Coordinating epidermal growth factor-induced motility promotes efficient wound closure

Richard C. Kurten,1,4 Parag Chowdhury,1 Ronald C. Sanders, Jr.,5 Laura M. Pittman,2 Laura W. Sessions,2 Timothy C. Chambers,3 Christopher S. Lyle,3 Bradley J. Schnackenberg,2,4 and Stacie M. Jones1,2,4

Departments of 1Physiology & Biophysics, 2Pediatrics, and 3Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, and 4Arkansas Children's Hospital Research Institute, Little Rock, Arkansas; and 5Department of Pediatrics, Shands Children's Hospital, University of Florida, Gainesville, Florida

Submitted 15 January 2003 ; accepted in final form 1 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Wound healing is a response to injury that is initiated to reconstruct damaged tissue. In skin, reepithelialization involves both epithelial cells and fibroblasts and contributes to the reformation of a barrier between the external environment and internal milieu. Growth factors including epidermal growth factor (EGF) play important roles in promoting this process. In the present studies we employed CV-1 fibroblasts in a tissue culture model of reepithelialization to develop strategies for optimizing wound closure stimulated by EGF. We found that EGF enhanced cell motility within 6–8 h of EGF treatment in serum-free medium but wounds failed to close within 24 h. However, if medium on these cultures was exchanged for medium containing serum, cells pretreated with EGF closed new scrape wounds more rapidly than did cells that were not pretreated. These results indicate that serum factors work in concert with EGF to coordinate cell motility for efficient wound closure. Indeed, EGF enhanced the rate of wound closure in the presence of serum, and this effect also persisted for at least 24 h after EGF was removed. This coordination of EGF-induced cell motility was accompanied by an increase in the transient phosphorylation of ERK1 and ERK2. The persistent effects of EGF were blocked by transient exposure to reversible inhibitors of transcription and translation, indicating that the expression of new proteins mediated this response. We propose that EGF-stimulated CV-1 fibroblast motility is coordinated by a serum component that induces cell-cell adhesive properties consistent with an epithelial phenotype, thereby enhancing the reepithelialization process.

wound healing; cell motility


THE RESPONSE OF A TISSUE TO INJURY includes the migration of cells into the denuded area to reconstitute the barrier between the organism and the environment. A similar behavior occurs during embryogenesis when sheets of cells migrate and fuse to generate specific structures (52). Both fibroblasts and epithelial cells participate in wound healing. In skin (28), proliferation and migration by dermal fibroblasts is responsible for the deposition of a collagen-rich matrix. Subsequent differentiation of fibroblasts into myofibroblasts provides tensile forces necessary for wound contraction (15). Early in vitro studies of cell migration in mucosal healing showed that cell migration is largely independent of cell proliferation, is dependent on actin polymerization, and is enhanced by extracellular matrix (31). In small circular wounds in epithelia, a coordinated polymerization of actin in cells at the wound margin forms a "purse string" (22) that begins to pull the cells into the wound area (26). Secondarily, the cells form lamellae that bind the extracellular matrix via {alpha}6-integrins to provide traction forces that pull the cells across the wounded area. Larger scrape wounds in Madin-Darby canine kidney (MDCK) epithelial cell sheets do not employ the actin purse-string contraction strategy but, instead, exhibit a coordinated crawling using lamellipodia whose formation is dependent on Rac and phosphoinositides (13). In this case, coordinated cell migration requires the activities of the small GTPases Rho and Cdc42 (8, 14) and also implies that signals generated at the wound edge are propagated through a distance of several cell widths from the wound margin.

Peptide growth factors provide signals that can enhance wound-healing responses. Epidermal growth factor (EGF), a prototypical growth factor, binds a plasma membrane receptor tyrosine kinase, the EGF receptor (9, 10, 46), and enhances cell proliferation and motility in both fibroblasts and epithelial cells. For example, EGF promotes healing of superficial tongue wounds in mice. The removal of submandibular salivary glands (the major source of salivary EGF) reduced the rate at which wounds healed, and the inclusion of EGF in drinking water restored the rate of wound healing to normal levels (35). Thus EGF appears to play a physiologically relevant role in wound healing in oral epithelium. Another EGF receptor ligand, TGF-{alpha}, enhances the repair of scrape-wounded alveolar epithelial cells (21). Similarly, in cultured guinea pig airway epithelial cells, EGF promotes the repair of scrape-wounded monolayers by stimulating cell migration with no effect on cell proliferation in the wound margin (23).

EGF also accelerates the repair of scrape wounds in human bronchial epithelial cell monolayers (16HBE14o_) (39) and enhances migration in the human bronchial epithelial cell line BEAS-2B (36). However, in the latter example, the wound-healing response is marginal. This contrast in migratory behavior between two different bronchial epithelial cell lines is not unique. In keratinocyte cell lines, EGF induces the dispersion of colonies at low cell density and the closure of scrape wounds in monolayer cultures (30). These contrasting behaviors prompt a question: How is the enhanced motility harnessed to generate the vectorial migration of cells necessary to close a wound? Conversely, how do differences between cell lines or in culture conditions such as cell density contribute to distinct cell behaviors? Although it is generally recognized that epithelial motility is coordinated, it is not well established that fibroblast motility is also coordinated (16, 19). Indeed, uncoordinated fibroblast motility is considered an advantage in studies of cell motility, because the analysis is not complicated by cell-to-cell adhesion (49).

