1 Universidad Autónoma de Puebla, Puebla, Puebla CP 72190; 2 Instituto Nacional de Enfermedades Respiratorias, México DF, CP 14080; 3 Instituto Nacional de Cancerología, México DF, CP 14000; 5 Facultad de Ciencias, Universidad Nacional Autónoma de Mexico, México DF, CP 04510, Mexico; and 4 Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During lung injury, fibroblasts migrate into the alveolar spaces where they can be exposed to pulmonary surfactant. We examined the effects of Survanta and surfactant protein A (SP-A) on fibroblast growth and apoptosis and on type I collagen, collagenase-1, and tissue inhibitor of metalloproteinase (TIMP)-1 expression. Lung fibroblasts were treated with 100, 500, and 1,000 µg/ml of Survanta; 10, 50, and 100 µg/ml of SP-A; and 500 µg/ml of Survanta plus 50 µg/ml of SP-A. Growth rate was evaluated by a formazan-based chromogenic assay, apoptosis was evaluated by DNA end labeling and ELISA, and collagen, collagenase-1, and TIMP-1 were evaluated by Northern blotting. Survanta provoked fibroblast apoptosis, induced collagenase-1 expression, and decreased type I collagen affecting mRNA stability ~10-fold as assessed with the use of actinomycin D. Collagen synthesis and collagenase activity paralleled the gene expression results. SP-A increased collagen expression ~2-fold and had no effect on collagenase-1, TIMP-1, or growth rate. When fibroblasts were exposed to a combination of Survanta plus SP-A, the effects of Survanta were partially reversed. These findings suggest that surfactant lipids may protect against intraluminal fibrogenesis by inducing fibroblast apoptosis and decreasing collagen accumulation.
pulmonary fibrosis; surfactant protein A
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FIBROBLAST AND MYOFIBROBLAST MIGRATION from the interstitium to the alveolar spaces is a common pathological feature in subacute and chronic interstitial lung disease (ILD), acute lung injury, and acute interstitial pneumonia (3, 14, 21, 37). In all these disorders, lung damage may result in plasma-derived fluid flows through the disrupted alveolar walls into the alveolar lumen. The fluid enhances the formation of an intraalveolar fibrin exudate that may serve as a scaffold for fibroblastic invasion, proliferation, and active collagen synthesis (34). In the process of migration through partially disrupted and denuded epithelial basement membranes, fibroblasts are exposed to the components of alveolar spaces including surfactant components.
Pulmonary surfactant is a complex mixture of lipids and specific proteins. Approximately 90% of the lipid fraction consists of a mixture of phospholipids, with phosphatidylcholine comprising about 80% of total surfactant phospholipids and approximately two-thirds of whole surfactant (12). A major constituent of the protein fraction is surfactant protein (SP) A, a sialoglycoprotein with an amino-terminal collagenous domain that belongs to a group of proteins known as collectins (22).
Earlier works on surfactant have focused on its surface tension-lowering properties and on its role in pulmonary mechanics, but in recent years, surfactant functions other than those related to the mechanics of ventilation have been reported (12). Now it is known that surfactant plays a role in the modulation of immune cell function in the lung including proliferation (18), cytokine production (16), and the expression of cell surface markers by macrophages (17).
There is still little known about the role of surfactant on lung fibroblast activity. There is evidence that an exogenous surfactant preparation devoid of SP-A can downregulate the synthesis of DNA and decrease the release of inflammatory mediators in cultured normal human lung fibroblasts (38). However, no information is available about the effects of surfactant lipids or SP-A on gene expression of extracellular matrix-related molecules by fibroblasts.
Normally, parenchymal lung fibroblasts are confined to the interstitium, and they have no contact with pulmonary surfactant. As mentioned before, however, in acute lung injury and ILD, fibroblasts and myofibroblasts migrate to the air spaces, contributing to the formation of intra-alveolar granulation tissue, and in these pathological conditions, these cells could be exposed to surfactant components.
