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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Therapies that mitigate the fibrotic process may be able to slow progressive loss of function in many lung diseases. Because cyclic adenosine monophosphate is known to regulate fibroblasts, the current study was designed to evaluate the activity of selective phosphodiesterase (PDE) inhibitors on two in vitro fibroblast responses: chemotaxis and contraction of three-dimensional collagen gels. Selective PDE4 inhibitors, rolipram and cilomilast, each inhibited the chemotaxis of human fetal lung fibroblasts (HFL-1) toward fibronectin in the blindwell assay system (control: 100% versus cilomilast [10 µM]: 40.5 ± 7.3% versus rolipram: [10 µM] 32.1 ± 2.7% cells/5 high-power fields; P < 0.05, both comparisons). These PDE4 inhibitors also inhibited contraction of three-dimensional collagen gels (control: 100% versus cilomilast: 167.7 ± 6.9% versus rolipram: 129.9 ± 1.9% of initial size; P < 0.05, both comparisons). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect in either system. Prostaglandin E2 (PGE2) inhibited both chemotaxis and gel contraction, and the PDE4 inhibitors shifted the PGE2 concentration-dependence curve to the left in both systems. The inhibition of endogenous PGE2 production by indomethacin diminished the effects of the PDE4 inhibitors in both chemotaxis and gel contraction, consistent with the concept that the PDE4 inhibitory effects on fibroblasts are related to the presence of cyclic adenosine monophosphate in the cells. In summary, these in vitro results suggest that PDE4 inhibitors may be able to suppress fibroblast activity and, thus, have the potential to block the development of progressive fibrosis.
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Introduction |
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Materials and Methods
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The accumulation of fibroblasts to the wound site is often a key event in repair following injury. However, an excess accumulation of fibroblasts results in replacement of the normal tissue parenchyma with fibrotic scar (1), thereby distorting normal tissue architecture and compromising organ function. Scars characteristically contract, and fibrosis of the small airways is associated with narrowing of the airway lumen, a process seen in asthma and chronic obstructive pulmonary disease (COPD) and which is thought to result from both fibroblast accumulation and their subsequent contraction of the surrounding matrix. In both asthma and COPD, narrowing of small airways secondary to fibrosis may lead to progressive loss of lung function. Reduction of tissue distortion due to fibrosis could represent a key therapeutic goal that might result from inhibition of fibroblast chemotaxis and/or contraction.
Both fibroblast chemotaxis and fibroblast-mediated contraction of collagen gels can be regulated by a variety of exogenous mediators. Interestingly, cyclic adenosine monophosphate (cAMP) appears to function as a secondary signal in response to several mediators that can inhibit both fibroblast chemotaxis (2) and fibroblast contraction (3). Modulation of cellular cAMP levels, therefore, may be a means of inhibiting both fibroblast activities.
cAMP levels are regulated by both the activity of the enzyme adenyl cyclase that forms cAMP and phosphodiesterases (PDE) that catalyze the hydrolysis of cAMP to AMP. The PDEs comprise a large and complex group of enzymes represented by at least seven distinct families (4). Of these, the PDE4 family of enzymes has attracted particular interest with regard to lung diseases. These enzymes appear to play a major role in regulating the activity of inflammatory cells relevant to asthma and COPD (5). A number of agents that inhibit PDE4, therefore, have been developed and are in clinical trials for these conditions. In particular, the action of PDE4 inhibitors on inflammatory cells, which may lead to an acute reduction in symptoms in asthma and COPD, offers the possibility of modulating the relentlessly progressive loss of lung function that also characterizes these disorders.
The current study evaluated the potential effects of PDE4 inhibitors on fibroblast-mediated repair responses by using two in vitro models of tissue remodeling: Boyden's blindwell chamber technique for assessing chemotaxis, and fibroblast-mediated contraction of collagen gels. In the latter culture system, human lung fibroblasts are cultured in three-dimensional collagen gels, a method that enables cells to contract the gels (9). Finally, any effect of a PDE4 inhibitor must depend on a baseline level of cAMP and hence activation of adenyl cyclase. For this reason, we also evaluated interactions between PDE4 inhibitors and prostaglandin E2 (PGE2), a known potent stimulator of fibroblast adenyl cyclase.
