Departments of 1 Biomedical Engineering, 2 Pediatrics, and 3 Medicine, Northwestern University, Evanston 60208; 4 Veterans Affairs Chicago Health Care System-Lakeside Division, Chicago, Illinois 60611; and 5 Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163
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ABSTRACT |
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Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E2 (PGE2) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE2 stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.
16HBE14o cells; prostaglandin receptors; iloprost; enprostil; prostaglandin G/H synthase
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INTRODUCTION |
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THE AIRWAY EPITHELIUM is the primary defense for the lung against inflammatory and physical insults that result from bacterial and viral infections, pollutants, and mechanical trauma. Injury to the epithelium can increase the sensitivity of the underlying airway smooth muscle to bronchoactive agents. The rapid and accurate regeneration of a continuous epithelium is critical in restoring normal lung function, maintaining barriers (7), and limiting airway hyperreactivity (33). The reepithelialization process involves several steps including spreading and migration of cells at the wound edge into the denuded surface and, eventually, proliferation to repopulate the wound site (44, 45). Each step may be regulated by external factors such as growth factors, extracellular matrix components, or eicosanoids.
Eicosanoids such as prostaglandins (PGs), prostacyclin
(PGI2), and thromboxane are the end products of
cyclooxygenase (COX) metabolism of arachidonic acid (AA) and are
actively secreted by airway epithelial cells (AECs) (3).
Prostanoids are involved in the wound closure process in a broad
variety of tissues including corneal endothelium (16),
keratinocytes (18), fibroblasts (18, 25),
skin (39), and intestinal epithelium (46). In particular, PGE2, which has been demonstrated to be the
predominant eicosanoid produced by guinea pig tracheae
(4), rat tracheal epithelial cells (13),
feline tracheal epithelial cells (35), and a cell line of
human airway epithelial cells (Calu-3) (35), has been
shown to stimulate corneal endothelial cell migration (15), murine fibroblast proliferation (25),
and rat glomerular epithelial cell proliferation (6) in
models of wound healing. The receptors for PGE2 have been
classified into four pharmacological subtypes called E-prostanoid (EP)
1, EP2, EP3, and EP4 based on their responsiveness to selective
agonists and antagonists (5, 28). The EP receptors mediate
a range of biological activities including contraction and relaxation
of smooth muscle, inflammatory mediator release, and neurotransmitter
release (5, 28). PGE2 and other prostanoids
have also been associated with the stimulation of wound closure
observed with growth factors such as epidermal growth factor (EGF)
(15, 46), hepatocyte growth factor (37, 46),
and transforming growth factor- (46).
In most cells, the conversion of AA to prostanoids is catalyzed by the COX enzyme isoform COX-1, although several cell types use the isoform COX-2 for AA conversion when stimulated with cytokines or growth factors. However, several studies of pulmonary epithelium (1, 11, 24) have suggested that COX-2 is the constitutive and dominant isoform in these cells. Through a different pathway, the lipoxygenases convert AA to hydroperoxyeicosatetraenoic acids (HPETEs), which are rapidly converted to leukotrienes and lipoxins. Lipoxygenase products may alter cell growth (2, 22, 36), stimulate chemotaxis (27, 43), or enhance adhesion (14), all of which are important in the wound repair process. Importantly, Moreno (25) found that wounding of cell monolayers increases AA mobilization and eicosanoid production, suggesting that AA and its metabolites may be important in modulating the adhesive properties of cells.
In this study, we examined the role of prostanoids, in particular
PGE2, in the regulation of airway epithelial wound closure with primary cat and human tracheal and bronchial epithelial cells and
an immortalized line of airway epithelium, 16HBE14o. We
show that COX products are essential for rapid wound repair in AECs.
Both isoforms of COX appear to function in this process, whereas
5-lipoxygenase products are not involved in these cells. We also show
that the prostanoid PGE2 provides a significant stimulation of wound closure and that its effects are likely mediated by the EP1
and EP4 receptor subtypes. The stimulation of closure by prostanoid metabolites occurs immediately after wounding and may stimulate spreading and migration.