In this study, we examined the effects of the exogenous surfactant
replacement preparation Survanta and of SP-A on human lung fibroblasts
with respect to 1) proliferation and apoptosis and 2) expression of 1 type I
[
1(I)] procollagen, interstitial collagenase [matrix
metalloproteinase (MMP)-1 or collagenase-1], and tissue inhibitor of
metalloproteinase (TIMP)-1. These three molecules were selected because
they play a critical role in tissue fibrogenesis. Lung fibrotic
response is primarily characterized by collagen accumulation and by an
imbalance between MMP-1, which mainly degrades fibrillar collagens, and
its tissue inhibitor TIMP-1 (30). Survanta was chosen
because it is a natural bovine lung extract containing phospholipids,
neutral lipids, fatty acids, and surfactant-associated hydrophobic
proteins B and C and because it is in widespread clinical use. Survanta
is devoid of SP-A and SP-D.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fibroblast isolation and culture. Primary human lung fibroblasts were obtained in our laboratory as previously described (5). Briefly, cells were harvested from individuals having lung resections for the removal of a primary lung tumor. No morphological evidence of disease was found in the tissue samples used for fibroblast isolation. Lung fibroblasts were isolated by trypsin dispersion, and cells were grown in Ham's F-12 medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GIBCO BRL). Fibroblasts (passages 5-8) were cultured at 37°C in 5% CO2-95% air in T-25-cm2 Falcon flasks containing Ham's F-12 medium supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin.
SP-A preparation. SP-A was prepared from alveolar proteinosis lavage material. The insoluble protein fraction of the lavage material, which contains SP-A, was dissolved in 3 M urea and 20% glycerol and subjected to preparatory isoelectric focusing. At the end of the focusing period, the SP-A-containing fractions were placed in a dialysis bag with 1 M NaCl and dialyzed against water. The salt is included in the initial dialysis to help dissociate the ampholines from the protein. Dialysis continued against multiple changes of water at 4°C for at least 4 days. After dialysis, the SP-A preparation was characterized by protein determination, electrophoretic analysis, SP-A ELISA, and lipopolysaccharide determination (and removal if necessary). We estimated the purity of the SP-A to be >98%.
Growth rate assay. Early confluent fibroblasts were trypsinized, harvested, resuspended in Ham's F-12 medium supplemented with 10% FBS, counted, plated in 96-well culture plates at a concentration of 15 × 103 cells/well, and incubated at 37°C in 5% CO2-95% air. After 24 h, FBS-supplemented medium was replaced with serum-free medium (SFM) with either 100, 500, or 1,000 µg/ml of Survanta (Abbott Laboratories, Columbus, OH) or 10, 50, or 100 µg/ml of purified human SP-A and incubated for 48 h.
Cell number was determined with the cell proliferation reagent WST-1 (Boehringer Mannheim) as previously described (40). WST-1 is a tetrazolium salt cleaved by the mitochondria of viable cells to yield a soluble formazan chromophore. The medium of corresponding wells was replaced with fresh medium, and the dye solution was added to each well according to the manufacturer's instructions (Boehringer Mannheim). Absorbance was read on an ELISA plate reader at a wavelength of 450 nm, with a reference wavelength of 620 nm. In a parallel experiment, growth rate was also evaluated in the presence of 1 nM of Z-Val-Ala-Asp fluoromethyl ketone, caspase-1 inhibitor (Calbiochem, La Jolla, CA). Cells were cultured for 40 h in the presence of 500 µg/ml of Survanta and 500 µg/ml of Survanta plus 1 nM caspase inhibitor, and growth rate was compared with control nontreated cells.Apoptosis assays. Nuclear morphology and chromatin condensation were assessed through fluorescence images of fibroblasts stained with ethidium bromide and by DNA end labeling.
Nuclear staining with ethidium bromide was performed as described elsewhere (24). Briefly, cells exposed to Survanta (500 µg/ml), Survanta plus SP-A (50 µg/ml), or vehicle for 36 and 48 h were fixed in 70% ethanol at-1(I) procollagen, interstitial collagenase
(MMP-1), and TIMP-1 gene expression.
When fibroblasts reached early confluence, the medium was replaced with
SFM that contained either 10, 50, or 100 µg/ml of SP-A; 500 µg/ml
of Survanta; or a combination of 50 µg/ml of SP-A plus 500 µg/ml of
Survanta. In all cases, cells were incubated for 48 h.
RNA isolation and Northern blot analysis.
