1 Mount Sinai Hospital, Toronto, Canada M5G 1X5; 2 University of Nebraska Medical Center, Omaha, Nebraska 68198; 3 Pulmonary Medicine, Tokyo Medical and Dental University, Tokyo 113-8519; 4 Department of Respiratory Medicine, University of Tokyo, Tokyo 113-8655, Japan; 5 Department of Respiratory Diseases, Jincheng Hospital, Lanzhou 730050, China; and 6 Department of Internal Medicine, Seoul Adventist Hospital, Seoul 130-650, Korea
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ABSTRACT |
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Cigarette smoke exposure has been
associated with a variety of diseases, including emphysema. The current
study evaluated the interaction of cell density and cigarette smoke
extract (CSE) on fibroblast contraction of collagen gels. Protein
levels of transforming growth factor (TGF)-1, fibronectin,
PGE2, and TGF-
1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 × 105 cell/ml), only 5% CSE augmented contraction in
higher-density cultures (3-5 × 105 cells/ml).
CSE also inhibited fibronectin and TGF-
1 production in low-density
cultures but stimulated fibronectin production in high-density
cultures. Active TGF-
1 was readily detectable only in higher-density
cultures and was markedly augmented by 5% CSE. In contrast, although
TGF-
1 mRNA expression was inhibited in high-density cultures by 10%
CSE, expression was increased in the presence of 5% CSE. These results
suggest that CSE-induced inhibition of low-density fibroblast
contraction is due to inhibition of fibronectin production, whereas
CSE's stimulatory effect on high-density cells is the result of
increased release of TGF-
1. These effects may help explain the
varied pathologies associated with exposure to cigarette smoke.
cigarette smoke extract; collagen gels; transforming growth
factor-; lung
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INTRODUCTION |
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CIGARETTE SMOKE EXPOSURE can lead to a variety of diseases, including those where tissue destruction occurs, such as in pulmonary emphysema (31) and osteoporosis (32). Also included among cigarette smoke-induced diseases, however, are disorders in which an excessive deposition of fibrotic scar occurs, such as in atherosclerosis (14) and idiopathic pulmonary fibrosis (11). How cigarette smoke interacts with tissue repair and remodeling, therefore, is important in understanding these diverse diseases.
The culture of fibroblasts in three-dimensional lattices composed of
native collagen has been used as a model of tissue repair and scar
formation (4, 21). When cultured under these conditions, fibroblasts attach to the collagen lattice. By exerting mechanical tension, fibroblasts can cause contraction of the three-dimensional lattices, a process thought to mimic the contraction that characterizes both scars and fibrotic tissues. Consistent with this model, mediators believed to play a role in tissue repair, including transforming growth
factor-beta (TGF-) and platelet-derived growth factor (PDGF),
augmenting fibroblast contraction of three-dimensional collagen gels
(20, 27).
Among the factors that modulate fibroblast contraction of three-dimensional collagen gels is the density of fibroblasts within the cultures. As originally demonstrated by Bell et al. (4), gels with higher densities of fibroblasts contract more rapidly and to a greater degree. Qualitative differences in contraction may also result from differences in density. In this context, Ehrlich and Rittenberg (12) have recently demonstrated that collagen gels containing fibroblasts at low density differ from those at high density in their sensitivity to a variety of exogenous agents.
A recent study has demonstrated that cigarette smoke extract (CSE)
inhibits the contraction of collagen gels populated with fibroblasts
cultured at low density (8). The current study, therefore,
was undertaken to determine whether the effects of cigarette smoke on
the contraction of fibroblast-populated collagen gels is dependent on
cell density. Not only have we demonstrated the density-dependent
effects, but we have also explored the mechanisms by which smoke exerts
differential effects by determining the effect of CSE on the release of
TGF-, fibronectin, and PGE2, mediators that could
function as paracrine regulators of collagen gel contraction.
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MATERIALS AND METHODS |
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Materials. Type I collagen gels were made from collagen extracted from rat tail tendons [rat tail tendon collagen (RTTC)] using previously published methods (13, 21). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were carefully removed. After repeated washing with Tris-hydroxymethyl aminomethane (Tris)-buffered saline (0.9% NaCl and 10 mM Tris, pH 7.5) and 95% ethanol, type I collagen was extracted in 6 mM hydrochloric acid. We determined protein concentration by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) routinely demonstrated no detectable proteins other than type I collagen.
