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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 375-381, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4830

PGD2 Modulates Fibroblast-Mediated Native Collagen Gel Contraction

Tadashi Kohyama, Todd A. Wyatt, Xiangde Liu, Fu-Qiang Wen, Tetsu Kobayashi, Qiuhong Fang, Hui Jung Kim and Stephen I. Rennard

Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan; University of Nebraska Medical Center, Omaha, Nebraska; and Department of Internal Medicine, Seoul Adventist Hospital, Seoul, Korea

Address correspondence to: Stephen I. Rennard, M.D., University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 68198-5125. E-mail: srennard{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Repair of tissues is a necessary step in restoring tissue function following injury consequent to inflammation. Many inflammatory mediators are capable of modulating not only the activity of "inflammatory cells" but also of modulating functions of parenchymal cells that may contribute to repair. Disordered repair is believed to contribute to tissue dysfunction in many inflammatory diseases, including bronchial asthma. The current study evaluated the ability of prostaglandin D2 (PGD2) to modulate fibroblast repair using the in vitro contraction of three-dimensional native collagen gels as a model system. PGD2 stimulated gel contraction in a concentration- and time-dependent manner. In contrast, the PGD2 analog BW245C inhibited contraction. Both effects were blocked by a DP-receptor blocker (AH6809). Neither TP receptor blocker SQ29548 nor protein kinase (PK) A antagonist KT5720 hand an effect on PGD2-stimulated contraction, suggesting action through a novel prostaglandin D receptor. PKC inhibitor calphostin-C (10-6 M) blocked the PGD2 stimulation of gel contraction. A calcium-independent PKC-{epsilon} inhibitor (Ro31-8220), but not calcium-dependent PKC-{alpha} and -ß inhibitors, also blocked the PGD2 effect on contraction, implying a role for a calcium-independent pathway. This study, therefore, supports a role for PGD2 in tissue repair and remodeling. These effects of PGD2 appear to be mediated through receptor-signal transduction pathways different from the cAMP-PKA pathways mediating the proinflammatory activity of PGD2, creating the possibility for selective therapeutic manipulation.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • dimethyl sulfoxide, DMSO • PGD2 receptors, DP • PGE2 receptor, EP • fetal calf serum, FCS • human fetal lung fibroblasts, HFL • interleukin, IL • prostaglandin D2, PGD2 • phospholipase C, PLC • rat tail tendon collagen, RTTC • sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE • Tris-buffered saline, TBS • transforming growth factor-ß, TGF-ß • thromboxane receptor, TP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute inflammatory processes frequently lead to tissue disruption and damage. Maintenance of tissue function after injury requires tissue repair. Many inflammatory mediators are now recognized as able to stimulate parenchymal cells, consistent with the concept that repair is an integral part of the inflammatory process.

Bronchial asthma is a chronic inflammatory disorder of the airways characterized by airway remodeling. Among the mediators believed to play a role in asthma is prostaglandin D2 (PGD2). This lipid mediator is an arachidonic acid product of the cyclo-oxygenase pathway derived from prostaglandin H. It is capable of inducing not only bronchoconstriction (1, 2) but also can mediate effects on vessels (3) and platelets (4). It is a product of dendritic cells (5), Th2 lymphocytes, and mast cells (6, 7). Both mast cell– and Th2-derived mediators, including transforming growth factor ß (TGF-ß), histamine, and the interleukins (ILs)-4 and -13, have been suggested to modulate fibroblast functions believed important in the repair and remodeling that develops in asthma (8–11). The current study was designed to evaluate the hypothesis that PGD2 can modulate remodeling specifically by altering the ability of fibroblasts to reorganize their surrounding extracellular matrix.

Fibroblasts are the major mesenchymal cells believed responsible for the formation of fibrotic scar tissue. Culture of fibroblasts in three-dimensional gels made from native type I collagen has been used as an in vitro model of the contraction that characterizes both normal wound repair and fibrosis (12). The current study utilized this in vitro system to evaluate the activity of PGD2 as a potential modulator of fibroblast repair activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collagen Preparation
Type I collagen was extracted from rat tail tendons (RTTC) using a previously published method (13). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were carefully removed. After repeated washing with tris-buffered saline (TBS), ethanol concentrations were gradually increased from 50% to 99%, and type I collagen was extracted in 6 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that no detectable proteins other than type I collagen were present.