Total cellular RNA from lung fibroblasts was isolated by TRIzol reagent
according to the manufacturer's specifications (GIBCO BRL, Life
Technologies). Total RNA (15 µg/lane) was fractionated on a 1%
agarose gel containing 0.66 M formaldehyde. rRNA was visualized with
ethidium bromide, and the fractionated RNA was electrophoretically transferred for 6 h at 16 V onto a Nytran transfer membrane. RNA was immobilized by baking at 80°C for 2 h and then prehybridized at 42°C for 18 h in 5× saline-sodium citrate (SSC), 50%
formamide, 5× Denhardt's solution, and 0.5% SDS containing 100 µg/ml of denatured salmon sperm DNA. Hybridization was carried out at
42°C for 18 h in hybridization buffer containing 50% dextran
sulfate plus heat-denatured 32P-labeled cDNA probes.
Membranes were washed in 2× SSC-0.1% SDS at 42°C for 25 min,
followed by 0.25× SSC-0.1% SDS at 55°C and 0.1× SSC-0.1% SDS at
65°C. After they were dried, membranes were exposed to Kodak BIOMAX
MS film at 70°C with an intensifying screen. Equal loading of RNA
samples was monitored by assessing the level of 18S rRNA. The cDNA
probes were radiolabeled with a [32P]deoxycytidine
triphosphate to a specific activity of 200 × 106
dpm/µg with a multiprime DNA labeling kit (GIBCO BRL, Life Technologies).
Complementary DNA probes.
cDNA clones for human collagenase-1 (MMP-1), TIMP-1,
1(I) procollagen, and 18S rRNA were obtained from the
American Type Culture Collection (Manassas, VA).
RNA stability experiments.
In parallel experiments, actinomycin D (at a final concentration of
12.5 µg/ml) was added to serum-free subconfluent cultures to stop
gene transcription (5). Fibroblasts were removed before actinomycin addition as a time 0 control.
Actinomycin-treated cells with and without exposure to 500 µg/ml of
Survanta were removed for total RNA extraction 6, 12, and 24 h
after the beginning of treatment. Total RNA from fibroblasts exposed to
500 µg/ml of Survanta without actinomycin was isolated at the same
time points. Northern blot analysis was carried out with the use of 1(I) procollagen cDNA. After quantitation by
densitometry, differences in RNA loading were normalized with a cDNA
probe for 18S rRNA. The normalized collagen signal is expressed as a
percentage of the 0-h control.
Collagenase activity assay.
Conditioned media derived from T-75 flasks of control and 24-h
Survanta-treated fibroblasts (500 µg/ml) were dialyzed against distilled water, lyophilized, and resuspended in 50 mM
Tris · HCl buffer, pH 7.5, with 10 mM CaCl2.
Collagenolytic activity was measured as reported elsewhere
(29), and native guinea pig skin collagen labeled with
[3H]acetic anhydride was used as a substrate. One hundred
microliters of a 1.2 mg/ml collagen solution with a specific activity
of 9.8 × 105 counts · min1
(cpm) · mg
1 were used for each assay. Activation
of latent collagenase was performed by preincubating the samples with
trypsin (1-4 µg) for 10 min at 37°C, followed by a fivefold
molar excess of soybean trypsin inhibitor (Sigma, St. Louis, MO).
Incubations were carried out for 18 h at 30°C. Collagenolytic
activity was calculated after background subtraction and is expressed
as micrograms of collagen degraded at 30°C for 18 h.
Collagen measurement. To analyze collagen production by lung fibroblasts, hydroxyproline content was measured in three independent experiments by a colorimetric assay. Conditioned media derived from T-75 flasks of control and 500 µg/ml Survanta-treated fibroblasts were lyophilized and resuspended in 1 ml of distilled water. After acid hydrolysis, hydroxyproline was measured as described by Woessner (41), and results are expressed as micrograms of OH-proline per 106 cells per 48 h.
Statistical analysis. Results are expressed as means ± SD. Comparisons were made with Student's t-test for paired observations. When more than two groups were compared, the Bonferroni correction for multiple comparisons was applied. Values of P < 0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of Survanta and SP-A on fibroblast growth rate.