One possible explanation for differences in the behavior of different cell types is that the level of expression of EGF receptors regulates the rate of wound healing by controlling motility. Indeed, in cultured keratinocytes transfected with EGF receptors, a positive correlation between EGF receptor levels and ligand-induced cell motility was demonstrated (30). Furthermore, the delivery of expression vectors encoding human EGF receptors into porcine skin with the use of a gene gun delivery system enhanced wound healing (32). Similarly, in asthmatic bronchiolar epithelium, where epithelial damage is a feature of the disease, EGF receptor expression is elevated compared with normal epithelium (12). Other mechanisms exist whereby erb2–4 (members of the EGF receptor family) are segregated on basolateral surfaces and are inaccessible to a ligand restricted to the apical surface (47). In contrast to the highly organized migration of epithelial sheets, fibroblast migration in wound repair is less coordinated because there are fewer of the cell-to-cell junctions that define epithelial cells (49). Nevertheless, EGF still plays an important role in wound repair by fibroblasts. For example, in senescent fetal lung fibroblasts (3) or in fibroblasts derived from older donors (40), reductions in EGF receptor levels and activation are correlated with reductions in cell motility.

Because EGF receptors are efficiently downregulated within an hour of EGF treatment (24) and wound closure may take many hours to complete, we reasoned that EGF must persistently activate some pathways to promote the motility essential to efficient wound closure. To begin to define these persistent pathways, we performed a quantitative analysis of wound healing in vitro using scrape-wounded African Green monkey kidney cell monolayers expressing endogenous EGF receptors. We used EGF alone or in combination with serum and monitored wound closure. EGF treatment in the absence of calf serum led to an uncoordinated increase in the motility of individual cells with fibroblastic morphology but did not lead to efficient wound closure. By contrast, we found that EGF treatment in the presence of calf serum accelerated the rate of CV-1 cell monolayer wound healing by promoting a coordinated or syncytial migratory behavior more reminiscent of epithelial cells. The promotion of wound healing by EGF persisted even in the absence of exogenous EGF, provided that serum was present. This response was not simply due to the presence of low levels of EGF in serum and was dependent on transcription and translation. Thus EGF promotes the stable expression of a set of proteins mediating enhanced motility, and serum factors promote cell-to-cell adhesion resulting in the coordinated migration of cells necessary for efficient wound closure. Together, our results also define a fibroblast-to-epithelium differentiation pathway complementary to the epithelium-to-mesenchyme transition.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. African Green monkey kidney cells (CV-1) (18) were purchased from the American Type Culture Collection (Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium/F-12 (50:50) supplemented with 15 mM HEPES, 2.5 mM L-glutamine, 5–10% calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B in a 5% CO2 incubator. Cells were treated with recombinant human EGF and/or actinomycin D or cycloheximide purchased from Sigma (St. Louis, MO). Serum-free medium was supplemented with 0.01% bovine serum albumin. IMR-90 cells (34) purchased from American Type Culture Collection were maintained in Eagle's minimum essential medium with 2 mM L-glutamine and Earle's balanced salt solution adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. 16HBE cells were the generous gift of Dr. D. C. Gruenert (University of Vermont, Burlington, VT). These cells are from a SV40 large T-antigen transformed epithelial cell line (16HBE14o_) derived from human bronchial epithelium that retains differentiated epithelial morphology and function (11). The cells were cultured in minimum essential medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 15% fetal calf serum on plastic dishes coated with a solution of bovine collagen I (29 µg/ml), human fibronectin (10 µg/ml), and bovine serum albumin (100 µg/ml) in LHC basal medium (11).

Video microscopy and image analysis. Cells were cultured in Bioptechs (Butler, PA) dishes with 0.5-mm-thick coverglass bottoms fitted to Zeiss Axiovert microscopes (Carl Zeiss, Thornwood, NY). The dishes were maintained at 37°C with a Bioptechs {Delta}TC controller under a 1 liter/min stream of 5% CO2 and 95% air. To prevent evaporation, the medium was overlaid with Serva silicone DC200 fluid (Crescent Chemical, Hauppauge, NY). Images were collected at 6-min intervals for 24 h using a chilled charge-coupled device camera (C5985; Hamamatsu Photonic Systems, Bridgewater, NJ) and a CG-7 frame grabber (Scion, Frederick, MD) installed in a personal computer. The monolayers were "wounded" by being scratched with a sterile, disposable 200-µl plastic pipette tip (Fisher, St. Louis, MO). A macro was written in Scion Image to control image acquisition and storage from two or three microscopes operated in parallel. The macro operated Uniblitz shutters (Vincent Associates, Rochester, NY) so that cultures were only illuminated for image acquisition. Once collected, the series of TIFF files were converted into AVI files using Adobe Premier (San Jose, CA), and plates were generated using Adobe Photoshop (San Jose, CA). Quantitative analysis of wound healing was also performed using a custom Scion Image macro. The denuded area in every file in a series was measured by applying an edge filter and by selecting a range of densities (density slice) corresponding to the denuding area. This processed image was converted into a binary image, and the number of pixels (black) corresponding to the denuded area were counted. The resulting pixel counts were exported to Microsoft Excel (Redmond, WA), converted to areas, and plotted as a function of time. This analysis yields an "offset" attributed to the presence of areas (including spaces between cells) with a density identical to that of the wound. The offset is about 10% of the starting wound area and is noted in the figures. To control for the quality of the automated analysis, the binary images were saved and examined to ensure that the denuded areas were actually being measured. Scion Image is available free of charge at http://www.scioncorp.com/.

