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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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By interfering with the ability of airway epithelial cells to support repair processes, cigarette smoke could contribute to alterations of airway structures and functions that characterize chronic obstructive pulmonary disease (COPD). The current
study assessed the ability of cigarette smoke extract (CSE) to
alter human airway epithelial cell chemotaxis, proliferation,
and contraction of three-dimensional collagen gels, a model
of extracellular matrix remodeling. The volatile components
contained in cigarette smoke, acetaldehyde and acrolein, were
able to inhibit all three processes. Nonvolatile components
contained within lyophilized CSE also inhibited chemotaxis
but displayed no activity in the other two bioassays. CSE also
inhibited the ability of airway epithelial cells to release transforming growth factor (TGF)-
and fibronectin. Exogenous fibronectin was unable to restore epithelial cell contraction of
collagen gels. Exogenous TGF- partially restored the ability of airway epithelial cells to contract collagen gels and to produce fibronectin. This supports a role for inhibition of TGF-
release in mediating the inhibitory effects of cigarette smoke.
Taken together, the results of the current study suggest that
epithelial cells present in the airways of smokers may be altered in their ability to support repair responses, which may
contribute to architectural disruptions present in the airways
in COPD associated with cigarette smoking.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The normal epithelium lining the airways provides a barrier to the external environment and is capable of initiating a variety of responses when injured, such as rapidly supporting repair processes. It is likely that the recruitment, proliferation, and redifferentiation of airway epithelial cells that occur following injury is essential in maintaining normal airway function throughout life. In this context, alterations in epithelial repair may be important contributors to the architectural changes that can lead to fixed airflow limitation in diseases such as chronic obstructive pulmonary disease (COPD).
The major risk factor for airflow limitation in COPD is cigarette smoking (1). It is likely that cigarette smoke leads to disease by a variety of mechanisms. Considerable evidence supports the importance of cigarette-smoke-initiated inflammation (2, 3). The possibility that cigarette smoke might also alter the ability of the airway epithelium to support repair suggests another mechanism by which cigarette smoke could disrupt the mechanisms that serve to maintain normal airway structure and function. In this context, acute exposure to cigarette smoke has been demonstrated to inhibit the ability of bovine bronchial epithelial cells to attach to extracellular matrix and to migrate in response to chemotactic stimuli (4).
The current study was designed, therefore, to evaluate the hypothesis that cigarette smoke inhibits the ability of epithelial cells to participate in airway repair. Using epithelial cells derived from normal human airways, the ability of cigarette smoke to affect chemotaxis, proliferation, and remodeling of extracellular matrix by epithelial cells was assessed. In addition, the mechanisms by which cigarette smoke modulates the ability of epithelial cells to remodel connective tissue were explored.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Type I collagen (rat-tail tendon collagen, RTTC) was extracted from rat-tail tendons by a previously described method (5). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were removed carefully. After repeated washing with Tris-buffered saline (0.9% NaCl, 10 mM Tris, pH 7.5), followed by dehydration and sterilization with 50%, 75%, 95%, and pure ethanol, the collagen was extracted in 6 mM hydrochloric acid. The supernatant was harvested by centrifugation at 2,000 × g for 1 h at 4°C. Collagen concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecyl sulfate polyacrylamide gel electrophoresis routinely demonstrated no detectable protein other than type I collagen. The RTTC was stored at 4°C until use.
Cigarette smoke extract (CSE) was prepared by a modification of a previously described method (6). Briefly, one filterless cigarette was combusted using a modified syringe-driven apparatus. The smoke was bubbled through 25 ml of basal medium, which is 1:1 (vol/vol) mixture of LHC-D (Biofluids, Rockville, MD) and RPMI-1640 (Gibco Life Technologies, Grand Island, NY). The resulting suspension was adjusted to pH 7.4 with concentrated NaOH and filtered through a 0.22-µm-pore filter to remove bacteria and large particles. CSE was applied to human bronchial epithelial cells (HBECs) within 30 min of preparation. To examine the effect of volatilization, CSE was lyophilized and reconstituted to the initial volume with distilled water.