PGD2, the PGD2 receptor (DP) agonist BW245C, the DP antagonist AH6809, and the thromboxane receptor (TP) antagonist SQ29,548 were purchased from Cayman Chemical (Ann Arbor, MI). Calphostin C and TMB-8 were purchased from Sigma (St. Louis, MO). Rottlerin, Gö6976, LY379196, Ro31-8220, and KT5720 were purchased from Calbiochem (San Diego, CA). Rp-8-pCPT-cGMPS, the phospholipase C (PLC) inhibitor U-73122, and its inactive analog U-73343 were purchased from BioMol (Plymouth Meeting, PA). PGD2 was dissolved in H2O to a stock solution of 5 x 10-3 M.

Calphostin C and KT5720 (both 10-2 M) were dissolved in dimethylsulfoxide (DMSO). BW245C (10-2 M), AH6809 (10-3 M), SQ29,548 (2 x 10-3 M), and Rp-8-pCPT-cGMPS (10-2 M) were dissolved in ethanol. TMB-8 (10-4 M) was dissolved in ddH2O. U-73122 and U-73343 (both 10-2 M) were dissolved in DMSO immediately before use. Tissue culture supplements and media were purchased from Gibco-BRL (Life Technologies, Grand Island, NY). Fetal calf serum (FCS) was purchased from Biofluid (Rockville, MD).

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) with Dulbecco's modified Eagle's medium (DMEM), which contained 0.4 mM L-arginine, supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.2 µg/ml fungizone. The fibroblasts were passaged every 3–5 d. Subconfluent fibroblasts were trypsinized (trypsin-EDTA; 0.05% trypsin, 0.53 mM sodium EDTA) and used for experiments. Fibroblasts used in these experiments were between cell passages 16 and 20.

Preparation of Collagen Gels
Fibroblasts were cast into collagen gels using previously reported methods (14). Briefly, RTTC, distilled water, 4x concentrated DMEM, and suspended cells were gently mixed. Volumes were adjusted so that collagen concentration was 0.75 mg/ml and cell density was 3 x 105 cells/ml, with a physiological ionic strength equaling 1x DMEM. Fibroblasts were always added last. The mixture (0.5 ml) was cast into each well of 24-well tissue culture plates (Falcon). Gelation occurred in 20 min at room temperature. Gels were then detached and transferred to 60-mm tissue culture dishes containing 5 ml of media with designed concentrations of reagent and incubated with 95% O2 and 5% CO2 at 37°C. Gel size was determined daily for 5 d using an image analysis system (Optomax, Burlington, MA).

Protein Kinase C Activity
Protein kinase (PK) C activity was determined in HFL-1 cells. After pretreatment of HFL-1, with or without various PKC isoform–specific inhibitors, cells were fractionated into membrane and cytosolic fractions before measurement of PKC activity as previously reported (15). Translocation of any PKC activity to membrane fractions could then be compared with cytosolic PKC activity or total PKC activity. The assay used was a modification of procedures previously described (16) with the use of 900 µM PKC substrate peptide (Peninsula Laboratories, Belmont, CA), 12 mM calcium acetate, 8 µM phosphatidyl-L-serine, 24 µg/ml of phorbol-12-myristate-13-acetate (PMA), 30 mM dithiothreitol, 150 µM adenosine triphosphate (ATP), 45 mM magnesium acetate, and 10 µCi/ml [{gamma} 32P]ATP in a Tris-HCl buffer (pH 7.5). For PKC-{varepsilon} activity determination, a PKC-{varepsilon}–specific substrate peptide was used (Calbiochem). Samples (20 µl) were added to 40 µl of the above reaction mixture and incubated for 15 min at 30°C. Spotting of 50 µl of each sample onto P-81 phosphocellulose papers (Whatman, Clifton, NJ) halted incubations. Papers were subsequently washed five times for 5 min each in phosphoric acid (75 mM), washed once in ethanol, dried, and counted in nonaqueous scintillant as previously described (17). Kinase activity was expressed in relation to total cellular protein assayed and calculated in picomoles per minute per milligram.