To quantify wound closure, we measured the rate of migration of the monolayer. To ensure that the size of the wound, or the size of the microscope field, did not influence the measurements, all experiments were performed in parallel with one control culture and one experimental culture. All wounds were made with the same tool and were similar in width. To permit comparison between different sets of experiments, the instantaneous rate of closure (µm2/h) was calculated using differentials and was normalized to the length of the wound sampled. Depending on the objective used, the length was either the width of the camera frame (310 µm for a x20 objective) for wound closure from only one side of the field or twice the width of the camera frame (620 µm for a x10 objective) if the wounds closed from both the top and the bottom of the field. This calculated parameter corresponded to the rate of movement of the monolayer across the substrate. To reduce the variability in this parameter, it was calculated as a rolling average over nine intervals of 6 min each, and the mean and standard deviation were calculated. A maximal rate of monolayer migration was similarly calculated using linear regression where the rates were constant. Both calculations gave equivalent results over intervals, provided the rate of migration was constant. In EGF-pretreated cells displaying a robust wound-healing response, the linear migration rate was ~20 µm/h. By contrast, in cells cultured in 10% serum, the linear migration rate peaked at ~10 µm/h. Despite the utility of the time-lapse assay, there are limitations in the cost and availability of the instrumentation and, thus, in the number of assays that can be performed simultaneously. Therefore, static assays were also performed in which the wound areas were simply measured after intervals of 6–24 h.

Statistical comparisons were made using SigmaStat for Windows (version 3.0; SPSS, Rochester, MN). Significance was assessed using one-way analysis of variance followed by the Holm-Sidak method of evaluating all pairwise multiple comparisons, using P < 0.05 to define significant differences.

Western blot analysis. CV-1 cells were grown to confluence and serum-starved for at least 24 h. Cells were treated with 10 nM EGF and harvested by scraping in boiling lysis buffer (1% SDS, 10 mM Tris, pH 7.4, and 1 mM sodium orthovanadate, supplemented with protease inhibitors). After the viscosity was reduced via several passages through a tuberculin syringe, lysates were clarified by centrifugation at 13,000 g and the protein concentrations were determined with the BCA assay (Pierce, Rockford, IL), using bovine serum albumin as the standard. The total mass of EGF receptors, Shc, E-cadherin, N-cadherin, {alpha}-catenin, {beta}-catenin, {gamma}-catenin, integrin {beta}1, integrin {beta}2, integrin {beta}3, integrin {beta}4, integrin {alpha}5, and fibronectin in the lysates was measured with the use of monoclonal antibodies from BD Transduction Laboratories (San Diego, CA) by performing Western blotting as previously described (20). An ERK1/2 antibody was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA), and the phosphospecific ERK1/2 antibody was obtained from Cell Signaling Technology (Beverly, MA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To determine the impact of serum and growth factors on CV-1 cell wound healing, we cultured confluent growth-arrested CV-1 monolayers in serum-free medium (serum starved) overnight before scrape wounding. The extent of wound closure was monitored in a representative sample (corresponding to a microscope field) from a ~0.05 x 3.5-cm wound over the next 24 h by using time-lapse video microscopy (Fig. 1A). Making the wound much longer than it was wide permitted a semiautomated measurement of the rate of migration of the monolayer into the wound. Individual video frames were processed by selecting a range of intensities corresponding to the wounded area and generating binary images (Fig. 1B). The black pixels were counted in each frame in the series, converted to area, and plotted as a function of time (Fig. 1C). From these measurements, the "instantaneous" rate of migration of the wound front was calculated on the basis of differentials (Fig. 1D) or the sustained maximal rate was calculated using linear regression. Over the 24-h course of this experiment, the monolayer migrated across the dish at a rate of 3.04 ± 1.02 µm/h (n = 231) as assessed by calculations based on differentials and 2.94 µm/h (r2 = 0.99) on the basis of linear regression. The differential plots are useful for defining when changes in cell behavior occur, whereas the rates calculated using linear regression are most useful for comparing the sustained rates of migration between different experiments. Importantly, the rate of monolayer migration measured in these experiments is not sensitive to small differences in the width of the wounds as assessed by the reproducibility of the measurements in the experiments we describe subsequently. Although not analyzed, we do anticipate that the rate of monolayer migration across wider wounds would decline as the cells become spread out across the dish. Eventually, cell division would also begin to contribute to the rate of monolayer migration, but our scrape wounds were sufficiently narrow such that increases in cell division were not apparent until after the completion of wound closure.



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Fig. 1. Automated quantitative time-lapse analysis of scrape wound closure in CV-1 cells that were serum starved for 16 h. Cultures were scrape wounded on the microscope stage, and images were collected every 6 min for 24 h. A: excerpts from the image series at 3-h intervals. Numbers indicate the time (in h) after scrape wounding. Each image is 310 µm wide. B: processed binary images derived from the 8-bit images shown in A. These images were generated by applying an edge (Sobel) filter, selecting a density slice (in most cases 1–49), and performing a binary conversion using Scion Image. Binary images were generated for the indicated intervals for all data sets analyzed to confirm selection of the appropriate density slice. C: the number of black pixels in each frame of the series was counted. These counts were imported into an Excel spreadsheet, converted to wound area in µm2, and plotted as a function of time to provide a quantitative measure of wound healing. D: the data in C were used to calculate an instantaneous linear migration rate of the monolayer. The mean was calculated as a rolling average from 9 intervals (6 min each) and the means ± SD are plotted.