This study provides evidence that PDE4 inhibitors have the ability to modulate fibroblast recruitment and activity.
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Materials and Methods |
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Materials and Methods
Results
Discussion
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Materials
Cell culture media except fetal calf serum (FCS) were purchased
from GIBCO (Life Technologies, Grand Island, NY). FCS was purchased from Biofluid (Rockville, MD). Rolipram and cilomilast were kind gifts from Glaxo Smith Kline (King of Prussia, PA)
and were dissolved in 100% ethanol. Zaprinast (Sigma, St. Louis,
MO) was dissolved in 100% ethanol to a stock solution of 10 mM.
Amrinone was purchased from Sigma and dissolved in dimethylsulfoxide (Sigma) to a stock solution of 10 mM. Isoproterenol was purchased from Sigma and was dissolved in sterile, distilled water.
PGE2 and indomethacin were purchased from Sigma and dissolved
in 100% ethanol. Preliminary experiments with MTT and DNA
quantification demonstrated that the concentrations of the drug,
as well as those of ethanol and dimethylsulfoxide used in this
study, did not show any significant cytotoxicity on fibroblasts
(data not shown). Tris was purchased from GIBCO. Ethylenediamine tetraacetic acid was purchased from J. T. Baker Inc. (Philipsburg, NJ). Ethyleneglycol-bis-(
-aminoethyl ether)-N,N'-tetraacetic acid, benzamidine, dithiothreitol, soybean trypsin inhibitor,
Iosyl-L-lysine chloromethyl ketone, bacitracin, leupeptin, and
phenylmethylsulfonyl fluoride were purchased from Sigma.
Human Fetal Lung Fibroblasts
Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in 100-mm tissue culture dishes (FALCON; Becton-Dickinson Labware, Lincoln Park, NJ) in Dulbecco's modified Eagle's medium (DMEM; GIBCO), supplemented with 10% FCS, 50 U/ml penicillin G sodium, 50 µg/ml streptomycin sulfate (penicillin-streptomycin; GIBCO), and 1 µg/ml amphotericin (Parma-Tek, Huntington, NY) in a humidified, 5% CO2 atmosphere at 37°C. The fibroblasts were routinely passaged every 4 or 5 d, and cells used were between passages 13 and 20. Confluent fibroblasts were removed from dishes by treatment with 0.05% trypsin in 0.53 mM EDTA and resuspended in DMEM without serum.
Human Fibronectin
Human fibronectin was prepared from human plasma by gelatin-Sepharose affinity chromatography, as previously described (10). Following elution with 4 M urea, the fibronectin was further purified by heparin-agarose affinity chromatography and eluted with
500 mM NaCl. Samples were frozen at 
80°C until use.
Fibroblast Chemotaxis
HFL-1 chemotaxis was assessed by the Boyden blindwell chamber technique (11). In experiments with PGE2, the cells were not preincubated. In experiments with isoproterenol and indomethacin, after achieving confluence, HFL-1 cells were rinsed and incubated for 60 min with the desired concentration of reagent prior to trypsinization. Human fibronectin (20 µg/ml) was placed in the bottom chamber as the chemoattractant. The two wells were separated by an 8-µM pore filter (Nucleopore, Pleasanton, CA), coated with 0.1% gelatin (Bio Rad, Hercules, CA). HFL-1 (106/ml in DMEM without serum) were loaded into the upper wells of the chamber. The chamber was incubated at 37°C in a moist, 5% CO2 atmosphere. Except as designated, chambers were incubated for 6 h, after which the cells on top of the filter were removed by scraping. The filter was then fixed, stained with PROTOCOL (Biochemical Science, Swedesboro, NJ), and mounted on a glass microscope slide. Migration was assessed by counting the number of cells in five high-power fields. Replica experiments were performed in triplicate with separate cell cultures for every condition on separate occasions.