Statistical Analysis
Results are expressed as the means ± SEM of at least three separate experiments, each performed with triplicate gels. Paired data were analyzed using Student's t test. Multiple group data were analyzed for significance using ANOVA. Where ANOVA indicated significant differences between groups, Tukey correction was performed and P < 0.05 was taken as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PGD2 stimulated HFL-1–mediated gel contraction concentration-dependently (Figure 1) . The effect was significant from 10-6 M PGD2. The stimulatory effect of PGD2 (10-5 M) on gel contraction was observed at every time point during the 5 d of culture (Figure 2) .



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  		Figure 1. Concentration-dependent effect of PGD2 on collagen gel contraction mediated by human lung fibroblasts. Gels were cultured for 5 d with serial concentrations of PGD2 (10–7 M to 10–5 M) in the surrounding media, and gel sizes were measured on Day 5. Vertical axis: size expressed as percentage of initial area; horizontal axis: PGD2 concentrations. Data are means ± SEM for three separate experiments, each performed in triplicate, *P < 0.05.

 


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  		Figure 2. Effect of PGD2 on collagen gel contraction mediated by human lung fibroblasts (time course). Gels were released into 60-mm tissue culture dishes containing 5 ml of DMEM with (diamonds) or without (squares) PGD2 (10–5 M). The area of each gel was assessed daily for 5 d. Vertical axis: expressed as percentage of initial area. Horizontal axis: time. Shown are means ± SEM for three separate experiments, each performed in triplicate. *P < 0.05.

 
To elucidate if the effects of PGD2 were mediated through the PGD2 receptor, the DP antagonist AH6809 (10-5 M) was added to the surrounding media. Alone, AH6809 slightly stimulated fibroblast-mediated gel contraction; the antagonist, however, completely blocked the stimulating effect of PGD2 (Figure 3A) . In contrast, the DP agonist BW245C inhibited fibroblast-mediated gel contraction concentration-dependently, and AH6809 blocked this effect (Figure 3B). The bronchoconstrictive effect exerted by PGD2 is believed to be mediated through the TP (1). To elucidate whether the effect of PGD2 on gel contraction is mediated through the TP, the TP antagonist SQ29,548 was added to the media surrounding gels. SQ29,548 alone had no effect on HFL-1–mediated gel contraction nor on contraction modulated by PGD2 (Figure 4) .




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  		Figure 3. Effects of the DP receptor antagonist AH6809 on HFL-1 gel contraction, modulated by PGD2 or BW245C. Gels were released into 60-mm tissue culture dishes containing 5 ml of DMEM with (diamonds) or without (squares) AH6809 (10–5 M) in the presence of varying concentrations of PGD2 (10–7 to 10–5 M; A) or BW245C (10–7 to 10–5 M; B). The size of the gels was determined by quantifying the area of the gels after 3 d. Vertical axis: expressed as percentage of initial area. Horizontal axis: conditions. Data are means ± SEM for three separate experiments, each performed in triplicate, *P < 0.05.

 


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  		Figure 4. Effects of the TP receptor antagonist SQ29,548 on HFL-1 gel contraction induced by PGD2. Gels were released into 60-mm tissue culture dishes containing 5 ml of DMEM with or without SQ29,548 (10–5 M) and/or PGD2 (10–6 M). The contraction of the gels was determined by quantifying the area of the gels after 3 d. Vertical axis: expressed as percentage of initial area. Horizontal axis: conditions. Data are means ± SEM for three separate experiments each performed in triplicate, *P < 0.05.

 
To determine if the PGD2 effect is mediated through the cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calcium-dependent protein kinase (PKC) pathways, HFL-1 cells were treated with the PKA inhibitor KT5720 (10-7 M), the PKG inhibitor Rp-8-pCPT-cGMP (10-7 M), or the PKC inhibitor calphostin-C (10-6 M). Neither the PKA nor the PKG inhibitor had an effect on fibroblast-mediated gel contraction modulated by PGD2. In contrast, the PKC inhibitor calphostin-C (10-6 M) blocked the prostanoid's effect (Table 1).