Anti-TGF-CSE. CSE was prepared by a modification of the method of Carp and Janoff (9). Briefly, one cigarette without filter was combusted with a modified syringe-driven device. The smoke was bubbled through 25 ml of serum-free DMEM. The resulting suspension was adjusted to pH 7.4 with concentrated NaOH and then filtered through a 0.22-µM-pore filter (Lida Manufacturing, Kenosha, WI) to remove bacteria and large particles. CSE was applied to culture medium within 30 min of preparation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays demonstrated that there was not significant CSE (up to 10%) cytotoxicity on fibroblasts.
Cell culture. Human fetal lung fibroblasts were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured on tissue culture dishes (Falcon; Becton-Dickinson Labware, Lincoln Park, NJ) with DMEM supplemented with 10% FCS, 100 µg/ml penicillin, 250 µg/ml streptomycin, and 2.5 µg/ml Fungizone. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and passaged once a week at a 1:3 ratio. Fibroblasts were used between the 14th and 20th passages.
Collagen gel contraction assay. Collagen gels were prepared as described previously (21). Briefly, RTTC, distilled water, and 4× concentrated DMEM were combined so that the final mixture resulted in 0.75 mg/ml collagen, with a physiological ionic strength of 1× DMEM. Cell suspensions, routinely added last, were added to achieve several fibroblast cell densities. One-half-milliliter aliquots of each mixture were placed into 24-well tissue culture plates (Falcon) and allowed to gel. This was usually completed within 20 min at 37°C. Gels then were released and floated in 60-mm dishes containing 5 ml of DMEM supplemented with 1% FCS and 5 or 10% CSE.
To investigate the effect of anti-TGF-TGF-1, fibronectin, and PGE2 measurement.
TGF-
1 and fibronectin concentrations were measured by an
enzyme-linked immunoabsorbent assay. In general, cultures were
maintained for 48 h in the presence of various concentrations of
CSE before fibronectin or PGE2 was quantified. We collected
the media in which the gels were floated and the supernatant solutions
after the gels were solubilized by collagenase. To measure TGF-
1,
samples were assayed both with and without acidification and
neutralization to convert the latent form of TGF-
1 to active forms.
TGF-
1 was quantified by an ELISA with commercially available
materials (R&D Systems) that detect an epitope expressed on the active
form. Fibronectin was assayed with an ELISA that specifically detects human but not bovine fibronectin (28). PGE2
production from cells was determined by enzyme immunoassay (EIA; Cayman
Chemical, Ann Arbor, MI) following the manufacturer's instructions.
Quantification of TGF- mRNA.
Total RNA was extracted from fibroblasts in three-dimensional cultures
as described previously (2). Briefly, cell pellets were
obtained by digesting the collagens by RNase-free collagenase and
suspended in solution D [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7), 0.05% sarcosyl, and 0.1 M
2-mercaptoethanol]. Total RNA was extracted using the
single-step method. The amount of RNA was quantified by
spectrophotometric absorbance at 260/280 nm. Genomic DNA was removed
from total RNA samples with DNase I (BRL reagent; Invitrogen),
according to the manufacturer's instructions. Reverse transcription
was conducted following kit instructions (PerkinElmer Instruments,
Branchburg, NJ). The reaction solution contained 10× buffer, 2 mmol/l
dNTPs (500 µM for each dNTP), 5.5 mM MgCl2, 2.5 µM
random Hexamer, and 1.25 U/µl murine leukemia virus RT. The
master mix and 4-µl RNA (containing 200 ng total RNA) samples were
added to a final volume of 20 µl. The mixture was incubated at 25°C
for 10 min, 48°C for 30 min for reverse transcription, and 95°C for
10 min for RT inactivation. For TGF-
1, the cDNA was quantified by
real-time quantitative PCR assay by using the TaqMan Gold PCR kit
(Applied Biosystems, Foster City, CA) and Sequence Detection System.
Briefly, 25 µl master mix, 5 µl distilled and deionized
H2O, 10 µl primers (5 µl each), 5 µl probe,
and 5 µl cDNA sample were added to each well of a 96-well plate. For
ribosome RNA detection, ribosomal RNA control reagents (part no.