 
Serum stimulates wound closure. To examine the contribution of serum to wound healing, we cultured CV-1 cells in serum-free medium (serum starved) for 24 h. When these cells were transferred to the microscope, scrape wounded, and cultured in serum-free medium, there was little closure of the wound (Fig. 2A), because the rate of migration was low. In serum-free medium on day 1, the migration rate was significantly lower (2.13 ± 0.48 µm/h, n = 8) than the rate when serum was added to the medium (9.77 ± 1.16 µm/h, n = 11) (Fig. 2B). The maximal rate of wound healing in cultures treated with serum during both the 24 h preceding the experiment and the 24 h of the experiment (8.54 ± 0.60 µm/h, n = 3) was not significantly different from that in cells treated with serum only during the 24 h of the experiment. Thus serum enhances the wound-healing response more than fourfold. Culture of CV-1 cells for prolonged periods in serum-free medium was not toxic, because the cells consistently closed the scrape wounds if serum was subsequently added to the culture. The wound-healing response to serum gradually declined over a period of several days of culture in serum-free medium. For example, the rate of migration for cells serum starved for 16 h (2.94 µm/h, Fig. 1) was higher than for cells serum starved for 24 h (2.13 ± 0.48 µm/h, Fig. 2). In all remaining experiments, cells were serum starved for at least 48 h before the scrape wound assay was initiated, to ensure maximal suppression of the serum-dependent wound-healing response.



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Fig. 2. Serum enhances the closure of scrape wounds in CV-1 cells. A: serum-starved CV-1 cell cultures were scrape wounded on the microscope stage, and images were collected at 6-min intervals for 24 h. The area of the scrape wound was measured for each image and is plotted for cells cultured in serum-free medium for the first 24 h (serum free). Next, a different area in the same culture was scrape wounded and the serum-free medium was replaced with serum-containing medium, and images were collected at 6-min intervals for 24 h. The area of the scrape wound was measured for each image and is plotted (10% serum). B: the linear rate of wound closure was calculated by linear regression from replicate experiments, including that depicted in A. The means ± SE are plotted for independent experiments performed in serum-free medium (n = 8), in serum-containing medium during the experiment (1 day serum, n = 11), or in serum-containing medium for 24 h before and during the experiment (2 days serum, n = 3). *Significant (P < 0.05) enhancement in the rate of monolayer migration compared with serum-free cultures.

 
EGF persistently enhances motility in serum-free medium. Cultures were treated with 10 nM EGF to determine whether this growth factor would also promote wound closure. Compared with scrape-wounded serum-starved cultures (Fig. 3A), EGF enhanced cell motility (Fig. 3B) but failed to promote complete wound closure within 24 h (Fig. 3C). Quantitative analysis indicates that EGF enhanced the migration rate 3.0 ± 0.9-fold, but the motility of individual cells was not coordinated and the morphology was that of migrating fibroblasts (Fig. 3B and Video 1, available as Supplemental Material online).1 EGF-enhanced fibroblast motility has been well studied, so this is not a surprising observation. However, we were surprised to find that if EGF-treated cultures were washed (to remove the exogenous EGF) and fed serum-containing medium and scrape wounded a second time, that the EGF-pretreated scrape wounds closed much faster (19.6 ± 2.9 µm/h, n = 5) than did serum-treated scrape wounds (8.6 ± 0.7 µm/h, n = 5) (Fig. 4 A and Video 2). In EGF-pretreated cultures, instantaneous linear migration rates as high as ~25 µm/h were measured compared with a maximum of ~9 µm/h in serum-containing control cultures (Fig. 4B and Video 2). Importantly, in these experiments, EGF enhanced wound closure 2.3 ± 0.9-fold even though no exogenous EGF was present during the second 24 h of the experiment.



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Fig. 3. EGF promotes only modest enhancement of CV-1 cell scrape wound healing in serum-free medium. Parallel serum-starved CV-1 cell cultures were scrape wounded on the microscope stage, images were collected at 6-min intervals for 24 h, and the wound area was measured. A: image of cells 24 h after scrape wounding and culture in serum-free medium. B: image of cells 24 h after scrape wounding and culture in serum-free medium supplemented with 10 nM EGF. Each image is 310 µm wide. C: the area of the scrape wound was measured for each image and plotted for cells cultured in serum-free medium (control) or in serum-free medium supplemented with 10 nM EGF. See also Video 1 (available online as Supplemental Material).

 


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Fig. 4. EGF treatment on day 1 enhances serum-dependent closure of CV-1 cell scrape wounds on day 2 in the absence of EGF. Parallel CV-1 cell cultures were scrape wounded on the microscope stage in the presence of 10% calf serum, and images were collected at 6-min intervals for 24 h. A: the area of the scrape wound was measured for each image and is plotted for cells cultured in either serum-free medium (control) or in serum-free medium supplemented with 10 nM EGF for 24 h before the initiation of the experiment. The arrow to the right of the plot indicates the point of complete wound closure. B: the instantaneous linear rate of wound closure was calculated from the data plotted in A. The means ± SD calculated over a 54-min interval are plotted as a function of time. C: diagram of the experimental design. This experiment has been repeated 5 times with similar results. See Video 2 for an example.

