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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Airway smooth muscle proliferation may contribute to the airway wall remodeling seen in asthma. In this study we tested for the presence of airway smooth muscle mitogenic activity in bronchoalveolar lavage (BAL) fluid obtained from 12 atopic asthmatics before and serially after segmental allergen challenge, and from four normal subjects who did not undergo allergen challenge. Mitogenic effect was assessed by coincubating BAL fluid with human airway smooth muscle cells, and measuring its effect on 3[H]thymidine incorporation and cell number. Induction of ERK phosphorylation and cyclin D1 protein abundance were also assessed. Compared with serum-free medium alone, BAL fluid obtained from normal subjects increased thymidine incorporation, cell number, ERK phosphorylation, and cyclin D1 abundance. BAL fluid from asthmatic subjects prior to allergen challenge induced even greater increases in all measures, except for cell number, which was similar to that observed with normal subjects' BAL fluid. Incubation with lavage fluid obtained 48 h after segmental allergen challenge in atopic asthmatics caused yet further increases in thymidine incorporation, cell number, and cyclin D1 protein abundance. Molecular sieving of prechallenge BAL fluid from three asthmatic subjects demonstrated that mitogenic activity was present exclusively in the > 10 kD fraction. These results provide the first direct demonstration that fluid lining the airways of asthmatics contains excess mitogenic activity for human airway smooth muscle, and that this activity increases further after allergen challenge. Naureckas ET, Ndukwu IM, Halayko AJ, Maxwell C, Hershenson MB, Solway J. Bronchoalveolar lavage fluid from asthmatic subjects is mitogenic for human airway smooth muscle.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Excess accumulation of bronchial smooth muscle is a prominent feature of the abnormal architecture of asthmatic airways. This pathologic increase in smooth muscle mass probably plays a functional role in the constrictor hyperresponsiveness that is a hallmark of asthma (1). As such, identification of mechanisms that contribute to excessive smooth muscle accumulation in asthma should provide useful insights into the pathogenesis of this common disease.
Morphologic studies suggest that both myocyte hypertrophy and myocyte hyperplasia (4) increase airway smooth muscle mass in asthma. Cell culture studies have disclosed a wide range of soluble factors (5) that can promote proliferation of human airway smooth muscle in vitro. Previously, we recovered smooth muscle mitogens from bronchoalveolar lavage (BAL) fluid in an animal model of airway smooth muscle remodeling (10). On the basis of these findings, we hypothesized that the airways of asthmatic subjects might also contain a surfeit of smooth muscle mitogens, and that these factors might be recovered by bronchoalveolar lavage. To test this hypothesis, we evaluated the smooth muscle growth-promoting activity in BAL fluid from atopic asthmatic subjects obtained before and serially after intra-airway segmental allergen challenge. Our results show that airway fluid from atopic asthmatics contains excessive mitogenic activity for human airway smooth muscle, and that the recoverable mitogenic activity increases 2 to 4 d after segmental allergen challenge.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Subject Characterization
Atopic asthmatic subjects and nonatopic, nonasthmatic subjects were
recruited by advertisement. Twelve atopic asthmatic subjects (18 to 35 yr of age, four women, eight men) participated in this study, and had
the following characteristics: (1) skin sensitivity to at least one of 14 allergen extracts; (2) prior physician diagnosis of asthma; (3) no
asthma medication use other than inhaled 
2-agonists (one subject
used nasal beclomethasone); (4) no smoking history, except for one
subject who reported smoking five cigarettes per day for 18 mo ending 1 mo prior to participation in this study; and (5) no history of other
major medical illness. Four nonatopic, nonasthmatic subjects (19 to 33 yr of age, three women, 1 man) also participated; these subjects exhibited no skin sensitivity to the allergen panel, and they reported no history of asthma, other respiratory disease or major medical illness, current medication use, or current or past cigarette use. Data concerning
the time course of cellular migration, derived from other analyses of
these subjects' blood and bronchoalveolar lavage fluid, have been reported separately (11).