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METHODS |
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Materials. Phosphate-buffered saline (PBS), minimum essential medium (MEM), Ham's F-12 medium, fetal bovine serum (FBS), gentamicin, trypsin-EDTA, and nonessential amino acids were obtained from GIBCO BRL (Life Technologies, Grand Island, NY). Basal epithelial growth medium was obtained from Clonetics (San Diego, CA). Collagenase was obtained from Worthington Biochemical (Freehold, NJ). PGE2, NS-398, and valeryl salicylate were purchased from Cayman Chemical (Ann Arbor, MI). Iloprost (EP1 agonist), enprostil (EP3 agonist), AH-6809 (EP1 antagonist), and AH-23848B (EP4 antagonist) were a gift from Dr. Steve White (University of Chicago, Chicago, IL). BAY X 1005 [inhibitor of 5-lipoxygenase activating protein (FLAP)] was from Bayer Pharmaceuticals, and A-79175 (inhibitor of 5-lipoxygenase) was from Abbott Laboratories (North Chicago, IL). All other chemicals were purchased from Sigma (St. Louis, MO).
Cell culture. Cat tracheal epithelial (CTE) cells were isolated from healthy cats (courtesy of Dr. Robert TenEick, Dept. of Molecular Pharmacology, Northwestern University, Evanston, IL) euthanized with an intravenous injection of pentobarbital sodium (Abbott Laboratories). The isolation has previously been described in detail (35). Briefly, the trachea was dissected from the surrounding tissue, cut longitudinally, and pinned open on a Styrofoam mat. Strips of epithelium pulled away from the underlying tissue were rinsed two to three times in sterile PBS containing antibiotics and were then placed in a solution of 0.02% (wt/vol) type II collagenase, 5 mM dithiothreitol, and 200 U/ml of DNase in Ca2+- and Mg2+-free PBS containing antibiotics at 37°C. After 60 and 120 min, the cell suspension was removed and washed twice in CTE cell medium (Ham's F-12 medium with 10 µg/ml of insulin, 7.5 µg/ml of endothelial cell growth supplement, 0.5 µg/ml of transferrin, 0.4 µg/ml of hydrocortisone, 2 µg/ml of triiodothyronine, 1% antibiotic-antimycotic solution, and 2% sodium bicarbonate). Cells were seeded on 12-well plates, 2.5-3 × 105 cells/well, and cultured in CTE cell medium with 10% FBS (200,000 cells/well at confluence). The medium was changed every 2 days, and the cells were used in experiments on day 6 or 7 of culture.
AECs transformed with the SV40 virus (16HBE14oExperimental protocol. Cells were grown to confluence on plastic 12-well plates, and linear wounds with a width of ~500 µm were produced by scraping the monolayers with a pipette tip across the diameter of the well. Before the initiation of the experiment, the cells were rinsed once with PBS to remove cellular debris. Two milliliters of complete growth medium were added to the wells; this medium was not removed during the course of the experiment unless otherwise indicated. Indomethacin, AH-23848B, and PGE2 were dissolved in ethanol before dilution; A-79175 (30 µM), BAY X 1005 (30 µM), NS-398 (10 µM), and valeryl salicylate (500 µM) were dissolved in DMSO before dilution (wound healing was not affected by the low concentrations of ethanol and DMSO; data not shown). Optimal concentrations of EP agonists and antagonists for maximal stimulus in trachealis were used (29), and COX-1, COX-2, and 5-lipoxygenase inhibitors were used at concentrations optimal for inhibition as suggested by Asano et al. (1). Images were obtained at the initial time of wounding and at various times up to 48 h after wounding. Images of the wounds in all wells were obtained at two points in each well; measurements from these two images were averaged, and the results are presented as a single data point for that experiment.
Imaging the wound.
Images of the wounds were collected at specified times with a Nikon
Diaphot 300 inverted microscope equipped with a Hamamatsu integrating
charge-coupled device camera, an Argus-20 real-time digital image
processor, and a pentium DataStor computer with a frame grabber (Fryer,
Huntley, IL). Images were analyzed with the Metamorph image analysis
program (Universal Imaging, West Chester, PA). After the image was
acquired, it was converted from pixels to micrometers with a
calibration image. With the use of Metamorph, an outline of the wound
was manually drawn, and the mean wound width (W) was
tabulated by a Metamorph-defined parameter. W was given by
W = 0.25[P
],
where P and A are the perimeter and
area, respectively, of the wound in the image. This equation is derived from the relationship between length, width, area, and perimeter. Data
are expressed as a percentage of the time 0 wound width in order to normalize variability in wounding from well to well and experiment to experiment, although initial wounds of similar size were
consistently observed.
