35 6 17 yr [mean 6 SD; range, 14–54 yr]) or without asthma (n = 4; 50% male; age, 58 6 15 yr [range, 38–70 yr]) who underwent lung resection surgery (14). All procedures were approved

Human ASM cells (60–70% monolayer confluence) were serum deprived (24 h) in DMEM with 1% ITS (insulin 5 mg/ml; transferrin 5 mg/ml; selenium 5 mg/ml) and 1% penicillin streptomycin. Various mitogens were used to assess whether the effects of artesunate were mitogen dependent. ASM cells were pretreated (1 h) with dexamethasone (100 nM) (15) (Sigma-Aldrich, St. Louis, MO) or artesunate (0.3–30 mM) (SigmaAldrich). Cell Number Enumeration

After incubation with mitogens for 48 hours, cell counting by trypan blue exclusion method was performed (14). Immunoblot Analysis

After incubation with mitogens, the cells were lysed, and proteins (6–12 mg) were separated on a 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) as previously described (16). Antibodies used were cyclin D1, phospho-ERK1/2, total ERK, phosphoAkt (S473), total Akt, phospho-p70S6K (T389), total p70S6K (all 1:1,000 dilution) (Cell Signaling, Danvers, MA), or b-actin (1:15,000 dilution) (Sigma-Aldrich). Animals

Figure 1. Effect of increasing concentrations of artesunate on primary human airway smooth muscle (ASM) cell number. (A) Quiescent cells were stimulated for 48 hours with FBS (5%) and counted using trypan blue exclusion to identify nonviable cells. Dexamethasone (Dex, 100 nM) and artesunate (ART, 0.3–30 mM) were added 1 hour before stimulation. (B) Quiescent cells were stimulated for 48 hours with thrombin (Thr) (0.3 U/ml), basic fibroblast growth factor (bFGF) (0.3 nM), and epidermal growth factor (EGF) (10 ng/ml) and counted using trypan blue exclusion to identify nonviable cells. ART (30 mM) was added 1 hour before stimulation. Data are expressed as fold increment over basal (unstimulated cells). *P , 0.05 compared with basal. †P , 0.05 compared with FBS, Thr, bFGF, or EGF (n = 4).

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All animal experiments were performed according to the Institutional Guidelines for Animal Care and Use Committee (National University of Singapore). Female BALB/c mice (6–8 wk old) were used in these experiments (Centre for Animal Resource, Australia). Briefly, mice were sensitized by intraperitoneal injections of 20 mg OVA and 4 mg Al (OH)3 suspended in 0.1 ml saline on Days 0 and 14. On Days 22, 23, and 24, mice were challenged with 1% OVA aerosol for 30 minutes (17). Artesunate (30 mg/kg) or vehicle (5% DMSO) in 0.1 ml saline was given by intraperitoneal injection 1 hour before each OVA aerosol challenge. Saline aerosol was used as a negative control. Mice were killed on Day 25, and lung tissues were collected for histological analysis and immunoblotting (20 mg).

American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

ORIGINAL RESEARCH (Figure 1A). There was no difference in the percentage of trypan blue positive cells across all treatment groups, with an average percentage of trypan blue–positive cells of 15 6 4% (n = 4). Moreover, our preliminary studies show that artesunate alone had no significant effect on basal human ASM cell number (basal, 1.0 6 0.0; 100 nM dexamethasone alone, 1.0 6 0.2; 30 mM artesunate alone, 1.0 6 0.1), suggesting that the effects of artesunate on ASM cells were not due to drug-induced cytotoxicity. To examine whether this phenomenon was a general inhibitory effect of artesunate, we tested other wellcharacterized mitogens of ASM cells. We showed that artesunate was able to inhibit ASM cell proliferation induced by thrombin (Thr) (Sigma-Aldrich), basic fibroblast growth factor (Promega, Madison, WI), and epidermal growth factor (EGF) (SigmaAldrich) (Figure 1B), whereas previous studies indicate that EGF is relatively resistant to modulation by dexamethasone (19). Taken together, these results demonstrate that artesunate inhibits primary human ASM cell number and that the effects appear to be non–mitogen dependent.