Subjects visited the laboratory on five occasions. On an initial screening visit, skin allergy testing was performed using a modification of the procedure of Friedhoff and colleagues (12), in which a panel of standard allergen extracts was applied using a sterile Wyeth bifurcated needle. Wheal diameter was measured 15 min thereafter, and the allergen yielding the greatest reaction (other than Felis domesticus) was chosen for eventual intra-airway instillation. Increasing 10-fold dilutions in normal saline were administered to find the least concentration that induced a detectable skin wheal. This allergen extract was further diluted 100-fold for intra-airway allergen challenge.
The second study day was scheduled at least 1 d after skin testing.
The subject performed spirometry, and then underwent bronchoscopy only if FEV1 was at least 60% of the predicted value (13). Prior to bronchoscopy, each patient was pretreated with 0.5 ml 0.5% albuterol in 3 ml 1% lidocaine by aerosol. After further topical anesthesia with lidocaine and conscious sedation with midazolam and morphine, a large channel video bronchoscope was advanced to a wedge
position in the left lower lobe, and the isolated basilar segment was
lavaged with two 60-ml aliquots of normal saline at 37° C. The bronchoscope was then repositioned into a basilar segment of the right
lower lobe and wedged, and an occlusion balloon pulmonary catheter
(PRO-BAL; Mill-Rose Laboratories, or Balloon Catheter B5-2C; Olympus America, New Hyde Park, NY) was passed through the lumen of
the bronchoscope and positioned distal to the bronchoscope tip. The
balloon was inflated, isolating an airway segment between the balloon
and the bronchoscope, and allergen was instilled through the bronchoscope channel in 5-ml aliquots. Nonasthmatic subjects did not receive an allergen challenge. During subsequent visits, asthmatic subjects returned for serial bronchoscopies using the methods described
above at 5 or 24 h, and 48 and 72 or 96 h after allergen challenge, at
which times BAL was performed within the challenged segment. BAL
fluid was immediately placed on ice and centrifuged at 200 × g for
5 min. Supernatants were aliquoted and frozen at 
70° C for subsequent analysis.
Human Airway Smooth Muscle Culture
Human bronchi were obtained from resected specimens or from donor lungs found unsuitable for human transplantation. The smooth muscle layer was isolated under a dissecting microscope in ice-cold HBSS solution at pH 7.4 containing antibiotic/antimycotic solution (Gibco, Grand Island, NY), then washed four times in the same solution. The tissue was minced into 1 to 2-mm cubes, then digested for at least 20 min in HBSS containing antibiotic/antimycotic, collagenase (Gibco), Elastase Type IV (Sigma Chemical, St. Louis, MO), and Protease Type XXVII (Sigma). The digestate was then triturated and cells allowed to settle. The supernatant was transferred to a conical tube and centrifuged at 750 × g for 5 min, after which cells were plated at 5,000 cells/cm2 in 150-mm dishes in DMEM:F12 medium (Gibco) containing 10% FBS (Hyclone, Logan, UT), NEAA (Gibco), and penicillin-streptomycin solution (Sigma). Human airway myocytes were passaged at confluence and passages 1 to 4 were used for subsequent experiments.
Thymidine Incorporation
Human airway smooth muscle cells were seeded in 96-well plates (5,000 cells/well) and allowed to grow to confluence (approximately 3 d). Cells were growth-arrested for 24 h in DMEM:F12 solution containing insulin, transferrin, and selenium (ITS; Collaborative Biomedical Products Research), after which old medium was aspirated and replaced with 100 µl of filter sterilized (0.2 µm) BAL fluid (or PBS or 10% FBS control) plus 100 µl of DMEM:F12:ITS. After 16 h, 0.1 µCi [3H]thymidine (Amersham, Arlington Heights, IL), was added. Eight hours later, the cells were washed twice with PBS, trypsin-digested, and harvested onto a glass filter. [3H]thymidine incorporation was measured using a 96-well liquid scintillation counter. BAL samples were tested in triplicate and averaged to yield each datum.
Cell Proliferation
Human airway smooth muscle cells were seeded into two 96-well plates at 5,000 cells/well and incubated in DMEM:F12 with 10% FBS for 24 h. The cells were then growth-arrested in DMEM:F12:ITS for 24 h. Cells from one plate were trypsinized, harvested, and counted by hemacytometer, and cells on the other plate were exposed for an additional 48 h to 100 µl of BAL fluid (or PBS or 10% FBS control) plus 100 µl of DMEM:F12:ITS per well, harvested, and counted. Each incubation condition was performed in triplicate wells and the results averaged for each datum.