 
The temporal relationship between EGF stimulation and serum-promoted scrape wound closure was examined by adding 10 nM EGF directly to serum-containing medium. As already shown in Figs. 2 and 4, a robust wound-healing response was observed with CV-1 cells treated with serum alone, although the scrape wounds usually failed to close completely within 24 h (Fig. 5A). The addition of 10 nM EGF to a parallel culture containing serum enhanced the rate of wound closure. Initially, wound closure proceeded at the same rate in each culture. However, the rate of wound closure in EGF-treated cultures began to increase 4–5 h after EGF was added and remained elevated until the wound closed. Furthermore, the behavior of the monolayer in the presence of EGF plus serum (EGF+serum) was more reminiscent of an epithelial cell sheet than of the migratory fibroblasts seen in the presence of EGF alone (compare Fig. 5F with Fig. 3B; Video 3). EGF+serum-treated cells appeared to retain contacts with neighboring cells and behaved as a syncytial mass migrating vectorially into the wound, whereas cells treated with EGF alone migrated autonomously and in any direction.



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Fig. 5. EGF enhances serum-dependent closure of CV-1 cell scrape wounds. A: parallel serum-starved CV-1 cell cultures were scrape wounded on the microscope stage, and images were collected at 6-min intervals for 24 h. The area of the scrape wound was measured for each image and is plotted for cells cultured in 10% calf serum-containing medium (control) or in 10% calf serum-containing medium supplemented with 10 nM EGF. B: at the end of the experiment shown in A, the medium in both cultures was replaced with 10% calf serum and the assay was repeated. No EGF was present on day 2 of the experiment. The arrows to the right of the plots in A and B indicate the point of complete wound closure. C: the sustained maximum migration rates were calculated using linear regression (means ± SE). *Significant increase (P < 0.05) in the monolayer migration rate compared with time-matched controls. D: cells cultured in calf serum-containing medium were treated with EGF during the indicated intervals on day 1, and wound closure on day 2 was measured using a static assay. Values are means ± SE from triplicate determinations in 4 independent experiments. *Significant reduction (P < 0.05) in wound area compared with the time-matched control treated with calf serum only. The morphology of wound closure after 15 h is shown for cells cultured in serum (E) or in serum plus 10 nM EGF (F). See Video 3 for an example of time lapse.

 
As was the case for EGF pretreatment in serum-free medium observed in Fig. 4, the effect of EGF to promote wound closure persisted even after the exongenous EGF was removed (Fig. 5B and Video 3). When the medium in both cultures was replaced with serum-containing medium and a new set of scrape wounds was made, the EGF-pretreated scrape wounds closed much faster than did serum-treated scrape wounds. Although the linear migration rate was only 2.4-fold higher in the EGF-pretreated cultures than in the serum-treated cultures (Fig. 5C), there was no lag phase and the wounds closed within ~10 h on the second day (Fig. 5B) rather than within ~18 h in the presence of EGF on the first day (Fig. 5A). To determine the minimum duration of EGF treatment necessary to elicit this persistent wound-healing response, cells cultured in serum-containing medium were treated with EGF for 0–1, 0–6, 0–12, or 0–24 h and were scraped at 24 h, and wound closure was measured during the next 24 h (Fig. 5D). Compared with untreated cultures, there was a significant enhancement (P < 0.05) of wound closure 6 h after the scrape in cultures treated with EGF for 0–6, 0–12, and 0–24 h. There was a significant enhancement of wound closure 12 h after scrape wounding in cultures treated with EGF for 0–12 and 0–24 h. Thus 6- to 12-h treatment with EGF is sufficient to elicit a persistent wound-healing response, whereas treatment with EGF for only 1 h is inadequate.

We considered that our results might be compromised by the presence of EGF in serum. Using an estimate for the serum concentration of EGF of ~1 nM (45), we calculated that the final concentration of endogenous EGF in medium supplemented with 10% serum is ~0.1 nM. Although this is low compared with the 10 nM EGF we used in our experiments, it is high enough to stimulate half-maximal thymidine incorporation in a fibroblast growth assay and is at the low end of the 1–10 nM range used for EGF as a growth supplement (www.roche-applied-science.com/pack-insert/1376454a.pdf). Nevertheless, we observed a fourfold enhancement in the rate of wound healing by adding 10 nM EGF to serum for a final concentration of ~10.1 nM. We typically use 10 nM EGF in our experiments, but we have observed similar effects with concentrations as high as 100 nM EGF. To determine the potential effects of ~0.1 nM serum-derived EGF, we tested IgG1 MAb-225, a neutralizing monoclonal antibody specific for the EGF receptor (44), and found that it attenuated both EGF- and serum-induced wound closure (Fig. 6A). When assays were performed in the presence of the EGF receptor tyrosine kinase inhibitor AG-494, the stimulation of wound closure by both serum alone and EGF+serum was blocked (Fig. 6B). A structurally related benzylidenecyanothioacetamide inhibitor specific for the NGF receptor tyrosine kinase (AG-879) was without effect when used at the same concentration. Thus serum-derived EGF does stimulate wound closure, and this is mediated via activation of the EGF receptor kinase. However, wound closure stimulated by serum-derived EGF is not maximal, because it is enhanced by the addition of exogenous EGF. Our experimental observations are most consistent with a role for EGF in stimulating cellular motility and with a role for another serum-derived factor in coordinating EGF-induced motility.