Figure 2. Immunoblot analysis showing the effect of ART on cyclin D1 protein expression in primary human ASM cells. Cells were stimulated for 20 hours with FBS (5%) (A) or Thr (0.3 U/ml) (B). Dex (100 nM) and ART (3, 10, and 30 mM) were added 1 hour before stimulation. Data are expressed as fold increment over basal (unstimulated cells) relative to b-actin protein abundance. *P , 0.05 compared with basal. †P , 0.05 compared with FBS or Thr (n = 4–5).

Harris Hematoxylin and Eosin Staining

Lung sections (5 mm) were deparaffinized with xylene, rehydrated with graded alcohol, and subjected to Harris hematoxylin staining, differentiation in 0.5% acid alcohol, and counterstaining with eosin.

(GraphPad Prism 6; GraphPad Software, La Jolla, CA) followed by Bonferroni’s post hoc test (for cell number and immunoblot analysis) or uncorrected Fisher’s least significant difference test (for a-SMA quantification). A P value , 0.05 was deemed significant.

Immunohistochemistry

Immunohistochemical staining of a-smooth muscle actin (a-SMA) (1:100) was performed (16). The area of a-SMA–positive cells (measured by a single observer in a blinded fashion) was corrected for airway basement membrane length (18). CellSens dimension software (Olympus, Hamburg, Germany) was used for analysis. Digital photographs of lung sections were analyzed at a magnification of 203. Statistical Analysis

Differences were determined using one-way ANOVA with repeated measures

Results Artesunate Inhibits Primary Human ASM Cell Number Independent of Mitogen Used

Incubation of quiescent primary human ASM cells with FBS for 48 hours significantly increased ASM cell number by approximately 1.8-fold when compared with basal (unstimulated ASM cells) (Figure 1A). Pretreatment of ASM cells with dexamethasone or artesunate significantly inhibited the effects of FBS on cell number


Artesunate Decreases Cyclin D1 Protein Abundance but Not ERK1/2 Phosphorylation

We next investigated the effect of artesunate on a number of biomarkers of cell proliferation using immunoblot analysis (i.e., namely cyclin D1, ERK1/2, Akt, and p70S6K). The incubation period of ASM cells with the respective mitogens for the measurement of ERK1/2, Akt, p70S6K phosphorylation, and cyclin D1 are chosen on the basis of published work by our group and others (14, 20–23). FBS caused a significant increase in the protein levels of cyclin D1 when compared with basal (Figure 2A). FBS-induced increase in cyclin D1 protein abundance was significantly inhibited when cells were pretreated with dexamethasone and artesunate (Figure 2A). A similar pattern was observed when the mitogen was switched to thrombin, with artesunate exerting similar inhibitory effects on cyclin D1 protein abundance (Figure 2B). Because artesunate influences cyclin D1 protein levels, we investigated whether artesunate regulates ERK1/2 phosphorylation. FBS increased the phosphorylation of ERK1/2 (Table 1). However, the effects of FBS on ERK1/2 phosphorylation were not significantly 453

ORIGINAL RESEARCH Table 1: Effect of Artesunate on Phospho-Erk1/2 Protein Abundance in Primary Human Airway Smooth Muscle Cells* FBS (5%) Basal 1FBS/thrombin 1Dex (100 nM) 1ART (30 mM)

1.0 9.6 13.7 8.0

6 6 6 6

0.0 8.0† 10.2 5.8

Thrombin (0.3 U/ml) 1.0 4.0 4.1 3.3

6 6 6 6

0.0 2.2† 1.1 1.7

Definition of abbreviations: ART, artesunate; Dex, dexamethasone. *Quiescent cells were stimulated for 6 h with FBS or thrombin. Dex and ART were added 1 h before stimulation. Data are expressed as fold increment over basal relative to total ERK1/2 protein abundance. † P , 0.05 compared with basal (n = 4).