Extracellular Signal-regulated Kinase (ERK) Phosphorylation and Cyclin D1 Protein Abundance
ERK phosphorylation and cyclin D1 protein abundance were assessed
in human airway smooth muscle cells in response to BAL fluid from
five asthmatic and four nonasthmatic subjects. Myocytes were seeded
at 50,000 cells/well into 6-well plates, grown to confluence, serum-
deprived for 24 h, then coincubated with BAL fluid/DMEM:F12:ITS as above, with exposure times of 5 min for ERK phosphorylation and
20 h for cyclin D1 content. After incubation, cells were washed with
cold phosphate-buffered saline (PBS; 0.1 M phosphate at pH 7.5) and
suspended in 100 µl homogenization solution (50 mM -glycerophosphate at pH 7.4, 1 mM EGTA, 1 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mM sodium orthovanadate). Homogenates were lysed by passing through a 26-gauge needle, centrifuged
(14,000 rpm for 10 min at 4° C), and the supernatant resolved on a
10% SDS-polyacrylamide gel.
Bands were electroblotted onto nitrocellulose by semidry transfer (Hoefer, San Francisco, CA) and incubated with polyclonal antibodies against either phospho-ERK (Promega, Madison, WI) or cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were visualized using antirabbit IgG (Sigma Chemical) and enhanced chemiluminescence (Amersham Life Science). Individual bands were scanned and their intensities quantified as described previously (14, 15). The antiphosphoERK antibody recognizes ERKs only when phosphorylated at Thr183 and Tyr185, which are required for full enzymatic activity (16). Most samples were assayed in duplicate and their values averaged to yield the corresponding datum.
Molecular Weight Separation
Approximate molecular weight of the mitogenic activity was determined using BAL obtained prior to allergen challenge in three asthmatic subjects. Each BAL sample was fractionated through a Centricon concentrator (Amicon, Beverly, MA) with a 10-kD exclusion limit. The > 10 kD and < 10 kD fractions were reconstituted with PBS to the original sample volume and assayed for ability to induce thymidine incorporation.
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RESULTS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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BAL fluid obtained either from nonasthmatic subjects or from atopic asthmatic subjects prior to allergen challenge increased thymidine incorporation by cultured human airway smooth muscle cells (p < 0.05 each BAL fluid versus DMEM alone) (Figure 1). Compared with BAL fluid from normal subjects, BAL fluid from asthmatic subjects had the larger effect (p < 0.05). Segmental allergen challenge in atopic asthmatic subjects further increased BAL fluid mitogenic activity within the challenged segment, with significant increases in thymidine incorporation observed using BAL fluid obtained at 48 h postchallenge and beyond.
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Incubation of human airway myocytes with BAL fluid from either normal subjects or asthmatics preallergen challenge also caused proliferation. The number of cells counted after 24 h incubation with BAL fluid was greater than that in DMEM- exposed cultures (p < 0.05) (Figure 2). BAL fluid from normal or asthmatic subjects induced similar changes in cell number. However, BAL fluid obtained 48 h after allergen challenge in asthmatic subjects induced yet greater cellular proliferation than its prechallenge counterpart (p < 0.05).
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Changes in ERK phosphorylation (which reflect changes in ERK activity [17]) induced by exposure BAL fluid from five asthmatic (prechallenge) and four nonasthmatic subjects are demonstrated in Figure 3. BAL fluid from both groups stimulated human airway myocyte ERK phosphorylation several-fold above the level seen with exposure to DMEM alone, and prechallenge BAL from asthmatics had a greater effect than that of normal subjects. BAL fluid obtained after allergen challenge did not increase ERK phosphorylation further.
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Like the measures of mitogenic activity, cyclin D1 content (Figure 4) was significantly increased by incubation with BAL fluid, with fluid from asthmatics (prechallenge) causing more increase than BAL fluid from normal subjects. Again, BAL fluid obtained after segmental allergen challenge was even more effective than prechallenge BAL fluid in this regard.