4308329; Applied Biosystems) were used following the manufacturer's
instruction. ABI Prism 7700 Sequence Detection System (Applied
Biosystems) was used to monitor the reaction. Thermal cycling
parameters were 50°C × 2 min and 95°C × 10 min for the
initial step and 95°C × 15 s and 60°C × 1 min for
40 cycles. The master mixture without sample was used as the
no-template control. Human rRNA was used as an internal control. Values
are expressed 105 rRNA units. The sequences of primers used
are as follows: human TGF-
1 forward, 5'-CGA GCC TGA GGC CGA CTA C;
human TGF-
1 reverse, 5'-AGA TTT CGT TGT GGG TTT CCA; human TGF-
1
probe, 6FAM-CCA AGG AGG TCA CCC GCG TGC-TAMRA.
Statistical analysis. Groups of data were evaluated by analysis of variance followed by Tukey's procedure using the Stat View Package. Student's t-test was performed to compare paired group data. Values of P < 0.05 after correction for multiple comparisons were considered significant. Each experiment was repeated at least three times, and the data presented are means ± SE of separate determinations, except as described otherwise.
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RESULTS |
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Variable effect of CSE on fibroblast-mediated collagen gel
contraction dependent on cell density.
Consistent with previous results (8), 5% CSE
significantly inhibited contraction in gels populated with fibroblasts
at a density of 1 × 105 cells/ml. In contrast, 5%
CSE had no effect on contraction of gels populated with fibroblasts at
a density of 2 × 105 cells/ml and augmented gel
contraction in gels populated at a density of 3 × 105
cells/ml or higher. Ten percent CSE inhibited gel contraction, but only
at cell densities below 4 × 105 (Fig.
1). On average in five experiments, 5%
smoke extract inhibited contraction 34.5 ± 6.8% (compared with
control, P < 0.05) in low-density gels but augmented
contraction (32.04 ± 3.20%; P < 0.05) in
high-density gels. No changes were observed in DNA amounts at the end
of the study in groups with or without CSE.
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Effect of CSE on fibronectin production by human lung fibroblasts:
cell density dependency.
Previous studies have demonstrated that cigarette smoke inhibits
fibroblast-mediated collagen gel contraction at low cell density, at
least in part by inhibiting fibronectin production (8). To
determine whether fibronectin production was affected differentially in
high- and low-density cultures, we measured fibronectin concentration
both within the collagen gels and in the surrounding media for gels
prepared with low (1 × 105/ml) and high (5 × 105/ml) cell densities. At low cell density, 5% CSE
significantly inhibited fibronectin production (Fig.
2, P < 0.05), whereas it significantly augmented fibronectin production at high cell density (P < 0.05). In contrast, 10% CSE inhibited
fibronectin production in both low cell and high cell density gels
(Fig. 2, P < 0.05, both comparisons).
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Effect of CSE on fibroblast PGE2 production in collagen
gel culture: cell density dependency.
It has been suggested that PGE2 can inhibit
fibroblast-mediated collagen gel contraction and could function as an
autocrine regulator of this process (35). To determine
whether the differential effect of cigarette smoke on collagen gel
contraction at high and low cell densities could be mediated through a
differential effect on PGE2 production, we specifically
quantified PGE2 release (Fig.
3). At low cell density, only 10% CSE
significantly inhibited PGE2 release (40.1 ± 8.2%
inhibition) compared with control (P < 0.05). In
contrast, under conditions of high cell density, both 5 and 10% CSE
significantly inhibited PGE2 production (53.0 ± 1.2 and 55.8 ± 1.1%, respectively, compared with control,
P < 0.01).
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Effect of CSE on TGF-1 production by human lung fibroblasts:
cell density dependency.
Because TGF-
1 can augment both fibroblast-mediated collagen gel
contraction and fibronectin production, the possibility that cigarette
smoke differentially modulates TGF-
1 production as a function of
cell density was evaluated. In low cell density gels, total TGF-
1
release was significantly inhibited by 5% CSE and was further
inhibited by 10% CSE (Fig.
4A). In contrast, CSE did not
affect the total TGF-
1 release in high-density gels. The
differential affect of CSE on TGF-
1 release was more pronounced when
endogenously activated TGF-
1 was quantified (Fig. 4B).