inhibited by pretreatment with dexamethasone or artesunate. Given that each primary human ASM culture was derived from an individual patient, we attribute the large variation in ERK phosphorylation responses to interpatient variability. For example, the magnitude of

ERK phosphorylation to FBS stimulation ranged from 2- to 20-fold. However, the use of a paired test analysis enabled interdonor variation to be removed from the analysis, and on this basis it is clear that neither dexamethasone nor artesunate influenced ERK phosphorylation (normalized to FBS

100%, dexamethasone 151.4%, and artesunate 96.8%). Similar results were obtained in experiments using thrombin as the mitogen (normalized to thrombin 100%, dexamethasone 115.3%, and artesunate 93.8%) (Table 1). Artesunate, but Not Dexamethasone, Decreases Akt and p70S6K Phosphorylation

Given that the inhibition of cell number and cyclin D1 by artesunate was nonmitogen dependent, we focused our subsequent studies on FBS as a stimulus to study the effects of artesunate on downstream signaling pathways that regulate human ASM proliferation. The PI3K/Akt pathway works in parallel to that of the ERK1/2 pathway to mediate ASM cell proliferation (24–26). Downstream of Akt, p70S6K has been shown to mediate DNA synthesis in human (26) and bovine (27) ASM cells. Inhibition of p70S6K activity inhibits cyclin D1 protein expression (28). We showed that FBS significantly increased the levels of phosphoAkt and phospho-p70S6K relative to basal (Figure 3). Pretreatment with artesunate, but not dexamethasone, inhibited the FBSstimulated increase in phospho-Akt and phospho-p70S6K (Figure 3). Effects of Artesunate on Primary Human ASM Cells Derived from Donors with Asthma

As a more pathophysiologically relevant in vitro model, we examined whether the antiproliferative effects of artesunate were observed in ASM cells taken from subjects with asthma. Similar to that of nonasthmatic donors, incubation with FBS for 48 hours significantly increased ASM cell number by approximately 2-fold when compared with basal (Figure 4A). FBS also caused a significant increase in the protein levels of cyclin D1 and phospho-Akt when compared with basal (Figures 4B and 4C). Pretreatment with artesunate, but not dexamethasone, inhibited the effects of FBS on cell number, cyclin D1, and phospho-Akt protein abundance (Figures 4A–4C). These results demonstrate that artesunate exerts antiproliferative effects on primary human ASM cells derived from nonasthmatic donors and from subjects with asthma. Figure 3. Immunoblot analysis showing the effect of ART on phospho-Akt (Ser473) (A) and phosphop70S6K (Thr389) (B) protein expression in primary human ASM cells. Human ASM cells were stimulated for 20 hours with FBS (5%). Dex (100 nM) and ART (30 mM) were added 1 hour before stimulation. Data are expressed as fold increment over basal (unstimulated cells) relative to total protein abundance. *P , 0.05 compared with basal. †P , 0.05 compared with FBS (n = 5–8).

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Effects of Artesunate in a Murine Model of Allergic Asthma

To verify the antiproliferative effects of artesunate in vivo, we used a murine model


ORIGINAL RESEARCH

Figure 4. Effect of ART on primary human asthmatic ASM cell number and on cyclin D1 and phospho-Akt (Ser473) expression. (A) Quiescent cells were stimulated for 48 hours with FBS (5%) and counted using trypan blue exclusion to identify nonviable cells. Dex (100 nM) and ART (3, 10, and 30 mM) were added 1 hour before stimulation. Data are expressed as fold increment over basal (number of unstimulated cells). (B and C) Cells were stimulated for 20 hours with FBS (5%). Data are expressed as fold increment over basal (unstimulated cells) relative to b-actin protein abundance. *P , 0.05 compared with basal. †P , 0.05 compared with FBS (n = 3).

of allergic asthma as previously described (13, 17). When compared with salinechallenged mice, ovalbumin (OVA)sensitized and -challenged mice showed an increase in the area of a-SMA–positive cells (Figures 5A and 5B). This increase was significantly inhibited with artesunate pretreatment (Figures 5A and 5B). Moreover, compared with saline-challenged mice, mouse lung lysates from the OVAchallenge group showed increased cyclin D1

protein abundance, and this increase was significantly inhibited with artesunate pretreatment (Figure 5C).