Cell Culture
HBECs were obtained from bronchial brush biopsies by a modification of a previously published method (7). HBECs were cultured under serum-free conditions using a 1:1 mixture of LHC-9 (8) and RPMI-1640 media (LHC-9/RPMI-1640) (9). Cells were plated on collagen-coated tissue culture dishes (Vitrogen 100; Collagen, Palo Alto, CA) at 37°C in a humidified atmosphere of 5% CO2. Cells were passaged once a week at a 1:3 ratio. Cells between the 4th and 10th passage were used for experiments.
Proliferation Assay
To quantify cell proliferation, HBECs (5 × 104/well) were plated into 12-well tissue culture plates in LHC-9/RPMI-1640 media with or without various reagents, including fresh CSE, lyophilized CSE, acetaldehyde, or acrolein. Cultures were maintained for 6 d with fresh media changes every other day.
For recovery experiments, cells were initially plated as described above. After 24 h, some cultures were changed from media supplemented with CSE to CSE-free LHC-9/RPMI. Cultures were then maintained as described above. Cell numbers for recovery experiments were determined on Days 1, 3, and 6.
Collagen Gel Contraction Assay
Collagen gels were prepared using a previously described method (10). Gels were mixed with the appropriate amount of RTTC, distilled water, and 4× concentrated Dulbecco's modified Eagle's medium (DMEM), so that the final mixture resulted in 0.75 mg/ml of collagen, a physiologic ionic strength, and 1× DMEM and 0.5-ml aliquots of the mixture were cast into each well of 24-well tissue culture plates (FALCON; Becton-Dickinson, Lincoln Park, NJ). After polymerization was complete, generally within 20 min at room temperature, freshly trypsinized HBECs suspended in LHC-9/RPMI-1640 were plated on the top of the gels at a cell density of 5 × 104/gel. The gels were then incubated for 3 h at 37°C in a 5% CO2 atmosphere to allow the HBECs to attach to the gel surface. After this incubation, the gels were gently released from the 24-well tissue culture plates using a sterile spatula. The gels were transferred to 60-mm tissue culture dishes (FALCON) that contained 5 ml of prewarmed LHC-9/RPMI-1640 with or without additional reagents. Gels were then incubated at 37°C in a 5% CO2 atmosphere. The area of each gel was measured with a V image analyzer (Optomax, Burlington, MA) at various times. Data are expressed as the percentage of gel area compared with the original gel size, and each data point within an experiment was calculated as the mean ± SE for three identically treated gels. For the reversibility assay, medium was removed on Day 1 and changed to fresh CSE-free LHC-9/RPMI-1640.
Assay of HBEC Migration
HBEC chemotaxis was examined by a modification of Boyden's blindwell chamber technique as described by Postlethwaite (11), using a 48-well chamber (Neuro Probe, Cabin John, MD). An 8-µm-pore size filter membrane (Neuro Probe) coated with 0.1% gelatin (Bio-Rad, Richmond, CA) was used. Human fibronectin (40 µg/ml) was placed into the bottom of each well as the chemoattractant. HBECs suspended at 1 × 106 cells/ml in LHC-D/RPMI-1640 containing various reagents were placed into the top wells (50 µl/well). The chambers were then incubated at 37°C under 5% CO2 for 6 h. After the incubation, cells on the top of the membrane were removed by scraping, and the membranes were stained with a modified Wright stain (LeukoStat; Fisher, Pittsburgh, PA). HBEC chemotactic activity was calculated as the total number of migrated HBECs counted in 10 high-power fields using a light microscope (Olympus, Lake Success, NY) at 400× magnification.
To investigate the reversibility of CSE on HBEC migration, the fence assay method was used (12). HBECs were plated in the middle hole of the fence, which was placed in a 35-mm dish coated by 1% Vitrogen. After 3 h for attachment, the fences were removed and the cultures re-fed with fresh medium with and without CSE. After 8 h, cells were re-fed with fresh LHC-9/ RPMI-1640 with or without CSE. Cell migration was evaluated by measuring the diameter of cell layer 24 h later.