Type I Collagen
Type I collagen (rat tail tendon collagen, RTTC) was extracted from rat tail tendons, as previously described (9). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissue was carefully removed. After repeated washing with Tris-buffered saline (0.9% NaCl, 10 mM Tris, pH 7.5) and 95% ethanol, collagen was extracted in 6 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each batch of collagen. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) routinely demonstrated no detectable proteins other than type I collagen. The RTTC was stored at 4°C until use.
Collagen Gel Contraction Assay
The ability of PDE inhibitors to affect fibroblast-mediated gel contraction was measured according to a method developed by Bell and coworkers (12). Collagen gels were prepared as described previously (13). Briefly, RTTC, distilled water, 4× DMEM, and fibroblast suspensions were mixed by pipetting so that the final mixture resulted in 0.75 mg/ml of collagen, 3× 105 fibroblasts/ ml gel, and a physiologic ionic strength of 1× DMEM. A 500-µl portion of the gel solution was then cast into each well of a 24-well tissue culture plate with a 2-cm2 growth area (FALCON). Gelation occurred within 15 min at room temperature, after which the gels were released from the surface of the culture well using a sterile spatula, and transferred into 60-mm tissue culture dishes (FALCON) containing 5 ml of 0.5% serum DMEM with designated concentrations of PDE inhibitors with or without PGE2. The floating gels were cultured for up to 5 d, and the ability of the fibroblasts to contract the gels was determined by quantifying the area of the gels daily using an Optomax V image analyzer (Optomax, Burlington, MA).
Cytosolic Protein Extraction
HFL-1 cells (3 × 105 cells/ml) were cultured for 24 h either as a
monolayer in DMEM supplemented with FCS (10%) or grown in collagen gels as described above. After 24 h, cells grown as a monolayer were removed from the plates by scraping using a rubber policeman. For cells grown in collagen, the gels were digested with
collagenase (0.25 mg/ml), and cells were recovered and washed twice with phosphate-buffered saline. Cells were resuspended at 10 × 106 cells/ml in homogenization buffer of the following composition: Tris HCl (20 mM, pH 7.4); ethylenediamine tetraacetic
acid (1 mM); ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (2 mM); benzamidine (2 mM); dithiothreitol (2.5 mM);
soybean trypsin inhibitor (20 µg/ml); Iosyl-L-lysine chloromethyl
ketone (100 µM); bacitracin (100 µg/ml); leupeptin (100 nM); and
phenylmethylsulfonyl fluoride (40 µg/ml). Cells were disrupted by
dounce homogenization on ice and the cell suspension centrifuged at 2,000 × g for 15 min to remove nuclei and cell debris.
The supernatant fraction was then centrifuged at 100,000 × g for
10 min to collect the cytosolic and particulate fractions.
PDE Activity Assay
The PDE activity assay was conducted in 100 µl of a standard reaction mixture containing (final concentration): 50 mM Tris HCl buffer, pH 7.5, 5 mM MgCl2, 0.05% bovine serum albumin, 5 mM [14C]5'-AMP (~ 400 dpm/nmol) as carrier and also for determination of percent recovery of the product, 1 µM [3H]cAMP as substrate (~ 2,000 dpm/pmol), and enzyme. Generally, appropriate concentrations of cytosol and particulate fractions (see above) were preincubated in the absence or presence of various selective PDE isozyme inhibitors for 10 min at room temperature. The PDE reaction was then initiated by the addition of the substrate and allowed to proceed at 30°C for 30 min. The reaction was terminated with 100 µl of 0.1 M Tris, pH 8.0, containing 25 mM EDTA. Following a method for separating the cyclic nucleotide substrate from the 5' nucleotide product as described by Smith (14), the entire terminated reaction mixture was applied to 0.3 g of dry, unwashed neutral alumina contained within a disposable column. The cyclic nucleotide substrate was eluted with 12 ml 0.1 M TES (pH 8.0) followed by elution of the 5'-nucleotide product with 2 ml 2 M NaOH into a scintillation vial containing 15 ml of Gold Ultima XR liquid scintillation cocktail (Sigma L-8411) and the radioactivity measured using a liquid scintillation counter.