Discussion Using in vitro and in vivo approaches, we showed for the first time that artesunate inhibited primary human cultured ASM cell number and decreased cyclin D1


protein levels in a non–mitogen-dependent manner. This was evident in primary human ASM cells derived from nonasthmatic and asthmatic donors. Furthermore, artesunate, but not dexamethasone, decreased Akt and p70S6K phosphorylation, suggesting that artesunate exerts antiproliferative effects in human ASM cells via actions on this pathway. We showed, in a murine model of allergic asthma, that artesunate treatment decreased the area of a-SMA–positive cells and cyclin D1 protein levels in the lungs of OVAsensitized and -challenged mice. The PI3K/Akt pathway and the ERK pathway work in parallel to mediate proliferation of ASM cells. We observed that dexamethasone did not decrease the level of Akt phosphorylation. In certain cell types, such as auditory hair cells, dexamethasone activated the PI3K/Akt pathway (29). Unlike dexamethasone, artesunate significantly inhibited FBS-stimulated Akt and p70S6K phosphorylation. This is supported by our previous studies, which showed that artesunate blocked EGF-induced phosphorylation of Akt and p70S6K in normal human bronchial epithelial cells (13). PI3K/Akt regulates p70S6K activity, which in turn regulates cyclin D1 protein levels to promote ASM cell proliferation (26–28). Stewart and colleagues (22) have shown that dexamethasone inhibits ASM proliferation by regulating cyclin D1 at the mRNA level. Taken together with current findings indicating a lack of impact of dexamethasone on PI3K, these observations are consistent with the notion that dexamethasone works via mechanisms that are downstream of PI3K/Akt. Thus, dexamethasone and artesunate appear to work through different mechanisms to inhibit cell proliferation. The mechanism by which artesunate inhibits the PI3K/Akt/p70S6K pathway remains to be fully elucidated. Cheng’s recent findings in mast cells suggest that Syk and phospholipase C g are potential targets of artesunate that are upstream of PI3K (30). Further investigation is warranted to determine whether these two proteins upstream of PI3K are involved in human ASM cell proliferation. Nonetheless, our study is the first to show that artesunate exerts inhibitory effects on the PI3K/Akt/p70S6K/cyclin D1 pathway in human ASM cells in vitro and in vivo. This is favorable in asthma treatment because it suggests that artesunate targets one of the dominant pathways by which ASM cells proliferate, which is not targeted by 455

ORIGINAL RESEARCH

Figure 5. Effect of ART in a murine model of allergic asthma. Female BALB/c mice were sensitized with ovalbumin (OVA) and challenged with saline aerosol (O/S), OVA aerosol (O/O) with or without DMSO, or ART (30 mg/kg). Lung tissues were collected 24 hours after last challenge. (A) Hematoxylin and eosin and immunohistochemistry staining of a-smooth muscle acrtin (a-SMA) were performed on the lung tissue sections. (B) Quantification of a-SMA– positive staining (1:100) in mouse lung tissues (O/S sections, n = 6; O/O sections, n = 5; DMSO and artesunate sections, n = 4). Data are expressed as