Cytotoxicity
To assess the cytotoxic effect of various reagents on HBECs, lactic dehydrogenase (LDH) release was used. HBEC monolayers were exposed to various concentrations of reagents. Media were then collected and centrifuged at 500 × g for 10 min, and the LDH concentration in the supernatant was examined using a commercially available kit (LDH-20; Sigma, St. Louis, MO).
Measurement of Fibronectin and TGF- by ELISA
For quantification of fibronectin and TGF- production, the supernatants were harvested from HBEC cultures and frozen at
80°C until assayed. Fibronectin was quantified by an ELISA
specific for human fibronectin (13). TGF-1, TGF-2, and TGF-3
levels were determined by ELISA using specific antibodies (R&D
Systems, Minneapolis, MN). For capture, monoclonal antihuman
TGF-1, 2, or 3 (clone: 9016.2, 8607.211, and 20724.1) antibodies, which recognize the active forms of the respective TGF- isoforms, were used. Therefore, all supernatant media assayed for
TGF- were assayed both with and without acidification and
neutralization so that both latent and spontaneously activated
TGF- could be quantified.
Statistical Analysis
Groups of data were evaluated by ANOVA. Data pairs that appeared to be statistically significant were compared by Student's t test. Values of P < 0.05 were considered significant. Each experiment repeated on separate occasions contained independent triplicates. Graphic representations of individual experiments represent the mean ± SEM for these triplicates.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cytotoxic Effect of CSE, Acrolein, and Acetaldehyde on HBECs
LDH was used as a measure of cellular cytotoxicity. Exposure of HBECs for 48 h to LHC-9/RPMI-1640 with concentrations of 20% fresh or lyophilized CSE revealed no significant increase in LDH release. Neither acrolein at 10 µM nor acetaldehyde at 5 mM caused LDH release. Azide (1%) was used as positive control (Figure 1). To further confirm cell viability, MTT assay was performed in which similar results to the LDH measurements was observed (data not shown).
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Inhibition of HBEC Proliferation, Chemotaxis, and Collagen Gel Contraction by CSE
Fresh CSE inhibited HBEC proliferation in a concentration-dependent manner (Figure 2A). Lyophilized CSE had no effect. On Day 0, ~ 60% of plated cells were attached under normal culture conditions. During the 6 d of culture, cells without CSE exposure increased over 10-fold. However, 5% fresh CSE inhibited cell proliferation over 50%, and 10% CSE completely inhibited the cell proliferation. On average, in three separate experiments, 5% CSE inhibited proliferation 51.46 ± 2.89% (P < 0.01).
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Both fresh and lyophilized CSE inhibited HBEC migration to human fibronectin in a concentration-dependent manner (Figure 2B). In six separate experiments, 5% fresh CSE inhibited chemotaxis 34.80 ± 1.83%, and 10% fresh CSE inhibited chemotaxis 63.70 ± 7.01% (P < 0.01, both comparisons).
CSE also inhibited HBEC-mediated contraction of three-dimensional collagen gels in a concentration-dependent manner. As previously noted, HBECs plated on the surface of three-dimensional collagen gels contracted gels rapidly, with the majority of the contraction complete by 24 h. Both 5% and, more potently, 10% CSE inhibited this contraction (Figure 2C). Again, this result was consistently observed. Over seven experiments, 5% fresh CSE inhibited contraction, on average, 34.4 ± 1.8%, and 10% fresh CSE inhibited contraction 50.5 ± 2.0% at 48 h (P < 0.01). Lyophilized CSE had no effect on gel contraction.