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Fig. 6. EGF receptor activation is required for efficient wound closure. A: inhibition of EGF-induced motility and serum-induced motility by anti-EGF receptor antibody (IgG1 MAb-225). CV-1 monolayers cultured in multiwell plates were scrape wounded, treated for 24 h, fixed, and stained with crystal violet, and the area of the wounds was measured. Values are means ± SD measured from 3–4 wells (SF, serum-free; CS, calf serum). The appearance of the scrape wounds is shown above the plot. B: confluent, serum-starved CV-1 cell cultures were pretreated with an EGF receptor (AG-494) or a NGF receptor tyrosine kinase inhibitor (AG-879) for 60 min and scrape wounded, and wound areas were measured after 9, 16, and 24 h. Values are means ± SE derived from 4 independent experiments. C: Western blot analysis of the time course and magnitude of ERK activation by EGF or EGF+serum. An immunoblot using phophospecific ERK1/2 antibody is shown. D: LPA enhances wound closure when combined with EGF+serum but will not substitute for serum. CV-1 monolayers cultured in multiwell plates were scrape wounded and treated in the presence and absence of LPA as indicated, and the area of the wounds was measured after 6, 12, and 24 h.

 
We examined the activation of ERK1 and ERK2 in CV-1 cells (Fig. 6C) maintained in serum and found that levels of activated ERK1/2 were low (albeit not undetectable). Treatment with EGF resulted in a transient activation of ERK1/2. Treatment with EGF+serum resulted in even more robust activation of ERK1/2 than was observed with EGF alone. We have already shown that EGF alone does not promote efficient wound closure and that the addition of EGF to serum-containing medium (EGF+serum) results in a significant enhancement of scrape wound closure. Thus, although serum contains EGF, and EGF receptor binding and activation are necessary for serum to stimulate wound closure, a serum component other than EGF must be required to coordinate cell migration. Lysophosphatidic acid (LPA), an abundant serum factor, has been shown to stimulate wound closure (4, 25, 43). We tested LPA and found that wound closure in EGF+LPA-treated cultures was significantly less than wound closure in cultures treated with EGF+serum (Fig. 6D). When LPA was combined with EGF+serum, wound closure was enhanced further. Thus, although LPA does stimulate wound closure in CV-1 cells, it will not substitute for serum.

Potential mechanisms for persistent effects of EGF + serum. Video microscopy indicates that cell migration is coordinated by an enhancement of cell-to-cell contacts in serum-treated cultures. Given the morphological conversion of CV-1 cells from a fibroblastic to an epithelial behavior in EGF+serum-treated cultures compared with cultures treated with EGF alone, the abundance of a variety of proteins involved in cell adhesion and signaling was determined by Western blot analysis of whole cell extracts (Fig. 7A). Compared with cultures maintained in serum-free medium, no detectable differences in the total mass of E- or N-cadherin, {alpha}-, {beta}-, or {gamma}-catenin, or integrin {beta}1, {beta}3, or {beta}5 were observed in cultures treated with EGF, serum, or EGF+serum. There was a modest increase in the abundance of integrin {alpha}5 and an apparent stabilization of fibronectin and {gamma}-catenin to proteolysis by EGF, but this does not explain the coordination of cell motility by serum.



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Fig. 7. Immunoblot analysis of protein levels in CV-1 cells after 24–48 h. A: whole cell extracts were prepared after 24-h treatments with serum-free medium, 10 nM EGF, 10% calf serum, or 10% calf serum and 10 nM EGF (CS+EGF) that paralleled the wound closure studies, and 50 µg of total cellular protein were used for Western blotting with the indicated monoclonal antibodies. B: EGF downregulates EGF receptors and Shc isoforms in CV-1 cells. Whole cell extracts were prepared after the indicated treatments that paralleled the wound closure studies, and 100 µg of total cellular protein were used for Western blotting with monoclonal antibodies. This experiment was repeated 4 times with equivalent results. C: treatment with serum+EGF sustains phospho-ERK2 activation for up to 48 h. Cell extracts were subjected to immunoblotting with phosphospecific ERK1/2 antibody (top) or a phosphorylation-independent ERK1/2 antibody (bottom).

 
Together, the experiments described in Figs. 35 indicate that EGF promotes a stable alteration in the cells that enhances their wound-healing responses to serum. It took 4–5 h for EGF to enhance the rate of wound closure in both the absence and the presence of serum (Figs. 3A and 5A) and 6–12 h for EGF to induce a persistent enhancement of wound closure (Fig. 5D), indicating that the expression of new genes and proteins must be induced by EGF. Such an induction of new proteins could account for the fact that accelerated wound closure was instantaneous in EGF-pretreated cells (Figs. 4A and 5B). An analysis of total EGF receptor mass by Western blotting indicated that there were not many EGF receptors in CV-1 cells and that those were downregulated by treatment with EGF for 24 h (Fig. 7B). Thus it seems unlikely that residual active EGF receptors mediate the persistent wound-healing response. We also examined the content of the signaling intermediary Shc and the phosphorylation status of ERK1 and ERK2 in whole cell lysates and found that like the EGF receptor, all three isoforms of Shc were also downregulated by treatment with EGF for 24 h. However, there was a persistent low, sustained phosphorylation (activation) of ERK2 in 48-h EGF+serum-treated cultures that was not observed for EGF or serum treatment alone (Fig. 7C). It should be noted that the blot shown in Fig. 7C was exposed for longer than the blot in Fig. 6C to detect the lower levels of sustained ERK phosphorylation.