Immunoblotting.
Before investigation, cells were washed twice with serum-free medium,
and 2 ml/well of fresh medium were added for the duration of the
experiment. After 24 h, cells were lysed with homogenization buffer (100 mM NaCl, 50 mM potassium phosphate buffer, pH 7.1, 2 mM
EDTA, 2 mM glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml of
soybean trypsin inhibitor, 1 µg/ml of leupeptin, and 1 mM
dithiothreitol). Cells were scraped off the membranes with a rubber
policeman, and lysates were collected and stored at 80°C for
subsequent COX analysis. The protein concentration in the cell lysates
was determined with the BCA protein assay from Pierce (Rockford, IL).
Statistical analysis. Data are means ± SE for the indicated number of experiments (n). Wound widths from two peripheral regions of each static well were averaged, and the mean was treated as a single data point. Comparisons between mean values were made with a repeated-measures ANOVA and Tukey's modified t-test (the Bonferroni criterion). A P value < 0.05 was considered significant. All statistical comparisons were performed with the SigmaStat program (Jandel Scientific, San Rafael, CA).
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RESULTS |
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Inhibition of COX limits wound closure.
Based on recent reports (8, 10, 26, 38) that AA
metabolites contribute to epithelial repair and proliferation in other
organs, we hypothesized that COX products may regulate wound healing in
AECs. To investigate this role, we inhibited COX activity with
indomethacin. Figure 1 shows a
dose-dependent decrease in the rate of wound closure in CTE (Fig.
1A) and 16HBE14o (Fig. 1B) cells.
Indomethacin also inhibited wound closure in NHBE cells (Fig.
1C). Similar results were observed with a cell line of human
tracheal cells (Calu-3; data not shown). High concentrations of
indomethacin may be toxic to some cell types; when CTE cells were
treated with 3 µM indomethacin, some cells lifted off the plates and
appeared more rounded after 10 h in culture. This did not occur in
16HBE14o
or NHBE cells or at lower doses of indomethacin
in CTE cells.
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PGE2 stimulates wound closure.
Because PGE2 is one of the major eicosanoids produced by
AECs (4, 13, 35), we hypothesized that application of
exogenous PGE2 could overcome the inhibitory effect of
indomethacin. Figure 3 shows that the
addition of exogenous PGE2 was sufficient to offset the
inhibition of closure caused by indomethacin in CTE (Fig.
3A) and 16HBE14o (Fig. 3B) cells.
Inhibition of all COX products nearly abrogated wound closure (Fig. 1),
whereas exogenous PGE2 alone reversed the inhibition of
wound closure in this system (Fig. 3). Also note that, as shown in Fig.
3A, wound closure occurred in the presence of 3 µM
indomethacin when PGE2 was present in the medium, suggesting that the inhibition shown in Fig. 1 was not likely due to
the toxicity of indomethacin but rather to the absence of
PGE2. Although it is possible that other prostanoids may
contribute to the stimulation of wound healing, we did not investigate
them in this study.
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PGE2 acts through the EP1 and EP4 receptors.
To determine which receptors were stimulated by PGE2
to promote wound closure, wounded monolayers of
16HBE14o cells were treated with agonists and antagonists
of EP1, EP3, and EP4 receptors. Figure 5
shows that both the EP1 receptor antagonist AH-6809 and the EP4
receptor antagonist AH-23848B significantly inhibited wound repair
compared with that in untreated control monolayers. The EP1 agonist
iloprost stimulated wound closure to the same extent that exogenous
PGE2. The EP3 agonist enprostil did not accelerate closure
(Fig. 5). Antagonists for EP2 and EP3 receptors are currently not
available commercially nor is a receptor agonist for EP4.
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Time course of PGE2 and indomethacin effects.