To investigate whether Survanta or SP-A affect cell growth, fibroblasts
were exposed to 100, 500, or 1,000 µg/ml of Survanta or 10, 50, or
100 µg/ml of SP-A in SFM for 48 h. As shown in Fig. 1A, Survanta induced a
dose-dependent loss of cell density as measured by the WST-1 assay. The
decrease in cell number was statistically significant when fibroblasts
were exposed to 500 and 1,000 µg/ml of Survanta (P < 0.0002 and P < 0.0001, respectively), suggesting that
Survanta provoked fibroblast death. To determine whether apoptosis was
playing a role in this effect, growth rate was additionally examined in
the presence of a highly specific, irreversible inhibitor of
caspase-1-like proteases. At 40 h of culture, 1 nM inhibitor reduced by ~70% the decrease of growth rate induced by 500 µg/ml of Survanta [control cells, 1.735 ± 0.13; Survanta-treated
fibroblasts, 1.128 ± 0.08; and Survanta plus caspase
inhibitor-treated fibroblasts, 1.552 ± 0.1 optical density
(OD); P < 0.01].
|
Effect of Survanta and SP-A on fibroblast apoptosis.
To further evaluate whether Survanta induced apoptosis, nuclear
morphology was assessed by fluorescence images of fibroblasts stained
with ethidium bromide and by DNA end labeling. Figure 2 depicts ethidium bromide-stained
cultured fibroblasts incubated with 500 µg/ml of Survanta for 36 h. Microscopy of adherent cells revealed that exposure to Survanta
increased the number of typical apoptotic nuclei, (i.e., nuclei with
condensed and fragmented morphology; Fig. 2, B and
C). By TUNEL, cleaved DNA within discrete nuclear fragments
in a variety of sizes and numbers was also clearly detected (Fig.
3).
|
|
|
Effect of Survanta and SP-A on 1(I)
procollagen expression.
Human normal lung fibroblasts grown at early confluence were incubated
for 48 h in SFM containing either 50 µg/ml of SP-A, 500 µg/ml
of Survanta, or both. As shown in Fig. 5,
incubation of fibroblasts with 500 µg/ml of Survanta produced a
marked downregulation of
1(I) procollagen gene
expression, whereas SP-A raised the levels of this transcript. When the
signal for
1(I) procollagen mRNA was normalized to the
level of 18S rRNA and quantified by densitometry, an ~10-fold
decrease was noticed with Survanta; SP-A caused an ~2-fold increase.
Similar results were obtained with 100 µg/ml of SP-A, whereas a dose
of 10 µg/ml of SP-A had no effect (data not shown). When fibroblasts
were exposed to both Survanta and SP-A,
1(I) procollagen
mRNA expression also revealed a significant decrease, but an
attenuation of the effect produced by Survanta alone was observed.
|
|
Effect of Survanta and SP-A on MMP-1 and TIMP-1 gene expression. By Northern hybridization, MMP-1 transcript was not detected in untreated fibroblasts incubated in SFM. As shown in Fig. 5, Survanta strongly induced MMP-1 mRNA expression, whereas SP-A had no appreciable effect on the expression of this transcript. When both substances were present in the same culture, MMP-1 mRNA expression showed an increase but to a lesser extent than when treated with Survanta alone.
Collagenolytic activity of labeled type I collagen was measured in control and 24-h Survanta-treated cells. After trypsin activation, collagen digestion by conditioned media derived from fibroblasts exposed to Survanta showed a significant increase compared with that in control cells, which had no significant activity over background (8.8 ± 0.92 vs. 2.4 ± 0.23 µg of collagen degraded at 30°C for 18 h; P < 0.01). Collagenolytic activity was inhibited by 80 mM 1,10-o-phenanthroline. Under basal conditions, normal human lung fibroblasts expressed a 0.9-kb TIMP-1 transcript. No significant effect on its expression was found when cells were stimulated with Survanta, SP-A, or both. ![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pulmonary structural remodeling in ILD has been regarded as primarily consisting of thickening of the alveolar walls by interstitial fibrosis. However, in both chronic ILD and acute lung injury, intra-alveolar fibrosis, characterized by fibroblast proliferation and connective tissue accumulation that can obliterate the air spaces, plays a major pathogenic role (3, 7, 8, 14, 21, 37).
A number of cytokines that promote the fibroproliferative response have
been identified, including transforming growth factor-, platelet-derived growth factor, insulin-like growth factor molecules, and tumor necrosis factor-
. These fibrogenic cytokines enhance fibroblast migration and proliferation and collagen accumulation (1, 2, 15, 25, 27, 36). Conversely, some cytokines, such
as interferon-
and fibroblast growth factor-1, are able to induce an
antifibrogenic phenotype in fibroblasts (5, 13) and may
reduce the fibrotic response (11).