Effect of Acrolein and Acetaldehyde on HBECs
The volatile components of cigarette smoke, acetaldehyde and acrolein, have been demonstrated to have inhibitory effects on a variety of fibroblast cellular functions. To determine whether these reactive moieties may also have similar effects on HBECs, the ability of purified acetaldehyde and acrolein to affect HBEC proliferation, chemotaxis, and gel contraction was assessed. Both acrolein (Figure 3A) and acetaldehyde (Figure 3D) inhibited HBEC proliferation, chemotaxis (Figures 3B and 3E), and contraction of three-dimensional collagen gels (Figures 3C and 3F) (P < 0.01 in all comparisons). Both aldehydes showed steep concentration-dependent relationships regarding both inhibition of cell proliferation and inhibition of collagen gel contraction. The concentration dependence for inhibition of epithelial cell chemotaxis was slightly less steep.
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Reversibility of Cigarette Smoke Inhibition of HBEC Proliferation, Chemotaxis, and Contraction of Three-Dimensional Collagen Gels
To determine whether the inhibitory effect of CSE on HBEC proliferation was reversible, HBECs were exposed to cigarette smoke for 24 h, following which media were changed to fresh media with and without fresh CSE (Figure 4A). Cell proliferation was then monitored over time. Both 5% and 10% CSE caused marked inhibition of proliferation. Following refeeding with fresh CSE-free medium after 24 h, however, both cells exposed originally to 5% and to 10% CSE began to proliferate. Between Days 1 and 6 in culture, cells re-fed with fresh CSE-free medium resulted in 45.38 ± 0.51% and 54.23 ± 3.30% recovery compared with continuous 5% and 10% CSE exposure (P < 0.01, both comparisons).
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The ability of CSE to inhibit HBEC migration was also readily reversible. To assess this, the fence assay was used. CSE inhibited HBEC migration in the fence assay system (Figure 4B). When cells in the fence assay were initially exposed to CSE but then re-fed with fresh medium, their ability to migrate was at least partially restored. It was not feasible to remove media from cells incubated in Boyden's blindwell chamber assay. However, it was possible to expose cells in monolayer culture to smoke and then perform chemotaxis in the absence of added smoke. Under these conditions, HBEC migration was markedly greater than when HBECs were exposed to CSE during the 6 h of the migration assay (data not shown), consistent with some reversal of smoke inhibition of HBEC chemotaxis.
CSE inhibition of HBEC-mediated collagen gel contraction was also at least partially reversible (Figure 4C). When HBECs were incubated in the presence of 5% and 10% cigarette smoke for 8 h, following which media were changed, cells changed to fresh media contracted more than those exposed continuously to CSE. This suggests that inhibition of contraction is also at least partially reversible following removal of the CSE.
Role of Fibronectin and TGF- in CSE, Acrolein,
and Acetaldehyde Inhibition of HBEC Collagen
Gel Contraction
Previous experiments have suggested that CSE inhibition of fibroblast-mediated gel contraction was due partly to a decrease in fibroblasts' fibronectin production. In the current study, we examined fibronectin production of HBECs when exposed to fresh and lyophilized CSE, as well as components acrolein and acetaldehyde. CSE, acrolein, and acetaldehyde were found to inhibit the production of fibronectin by HBECs (Figure 5A) (P < 0.05). Lyophilized CSE had no effect on fibronectin production by HBECs (Figure 5A).
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Because TGF-2 can both stimulate HBEC fibronectin
production and modulate contraction of collagen gels, the
ability of cigarette smoke to alter HBEC function by altering
TGF-2 production was studied. HBECs produced TGF-2,
whereas CSE, acrolein, and acetaldehyde were all able to
inhibit TGF-2 release (Figure 5B) (P < 0.01). TGF-1 and
TGF-3 were not detected under any condition (data not shown).
Role of TGF-2 in CSE Inhibition of HBEC Contraction of
Collagen Gels and Fibronectin Production
Consistent with a role for TGF-2 in mediating the effects
of SCE, exogenous TGF-2 was able to augment both
basal contraction of HBECs and to partially reverse the
CSE-induced inhibition of collagen gel contraction (Figure 6A) (P < 0.05, all comparisons). Similarly, TGF-2
augmented both basal production of fibronectin by HBECs
and partially reversed the cigarette-smoke-induced inhibition of fibronectin production (Figure 6B) (P < 0.01, all
comparisons).