To investigate whether the effects of COX inhibition on wound closure
were due to initial events in the process, the wounded monolayers were
treated with indomethacin at the beginning of the time course followed
by removal of indomethacin and replacement with unsupplemented
medium. Control experiments that involved the switching of the
medium demonstrated no effect on wound closure due to the addition of
fresh medium (data not shown). When indomethacin was replaced with
medium after only 2 h, the inhibition of wound closure was nearly
the same as when indomethacin was applied for the entire time (Fig.
7A). With longer application
(4 or 6 h), there was no difference observed. These results
suggest that the role of COX metabolites may be most critical in the
early stages of repair. Proliferation does not begin in AECs for
several hours. In 16HBE14o cultures, incorporation of
5-bromo-2'-deoxyuridine into cells was not seen until after 10 h
(data not shown). This indicates that in the absence of significant
proliferation at the early stages, indomethacin inhibits spreading and
migration, the only other processes available for wound closure.
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DISCUSSION |
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In this study, we found that COX metabolites of the AA cascade can regulate wound closure in cultured AECs. The prostanoid PGE2 was shown to accelerate wound closure, and the stimulation occurred immediately on exposure to PGE2. The effects of PGE2 were shown to stimulate closure through both the EP1 and EP4 receptor subtypes. Inhibition of the synthesis of leukotrienes by either the 5-lipoxygenase inhibitor A-79175 or the FLAP inhibitor BAY X 1005 had no effect on AEC wound closure (Fig. 2C).
Treatment with indomethacin to block COX-1 and COX-2 resulted in a
significant decrease in wound closure in CTE, 16HBE14o,
and NHBE cells (Fig. 1), and both isoforms of COX were shown to be
involved in AEC repair (Fig. 2). Despite a recent report (1) that COX-2 may be the dominant isoform in these cells, we have shown that both isoforms are important in mediating wound closure in these cells. Although 5-lipoxygenase was not shown to be of
importance for early AEC repair in our system, a previous report
(22) has suggested that cysteinyl leukotrienes stimulate growth of human AECs in culture and thus may play a role in the later
proliferation response. It is also possible that metabolites of the 12- or 15-lipoxygenase cascade may play a role in AEC wound healing, but
this was not investigated in this study.
AA and its metabolites play important roles in cell protection, growth, angiogenesis, and extracellular matrix production. Several studies have demonstrated the importance of AA metabolites in these roles pertaining to wound closure. In the stomach, ulcers are often exacerbated by treatment with aspirin or other nonsteroidal anti-inflammatory drugs (COX inhibitors). In rats with gastric ulcers, treatment with indomethacin or aspirin significantly delayed ulcer healing through an inhibition of mucosal regeneration (32). In corneal epithelium, both lipoxygenase (10) and COX (26) inhibitors significantly retarded wound closure. Together with these studies, our results suggest that restitution of airway epithelium after injury may be significantly impaired in patients treated with COX inhibitors.
Besides the autocrine role of prostanoids in wound healing, others have proposed that prostanoids act in a paracrine fashion in wound repair. Takahashi et al. (37) demonstrated that PGs produced by gastric epithelium stimulated hepatocyte growth factor expression by the gastric fibroblasts and that the proliferative effects of PGE1 were inhibited by an anti-hepatocyte growth factor antibody in a coculture model. In contrast, the migration of human corneal endothelial cells was stimulated by EGF, but stimulation occurred only when PGE2 was present in the medium (15). EGF has also been shown to stimulate migration of guinea pig AECs (19), but the potential role of prostanoids in this mechanism has not been investigated. Waters and Savla (41) previously demonstrated that keratinocyte growth factor (KGF) significantly accelerates wound closure in AECs, but in that study, they did not report a role for prostanoids in the stimulation. However, we have performed AEC wound healing assays with KGF in the presence of indomethacin and found that KGF stimulated wound closure regardless of the concentration of indomethacin (data not shown). Thus although the inhibition of COX activity significantly retarded wound closure, the effects of KGF were independent of COX activity. This suggests that prostanoids (e.g., PGE2) and KGF may stimulate wound closure initially through similar but independent pathways.
An important regulatory factor in spreading, migration, and
proliferation is adhesion to the substrate and to other cells. Adhesion
to a solid substrate results in the release of AA, AA mobilization, and
AA metabolism through COX in 3T6 fibroblasts (23).