Recovery of [3H]5'-AMP, as determined with the [14C]5'-AMP carrier, was ~ 80%. All assays were conducted in the linear range of the reaction, where less than 25% of the initial substrate was hydrolyzed.
Cyclic GMP hydrolysis was assayed using a protocol identical to the one described above, using [3H]cGMP as the substrate and [14C]5'-GMP as carrier and for recovery determination of the product.
PDE Isozyme Expression in HFL-1 Cells by Reverse Transcriptase-Polymerase Chain Reaction
Cells grown as monolayer or in collagen gels as described above were isolated and RNA extracted with solution D. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using a RNA-PCR kit (Applied Biosystems, Foster City, CA). First strand cDNA was generated from total RNA using random hexamers to prime the reverse transcription and was directly amplified by PCR following the addition of specific primer pairs (20 µM) and Ampli-taq DNA polymerase. Oligonucleotide primers were: PDE3A, 5'-TCACCTCTCCAAGGGACTCCT-3' and 3'-GGTGACTACAAAATGTACGAC-5', defining a 708 bp product containing a HindIII site (15); PDE3B, 5'-AATTCTTCCAA CCATGGACC-3' and 3'-CGGTGTCTACACGATGTACG-5', defining a 694 bp product containing a BglII site (16); PDE4A5, 5'-AACAGCCTGAACAACTCTAAC-3' and 3'-TCAGAGTCC ACCCAAAATAAC-5', defining a 907 bp product containing a Xho site (17); PDE4B2, 5'-AGCTCATGACCCAGATAAGTG-3' and 3'-CTGTGAGTCCTTCTACCAATA-5', defining a 625 bp product containing a SaltI site (18, 19); PDE4C1, 5'-CTTTGC CCAGGTCCTGGCCAGT-3' and 3'-GCGAGGCCCTTGGTC CACAGG-5', defining a 315 bp product containing a AvrII site (17); PDE4D3, 5'-CGGAGATGACTTGATTGTGAC-3' and 3'-CGTGTGGTAAAAAGTCCTTGC-5', defining a 641 bp product with a StuI site (17, 20); and PDE7, 5'-ATAATGGACAA GCCAAGTGT-3' and 3'-CGACTTATTTCGGTCGACCT-5', defining a 936 bp product containing a PstI site (21). A commercially available human glyceraldehyde 3-phosphate dehydrogenase control primer set (Clontech #5406-1) was used in the presence and absence of reverse transcriptase as a control for each RNA sample. PCR reactions using the Perkin Elmer Geneamp PCR system (Wellesley, MA) 9,600 were as follows: an initial holding step for 105 s at 95°C, followed by 35 cycles for 15 s at 95°C and 30 s at 60°C, and a final 7-min hold at 72°C, after which they were cooled and held at 4°C. Products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.
Statistical Analysis
Unless otherwise indicated, results are expressed as the means of the determinations ± standard error of the mean (SEM). Grouped data were evaluated by one-way analysis of variance (ANOVA). Differences between series of data that appeared statistically different were corrected by Tukey test. Comparisons were considered statistically significant if P < 0.05.
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PDE Activity and Profile of Human Lung Fibroblasts
Initial experiments were conducted to partially determine in a qualitative manner the spectrum of PDE expressed by cultured human lung fibroblasts. To accomplish this, two approaches were taken. First, cytosolic and particulate fractions isolated from homogenates of human lung fibroblasts were assessed for PDE isozyme activity using either cAMP or cGMP as a substrate. In addition, the effect of several selective isozyme inhibitors was determined. These studies indicated that cyclic GMP-hydrolyzing activity was present, 80% of which could be accounted for by PDE5, based on inhibition by zaprinast (data not shown). In addition, cAMP-hydrolyzing activity was also present. Based on inhibitors, PDE3, PDE4, and PDE7, all accounted for most of the cAMP hydrolytic activity (Table 1). Most of the PDE3 activity was localized to the particulate fraction, whereas the majority of PDE4 appeared soluble. PDE7 appeared equally distributed. The distribution of PDEs was similar for fibroblasts cultured in monolayer and in three-dimensional collagen gels.