"Active" TGF-
1 was undetectable in low-density cultures under
all conditions studied. In contrast, TGF-
1 was readily detectable by
the antibody that recognizes active TGF-
in high-density cultures,
and this was markedly augmented by 5% CSE. Ten percent CSE also
augmented active TGF-
1 release but to a lesser degree than the lower
concentration of smoke.
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Effect of CSE on TGF-1 mRNA expression in three-dimensional
collagen gel culture: cell density dependency.
To determine whether CSE modulated TGF-
1 gene expression, we
quantified TGF-
mRNA expression. Because a number of studies have
reported that glyceraldehyde-3-phosphate dehydrogenase is not an
adequate control RNA against which other RNA values can be normalized
(6, 34), ribosomal RNA was quantified with TGF-
1 mRNA
simultaneously. Consistent with protein release, CSE significantly
inhibited TGF-
1 mRNA expression in low-density cultures in a
concentration-dependent manner (Fig. 5).
Alternately, in high-density cultures, TGF-
1 mRNA expression was
increased in the presence of 5% CSE (23.5 ± 6.1%), whereas 10%
CSE inhibited expression (42.3 ± 6.9%, both P < 0.05, compared with control).
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Effect of anti-TGF-1 antibodies on CSE modulation of collagen
gel contraction and fibronectin production.
To confirm a mechanistic role of the cell density-dependent modulation
of TGF-
1 production in response to CSE's differential effects on
gel contraction, we incubated cultures in the presence and absence of
anti-TGF-
1 antibodies.
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DISCUSSION |
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The current study evaluated the interaction of cell density and
CSE on fibroblast contraction of three-dimensional collagen gels. Five
percent CSE inhibited the contraction of collagen gels populated by
fibroblasts at low density but augmented contraction of those populated
by fibroblasts at high density. The inhibitory effects of 10% CSE were
greater than that of 5%, but much more notably so in the low-density
cells than in the high-density cells. CSE inhibited production of
fibronectin in low-density cultures but stimulated fibronectin
production in high-density cultures. Similarly, TGF-1 release was
inhibited in low-density cultures but trended toward stimulation in
high-density cultures. Perhaps more importantly, 5% CSE appeared to
augment the release of active TGF-
in high-density cultures, while
having no detectable effect on active TGF-
in low-density cultures.
The effects on TGF-
production were paralleled by effects on TGF-
mRNA. Finally, the augmented contraction observed in high-density
cultures is likely due to activity of TGF-
as antibodies to TGF-
blocked this response.
Contraction of gels composed of native collagen fibers in which
fibroblasts are cultured has been used as a model of wound repair and
tissue fibrosis (4). Like both scars and fibrotic tissues,
fibroblast-populated collagen gels contract. The degree of contraction
depends on a number of factors, including the concentration of collagen
in the gel, the presence of serum or exogenous growth factors
(24), and, importantly, the density of fibroblasts within the gels (12). Gels cultured with a higher density of
fibroblasts contract to a greater degree. In addition, Ehrlich and
Rittenberg (12) have demonstrated that contraction of gels
containing low densities of fibroblasts is sensitive to disruption of
microtubules, uncoupling of gap junctions, and inhibition of tyrosine
kinases, and requires the 2
1-integrin.
Gels containing high densities of fibroblasts, in contrast, lacked
those sensitivities but responded to PDGF. The current study
demonstrates that the effect of cigarette smoke on fibroblast
contraction of collagen gels is also density dependent and suggests a
mechanism to explain the differential effects at various densities.
As previously reported, cigarette smoke inhibited contraction of
collagen gels populated with fibroblasts at low density
(8). This effect is likely due to inhibition of
fibronectin production and can be largely reversed by the addition of
exogenous fibronectin (8). As demonstrated in the current
study, the reduction in fibronectin release may be due, at least in
part, to inhibition of TGF- production that appears to result from a
decrease in TGF-
mRNA expression in the presence of CSE in
low-density cultures. At low density, CSE also inhibited
PGE2 production. Because PGE2 functions as an
autocrine or paracrine inhibitor of collagen gel contraction, this CSE
effect might be expected to increase contraction. The net inhibition of
contraction that occurs at low density, therefore, appears to occur
because of factors that outweigh inhibition of PGE2 production.
