Prostacyclin analogs inhibit fibroblast migration

Tadashi Kohyama1, Xiangde Liu1, Hui Jung Kim2, Tetsu Kobayashi1, Ronald F. Ertl1, Fu-Qiang Wen1, Hajime Takizawa3, and Stephen I. Rennard1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (10-6 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (10-2 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibronectin directed HFL-1 chemotaxis concentration dependently. When added to HFL-1, carbaprostacyclin (10-6 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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Fig. 1.   Inhibition of fibroblast chemotaxis by the stable prostacyclin (PGI2) analog carbaprostacyclin. Chemotaxis was performed with or without the addition of carbaprostacyclin (10-6 M). Y-axis: fibroblast chemotaxis expressed as percentage to 20 µg/ml fibronectin without analog; x-axis: concentration of fibronectin. Data shown are means ± SE from 3 separate experiments, each performed in triplicate (*P < 0.05 by t-test at each paired concentration).



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Fig. 2.   Inhibition of fibroblast chemotaxis by the PGI2 analog carbaprostacyclin: concentration dependence. Fibronectin (20 µg/ml) was used as the chemoattractant. Various concentrations of carbaprostacyclin were added to the fibroblasts immediately before the cells were placed in the top wells of the chamber. Y-axis: fibroblast chemotaxis expressed as a percentage of control; x-axis: carbaprostacyclin concentration. Data shown are means ± SE from 3 separate experiments, each performed in triplicate (*P < 0.05 by Tukey procedure).



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Fig. 3.   Inhibition of fibroblast chemotaxis by the PGI2 analog carbaprostacyclin: time course. Fibronectin (20 µg/ml) was used as the chemoattractant. Various concentrations of carbaprostacyclin were added to the fibroblasts in the top wells of the chemotaxis chamber. Cells in the chamber were incubated for designated time periods, then removed for staining and counting. Y-axis: fibroblast chemotaxis expressed as a percentage to 12-h time course without analog; x-axis: time in hours. Data are expressed as means ± SE from 3 experiments, each performed in triplicate (*P < 0.05 by Tukey procedure at each time period).



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Fig. 4.   Inhibition of fibroblast chemotaxis by 3 PGI2 analogs. Fibronectin (20 µg/ml) was used as chemoattractant. Carbaprostacyclin (10-6 M), ciprostene (10-6 M), or dehydro-15-cyclohehexyl carbaprostacyclin (DHCC, 10-6 M) were added to fibroblasts immediately before cells were placed in the top wells. Y-axis: fibroblast chemotaxis as percentage of control; x-axis: PGI2 analogs in the presence of fibronectin. Data shown are means ± SE from 3 experiments, each performed in triplicate (*P < 0.05 by t-test).

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 (10-6 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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Fig. 5.   Inhibition of fibroblast chemotaxis toward platelet-derived growth factor (PDGF) by the PGI2 analog carbaprostacyclin. A: various concentrations of PDGF-BB were used as chemoattractant either with or without the addition of carbaprostacyclin (10-6 M). Y-axis: fibroblast chemotaxis expressed as number of cells migrated/5 high-power fields; x-axis: concentration of PDGF-BB chemoattractant. B: PDGF-BB (10 ng/ml) was used as chemoattractant. Various concentrations of carbaprostacyclin were added to the fibroblasts immediately before cells were placed in the top wells of the chemotaxis chamber. Y-axis: fibroblast chemotaxis expressed as percentage of control; x-axis: carbaprostacyclin concentration. Data shown are means ± SE from 3 experiments, each performed in triplicate (*P < 0.05 by t-test at each paired concentration for A; by Tukey procedure for B).

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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Table 1.   Checkerboard analysis of cell migration stimulated by fibronectin: effect of the prostacyclin analogue carbaprostacyclin

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 (10-7 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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Fig. 6.   Effect of inhibition of PKA on carbaprostacyclin modulation of fibroblast chemotaxis toward fibronectin. Human fetal lung-1 fibroblasts were preincubated with KT-5720 (10-7 M) in monolayer culture for 1 h and then harvested for chemotaxis. Fibronectin (20 µg/ml) was used as chemoattractant. Carbaprostacyclin (10-6 M) was added to fibroblasts in the top wells. Y-axis: fibroblast chemotaxis expressed as percentage of control. Data are means ± SE from 3 experiments, each performed in triplicate (*P < 0.05 by Tukey procedure).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

The authors acknowledge the excellent secretarial support of Lillian Richards and the editorial assistance of Mary C. Tourek.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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