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As a second means to characterize fibroblast expression of PDEs, mRNA expression for various PDE subtypes was determined using PCR. Human lung fibroblasts expressed mRNA for both subtypes of PDE3 and all four subtypes of PDE4. PDE7 could be readily detected in some cultures in the presence of dimethyl sulfoxide, but not in its absence (Figure 1). Taken together, these two experiments demonstrate qualitatively the expression of several PDE isoforms in cultured lung fibroblasts. Importantly, all four PDE4 isozymes appear to be present and together account for a large portion of the total PDE activity.
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Effects of PDE Inhibitors on Fibroblast Chemotaxis and Fibroblast-Mediated Gel Contraction
The PDE4 inhibitors rolipram and cilomilast, at a concentration of 10 µM, significantly inhibited fibroblast chemotaxis toward 20 µg/ml of human fibronectin (P < 0.05 for both rolipram and cilomilast, compared with control). However, the selective PDE3 inhibitor, amrinone, and the selective PDE5 inhibitor, zaprinast, had no effect (Figure 2A). Rolipram and cilomilast at concentrations of 10 µM each also significantly inhibited fibroblast-mediated gel contraction (P < 0.05 for both rolipram and cilomilast, compared with control). Again, neither amrinone nor zaprinast had an effect on gel contraction (Figure 2B).
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Concentration-Dependence Effects of PDE Inhibitors on Fibronectin-Mediated Fibroblast Chemotaxis
Both PDE4 inhibitors, rolipram and cilomilast, inhibited fibronectin-induced fibroblast chemotaxis concentration dependently. In contrast, neither amrinone nor zaprinast had a significant effect at any concentration tested (Figure 3). PDE inhibitors function by preventing the hydrolysis of cAMP to AMP. Because inhibitors of chemotaxis and gel contraction are mediated by cAMP, an endogenous source of cAMP is required. The hypothesis is that PGE2, an agonist capable of stimulating cAMP formation within fibroblasts, should interact with the PDE inhibitor and shift the concentration- dependence curves for fibroblast responses. This hypothesis was tested. Both cilomilast (Figure 4A) and rolipram (Figure 4B) shifted the PGE2 curve to the left. Neither amrinone (Figure 4C) nor zaprinast (Figure 4D) had an effect on PGE2-modulated HFL-1 chemotaxis, consistent with the lack of a role for PDE3 and PDE5 in regulating these responses.
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P < 0.05 compared without PDE inhibitor.
Effects of Indomethacin or Isoproterenol on PDE4 Inhibitor-Modulated, Human Fibronectin-Induced Chemotaxis of Fibroblasts
To further confirm the role of endogenous PGE2 on PDE4
inhibitor-modulated, fibronectin-induced fibroblast chemotaxis, 2 × 10
6 M indomethacin was placed in the upper
wells with the HFL-1 cells. Indomethacin stimulated chemotaxis. In the presence of indomethacin, PDE4 inhibitors inhibited chemotaxis only at the highest concentrations (Figure 5A). In contrast, isoproterenol, an agent that can elevate cAMP, inhibited the chemotaxis of fibroblasts. Moreover,
in the presence of isoproterenol, cilomilast shifted the isoproterenol concentration curve down and to the left, consistent with a synergistic effect (Figure 5B).
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Concentration-Dependence Effects of PDE Inhibitors on Gel Contraction Mediated by Fibroblasts
Both of the PDE4 inhibitors inhibited gel contraction mediated by HFL-1 cells concentration dependently (Figure 6).
Neither amrinone nor zaprinast had any significant effect (Figure 6). PGE2 also inhibited gel contraction in a concentration-dependent manner, and both cilomilast (Figure 7A) and rolipram (Figure 7B) shifted the PGE2 curve to the left, consistent
with a synergistic effect. In contrast, neither amrinone nor zaprinast had any effect on gel contraction mediated by HFL-1,
even in the presence of 107 M PGE2 (Figures 7C and 7D).