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  		TABLE 1 Effect of PKA, PKG, and PKC on PGD2-augmented collagen gel contraction*

 
Because the stimulatory effect of PGD2 on gel contraction was blocked by calphostin-C, the role of calcium and PLC, activators of PKC, were tested. The PLC inhibitor U-73122 (10-6 M) and its inactive analog U73343 (10-6 M), TMB-8 (10-6 M), an inhibitor of intracellular Ca2+ mobilization, or EGTA (10-2 M), an extracellular Ca2+ chelator, were added together with PGD2. None of these reagents modulated gel contraction directly or had an inhibitory effect on PGD2-augmented contraction (Table 2). To determine if the stimulation of fibroblast-mediated gel contraction by PGD2 occurs by way of a calcium-independent PKC pathway, specific PKC isozymes were investigated. The calcium-dependent PKC {alpha} and ß inhibitors Gö6976 and LY379196 had no effect on PGD2-enhanced gel contraction (Table 3). Similarly, the calcium-independent PKC-{delta} inhibitor rottlerin had no effect on the gel contraction induced by PGD2 (Table 3). In marked contrast, calcium-independent PKC-{varepsilon} inhibitor Ro31-8220 clearly blocked this effect on fibroblast-mediated gel contraction.


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  		TABLE 2 Effect of calcium chelator and PLC inhibitor on PGD2-augmented collagen gel contraction*

 

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  		TABLE 3 Effect of selective PKC isozyme inhibitor on PGD2-augmented collagen gel contraction*

 
To directly confirm the role of PKC-{varepsilon} as a mediator of the PGD effect, PKC-{varepsilon} activity was assayed in fibroblasts after stimulation with PGD2. PKC-{varepsilon} activity was primarily detected in the particulate fraction of HFL-1 cells. PGD2 stimulation resulted in a more than 2.3-fold increase in PKC-{varepsilon} activity (Figure 5A) . We observed no evidence for translocation of the novel PKC isoform PKC-{varepsilon} from cytosol to the membrane fraction consistent with current concepts regarding activation of novel PKC isoforms (18, 19). PKC-{varepsilon} activity was inhibited significantly by both the pan-specific inhibitor, calphostin-C, and the PKC-{varepsilon} inhibitor Ro31–8220, but was not inhibited by the PKC-{alpha} inhibitor Gö6976 (Figure 5A). PKC-{alpha} activity in the membrane fraction was also weakly stimulated by PGD2 1.7-fold (Figure 5B). PKC-{alpha} activity was inhibited by the PKC-{alpha} inhibitor Gö6976 and, to a lesser degree, by calphostin-C and Ro31–8220.




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  		Figure 5. Protein kinase activity in fibroblasts after stimulation with PGD2. Fibroblasts were exposed to PGD2 with and without various protein kinase inhibitors. Cells were harvested and fractionated as described in MATERIALS AND METHODS. The calcium-independent PKC-{varepsilon} (A) and the calcium-dependent PKC-{alpha} (B) were assayed with specific substrates. Vertical axes: protein kinase activity (expressed as percentage of unstimulated control). Horizontal axes: additions. Data are means + SEM of three separate experiments performed on separate occasions; *P < 0.05, **P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study demonstrates that PGD2 stimulates fibroblast-mediated contraction of native collagen gels. The effect of PGD2 is concentration- and time-dependent. In contrast to PGD2, BW245C, an agonist active at the DP, inhibited fibroblast-mediated collagen gel contraction. The DP antagonist AH6809 blocked both the stimulatory effect of PGD2 and the inhibitory effect of BW245C. Because PGD2 is known to act on the TP, the TP antagonist SQ29,548 was evaluated and found to have no effect on contraction induced by PGD2. The signal transduction pathways through which PGD2 mediates its effects were evaluated. Neither the PKA inhibitor KT5720 nor the PKG inhibitor Rp-8-pCPT-cGMP had any effect. In contrast, the PKC inhibitor calphostin-C blocked PGD2-induced contraction. However, neither inhibition of phospholipase C nor chelation of calcium had an effect. Consistent with a role for a calcium-independent, PKC-mediated effect, inhibition of the PKC-{varepsilon} isoform with Ro31–8220 also blocked PGD2-induced contraction. Further supporting a role for this PKC isozyme, PKC-{varepsilon} activity was directly assayed and found to increase significantly following PGD2 exposure. Taken together, these results demonstrate that PGD2 can result in augmentation of fibroblast-mediated contraction of native collagen gels. This effect appears to be mediated through a calcium-independent protein kinase C (such as PKC-{varepsilon}), and may involve a novel receptor.

