1 Pulmonary and Critical Care Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198-5125; 2 Pulmonary Division, Internal Medicine, Seoul Adventist Hospital, Seoul 130-650, Korea; and 3 Department of Laboratory and Pulmonary Medicine, Tokyo University, School of Medicine, Tokyo 113-8655, Japan
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ABSTRACT |
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The controlled accumulation of
fibroblasts to sites of inflammation is crucial to effective tissue
repair after injury. Either inadequate or excessive accumulation of
fibroblasts could result in abnormal tissue function. Prostacyclin
(PGI2) is a potent mediator in the coagulation and
inflammatory processes. The aim of this study was to investigate the
effect of PGI2 on chemotaxis of human fetal lung
fibroblasts (HFL-1). Using the blind well chamber technique, we found
that the PGI2 analog carbaprostacyclin (106
M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 µg/ml)
58.0 ± 13.2% (P < 0.05) and to platelet-derived
growth factor (PDGF)-BB (10 ng/ml) 48.7 ± 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin
inhibits both directed and undirected migration. The inhibitory effect
of the carbaprostacyclin was concentration dependent and blocked by the
cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that
a cAMP-PKA pathway may be involved in the process. Two other
PGI2 analogs, ciprostene and dehydro-15-cyclohexyl
carbaprostacyclin (both 10
6 M), significantly inhibited
fibroblast migration to fibronectin. In summary, PGI2
appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB.
Such an effect may contribute to the regulation of fibroblasts in wound
healing and could contribute to the pathogenesis of diseases
characterized by abnormal tissue repair remodeling.
prostaglandin I2; protein kinase; tissue repair; inflammation
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INTRODUCTION |
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AN IMPORTANT PART of the wound healing process is a balanced migration of fibroblasts from neighboring connective tissue to the site of injury. Whereas repair can be defective if fibroblast recruitment is inadequate, excess accumulation of fibroblasts can lead to the disruption of normal tissue function (8, 12, 27, 29). Mediators that regulate fibroblast movement, therefore, could be important targets for therapeutic intervention.
Prostacyclin (also known as PGI2), a metabolite of arachidonic acid, is a short-lived and short-acting prostaglandin (20). Produced by vascular endothelial cells, prostacyclin has been shown to modulate a variety of physiological responses, including vascular permeability (3, 21, 31), vasodilation, antiplatelet aggregation, decreased pulmonary vascular resistance, and inhibition of smooth muscle cell proliferation (19, 20). Prostacyclin receptor (IP) agonists have been shown to bind with the guanine nucleotide regulator protein (G protein)-coupled receptor and activate adenylate cyclase and elevate intracellular cAMP in human cells (6, 23).
The current study was designed to evaluate the effect of three stable analogs of prostacyclin, carbaprostacyclin, ciprostene, and dehydro-15-cyclohexyl carbaprostacyclin (DHCC), on fibroblast chemotaxis. Human plasma fibronectin and platelet-derived growth factor (PDGF)-BB were used as chemoattractants. The mechanism by which these analogs might exert their effect also was evaluated.
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MATERIALS AND METHODS |
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The prostacyclin analogs carbaprostacyclin, ciprostene, and DHCC
were purchased from Cayman Chemical (Ann Arbor, MI). The protein kinase
A (PKA) inhibitor KT-5720 was purchased from Calbiochem (San Diego,
CA). KT-5720 (102 M) was dissolved in DMSO, and
carbaprostacyclin (10
3 M), ciprostene (10
3
M), and DHCC (10
3) were separately dissolved in ethanol.
PDGF-BB, purchased from R&D (Minneapolis, MN), was dissolved in 4 mM
HCl with 0.1% BSA at 10 µg/ml. Tissue culture supplements and media,
except fetal calf serum (FCS), were purchased from Invitrogen (Grand
Island, NY). FCS was purchased from Biofluid (Rockville, MD).
Human lung fibroblasts. Human fetal lung fibroblasts (HFL-1) were obtained from 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; Invitrogen) and supplemented with 10% FCS, 50 U/ml penicillin G sodium, 50 µg/ml streptomycin sulfate (penicillin-streptomycin, Invitrogen), and 1 µg/ml amphotericin B (Parma-Tek, Huntington, NY) in a humidified atmosphere at 37°C with 5% CO2. Fibroblasts were routinely passaged every 4 or 5 days, and cells between passages 13 and 20 were used in all experiments. Confluent fibroblasts were removed from culture dishes by treatment with 0.05% trypsin in 0.53 mM EDTA and resuspended in serum-free DMEM.