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Effects of Indomethacin or Isoproterenol on PDE Inhibitor-Modulated Gel Contraction Mediated by Fibroblasts
To further confirm the role of endogenous PGE2 on PDE4
inhibitor-modulated, fibroblast-mediated gel contraction,
2 × 106 M indomethacin was placed into the media surrounding gels in floating culture. Indomethacin significantly
stimulated gel contraction (P < 0.05). In the presence of
indomethacin, PDE4 inhibitors blocked gel contraction of
HFL-1 only at the highest concentration tested (Figure
8A). In contrast, in the presence of isoproterenol, cilomilast shifted the isoproterenol curve up and to the left, consistent with a synergistic effect (Figure 8B).
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Discussion |
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The current study demonstrates that HFL-1 express several phosphodiesterases, including all four PDE4 isoforms. Two selective PDE4 inhibitors, rolipram and cilomilast, are capable of inhibiting fibroblast chemotaxis and fibroblast-mediated collagen gel contraction, both in a concentration-dependent manner. These PDE4 inhibitors also were able to enhance the ability of agents that increase intracellular cAMP, PGE2, and isoproterenol to inhibit both chemotaxis and gel contraction. In contrast, in the presence of indomethacin, a cyclooxygenase inhibitor that blocks production of endogenous PGE2, the inhibitory effects of rolipram and cilomilast decreased, suggesting that the effect of PDE4 inhibitors is dependent on the level of cAMP in the cells. Notably, inhibitors of PDE3 and PDE5 had no effect on chemotaxis or gel contraction, even in the presence of PGE2 or isoproterenol.
Specific 3'5' nucleotides act as second messengers in
regulating multiple functions in many cells (22). Included
among the functions regulated by cAMP are chemotaxis
and contraction of three-dimensional collagen gels. Increasing cAMP levels with either cAMP analogs or endogenous stimuli for cAMP production inhibits chemotaxis of
neutrophils (23), eosinophils (5, 24), and fibroblasts (25).
Similarly, both PGE2 and agonists, agents that stimulate
cAMP production, inhibit fibroblast-mediated contraction of collagen gels (26, 27), as do cAMP analogs (data not
shown). Cellular levels of cAMP are regulated not only by
agents that stimulate cAMP production, but also by enzymes that catalyze cAMP degradation.
cAMP degradation is mediated by PDEs. At least seven PDE isoenzyme families have been identified (4). The isoenzyme PDE4 is prominent in inflammatory cells, including neutrophils, eosinophils, monocytes, and macrophages (24). As a result, inhibitors of phosphodiesterase 4 are being developed to treat inflammatory lung diseases such as asthma and COPD. The current study suggests that PDE4 is also present in lung fibroblasts and that inhibitors of PDE4 might also attenuate the fibrotic process by slowing the migration of fibroblasts to the wound site and their consequent contraction of the surrounding matrix. The effectiveness of PDE4 inhibitors, however, depends on endogenous levels of cAMP. The PDE4 inhibitors, therefore, interact with agents that either stimulate or inhibit production of cAMP. For example, indomethacin, which blocks the production of PGE2, a major endogenous stimulus of cAMP, nearly eliminates any effect of the PDE4 inhibitors. In contrast, the PDE4 inhibitors shift the dose-response curves for PGE2 and isoproterenol, agents that increase cAMP and inhibit fibroblast chemotaxis and contraction, up and to the left, consistent with a positive-interacting effect.
During inflammation, bronchial epithelial cells and other cells in the airway produce PGE2 (4). A number of studies have suggested that PGE2 plays an important role as an autocrine or paracrine regulator of fibroblast responses (28, 29). PGE2 appears to regulate these responses by stimulating the production of cAMP. Given the effect of PGE2 on both inflammatory cells and fibroblasts, it is likely that PGE2 serves to regulate both the inflammatory response and the repair response that follows injury. By preventing the breakdown of PGE2-driven cAMP produced in an inflammatory milieu, it is likely that PDE inhibitors may have particularly potent effects during inflammation.