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Effect of Exogenous Fibronectin in Smoke Inhibition of Epithelial Cell Contraction of Three-Dimensional Collagen Gels
To determine the role of fibronectin in HBEC-mediated contraction of collagen gels in the presence of cigarette smoke, HBECs were plated atop native type I collagen gels, which were then released and cultured with exogenous fibronectin in media containing 5% or 10% CSE (Figure 7). Gel size was determined after 48 h. Exogenous fibronectin could neither enhance HBEC-mediated gel contraction under control conditions nor reverse the inhibitory effect of CSE (P > 0.2).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The current study demonstrates that cigarette smoke can
inhibit the ability of HBECs to participate in repair processes. Specifically, CSE inhibited HBEC proliferation and
chemotaxis as well as the ability of these cells to remodel
extracellular matrix, as assessed by contraction of three-
dimensional collagen gels. The effects of cigarette smoke
were, at least in part, reversible following removal of
smoke. Two volatile components contained in relatively high
concentration in cigarette smoke, acetaldehyde and acrolein, were each able to inhibit all three processes. In addition, nonvolatile components contained in lyophilized
CSE were also able to inhibit epithelial cell chemotaxis.
CSE, moreover, inhibited the ability of HBECs to release
both TGF-2 and fibronectin. Addition of exogenous fibronectin was unable to restore normal collagen gel contractility. In contrast, addition of exogenous TGF- was
able to partially restore both fibronectin production and
collagen gel contraction. These results suggest that cigarette smoke inhibition of TGF- production contributes to
the inhibition of collagen gel contraction but that this effect is not mediated through altered fibronectin production.
Taken together, these results support the concept that direct effects of cigarette smoke on the airway epithelium can alter the ability of the cells to participate in repair responses.
In a previous study, cigarette smoke was shown to inhibit bovine bronchial epithelial cell attachment and migration (4). The current study extends this previous observation in several ways. First, the current study utilized human airway epithelial cells. In addition, the current study demonstrated that airway epithelial cell proliferation is also inhibited by CSE. Migration of epithelial cells into a mechanical wound begins within minutes of injury. This is followed by marked stimulation of proliferation, which occurs ~ 24 h after the injury (14, 15). By inhibiting both these processes, cigarette smoke could have significant, adverse effects on epithelial repair.
Following recruitment and proliferation, normal epithelial repair is associated with redifferentiation of the airway epithelium, a process that takes several weeks (16, 17). Although the current study did not evaluate normal redifferentiation, it extends previous observations by evaluating the ability of CSE to alter the capacity of airway epithelial cells to modify the structure of their extracellular connective tissue matrix. In this regard, epithelial cells, like mesenchymal cells, can contract three-dimensional collagen gels (18). This in vitro process has been related to tissue remodeling, which can characterize both normal wound healing and the contraction of fibrotic scar tissues (21). The ability of airway epithelial cells to contract following injury may contribute to architectural alterations prominent in diseases such as COPD.
The current study demonstrates that CSE can inhibit the ability of airway epithelial cells to contract three-dimensional floating collagen gels when the cells are plated on the upper surface of the gels. Fibroblasts cast inside floating three-dimensional collagen gels are also able to cause contraction of the gels (22, 23). Cigarette smoke can inhibit fibroblast-mediated gel contraction (24). With fibroblasts, the mechanism appears to be due at least in part to inhibition of fibroblast fibronectin production. Specifically, the addition of exogenous fibronectin can partially restore the ability to contract collagen gels to fibroblasts that are exposed to cigarette smoke.
Different mechanisms appear to mediate the effect of smoke on epithelial cells. Similar to fibroblasts, cigarette smoke resulted in an inhibition of fibronectin production. In contrast to fibroblasts, however, the addition of exogenous fibronectin did not restore the ability of epithelial cells to contract collagen gels.