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ORIGINAL RESEARCH dexamethasone. Whether the effect of a combined treatment of artesunate with dexamethasone would result in greater antiproliferative effects on human ASM cells remains to be investigated. Although human ASM cell number was reduced with the pretreatment of artesunate at concentrations below 30 mM, only 30 mM of artesunate was sufficient to reduce cyclin D1 protein abundance. We reason that, at low concentrations of artesunate, other biomarkers of cell proliferation besides cyclin D1 may be affected. In support of this hypothesis, Chen and colleagues showed that derivatives of artemisinin, such as dihydroartemisinin, can down-regulate cyclin E and CDK2 and up-regulate the CDK inhibitor p27Kip1 (31). In normal human bronchial epithelial cells, artesunate also inhibits cyclin D3 (unpublished results). Dexamethasone did not inhibit FBSand thrombin-stimulated ERK1/2 phosphorylation. This is consistent with the work done by Fernandes and colleagues, who showed that glucocorticoids do not directly affect the phosphorylation of ERK1/2 (15). Similar to dexamethasone treatment, artesunate did not inhibit the effects of these mitogens on ERK1/2 phosphorylation. Previous studies have found that artesunate also had no effect on ERK phosphorylation in TNF-a– or hypoxia-stimulated human rheumatoid arthritis fibroblast-like synoviocytes (32). Although we cannot exclude the role of ERK1/2 in mediating ASM proliferation, the fact that inhibition of cyclin D1 is associated with a significant

decrease in cell number in vitro and with a marked decrease in the total area of a-SMA in vivo suggests a minor role for phospho-ERK1/2 in the regulation of ASM proliferation in this experimental model. We previously demonstrated that artesunate attenuated allergic airway inflammation and reduced airway hyperresponsiveness in a murine model of allergic inflammation (13). Using the same lung samples taken from this earlier study and as a proof-of-concept for the antiproliferative effects of artesunate in vivo, we now show that pretreatment of OVAsensitized and -challenged mice with artesunate decreased the area of a-SMA– positive cells and cyclin D1 protein levels. It is possible that the reduction in area of a-SMA and cyclin D1 protein in ASM cells could account for the reduction in airway reactivity that we previously reported (13). In further support, the levels of phospho-Akt and phosphop70S6K from OVA-sensitized and -challenged mice that were treated with artesunate was also reduced relative to untreated mice (13). We have shown in our previous study that treatment of OVA-sensitized and -challenged mice with artesunate significantly reduced eosinophil, neutrophil, and lymphocyte cell recruitment into the airways (13). Artesunate also reduced the levels of proinflammatory cytokines and chemokines in the bronchoalveolar lavage fluid of these mice. Thus, we cannot exclude the possibility that artesunate may be indirectly affecting ASM proliferation via inhibiting inflammation. Future

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time-course experiments to determine whether changes in ASM mass are consequential to reduction in inflammatory markers or vice versa are required. Because therapies for airway diseases such as asthma are based on symptom relief, further understanding of how artesunate modulates ASM function holds promise for the design of more effective therapies that target the underlying cellular and molecular mechanism(s) that govern AWR. The finding that artesunate can have antiproliferative properties via regulating the PI3K/Akt/p70S6K/cyclin D1 pathway in vitro and in vivo may offer an additional therapeutic strategy in combination therapy for use by patients who are insensitive to glucocorticoids due to an elevated PI3K/ Akt signaling pathway (33). These groups of patients who are glucocorticoid insensitive (often seen in severe asthma) make up more than half the total healthcare costs associated with asthma (34). Moreover, the findings from our study may be applicable to other disease states where ASM proliferation may play a role, such as in lymphangioleiomyomatosis, where hyperproliferation of abnormal ASM cells in the lungs of women of child-bearing age are targets. Finally, given the established safety and patient tolerability profile for artesunate in the treatment of malaria (35) and its low production cost, further research into its use for the treatment of other diseases such as lung disorders is warranted. n Author disclosures are available with the text of this article at www.atsjournals.org.

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Figure 5. (Continued). fold increment over O/S samples. BM = basement membrane. *P , 0.05 compared with O/S. †P , 0.05 compared with O/O. Digital photographs of lung sections were analyzed at a magnification of 203. (C) Immunoblot analysis showing the effect of ART on cyclin D1 protein expression in mouse lung tissue. Data are expressed as fold increment over O/S samples relative to b-actin protein abundance. *P , 0.05 compared with O/S. †P , 0.05 compared with vehicle control DMSO (n = 3).


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