Human fibronectin. Gelatin-sepharose affinity chromatography was used to prepare fibronectin from human plasma (11). Fibronectin was further purified by heparin-agarose affinity chromatography and eluted with 500 mM NaCl.
Fibroblast chemotaxis and chemokinesis. HFL-1 chemotaxis was assessed by the Boyden blind well chamber technique (7) using 48-well chambers (Nucleopore, Cabin John, MD). A chemoattractant, fibronectin or PDGF-BB, was placed in the bottom chamber. The wells of the chamber were separated with an 8-µm pore filter (Nucleopore, Pleasanton, CA), which was coated with 0.1% gelatin (Bio-Rad, Hercules, CA). Fibroblasts (1 × 106 cells/ml in DMEM without serum) were loaded into the upper well of the chamber with the desired concentration of prostacyclin analog and/or other additive. The chamber was then incubated at 37°C in a moist, 5% CO2 atmosphere. Except with the time course experiments, chambers were incubated for 6 h, after which the cells above 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. We assessed migration by counting the number of cells in five high-power fields under a light microscope.
A "checkerboard" analysis was performed to distinguish chemotaxis from chemokinesis. This system allows measurement of directional cell movement up a chemoattractant gradient (chemotaxis, the vertical columns, presented as a table), as well as undirected migration in the absence of a gradient (chemokinesis, the diagonal columns).Statistical analysis. We confirmed results by repeating experiments on separate occasions at least three times, each with triplicate chemotaxis wells. Samples with multiple comparisons were analyzed for significance by ANOVA. Pair comparisons were analyzed by independent two-sample t-tests. For multiple comparisons, critical values were adjusted by Tukey correction. Summary data are expressed as means ± SE. Values were considered statistically significant if P < 0.05.
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RESULTS |
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Fibronectin directed HFL-1 chemotaxis concentration dependently.
When added to HFL-1, carbaprostacyclin (106 M) inhibited
fibronectin-directed migration at all concentrations of fibronectin
assessed (Fig. 1). Carbaprostacyclin's
inhibition of migration directed by fibronectin (20 µg/ml) was
concentration dependent, reaching significance at 10
7 M
(Fig. 2). Inhibition of chemotaxis was
detectable at the highest concentrations after 4 h of incubation.
After 6 h, the number of migrating cells did not increase in the
presence of carbaprostacyclin but was still increasing in the control
(Fig. 3). Ciprostene and DHCC (both
10
6 M), other prostacyclin analogs, also significantly
inhibited fibroblast migration toward human fibronectin (Fig.
4). This inhibitory effect could not be
attributed to cytotoxicity, since cell viability was unaffected by up
to 10
5 M of the prostacyclin analogs examined, as
detected by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (data not shown).
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To examine whether the carbaprostacyclin effects were specific to
fibronectin, we also used PDGF-BB as chemoattractant for HFL-1
migration. PDGF-BB stimulated HFL-1 chemotaxis until the prozone level
for the chemoattractant was reached (100 ng/ml). Carbaprostacyclin
(106 M) added with HFL-1 inhibited migration toward a
range of PDGF-BB concentrations (Fig.
5A). Carbaprostacyclin
inhibition of PDGF-BB-induced migration of HFL-1, moreover, was
concentration dependent over a range similar to that which inhibited
chemotaxis toward fibronectin (Fig. 5B).
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To determine whether carbaprostacyclin inhibited chemokinesis, we used
checkerboard analysis: varying concentrations of fibronectin were
placed both above and below the filter in the blind well chamber.
Fibroblasts with or without carbaprostacyclin were placed in the upper
wells. The number of migrated cells increased when a gradient was
present (vertical columns), indicating chemotaxis. Similarly, the
number of cells migrating increased as the concentration of fibronectin
increased when the gradient was absent (diagonals), indicating
chemokinesis (Table 1). The addition of
carbaprostacyclin to HLF-1 fibroblasts inhibited both chemotaxis and
chemokinesis.