PDE inhibitors work by slowing the breakdown of cyclic nucleotides. PDE4 inhibitors also prevent the conversion of cAMP to AMP. Any action of these agents in cells depends on the source of cAMP. In this regard, Banner and colleagues have demonstrated that PDE4 inhibitors can have an antiproliferative effect on human mononuclear cells in the presence of PGE2 (30). Effects of PDE inhibitors, therefore, may be dependent on PGE2 production from fibroblasts or neighboring cells or, alternatively, on some other stimulus for cAMP production. Previous work from our group (25, 31) supports the concept that PGE2 can modulate both chemotaxis and contraction. As PGE is frequently produced by cells in an inflammatory milieu, these results suggest PGE may serve to regulate these functions in vivo.
The current study also demonstrated the presence of several PDEs in HFL-1. In addition to all four isoforms of PDE4, both isoforms of PDE3 and PDE7 were demonstrated by RT-PCR. PDE3, PDE7 and PDE5 also were demonstrated through inhibition of hydrolytic activity by pharmacologic inhibitors. These studies were conducted to confirm the presence of PDE4 in cultured lung fibroblasts and do not provide a quantitative estimate for the role of the various phosphodiesterases in regulating fibroblast functions. In the current study, neither the PDE3 inhibitor nor the PDE5 inhibitor affected fibroblast chemotaxis or fibroblast contraction of three-dimensional collagen gels. It is possible, however, that in the presence of other mediators, PDEs other than PDE4 may also contribute to the regulation of these and other fibroblast functions.
The current study evaluated a widely used normal human fetal lung fibroblast cell strain, HFL-1. It will be of interest to evaluate the expression and role of PDE enzymes in fibroblasts obtained from healthy adult individuals and in fibroblasts following stimulation by mediators likely present during pathologic processes. In this context, functions other than those assessed in the current study are also likely modulated by PDE inhibitors. Under the conditions of the assay used in the current study, fibroblasts do not proliferate (data not shown). PGE2, however, can both inhibit fibroblast proliferation and modulate fibroblast connective tissue matrix (28, 32). Both of these functions are known to differ as a function of age as well as of tissue origin.
Both asthma and COPD are associated with fibrosis of the small airways, which is characterized as the excess accumulation of fibroblasts and their subsequent contraction of the surrounding matrix. In this context, the narrowed airways in COPD are believed to contribute to progressive, fixed airflow limitation. A similar effect in asthma has been suggested. Inhibition of fibroblast-mediated chemotaxis and/or contraction could potentially reduce the tissue remodeling generated by fibrosis and thus have a beneficial effect on "fixed" airflow limitation. The current study used two in vitro models of tissue remodeling, Boyden's blindwell chamber technique for assessing chemotaxis, and fibroblast-mediated contraction of Type I collagen gels. The first is believed to model the recruitment of fibroblasts to sites of ongoing fibrosis (33). The latter assay system models the contraction that is part of the wound repair process and, in the airway, directly leads to narrowing (9, 13).
In summary, the current study demonstrates that PDE4 inhibitors can slow fibroblast chemotaxis and gel contraction, thereby potentially altering the tissue-remodeling process. These results could represent a therapeutic benefit of these agents.
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Footnotes |
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Address correspondence to: Stephen I. Rennard, M.D., Larson Professor, Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985125, Nebraska Medical Center, Omaha, NE 68198-5125. E-mail: srennard{at}unmc.edu
(Received in original form October 10, 2001 and in revised form January 24, 2002).
Abbreviations: one-way analysis of variance, ANOVA; cyclic adenosine monophosphate, cAMP; chronic obstructive pulmonary disease, COPD; Dulbecco's modified Eagle's medium, DMEM; fetal calf serum, FCS; human fetal lung fibroblasts, HFL-1; phosphodiesterase, PDE; prostaglandin E2, PGE2; reverse transcriptase-polymerase chain reaction, RT-PCR; rat tail tendon collagen, RTTC; standard error of the mean, SEM.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 a grant from GlaxoSmithKline.
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