PGD2 is a major prostanoid released by mast cells, Th2 lymphocytes, and dendritic cells (7, 20). It is also present in the brain, where it is believed to function in regulating sleep (21, 22). Peripherally, PGD2 can lead to a variety of effects, including increased vascular permeability, vasodilation (3), platelet aggregation (4), and bronchoconstriction (1, 2). Because of its effects and cellular sources, PGD2 has been suggested to play a pathogenetic role in the airways in asthma. Such a role is further supported by the finding that PGD2 levels are increased in bronchoalveolar lavage fluid from individuals with asthma (23, 24). Further supporting such a role, allergen stimulation in vitro and methacholine stimulation in vivo increase PGD2 release. Finally, mice genetically deficient for the DP are not as responsive to allergen in a murine model of asthma (25). The current study supports the concept that PGD2 may also modulate tissue remodeling.

Prostaglandins are generally believed to mediate their effects through seven membrane-spanning G-protein–coupled receptors. In this context, PGD2 is believed to interact with several receptors. The classic receptor for PGD2, the DP, activates adenylate cyclase and signals through increases in cAMP and intracellular calcium (26, 27). In addition, PGD2 can signal through the TP, so named because it also interacts with thromboxane (1). The effect of PGD2 on fibroblast-mediated collagen gel contraction does not appear to be mediated through either of these receptors. The TP antagonist SQ29,548 had no effect on PGD2-induced contraction, suggesting no role for the TP. This contrasts with the ability of this receptor antagonist to block the action of the thromboxane analog (Kohyama and coworkers, unpublished data). The DP agonist BW245C had the opposite effect of PGD2—namely, inhibited contraction. An inhibitory effect of this agonist is consistent with other agents known to increase or mimic cAMP, including ß-agonists, PGE2 , phosphodiesterase inhibitors, and dibutyryl cAMP, all of which also inhibit contraction (28–30). Taken together, these results suggest that the stimulatory effects of PGD2 on fibroblast-mediated collagen gel contraction may be mediated through a novel receptor.

Similar results suggesting a novel PGD2 receptor have been obtained by Monneret and colleagues evaluating eosinophil chemotaxis (31). In this study, PGD2 was noted to have the opposite effects from the DP agonist BW245C. A PGD2 action mediated through a novel receptor termed DP2 was suggested. A second receptor for PGD2 has been identified by Hirai and colleagues (32). This receptor, termed CRTH2, signals through Gi and intracellular calcium mobilization and modulates Th2 cell chemotaxis. Whether this receptor is the same as the DP2 receptor suggested functionally by the studies of Monneret or the receptor through which PGD2 effects are mediated in the current study remains to be determined.

The concentrations of PGD2 used in the current study are higher than eicosinoid concentrations used in many in vitro bioassays. Nevertheless, these concentrations are likely to be of biologic relevance. PGD2 concentrations have been reported in the range of 100–300 pg/ml in bronchoalveolar lavage fluid (33–35), corresponding to a concentration of ~ 10-9 M. Bronchoalveolar lavage, however, represents approximately a 1% dilution of epithelial lining fluid (36). Concentrations of PGD2 on the epithelial surface of the lung at steady state, therefore, are likely to be on the order 10-7 M. Following acute allergen challenge, however, increases in PGD2 concentrations of 100-fold have been reported (34, 37). These concentrations are likely to be ~ 10-5 M, similar to that used in the current study. Concentrations of PGD2 in the immediate vicinity of the cells producing this mediator are likely to be much higher. It is highly likely, therefore, that concentrations on the order of 10-5 to 10-6 M, which were found to be active in the current study, are present at least at sometimes in vivo. Presence of such high concentrations raises the possibility of interaction through lower affinity binding to a variety of receptors. Although the specific receptors through which PGD2 stimulates fibroblast-mediated collagen gel contraction remain to be identified, this action appears to be independent of the classical DP.