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The IP receptor is believed to elevate cAMP, followed by activation of
PKA. To determine whether the carbaprostacyclin effect on fibroblast
chemotaxis toward fibronectin occurs by way of PKA, we treated HFL-1
cells with the PKA inhibitor KT-5720 (107 M) for 1 h
before harvesting for chemotactic evaluation. KT-5720 alone had no
effect on chemotaxis toward fibronectin. In contrast, the PKA inhibitor
attenuated the carbaprostacyclin inhibition of fibroblast chemotaxis to
fibronectin (Fig. 6).
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DISCUSSION |
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The current study demonstrates that the prostacyclin analog carbaprostacyclin is capable of inhibiting fibroblast chemotaxis to both human fibronectin and PDGF-BB in a concentration-dependent manner. The inhibitory effect on fibroblast chemotaxis was further confirmed with other prostacyclin analogs, ciprostene and DHCC. Checkerboard analysis verified that both chemotaxis and chemokinesis were affected. Moreover, carbaprostacyclin's effect was blocked by KT-5720, an inhibitor of PKA, suggesting that inhibition is mediated by the PKA pathway.
The accumulation of fibroblasts is an important aspect of tissue repair after injury. This accumulation can occur through both chemotactic recruitment and proliferation within the wound. Several mediators, functioning as either stimulators or as inhibitors, can regulate these processes (24-26). In this regard, fibronectin, a multifunctional glycoprotein, and PDGF-BB are both potent fibroblast chemoattractants (24, 25), as well as contributors to fibroblast proliferation (4, 13). Both have been suggested to play important roles in normal wound healing and in the development of fibrotic scars. Inhibitors of fibroblast recruitment and proliferation have also been described, including cigarette smoke (22) and prostaglandin E2 (15). It is likely that whether repair processes result in restoration of normal tissue function or in excessive accumulation of fibroblasts with resulting scar depends on the balance between inhibitory and stimulatory signals. The current study demonstrates that prostacyclin can function as an inhibitor of fibroblast chemotaxis directed by either fibronectin or PDGF-BB.
Prostacyclin is an arachidonic acid metabolite released from a variety of cell types including mast cells, endothelial cells, and fibroblasts (28, 30, 31). Prostacyclin is a potent regulator of vascular functions. Its vascular effects are generally antagonized by thromboxane A2 (TxA2). The balance between prostacyclin and thromboxane, therefore, has been suggested to be an important determinant of coagulability (5, 17). This balance may also contribute to fibroblast recruitment to the site of injury. Although without chemotactic activity on its own, the TxA2 agonist U-46619 has been shown to potentiate fibroblast chemotaxis toward fibronectin (16).
The production of prostacyclin by endothelial cells is believed to play an important role in regulating acute vascular events. Physiological roles played by prostacyclin production from fibroblasts is less clearly defined. The current study suggests that prostacyclin may also play a role in the balance of mediators that regulate mesenchymal cell participation in repair responses.
Prostacyclin is capable of interacting with IP receptors causing activation of adenylate cyclase and increased levels of cAMP (6, 23). Carbaprostacyclin, ciprostene, and DHCC are stable analogs of prostacyclin and capable of inhibiting ADP-induced human platelet aggregation (1, 2, 14). In the current study, the effect of carbaprostacyclin on fibroblast migration was blocked by a PKA inhibitor, suggesting that the inhibitory effect of carbaprostacyclin is mediated through cAMP elevation, followed by activation of PKA. Several other agents that increase cAMP also inhibit fibroblast chemotaxis, including PGE2, forskolin, isoproterenol, and dibutyryl-cAMP (10, 15). This is consistent with a general mechanism by which cAMP may function as a downregulator of fibroblast recruitment. Interestingly, increasing intracellular levels of cAMP inhibits other "profibrotic" responses, including fibroblast proliferation (9), matrix production (32), and fibroblast-induced contraction of three-dimensional collagen gels (18), suggesting that cAMP may function as a common mediator serving to restrict profibrotic stimuli.
The current study, in summary, demonstrates that prostacyclin analogs, particularly carbaprostacyclin, can inhibit fibroblast chemotaxis and chemokinesis. Through such a mechanism, prostacyclin could contribute to the modulation of profibrotic stimuli and, therefore, play an important role in controlling fibrotic responses.
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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 C. Tourek.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. I. Rennard, Pulmonary and Critical Care Medicine, Univ. of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 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.
10.1152/ajplung.00432.2001
Received 5 November 2001; accepted in final form 21 March 2002.
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