CSE, however, had a dramatically different effect on fibroblasts
cultured in gels at high density. Under these conditions, 5% CSE
resulted in an increase in TGF- mRNA expression and a slight trend
toward an increase in total TGF-
release. Perhaps more important is
an increase in the release of active TGF-
1, which was spontaneously
detected by antibody that recognizes active TGF-
. Although TGF-
activity was not directly measured, this result is consistent with
smoke-induced endogenous activation of TGF-
. There was, moreover, a
parallel increase in the release of fibronectin in high-density
cultures exposed to CSE. Furthermore, a role for active TGF-
in
driving fibronectin production is also supported by the observation
that fibronectin release in smoke-exposed cultures, as well as
augmented contraction, was markedly reduced in the presence of
anti-TGF-
antibodies. TGF-
can be converted from its latent form
to its active form by a variety of mechanisms. These include
confirmational changes induced by binding to exogenous soluble factors
or to cell surface integrins and proteolytic cleavage mediated by the
action of secreted proteases (1, 16, 18, 22, 23, 33).
Which mechanisms account for TGF-
activation in our in vitro system
remains to be defined. Nevertheless, augmented contraction of
high-density cultures appears to result from increased release of
active TGF-
1, which leads to augmented fibronectin production and,
through this and/or other mechanisms, to augmented contraction.
Interestingly, antibodies to TGF-
augmented fibronectin production
in the presence of low concentrations of cigarette smoke in low-density
cultures. The mechanism by which this effect is mediated remains
unexplained. However, our study is consistent with the concept that the
effects of cigarette smoke on cells vary as a function of cell density
and that TGF-
production by cells is an important mediator of these
effects. Other mechanisms independent of TGF-
are, of course, also possible.
Density-dependent effects on TGF--mediated effects have been
described in other systems. Alteration in TGF-
production on a
per-cell basis has been reported in fibroblasts (19) and
in epithelial cells maintained in monolayer culture (30).
Density-dependent alterations have also been described for TGF-
receptor expression in fibroblasts in monolayer culture
(26). Alterations in TGF-
activity as a function of
cell density may account for many density-dependent responses, such as
fibronectin production and
-smooth muscle actin expression. Although
the mechanisms that regulate TGF-
production and responsiveness as
functions of density remain to be defined, the current study supports
the concept that fibroblast responsiveness to an exogenous agent such
as cigarette smoke may vary as a function of cell density.
In vivo cell densities are not well established. Within alveolar structures, interstitial cell densities are 19.4 × 106 cells/ml expressed by volume (10). However, the lung contains a considerable amount of air. When expressed per gram of tissue, cell densities within the alveolar interstitial structures are 1.7 × 108 cells/g. This corresponds to ~1-3 × 105 cells/ml in our in vitro cultures. However, when expressed relative to the density of connective tissue protein (1.3-4 × 105/mg in our cultures), interstitial lung cell densities are similar, 1.7 × 105 cells/mg. Undoubtedly, the in vitro culture system used in the current study is only an approximation of in vivo cell conditions. How these cell densities reflect the in vivo scenario remains to be defined. Perhaps more relevant are the marked structural differences between the airways and the alveolar structures. In the airways, fibroblasts exist within a dense connective tissue matrix where fibroblasts are in relatively close proximity to other fibroblasts. In contrast, the alveolar structures contain fibroblasts, but they are relatively dispersed. It is thus appealing to consider the alveolar structures as a "low density" area and the airways as a "high density" area. The current study suggests that the response of cells may differ between these sites, in part, because of differences in cell density.
Cigarette smoke is associated with a number of diseases. It is the
major risk factor for the development of pulmonary emphysema, a disease
characterized by net tissue destruction within the lungs (5). The ability of cigarette smoke to inhibit
fibroblast-mediated repair responses observed at low density may be one
mechanism that contributes to deficient repair. Cigarette smoke,
however, is also associated with development of fibrotic diseases such as idiopathic pulmonary fibrosis and atherosclerosis (15,
25). The increased production of active TGF- associated with
the augmented contraction that occurs with cells at high density could
contribute to these processes. The differing effects of cigarette smoke
dependence on cell density, therefore, could help explain the many
disease states that result from CSE that have seemingly opposite
manifestations. This may help explain the paradox of cigarette smoke
being related to both fibrotic and otherwise destructive diseases. The
effect of smoke may be to accelerate the fibrotic repair process in
tissues with high cell density, such as in the airways during
development of peribronchiolar fibrosis associated with chronic
obstructive pulmonary disease. Cigarette smoke also may be associated
with defective repair and, therefore, with the development of emphysema in alveolar structures where fibroblast cell density is considerably lower.