Furthermore, the extent of AA release and metabolism is dependent on
adhesive interactions with neighboring cells, the level of confluence,
and the extent of cell-cell adhesion (23). In a separate
study by the same group (25), wounding of 3T6 fibroblasts
resulted in the release of AA, which may be a stimulus for enhancing
adhesion after wounding. When a COX-2 inhibitor was used in this wound
model, closure was significantly inhibited despite the wound-induced
release of AA. This study suggests that prostanoids might be
immediately involved in regulation of adhesion and spreading after
wounding. Wound repair of 16HBE14o cells was recently
reported to be dependent on adhesion to the substrate via the
1-integrin subunit (42), but to our
knowledge, there have been no reports demonstrating a connection
between AEC adhesion via specific integrins and prostanoid metabolism.
Several studies have demonstrated the importance of COX metabolites in cell migratory events. In corneal endothelial cells, indomethacin treatment increased spreading (17) and caused a stellate-shaped elongation of the cells (30). Similar results were found in bovine aortic endothelium, where exogenous AA induced cellular elongation (34). Blocking COX metabolism of AA in corneal endothelial cells was shown to promote cell translocation through formation of long actin stress fibers (17), and endogenous synthesis of PGE2 was found to be necessary for the maintenance of normal polygonal endothelial shape (30). Increased PGI2 synthesis in these cells was accompanied by less spreading, fewer stress fibers, and rounded cells (17); however, treatment with PGE2 significantly increased migration (15), indicating that different prostanoids have variable effects on spreading and migration.
Because PGE2 is a major metabolite of these cells
(35), we hypothesized that application of exogenous
PGE2 could overcome the inhibitory effect of indomethacin.
Indeed, addition of exogenous PGE2 was able to offset the
inhibition of closure caused by indomethacin in CTE and
16HBE14o cells (Fig. 3). In addition to the restorative
effect of adding PGE2 to indomethacin-treated cells,
exogenous PGE2 produced a dose-dependent acceleration of
wound closure in these cells (Fig. 4). It is interesting to note that
the maximal stimulatory effect of PGE2 occurred at much
lower concentrations (0.001-0.1 µg/ml) in the primary cultures
of CTE and NHBE cells, whereas much higher levels of PGE2
were required to stimulate the transformed cell line
16HBE14o
(Fig. 4). This may reflect adaptation of
16HBE14o
cells to the cell culture environment or an
effect of the cellular transformation. Alternatively, these differences
may be related to differences in expression of receptor subtypes or
differences in affinity for PGE2. Such differences have
been observed across species (28) and may also occur in
transformed cells. These potential variations in expression and
affinity may lead to changes in downstream mechanisms that regulate
wound closure such as cell spreading and migration. Thromboxane and
PGI2 also are produced by AECs (35) and may
provide stimulatory effects in wound closure, but these were not
examined in the present study.
Several studies have investigated other mechanisms by which AA metabolites exert their stimulatory effects. Stable analogs of PGI2 promoted cell proliferation and cytokine production in human keratinocytes and fibroblasts (18). In cultured human fibroblasts, PGI2 stimulated wound closure through enhanced production of urokinase-type plasminogen activator (12). Urokinase-type plasminogen activator is involved in tissue remodeling and cell migration during both normal and pathological conditions (12). In ethanol-treated gastric cells, PGE2 prevented ultrastructural changes such as disruption of cell membranes and swelling of mitochondria (20). According to Konda et al. (21), PGE2 can prevent damage through a diacylglycerol/protein kinase C-mediated pathway, whereas other studies (18, 31) have shown that eicosanoids exert their protection through cAMP-mediated events.
We have shown that in AECs, the EP1 and EP4 receptor subtypes are important in transducing the stimulus from PGE2 (Figs. 5 and 6). Treatment with the EP3 receptor agonist enprostil resulted in neither stimulation nor inhibition of closure, and we were unable to determine the importance of the EP2 receptor subtype. It should also be pointed out that because of the limited specificity of the receptor agonists and antagonists, our studies suggest but do not definitively prove the involvement of the receptor subtypes. For example, although AH-6809 has been used previously as an antagonist for the EP1 receptor, it also binds to EP2 and PGD2 receptors (28). Also, iloprost binds to PGI1 receptors in addition to EP1 receptors, and this may account for the lack of complete inhibition in the presence of AH-6809 shown in Fig. 6B. Each receptor subtype manifests its effects through changes in different cellular pathways; whereas the EP1 receptor exerts its effects through increasing intracellular Ca2+, the EP2 and EP4 receptors increase intracellular cAMP levels and the EP3 receptor transduces its effects through a decrease in cAMP (5). It is possible that the EP2 receptor is also important in AEC repair because the EP4 receptor was shown to mediate the effects of PGE2 (Fig. 6), and both the EP2 and EP4 receptors stimulate through increases in cAMP.