However, other factors besides cytokines may be involved in the regulation of matrix remodeling in the lung, and our results, at least in vitro, suggest that pulmonary surfactant may be one of them. Although normally compartmentalized to the lung interstitium, fibroblasts may come into contact with the components of surfactant when these cells migrate and invade the air spaces.
Pulmonary surfactant is a complex comprised primarily of phospholipids (mainly dipalmitoylphosphatidylcholine and specific apoproteins) that is distributed as a layer along the surface of the alveolar hypophase at the air-fluid interface. However, the composition of surfactant is altered in a number of disease states, and changes in the relative amounts of the various surfactant lipids or proteins, particularly SP-A, have been reported (12). For example, decreased amounts of surfactant lipids are found in the bronchoalveolar lavage fluid of patients with acute lung injury or idiopathic pulmonary fibrosis (12).
It has previously been shown (38) that exogenous preparations of surfactant, either synthetic or from natural derivatives, inhibit DNA synthesis and the secretion of some inflammatory mediators by fibroblasts. These observations led us to hypothesize that surfactant had a possible antifibrogenic role. To address this issue, we used Survanta, a natural bovine extract enriched with tripalmitin and fatty acids, that has a proven clinical efficacy.
Our results showed that Survanta strongly upregulates collagenase gene
expression, downregulates 1(I) procollagen gene
expression, and induces programmed cell death in fibroblasts. Synthesis
of collagen and collagenase activity paralleled gene expression
results. These findings suggest that surfactant induces an
antifibrogenic phenotype in human lung fibroblasts and, consequently,
may play a role in preventing the accumulation of collagen-rich
extracellular matrix proteins in the peripheral air spaces.
On the other hand, SP-A induced a moderate increase in the expression
of 1(I) procollagen mRNA and showed no apparent effect on collagenase and TIMP-1 expression as seen by Northern hybridization.
Nevertheless, when Survanta and SP-A were mixed in the same culture in
a ratio similar to that found in normal surfactant, it seemed that the
presence of the protein partially attenuated the effects exerted by
Survanta on 1(I) procollagen expression as well as on
the induction of apoptosis. This balance of opposing actions when both
substances are present has also been noted in other cell systems
(18).
The mechanisms behind the observed changes induced by Survanta in
collagen and collagenase gene expression remain to be elucidated. Regarding collagen, the results obtained after inhibiting new transcription with actinomycin D suggest that Survanta downregulates the 1(I) procollagen gene at least partially through
posttranscriptional mechanisms, destabilizing the mRNA. Thus an
increased turnover of the preexisting
1(I) procollagen
mRNA in the cytoplasm appears to affect the steady-state level of the
message, decreasing its expression.
There are a number of factors that influence collagen production by
modulating the stability and efficiency of the utilization of mRNA.
Among these, ascorbic acid induces a greater increase in collagen
synthesis than can be attributed to the change in mRNA levels alone
(9). On the other hand, some cytokines and growth factors
can affect collagen production by altering mRNA stability. Thus, for
example, similar results to those obtained with Survanta have been
found with fibroblast growth factor-1, which also induces an
antifibrogenic phenotype in lung fibroblasts (5). The
steady-state level of 1(I) procollagen mRNA is also strongly decreased by retinoic acid (19) and by
alterations in the amount of intracellular nutrients, especially amino
acid deprivation (20).
Regarding MMP-1, several inflammatory and growth-promoting cytokines
have been shown to stimulate its expression, as observed with Survanta.
These include, among others, interleukin-1 and -1
, tumor necrosis
factor-
, platelet-derived growth factor, epidermal growth factor
(6), and fibroblast growth factor-1 (5).
Likewise, lipid mediators of inflammation such as leukotriene C4 and platelet-activating factor are able to induce
collagenase expression in different cell systems (4, 26).
It is possible that Survanta may exert its effect by several mechanisms. Lipids often act by influencing the fluidity of cell membranes. Actually, the effect of some lipids on the expression and/or synthesis of collagen and MMP-1 has been reported in other cell systems. Thus it has been demonstrated that dilinoleoylphosphatidylcholine decreases hepatic stellate cell activation as judged by the decrease in proliferative activity and procollagen I expression (32). Likewise, in rat lipocytes, polyunsaturated lecithin prevented the acetaldehyde-induced accumulation of collagen, increasing collagenase activity (23).