In the current study, two concentrations of CSE were evaluated. The
differential effects of CSE on fibroblasts based on cell density were
most notable with 5% CSE. Higher concentrations of smoke were
generally inhibitory, although somewhat less so. These data suggest
that cigarette smoke may have a biphasic exposure response curve that
may interact with cell density. At low cell densities, all
concentrations of smoke may have "inhibitory" effects. At higher
densities, however, low concentrations of smoke may have
"stimulatory" effects as noted in the present study, such as
stimulation of TGF-1 release with consequent augmentation of gel
contraction. Higher concentrations of smoke, however, may still be
inhibitory. Such complex exposure-response relationships may also
contribute to the heterogeneity of responses to cigarette smoke.
The current study evaluated the effect of CSE on fibroblasts in vitro. Cigarette smoke contains as many as 6,000 components, many of which are absorbed and capable of exerting toxic effects both within the lung and at distal sites (17). The current study used an aqueous extract of cigarette smoke. Although widely used in the pulmonary field since its development by Carp and Janoff (9), this extract of smoke undoubtedly differs from the smoke to which smokers are exposed. The current study did not define which components of cigarette smoke are capable of affecting fibroblast-mediated gel contraction, but Carnevali et al. (8) identified two volatile components of smoke, acetaldehyde and acrolein, both of which inhibited collagen contraction. This suggests that multiple components of cigarette smoke may have interacting toxic effects. The extent to which these components reach fibroblasts depends on their interaction with a variety of components present between the inhaled air stream and the tissue cells. This includes the surface layer, the epithelial cells, and components in the interstitial matrix, including factors derived from the circulation system. These lung structures have considerable capacity to detoxify cigarette smoke. Airway epithelial cells, for example, are capable of metabolizing xenobiotics (3). They are, moreover, potent sources of antioxidants including glutathione, which can be secreted into the extracellular space (7). Finally, a variety of molecules derived from plasma can also interact with and neutralize components present in cigarette smoke (29). The toxicity of smoke on fibroblasts in vivo, therefore, depends not only on the ability of smoke-derived toxins to injure fibroblasts, but also on the defense mechanisms present in the lung that can serve to mitigate the effects of smoke.
Remodeling of tissues is a process that likely requires collaborative
interaction among cells distributed within a tissue. Such processes are
ideally suited for regulation and coordination through a variety of
cell-cell communication mechanisms. The observation made by Ehrlich and
Rittenberg (12) that gap junction communication can
contribute to the regulation of collagen gel contraction supports this
notion. Production by fibroblasts of mediators that can function as
paracrine mediators, such as PGE2, fibronectin, and
TGF-1, also supports this concept. As the production of these
mediators will vary with cell density, it seems likely that paracrine
modulation of tissue repair will be highly dependent on cell density.
Finally, the fibroblasts are not the only source of TGF-
within the
lung, and fibroblasts can be modulated by many factors in addition to TGF-
. The effect of cigarette smoke within the tissue, therefore, will depend not only on the effect of cigarette smoke on fibroblasts but also on the actions of other cells within the lung.
In summary, the current study demonstrates that CSE modulation of
contraction of three-dimensional collagen gels populated by fibroblasts
depends on cell density. Inhibition of contraction occurs at low cell
density due to inhibition of fibronectin production. In contrast, in
high-density cultures, CSE augments contraction likely through
increased release of active TGF-. These density-dependent effects
may account for the varied types of pathology that can result from
cigarette smoke exposure.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent secretarial support of Lillian Richards and the editorial assistance of Mary Tourek.
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FOOTNOTES |
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This work was supported by the Larson Endowment, University of Nebraska Medical Center, Omaha, Nebraska.
Address for reprint requests and other correspondence: S. I. Rennard, Univ. of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, Nebraska 68198-5125 (E-mail: srennard{at}unmc.edu).
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.
September 27, 2002;10.1152/ajplung.00042.2002
Received 28 January 2002; accepted in final form 23 September 2002.
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