To more generally test the hypothesis that EGF promotes the expression of proteins facilitating wound healing, we performed experiments using the protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D to block gene expression. Cells were cultured in plastic dishes and scrape wounded, and the size of the wounds was measured after 24 h in the presence of varying concentrations of cycloheximide and actinomycin D. Transient treatment (6 h) and washout of the protein synthesis inhibitor cycloheximide (10 µg/ml) had little effect on the basal rate of closure of scrape wounds in the presence of calf serum (Fig. 8A). By contrast, sustained treatment with cycloheximide (24 h) blocked wound closure, demonstrating the efficacy of the inhibitor. Similarly, transient treatment and washout of the transcription inhibitor actinomycin D (5 µg/ml) also had little effect on the basal rate of closure of scrape wounds in the presence of calf serum, whereas sustained treatment with actinomycin D (24 h) blocked wound closure. These experiments demonstrate that these two compounds can be used to reversibly inhibit wound healing, presumably by blocking gene transcription (actinomycin D) and mRNA translation (cycloheximide). Time-lapse experiments were performed to determine whether gene transcription and mRNA translation are required for the persistent effects of EGF on wound healing. Cultured CV-1 cells were scrape wounded and cultured in calf serum-containing medium supplemented with 10 nM EGF in the absence or presence of actinomycin D or cycloheximide. Wound healing was monitored for 24 h and was blocked by both inhibitors, as expected (data not shown). The medium was then exchanged for calf serum without EGF or inhibitors, a new scrape wound was made, and its closure was monitored during the second 24 h of the experiment. Treatment with either inhibitor for 0–24 h blocked the EGF-enhanced increase in the rate of wound closure from 24 to 48 h without effect on the rate of basal wound closure. The rate of closure in cultures treated with EGF was 2.6-fold higher than in cultures treated with EGF and cycloheximide during the first 24 h and was 3.0-fold higher than in cultures treated with EGF and actinomycin D during the first 24 h (Fig. 8B and Video 4). Treatment intervals of from 6 to 24 h on day 1 were used with similar, albeit slightly lower, inhibitory effects on wound healing on day 2 (data not shown). Thus both gene transcription and de novo protein synthesis are required for the persistent stimulation of scrape wound closure by EGF in the presence of serum. Together, the results support the hypothesis that EGF induces the expression of a stable set of proteins responsible for enhanced cell motility.



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Fig. 8. Inhibitors of translation and transcription reversibly block wound healing. CV-1 cells were cultured for 48 h in serum-free medium before scrape wounding and culture in 5% calf serum. Biosynthetic inhibitors (CHX, 10 µg/ml cycloheximide; ActD, 5 µg/ml actinomycin D) were added for 6 h (0–6 h) or for 24 h (0–24 h) as indicated, and the wound areas were measured 24 h after scrape wounding. Also plotted is the area of the initial wound (scrape) and the closed wound (0–24 h CON) after incubation for 24 h in the presence of calf serum. Values are means ± SD from 9 measurements from a single dish for each treatment. *Significant reduction (P < 0.05) in wound closure compared with the 0–24 h CON cultures. B: biosynthetic inhibitors attenuate persistent stimulation of scrape wound closure by EGF. Sustained maximal migration rates in the presence of 5% calf serum were calculated by linear regression from time-lapse assays on day 2 after cells were treated as indicated on day 1. Values are means ± SE. *Significant enhancement (P < 0.05) in the rate of wound closure compared with the control cultures cultured in the presence of calf serum alone. See Video 4.

 
Comparison with epithelial cells. We have noted that CV-1 cells are classified as fibroblasts by the American Type Culture Collection, and hence, they might seem to be of little relevance to the process of reepithelialization. However, the morphology of CV-1 cells is dependent on culture density. At low cell densities, the cells are fibroblastic (Fig. 9A), whereas at high cell densities they are epitheliod (Fig. 9B). Indeed, the first published study using these cells describes them as epithelial (18), and we document the expression of E-cadherin in confluent cultures. The behavior of CV-1 cells is distinct from finite lifespan fibroblasts like IMR-90 (Fig. 9, C and D) that do not express E-cadherin (data not shown) and epithelial cell lines like 16HBE (Fig. 9, E and F) that do express E-cadherin (33) and grow as clusters at low cell density. At confluence, CV-1 cells’ growth arrests, and they do not pile up on top of one another as is the case for fibroblasts (Fig. 9D), but they are easily dispersed into single cell suspensions by calcium chelation and trypsinization and behave like fibroblasts when replated at subconfluent densities. Thus the CV-1 cell line appears capable of a reversible epithelial-to-mesenchymal cell transition that can be promoted by trypsinization and plating at low density (Fig. 9A) or by treatment with EGF in serum-free medium (Fig. 3B). To determine whether the persistent stimulation of scrape wound closure by EGF in CV-1 cells could be generalized to epithelial cells, we examined a bronchiolar epithelial cell line (16HBE) in which EGF is known to enhance wound closure (39). In these cells, treatment with 20 nM EGF also dramatically enhanced the rate of wound closure compared with cells cultured in the presence of fetal bovine serum alone (Fig. 9G). However, the behavior of these cells differed substantially from that of CV-1 cells. In 16HBE cells, there was a collective saltatory motion of the entire monolayer (Video 5) such that in the control culture, there was a transient opening of the wound after partial closure (Video 6). In addition, there was no persistent effect of EGF on wound closure in the 16HBE cells (Fig. 9H). In particular, wound healing after 24-h treatment with EGF was similar to that in untreated cells. A major difference between CV-1 and 16HBE cells is the degree of adherence between cells. In CV-1 cells, we did observe motility at the level of individual cells, especially in cultures treated with EGF alone, whereas in 16HBE cultures, the motility of individual cells was constrained by adjacent cells such that the monolayer moved as a sheet under all conditions. Treatment of CV-1 cells with EGF+serum conferred a motility pattern more similar to that of the airway epithelial cells.