Alternatively, a number of molecules that may be constituents of Survanta but that have not yet been characterized may also play a role. For example, the presence of platelet-activating factor in pharmacological quantities in some surfactant replacement preparations has recently been demonstrated (28). As mentioned previously, this lipid mediator stimulates collagenase expression (4) and may be at least partially responsible for the effect observed with Survanta.
Of particular interest is the induction of fibroblast apoptosis. Recovery from lung injury depends on prompt, orderly, and effective repair of the alveolar-capillary barrier and reestablishment of the air-lung interface through the elimination of intra-alveolar mesenchymal cells. Recent evidence strongly suggests that programmed cell death is the main process responsible for the removal of intra-alveolar lung fibroblasts in patients who successfully recover from acute lung injury, although the mechanisms are unknown (31). Our results support the notion that surfactant may play a role in this process.
Changes in the chemical composition and/or functional activity of surfactant have been described in patients with acute respiratory distress syndrome and some ILDs (10, 33). Serum proteins, such as fibrinogen, present in alveolar edema fluid are an important cause of the deterioration of surfactant activity in these patients (35). If, as we demonstrated in vitro, surfactant has an antifibrotic effect in vivo, alterations in the pulmonary surfactant surface film may eventually contribute to intra-alveolar fibrosis. In this context, it can be speculated that in addition to reducing the alveolar surface tension, the use of exogenous surfactant may have a beneficial role in avoiding the formation of intraluminal fibrosis.
In summary, our results show that Survanta promotes programmed cell death of normal human lung fibroblasts and induces an upregulation of interstitial collagenase (MMP-1) and a downregulation of type I collagen. Conversely, SP-A has the opposite effect on collagen expression and partially reverses the effects of Survanta. These findings suggest that surfactant lipids provide a hostile environment to fibroblasts in the alveolar lumen, shifting these cells toward an antifibrogenic phenotype in vitro. Whether decreased levels of surfactant lipids promote fibrosis and whether the administration of exogenous surfactant might be useful in vivo to prevent or lessen fibrosis is currently unknown.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was partially supported by Programa Universitario de Investigacion en Salud, Universidad Nacional Autónoma de México.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. Selman, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502; Col. Sección XVI, México DF, CP 14080, México (E-mail:mselman{at}mailer.main.conacyt.mx)
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.
Received 19 October 1999; accepted in final form 24 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Antoniades, NH,
Bravo M,
Avila R,
Galanopoulus T,
Neville J,
and
Selman M.
Platelet-derived growth factor in idiopathic pulmonary fibrosis.
J Clin Invest
86:
1055-1064,
1990[ISI][Medline].
2.
Aston, C,
Jagirdar J,
Lee TC,
Hur T,
Hintz RL,
and
Rom WN.
Enhanced insulin-like growth factor molecules in idiopathic pulmonary fibrosis.
Am J Respir Crit Care Med
151:
1597-1603,
1995[Abstract].
3.
Basset, F,
Ferrans VJ,
Soler P,
Takemura T,
Fukuda Y,
and
Crystal RG.
Intraluminal fibrosis in interstitial lung disorders.
Am J Pathol
122:
443-461,
1986[Abstract].
4.
Bazan, HEP,
Tao Y,
and
Bazan NG.
Platelet-activating factor induces collagenase expression in corneal epithelial cells.
Proc Natl Acad Sci USA
90:
8678-8682,
1993
5.
Becerril, C,
Pardo A,
Montaño M,
Ramos C,
Ramírez R,
and
Selman M.
Acidic fibroblast growth factor (FGF-1) induces an antifibrogenic phenotype in human lung fibroblasts.
Am J Respir Cell Mol Biol
20:
1020-1027,
1999
6.
Fini, ME,
Cook JR,
Mohan R,
and
Brinckerhoff CE.
Regulation of matrix metalloproteinase gene expression.
In: Matrix Metalloproteinases, edited by Parks WC,
and Mecham RP.. San Diego, CA: Academic, 1998, p. 299-356.
7.
Fukuda, Y,
Basset F,
Ferrans VJ,
and
Yamanaka N.
Significance of early intra-alveolar fibrotic lesions and integrin expression in lung biopsy specimens from patients with idiopathic pulmonary fibrosis.
Hum Pathol
26:
53-61,
1995[ISI][Medline].
8.
Fukuda, Y,
Ishizaki M,
Masuda Y,
Kimura G,
Kawanami O,
and
Masugi Y.