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Size fractionation of preallergen challenge BAL fluid from three asthmatic subjects demonstrated that the mitogenic activity was contained within the > 10 kD fractions (p < 0.05, ANOVA) (Figure 5). Thymidine incorporation induced by these fractions was equivalent to that induced by corresponding unfractionated BAL. The < 10 kD fractions did not stimulate thymidine incorporation in excess of that induced by DMEM. Fractionation of 48-h postallergen challenge BAL obtained from these three asthmatic subjects demonstrated similar results. Thymidine incorporation caused by the > 10 kD fraction averaged 1.8 times that induced by the < 10 kD fraction (p < 0.01, paired t test).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study is the first to demonstrate that airway lining fluid from both normal and atopic asthmatic subjects contains factors that are mitogenic for human airway smooth muscle in vitro. In our experiments, BAL fluid induced ERK activation, cyclin D1 expression, thymidine incorporation, and cellular proliferation in cultured human airway myocytes. These multiple indices of mitogenic response provide strong evidence for this conclusion. Furthermore, the mitogenic activity recovered from BAL fluid of atopic asthmatic subjects was greater at baseline than that contained within BAL fluid of normal subjects, and this disparity was further augmented by allergen challenge, which led to maximal mitogenic activity 2 to 4 d after challenge.
A number of cytokines and growth factors have been demonstrated to induce proliferation in airway smooth muscle in culture (5). These growth factors and cytokines have been detected in BAL fluid obtained from asthmatic subjects, and growth factor- or cytokine-encoding mRNAs have been found in their BAL-derived cells (18). However, the majority of these investigations have addressed the issue of T-cell function and chemokinesis of eosinophils, and none has tested whether the factors found actually stimulate human airway smooth muscle proliferation. The approach we employed parallels that used in fibrotic lung diseases, including scleroderma (22) and asbestosis (23), to identify mechanisms underlying the abnormal proliferation of fibroblasts in those diseases.
Although our data clearly demonstrate that smooth muscle mitogenic activity is present in both asthmatic and normal subjects, its source and identity are uncertain on a number of levels. First, the observed activity may represent one or more growth factors. Second, it is not known whether this factor (or factors) is the same in normal subjects as in asthmatics or, third, whether the increase in activity seen after allergen challenge represents an increase in the same activity observed at baseline, the addition of a second mitogenic activity, or the loss of an inhibitory substance already present. These questions remain to be answered and will require considerable further experimentation.
The molecular size characterization we performed does
help to narrow the range of candidate factors, however.
Smooth muscle mitogenic activity in the < 10 kD fraction of
prechallenge or 48-h postchallenge BAL fluid from asthmatics
was indistinguishable from that induced by medium alone. As
such, a number of small molecules, including histamine, endothelin, or leukotrienes, are excluded. As previously noted,
several potential growth factors have been found in BAL fluid
from asthmatics. For example, EGF immunoreactivity is increased in the airways of patients with asthma (24), and is a
potent mitogen for human airway smooth muscle cells in culture (5, 6). TGF-, which can have proliferative or antiproliferative effects, has been reported as increased in asthma (18).
TGF- appears to induce smooth muscle growth primarily by
affecting cellular proliferative response to other mitogens (25). PDGF has also been found in BAL fluid from asthmatics, but its abundance is not increased over the quantities seen
in normal volunteers (20). However, it remains unknown
whether BAL fluid PDGF increases after specific allergen
challenge. Because the latter is associated with plasma leak
into the airway wall, one might speculate that this could occur.
In our study, we used ERK phosphorylation as a surrogate measure for ERK activity, rather than a more direct measurement of its activity, such as an in vitro phosphorylation assay. The quantity of BAL fluid that could be obtained from each subject on each occasion was limited, and therefore we elected to use this method, which requires less cellular protein. ERK phosphorylation has been shown to correlate with ERK activity in previous studies (16). Also, immunoprecipitation may bring down other kinases, leading to artifactual increases in ERK activity. Using an antibody against dually phosphorylated ERK, we found that, unlike the three alternative measures of mitogen-activated responses, there was no further increase in ERK phosphorylation with antigen challenge. From these data, we speculate that antigen challenge may increase cyclin D1 expression via ERK-independent pathways. We have recently characterized one such pathway in bovine tracheal smooth muscle (26, 27). In these studies, overexpression of constitutively active forms of phosphatidylinositol 3-kinase and Rac1 were sufficient to induce transcription from the cyclin D1 promoter independent of ERK activity. The importance of this pathway for human airway smooth muscle proliferation will require further study.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Julian Solway, M.D., Professor of Medicine and Pediatrics, University of Chicago, 5841 S. Maryland Ave., MC 6026, Chicago, IL 60637. E-mail: jsolway{at}medicine.bsd.uchicago.edu
(Received in original form March 29, 1999 and in revised form June 4, 1999).