Epithelial-cell fibronectin production is regulated by a
number of mediators, including TGF- (25, 26). The current study demonstrates that cigarette smoke can inhibit
TGF- release from airway epithelial cells. This inhibition
appears to account, at least in part, for the inhibition of fibronectin release due to cigarette smoke because addition
of exogenous TGF- partially restored fibronectin release.
Exogenous TGF- also partially restored the ability of
airway epithelial cells to contract collagen gels. This supports the concept that TGF- is regulating epithelial-cell
collagen gel contraction (27) but that this process is not
dependent on fibronectin (18, 28). This result is completely consistent with the observation that fibronectin appears to play no role in epithelial-cell-mediated contraction of collagen gels under "basal" culture conditions.
Cigarette smoke, therefore, can inhibit both epithelial-cell- and fibroblast-mediated contraction of collagen gels. Smoke also inhibits fibronectin release from both cell types. The inhibition of fibronectin release appears to contribute to smoke-induced inhibition of fibroblast contraction. This mechanism does not appear to play a role in smoke-induced inhibition of epithelial cells. However, because epithelial-cell-derived fibronectin may contribute to fibroblast mediated repair responses, it is possible that smoke inhibition of epithelial-cell-derived fibronectin could lead to altered fibroblast mediated repair. Because repair of tissues in vivo likely involves interactions among various parenchymal cells, it appears that smoke may alter tissue repair by a number of different mechanisms.
Cigarette smoke contains over 6,000 moieties. It is likely that many of these can exert potentially toxic effects. In this context, both acetaldehyde and acrolein, two volatile components present in cigarette smoke in relatively high concentrations (24, 29, 30), were able to inhibit proliferation, chemotaxis, and collagen-gel contraction. The concentrations of acetaldehyde, which were active in the current study, were comparable with those previously measured in CSE. The concentration of acrolein, moreover, is also what would be expected based on the yield of this compound relative to acetaldehyde (4). To determine if the effects of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were due to direct effects on components of cigarette smoke or were mediated by effects on the fibroblasts, fibroblasts were pretreated with NAC or BSO for 24 h. Control cells were pretreated with serum-free media. In addition, nonvolatile components were able to inhibit chemotaxis, indicating that multiple components present in cigarette smoke could account for the effects observed in the current study. The effects are unlikely due to nonspecific cytotoxicity, however, as evidenced by two findings. First, at the concentrations used, no cytotoxicity was observed as assessed by LDH release. Second, the effects of CSE were, at least in part, readily reversible suggesting that cytotoxicity had not occurred.
In bovine cells, CSE was able to inhibit cell attachment, but neither acetaldehyde nor acrolein was able to mimic this effect (4). Similar to the result observed in human cells, acrolein inhibited chemotaxis, but, in contrast to the human cells, acetaldehyde had no effect. This suggests that not only are multiple components contained within cigarette smoke responsible for alteration in epithelial cell function but that different animal species may differ in their sensitivity to individual toxic components contained within cigarette smoke. It is likely that the coordinated events that mediate normal epithelial repair depend on many cells in addition to the airway epithelial cells themselves. The ability of cigarette smoke to alter responses relevant to epithelial repair raises the possibility that events that would be followed by restoration of normal tissue architecture and function may, in the face of cigarette smoke, lead to alterations in tissue structure and potentially to the compromise of airway function. The ability of airway epithelial cells to recover responses relevant to repair following removal of CSE raises the possibility that smoking cessation could be associated with restoration of normal repair processes in vivo.
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Footnotes |
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Address correspondence to: Stephen I. Rennard, M.D., Pulmonary and Critical Care Medicine, University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 68198-5125. E-mail: srennard{at}unmc.edu
(Received in original form December 1, 2000 and in revised form August 23, 2001).