The role of intraalveolar fibrosis in the process of pulmonary structural remodeling in patients with diffuse alveolar damage.
Am J Pathol
126:
171-182,
1987[Abstract].
9.
Geesin, JC,
Darr D,
Kaufman R,
Murad S,
and
Pinnell SR.
Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human skin fibroblasts.
J Invest Dermatol
90:
420-424,
1988[Abstract].
10.
Gregory, TJ,
Longmore WJ,
Moxley MA,
Whittset JA,
Reed CR,
Fowler AA,
Hudson LD,
Maunder RJ,
Crim C,
and
Hyers TM.
Surfactant chemical composition and activity in acute respiratory distress syndrome.
J Clin Invest
88:
1976-1981,
1991[ISI][Medline].
11.
Gurujeyalakshmi, G,
and
Giri SN.
Molecular mechanisms of antifibrotic effect of interferon- in bleomycin-mouse model of lung fibrosis: downregulation of TGF-
and procollagen I and III gene expression.
Exp Lung Res
21:
791-808,
1995[ISI][Medline].
12.
Hamm, H,
Kroegel C,
and
Hohlfeld DJ.
Surfactant: a review of its functions and relevance in adult respiratory disorders.
Respir Med
90:
251-270,
1996[ISI][Medline].
13.
Jaffe, HA,
Gao Z,
Mori Y,
Li L,
and
Varga J.
Selective inhibition of collagen gene expression in fibroblasts by an interferon-gamma transgene.
Exp Lung Res
25:
199-215,
1999[ISI][Medline].
14.
Katzenstein, ALA,
and
Myers JL.
Idiopathic pulmonary fibrosis. Clinical relevance of pathologic classification.
Am J Respir Crit Care Med
157:
1301-1315,
1998
15.
Khalil, N,
O'Connor RN,
Unruh HW,
Warren PW,
Flanders KC,
Kemp A,
Bereznay OH,
and
Greenberg AH.
Increased production and immunohistochemical localization of transforming growth factor- in idiopathic pulmonary fibrosis.
Am J Respir Cell Mol Biol
5:
155-162,
1991[ISI][Medline].
16.
Kremlev, SG,
and
Phelps DS.
Surfactant protein A stimulation of inflammatory cytokine and immunoglobulin production.
Am J Physiol Lung Cell Mol Physiol
267:
L712-L719,
1994
17.
Kremlev, SG,
and
Phelps DS.
Effect of SP-A and surfactant lipids on expression of cell surface markers in the THP-1 monocytic cell line.
Am J Physiol Lung Cell Mol Physiol
272:
L1070-L1077,
1997
18.
Kremlev, SG,
Umstead TM,
and
Phelps DS.
Effects of surfactant protein A and surfactant lipids on lymphocyte proliferation in vitro.
Am J Physiol Lung Cell Mol Physiol
267:
L357-L364,
1994
19.
Krupsky, M,
Fine A,
Berk JL,
and
Goldstein RH.
Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts.
Biochim Biophys Acta
1219:
335-341,
1994[ISI][Medline].
20.
Krupsky, M,
Kuang P,
and
Goldstein RH.
Regulation of type I collagen mRNA by amino acid deprivation in human lung fibroblasts.
J Biol Chem
272:
13864-13868,
1997
21.
Kuhn, C, III,
Boldt J,
King TE,
Crouch E,
Vartio T,
and
McDonald JA.
An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.
Am Rev Respir Dis
140:
1693-1703,
1989[ISI][Medline].
22.
Kuroki, Y,
and
Voelker DR.
Pulmonary surfactant proteins.
J Biol Chem
269:
25943-25946,
1994
23.
Li, J,
Kim C,
Leo MA,
Mak KM,
Rojkind M,
and
Lieber CS.
Polyunsaturated lecithin prevents acetaldehyde-mediated hepatic collagen accumulation by stimulating collagenase activity in cultured lipocytes.
Hepatology
15:
373-381,
1992[ISI][Medline].
24.
Maldonado, V,
Melendez J,
Gonzalez H,
and
Ortega A.
Internucleosomal DNA cleavage in HeLa cells exposed to cisplatin.
Biochem Mol Biol Int
37:
691-696,
1995[ISI][Medline].
25.
Marinelli, WA,
Henke CA,
Harmon KR,
Hertz MI,
and
Bitterman PB.