Acknowledgments: Supported by SCOR Grants HL-56399, HL-54685, HL-63314, and RR00055 from the National Institutes of Health, by the Foundation for Fellows in Asthma Research, Olympus America Inc., by Inspiraplex, by the Blowitz-Ridgeway Foundation, and by the Sprague Memorial Institute.
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References |
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DISCUSSION
REFERENCES
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R. Gosens, G. L. Stelmack, G. Dueck, K. D. McNeill, A. Yamasaki, W. T. Gerthoffer, H. Unruh, A. S. Gounni, J. Zaagsma, and A. J Halayko Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L523 - L534. [Abstract] [Full Text] [PDF]
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P. Borger, M. Tamm, J. L. Black, and M. Roth Asthma: Is It Due to an Abnormal Airway Smooth Muscle Cell? Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 367 - 372. [Abstract] [Full Text] [PDF]
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S. Lajoie-Kadoch, P. Joubert, S. Letuve, A. J. Halayko, J. G. Martin, A. Soussi-Gounni, and Q. Hamid TNF-{alpha} and IFN-{gamma} inversely modulate expression of the IL-17E receptor in airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1238 - L1246. [Abstract] [Full Text] [PDF]
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A. S. Gounni, Q. Hamid, S. M. Rahman, J. Hoeck, J. Yang, and L. Shan IL-9-Mediated Induction of Eotaxin1/CCL11 in Human Airway Smooth Muscle Cells J. Immunol., August 15, 2004; 173(4): 2771 - 2779. [Abstract] [Full Text] [PDF]
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M. Terada, E. A. B. Kelly, and N. N. Jarjour Increased Thrombin Activity after Allergen Challenge: A Potential Link to Airway Remodeling? Am. J. Respir. Crit. Care Med., February 1, 2004; 169(3): 373 - 377. [Abstract] [Full Text] [PDF]
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 A. M. Vignola, F. Mirabella, G. Costanzo, R. Di Giorgi, M. Gjomarkaj, V. Bellia, and G. Bonsignore Airway Remodeling in Asthma Chest, March 1, 2003; 123(2007): 417S - 422S. [Abstract] [Full Text] [PDF]
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 K. Page, J. Li, K. C. Corbit, K. M. Rumilla, J.-W. Soh, I. B. Weinstein, C. Albanese, R. G. Pestell, M. R. Rosner, and M. B. Hershenson Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-{delta} Am. J. Respir. Cell Mol. Biol., August 1, 2002; 27(2): 204 - 213. [Abstract] [Full Text] [PDF]
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P. R. A. JOHNSON, M. ROTH, M. TAMM, M. HUGHES, Q. GE, G. KING, J. K. BURGESS, and J. L. BLACK Airway Smooth Muscle Cell Proliferation Is Increased in Asthma Am. J. Respir. Crit. Care Med., August 1, 2001; 164(3): 474 - 477. [Abstract] [Full Text] [PDF]
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P. R. A. JOHNSON, J. L. BLACK, S. CARLIN, Q. GE, and P. ANNE UNDERWOOD The Production of Extracellular Matrix Proteins by Human Passively Sensitized Airway Smooth-Muscle Cells in Culture . The Effect of Beclomethasone Am. J. Respir. Crit. Care Med., December 1, 2000; 162(6): 2145 - 2151. [Abstract] [Full Text]
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C. F. Thomas Jr. and A. H. Limper Phosphatidylinositol Kinase Regulation of Airway Smooth Muscle Cell Proliferation Am. J. Respir. Cell Mol. Biol., October 1, 2000; 23(4): 429 - 430. [Full Text]
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A. R. Simon, S. Takahashi, M. Severgnini, B. L. Fanburg, and B. H. Cochran Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1296 - L1304. [Abstract] [Full Text] [PDF]
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