Abbreviations: chronic obstructive pulmonary disease, COPD; cigarette smoke extract, CSE; Dulbecco's modified Eagle's medium, DMEM; human bronchial epithelial cell, HBEC; lactic dehydrogenase, LDH; rat-tail tendon collagen, RTTC; transforming growth factor, TGF.Acknowledgments: The authors acknowledge the excellent secretarial support of Ms. Lillian Richards and the editorial assistance of Ms. Mary Tourek. This work was supported by the Larson Endowment, University of Nebraska Medical Center, Omaha, Nebraska.
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References |
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Introduction
Materials and Methods
Results
Discussion
References
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1. Buist, A. S., and W. M. Vollmer. 1994. Smoking and other risk factors. In Textbook of Respiratory Medicine. J. F. Murray and J. A. Nadel, editors. W. B. Saunders Company, Philadelphia. 1259-1287.
2. Hunninghake, G. W., J. E. Gadek, O. Kawanami, V. J. Ferrans, and R. G. Crystal. 1979. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am. J. Pathol. 97: 149-206 [Abstract].
3. Niewoehner, D. E.. 1988. Cigarette smoking, lung inflammation and the development of emphysema. J. Lab. Clin. Med. 111: 15-27 [Medline].
4.
Cantral, D. E.,
J. H. Sisson,
T. Veys,
S. I. Rennard, and
J. R. Spurzem.
1995.
Effects of cigarette smoke extract on bovine bronchial epithelial cell attachment and migration.
Am. J. Physiol.
268:
L723-L728
5. Mio, T., Y. Adachi, D. J. Romberger, R. F. Ertl, and S. I. Rennard. 1996. Regulation of fibroblast proliferation in three dimensional collagen gel matrix. In Vitro Cell Dev. Biol. 32: 427-433 .
6. Carp, H., and A. Janoff. 1978. Possible mechanisms of emphysema in smokers: in vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants. Am. Rev. Respir. Dis. 118: 617-621 [Medline].
7. Kelsen, S. G., I. A. Mardini, S. Zhou, J. L. Benovic, and N. C. Higgins. 1992. A technique to harvest viable tracheobronchial epithelial cells from living human donors. Am. J. Respir. Cell Mol. Biol. 7: 66-72 .
8. Lechner, J. F., and M. A. LaVeck. 1985. A serum-free method for culturing normal human epithelial cells at clonal density. J. Tissue Cul. Methods 9: 43-48 .
9. Beckmann, J. D., H. Takizawa, D. Romberger, M. Illig, L. Claassen, K. Rickard, and S. I. Rennard. 1992. Serum-free culture of fractionated bovine bronchial epithelial cells. In Vitro Cell Dev. Biol. 28A:39-46.
10.
Mio, T.,
X. Liu,
Y. Adachi,
I. Striz,
C. M. Skold,
D. J. Romberger,
J. R. Spurzem,
M. G. Illig,
R. Ertl, and
S. I. Rennard.
1998.
Human bronchial
epithelial cells modulate collagen gel contraction by fibroblasts.
Am. J. Physiol.
274:
L119-L126
11.
Postlethwaite, A. E.,
R. Snyderman, and
A. H. Kang.
1976.
The chemotactic
attraction of human fibroblasts to a lymphocyte-derived factor.
J. Exp.
Med.
144:
1188-1203
12. Horodyski, J., and R. J. Powell. 1996. Effect of aprotinin on smooth muscle cell proliferation, migration, and extracellular matrix synthesis. J. Surg. Res. 66: 115-118 [Medline].
13. Rennard, S. I., R. Berg, G. R. Martin, J. M. Foidart, and P. G. Robey. 1980. Enzyme linked immunoassay (ELISA) for connective tissue components. Anal. Biochem. 104: 205-214 [Medline].
14.
Kim, J. S.,
V. S. McKinnis,
K. Adams, and
S. R. White.
1997.
Proliferation
and repair of guinea pig tracheal epithelium after neuropeptide depletion
and injury in vivo.
Am. J. Physiol.