Mechanisms of alveolar fibrosis after acute lung injury.
Clin Chest Med
11:
657-672,
1990[ISI][Medline].
26.
Medina, L,
Pérez J,
Ramírez R,
Selman M,
and
Pardo A.
Leukotriene C4 upregulates collagenase expression and synthesis in human lung fibroblasts.
Biochim Biophys Acta
1224:
168-174,
1994[ISI][Medline].
27.
Miyazaki, Y,
Araki K,
Vesin C,
Garcia I,
Kapanci Y,
Whitsett JA,
Piguet PF,
and
Vassalli P.
Expression of a tumor necrosis factor- transgene in murine lung causes lymphocytic and fibrosing alveolitis.
J Clin Invest
96:
250-259,
1995[ISI][Medline].
28.
Moya, FR,
Hoffman DR,
Zhao B,
and
Johnston JM.
Platelet-activating factor in surfactant preparations.
Lancet
341:
858-860,
1993[ISI][Medline].
29.
Pardo, A,
Ramírez R,
Gutiérrez-Kobeh L,
Mendoza F,
Bauer E,
and
Selman M.
Purification of a procollagenase-activator present in medium of cultured guinea pig carrageenin granuloma.
Connect Tissue Res
26:
259-269,
1991[ISI][Medline].
30.
Pardo, A,
and
Selman M.
Matrix metalloproteinases in the pathogenesis of lung injury.
In: Collagenases, edited by Hoeffler W.. Georgetown, TX: Landes, 1999, p. 221-239.
31.
Polunovsky, VA,
Chen B,
Henke C,
Snover D,
Wendt C,
Ingbar DH,
and
Bitterman PB.
Role of mesenchymal cell death in lung remodeling after injury.
J Clin Invest
92:
388-397,
1993[ISI][Medline].
32.
Poniachik, J,
Baraona E,
Zhao J,
and
Lieber CS.
Dilinoleoylphosphatidylcholine decreases hepatic stellate cell activation.
J Lab Clin Med
133:
342-348,
1999[ISI][Medline].
33.
Robinson, PC,
Watters LC,
King TE,
and
Mason RJ.
Idiopathic pulmonary fibrosis abnormalities in bronchoalveolar lavage fluid.
Am Rev Respir Dis
137:
585-591,
1988[ISI][Medline].
34.
Ryu, JH,
Colby TV,
and
Hartman TE.
Idiopathic pulmonary fibrosis: current concepts.
Mayo Clin Proc
73:
1085-1101,
1998[ISI][Medline].
35.
Seeger, WN,
Elssner A,
Gunther A,
Kramer HJ,
and
Kalinowski HO.
Lung surfactant phospholipids associate with polymerizing fibrin: loss of surface activity.
Am J Respir Cell Mol Biol
9:
213-220,
1993[ISI][Medline].
36.
Snyder, LS,
Hertz MI,
Peterson MS,
Harmon KR,
Marinelli WA,
Henke CA,
Greenheck JR,
Chen B,
and
Bitterman PB.
Acute lung injury. Pathogenesis of intraalveolar fibrosis.
J Clin Invest
88:
663-673,
1991[ISI][Medline].
37.
Svee, K,
White J,
Vaillant P,
Jessurun J,
Roongta U,
Krumwiede M,
and
Johnson D.
Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44.
J Clin Invest
98:
1713-1727,
1996
38.
Thomassen, MJ,
Antal JM,
Barna BP,
Davis LT,
Meeker DP,
and
Wiedemann HP.
Surfactant downregulates synthesis of DNA and inflammatory mediators in normal human lung fibroblasts.
Am J Physiol Lung Cell Mol Physiol
270:
L159-L163,
1996
39.
Uhal, BD,
Joshi I,
True AL,
Mundle S,
Raza A,
Pardo A,
and
Selman M.
Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro.
Am J Physiol Lung Cell Mol Physiol
269:
L819-L828,
1995
40.
Uhal, BD,
Ramos C,
Joshi I,
Bifero A,
Pardo A,
and
Selman M.
Cell size, cell cycle and -smooth muscle actin expression by primary human lung fibroblasts.
Am J Physiol Lung Cell Mol Physiol
275:
L998-L1005,
1998
41.
Woessner, JF.
The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.
Arch Biochem Biophys
93:
440-447,
1961[ISI].