We investigated the time course of application of the COX metabolites to determine which processes were maximally inhibited or stimulated. Because changing the medium at each time point was not responsible for acceleration of wound closure, we were able to investigate changes due to staggered indomethacin and PGE2 application. Sustained application of a high concentration of indomethacin resulted in the largest inhibition of closure (Fig. 7A). However, when indomethacin was replaced with medium after 2 h, only a modest recovery was observed. There was no recovery observed in wells receiving indomethacin for 4 or 6 h. This indicates that with a large concentration of indomethacin, early removal of COX metabolites sustains the inhibition of wound closure. COX metabolites must be partially responsible for stimulating and maintaining early-response processes in wound closure.
We also investigated the effects of early removal of the stimulatory prostanoid PGE2. Application of PGE2 for times as short as 2 h resulted in an overall enhancement of wound closure (Fig. 7B). Wound closure in wells that received PGE2 for longer times (4-6 h) was not significantly decelerated after PGE2 was replaced with medium. These results suggest that PGE2 can stimulate early-response wound closure processes such as spreading and migration. If sustained for a certain amount of time, early prostanoid application may be sufficient for overall stimulation of wound closure. Similar results were found for dermal wounds in a study by Talwar et al. (39) that suggested that exogenous PGE2 would be most beneficial in the early stages of wound healing.
To further test the hypothesis that PGE2 stimulation of closure occurs early, we applied unsupplemented 10% serum medium to wounded monolayers and, after 2, 4, or 6 h, replaced the medium with 10 µg/ml of PGE2 (data not shown). Wounds in untreated wells followed the same time course until switched to PGE2-supplemented medium, at which time the lines dropped to follow the curve of the sustained application treatment. When PGE2 was applied after 2 h, there was no difference compared with the closure in wells with sustained PGE2. In wells that received PGE2 later (4 or 6 h), there was an immediate acceleration in wound closure. However, after 12 h, wounds had not closed to the same extent as in wells that had received PGE2 continuously. It is possible that PGE2 provokes an immediate response, stimulating spreading and/or migration.
We also investigated the effect of early inhibition of COX metabolites with indomethacin followed by supplementation with PGE2 alone (Fig. 7C). Addition of PGE2 after indomethacin treatment resulted in significantly greater wound closure. Wounds treated initially with indomethacin and later with PGE2 were not significantly impeded in closure compared with those in the untreated control monolayers after 12 h. Early inhibition of COX, as shown in Fig. 7A, did inhibit early responses to wounding (spreading and/or migration); however, some of this inhibition was recovered with the application of PGE2.
From these data, we can conclude that PGE2 is important at the onset of wound closure. Early inhibition with indomethacin resulted in a sustained inhibition of closure, and early application of exogenous PGE2 resulted in a sustained acceleration of closure. These changes occurred early, suggesting that spreading and migration were the targets of stimulation. This is the first report to demonstrate the importance of prostanoids in AEC wound closure. These results have potential importance for the care of patients being treated with aspirin or other nonsteroidal anti-inflammatory drugs or in patients with asthma.
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ACKNOWLEDGEMENTS |
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We thank Dr. Steve White (University of Chicago, Chicago, IL) for helpful discussions regarding these experiments.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant 1R01-HL-64981 and the Cornelius Crane Asthma Center.
U. Savla was supported by a National Science Foundation Graduate Fellowship.
Address for reprint requests and other correspondence: C. M. Waters, Dept. of Physiology, Univ. of Tennessee, Memphis, 894 Union Ave., 426 Nash, Memphis, TN 38163 (E-mail: cwaters{at}physio1.utmem.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.
Received 17 July 2000; accepted in final form 16 October 2000.
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