The effect of PGD2 on fibroblast contraction of three-dimensional collagen gels appears to be mediated through calcium-independent PKC signaling. Consistent with this, both calphostin-C, a general PKC inhibitor, and Ro31–8220, a PKC-{varepsilon} selective inhibitor, blocked the PGD2 effect. In contrast, inhibitors of phospholipase C, of intracellular calcium mobilization, and of the calcium-dependent PKC isoforms {alpha} and ß were without effect. It is likely, however, that PGD2 leads to activation of calcium-dependent PKC isoforms. Consistent with this, a 1.7-fold increase in PKC-{alpha} was observed in response to PGD2. This isoform, however, does not appear to account for the augmentation of collagen gel contraction. PKC-{varepsilon} is a reasonable candidate to mediate the effect on gel contraction, as its activity increased following PGD2 stimulation. It remains possible, however, that another calcium-independent PKC isoform in addition to PKC-{varepsilon} could also play a role.

PGE2 is another major prostaglandin that can be released by HFL in three-dimensional collagen gel (38, 39). In contrast to PGD2, PGE2 is a potent inhibitor of the collagen gel contraction mediated by HFL-1 cells, presumably through cAMP–PKA pathway (38). Regarding HFL-1 cell chemotaxis toward fibronectin, however, both PGD2 and PGE2 have an inhibitory effect through DP and EP respectively, as well as through the downstream cAMP–PKA pathway (40, 41). These results indicate that prostaglandins may be involved in the process of airway remodeling by either stimulating or inhibiting tissue repair through different receptors.

Inflammation frequently leads to tissue disruption, necessitating tissue repair. An increasingly large number of inflammatory mediators have been described as having effects that can modulate repair and remodeling responses. The current study suggests that PGD2, in addition to its proinflammatory actions, may also modulate tissue repair by increasing the ability of fibroblasts to contract their surrounding matrix. In this context, fibroblast contraction is believed to contribute to the contraction that characterizes scars and fibrotic tissues, as well as the resolution of granulation tissue. Fibroblast contraction, therefore, may be an important mechanism leading to tissue distortion, which compromises tissue function in some cases. In others, fibroblast contraction may lead to apoptosis of cells that accumulate in response to injury and thus can lead to restoration of normal tissue function. The current study suggests that, among the mediators that contribute to the regulation of these complex processes, PGD2 can play a role.

Inhibition of inflammatory processes has been a major therapeutic strategy in many disorders, including asthma. The possibility that repair responses to mediators such as PGD2 may be mediated through receptors and signal transduction pathways distinct from the inflammatory responses creates the possibility for selective therapeutic manipulation. Modulation of repair processes may be a particularly important strategy to alter the tissue remodeling that characterizes many disorders such as asthma.

In summary, the current study demonstrates that PGD2 stimulates fibroblast-mediated collagen gel contraction. This effect appears to be mediated through a novel receptor distinct from the DP and TP, and involves signaling through a calcium-independent, novel PKC, possibly PKC-{varepsilon}. By virtue of its ability to modulate mesenchymal cell restructuring of ECM, PGD2 may contribute to tissue remodeling characteristic of inflammatory diseases such as asthma.


   Acknowledgments
 
The authors acknowledge the excellent secretarial support of Ms. Lillian Richards and the editorial assistance of Ms. Mary Tourek. This work was funded by the Larson Endowment, University of Nebraska Medical Center, and grant HL64088-03 from the National Institutes of Health.

Received in original form February 1, 2002

Received in final form April 23, 2002


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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