273:
L1235-L1241
15. Keenan, K. P., J. W. Combs, and E. M. McDowell. 1982. Regeneration of hamster tracheal epithelium after mechanical injury. Virchows Arch. 41: 193-214 .
16. Barrow, R. E., C.-Z. Wang, R. A. Cox, and M. J. Evans. 1992. Cellular sequence of tracheal repair in sheep after smoke inhalation injury. Lung 170: 331-338 [Medline].
17. Shimizu, T., M. Nishihara, S. Kawaguchi, and Y. Sakakura. 1994. Expression of phenotypic markers during regeneration of rat tracheal epithelium following mechanical injury. Am. J. Respir. Cell Mol. Biol. 11: 85-94 [Abstract].
18.
Liu, X.,
T. Umino,
M. Cano,
R. Ertl,
T. Veys,
J. Spurzem,
D. Romberger, and
S. I. Rennard.
1998.
Human bronchial epithelial cells can contract type
I collagen gels.
Am. J. Physiol.
274:
L58-L65
19. Sakamoto, T., D. Hinton, H. Sakamoto, A. Oganesian, L. Kohen, R. Gopalakrishna, and S. Ryan. 1994. Collagen gel contraction induced by retinal pigment epithelial cells and choroidal fibroblasts involves the protein kinase C pathway. Curr. Eye Res. 13: 451-459 [Medline].
20. Schafer, I. A., A. Shapiro, M. Kovach, C. Lang, and R. B. Fratianne. 1989. The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices. Exp. Cell Res. 183: 112-125 [Medline].
21. Nishiyama, T., and N. Tominaga. 1988. Quantitative evaluation of the factors affecting the process of fibroblast-mediated collagen gel contraction by separating the process into three phases. Collagen Related Res. 8: 259-273 . [Medline]
22.
Bell, E.,
B. Ivarsson, and
C. Merrill.
1979.
Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different
proliferative potential in vitro.
Proc. Natl. Acad. Sci. USA
76:
1274-1278
23. Asaga, H., S. Kikuchi, and K. Yoshizato. 1991. Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin. Exp. Cell Res. 193: 167-174 [Medline].
24. Carnevali, S., Y. Nakamura, T. Mio, X. Liu, K. Takigawa, D. J. Romberger, J. R. Spurzem, and S. I. Rennard. 1998. Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction. Am. J. Physiol. 247: L591-L598 .
25. Romberger, D. J., J. D. Beckmann, L. Claassen, R. F. Ertl, and S. I. Rennard. 1992. Modulation of fibronectin production of bovine bronchial epithelial cells by transforming growth factor-beta. Am. J. Respir. Cell Mol. Biol. 7: 149-155 .
26.
Linnala, A.,
V. Kinnula,
L. A. Laitinen,
V.-P. Lehto, and
I. Virtanen.
1995.
Transforming growth factor- regulates the expression of fibronectin and
tenascin in BEAS 2B human bronchial epithelial cells.
Am. J. Respir. Cell
Mol. Biol.
13:
578-585
[Abstract].
27.
Montesano, R., and
L. Orci.
1988.
Transforming growth factor- stimulates
collagen-matrix contraction by fibroblasts: implication for wound healing.
Proc Natl Acad Sci.
85:
4894-4897
28.
Umino, T.,
H. Wang,
Y. Zhu,
X. Liu,
S. L. Manouilova,
J. R. Spurzem,
M. P. Leuschen, and
S. I. Rennard.
2000.
Modification of type I collagenous gels
by alveolar epithelial cells.
Am. J. Respir Cell Mol. Biol.
22:
702-707
29. Houlgate, P. R., K. S. Dhingra, S. J. Nash, and W. H. Evans. 1989. Determination of formaldehyde and acetaldehyde in mainstream cigarette smoke by high-performance liquid chromatography. Analyst 114: 355-360 [Medline].
30. Huber, G. L., M. W. First, and O. Grubner. 1991. Marijuana and tobacco smoke gas-phase cytotoxins. Pharmacol. Biochem. Behav. 40: 629-636 [Medline].
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