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Fig. 9. CV-1 cells have epithelial cell properties when grown to confluence. A comparison of CV-1 cell (A and B), IMR-90 lung fibroblast (C and D), and 16HBE lung epithelial cell (E and F) culture morphology at low density (A, C, and E) and at confluence (B, D, and F) shows that CV-1 cells behave like fibroblasts at low density (compare A with C) and like epithelial cells at high density (compare B with F). EGF enhances wound healing in 16HBE epithelial cells but does not exert a persistent effect (G and H). 16HBE cells were cultured to confluence on collagen- and fibronectin-coated dishes in fetal bovine serum. Parallel cultures were scrape wounded on the microscope stage, and images were collected at 6-min intervals for 24 h (G). The area of the wound was measured and plotted as a function of time for cells cultured in the presence fetal bovine serum (control) or fetal bovine serum supplemented with 20 nM EGF. On day 2, the medium was changed to fetal bovine serum on both cultures, another scrape was made, and images were collected at 6-min intervals for 24 h and analyzed (H). The arrows to the right of the plots in G and H indicate the point of complete wound closure. See also Video 6.

 

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The present results demonstrate that EGF promotes the closure of scrape wounds when added to serum-containing medium. Consistent with earlier studies on fibroblasts (6) and epithelial cells (29), EGF induces motility in CV-1 cells. In the absence of serum, this motility is not coordinated, whereas in the presence of serum it is, resulting in a functional synergy when scrape wound closure is used as an end point. A possible mechanistic explanation for the synergy is an enhancement of transient ERK1 and ERK2 activation by EGF when serum is present. Furthermore, sustained treatment with EGF+serum resulted in a persistent activation of ERK2 not observed with serum or EGF alone. The effects of EGF to promote wound closure in concert with serum are long-lived. The stimulation of motility by EGF persisted for at least 24–48 h after exogenous EGF was removed. Time-course and inhibitor experiments indicated that the expression of new proteins is responsible for the persistent enhancement of wound closure by EGF observed in the presence of serum.

The observation that serum coordinates EGF-induced motility has important implications. Confluent monolayers of CV-1 cells displayed a robust wound closure response in the presence of serum, and removal of serum for at least 24 h before scrape wounding largely abolished this response. EGF enhanced this response greater than twofold when combined with serum. Under the most optimal conditions we have defined, this reduced the time it took to close a scrape wound from ~24 h to ~8 h. Although low levels of EGF present in serum appear to be responsible for basal motility that results in scrape wound closure within 24 h, EGF alone does not promote efficient wound closure. The use of serum in combination with EGF in the present study contrasts many studies with growth factors in which the experiments were performed in serum-free medium. We used serum-free medium in our initial studies and encountered variability in the assays that was traced to differences in the length of culture in serum-free medium before the initiation of experiments. Thus, although basal signaling activity can be suppressed by overnight serum starvation, suppression of functional responses important for wound closure requires serum starvation for 24–48 h.

The present experiments are most relevant to the problem of reepithelialization of partial thickness wounds. In many ways, the behavior of CV-1 cells is similar to that of the basal keratinocyte in skin epithelia and MDCK cells. In the absence of serum, EGF induces CV-1 cell scattering, whereas in its presence, EGF promotes coordinated wound closure. In keratinocytes (30), EGF induces scattering in isolated colonies and closure of scrape wounds in confluent monolayers. In MDCK cells, hepatocyte growth factor (HGF) promotes scattering of isolated colonies and coordinated migration of wounded, polarized monolayers cultured on permeable substrates (1). The present findings prompt two questions for further study: 1) What is the factor (or factors) in serum that promotes a coordinated motile response to EGF?, and 2) what molecules are activated or induced by serum that mediate the coordinated motile response to EGF?

Serum factors that regulate wound healing include insulin-like growth factors, PDGF, and TGF-{beta}, among many others (51). In addition, LPA has been shown to enhance migration in an intestinal epithelial restitution model (43) and in scrape-wounded endothelial cell monolayers (25). Furthemore, EGF+LPA synergistically stimulate mitogenesis in airway smooth muscle cells (5) and LPA+PDGF-BB synergistically enhance human gingival fibroblast migration in scrape wound assays (4). Although LPA did stimulate wound closure in combination with EGF+serum, it failed to substitute for serum when combined with EGF alone. TGF-{beta} is a logical candidate to test in future experiments, given reported synergistic enhancements of EGF-stimulated hepatocyte motility (42). However, TGF-{beta} is also reported to promote an epithelium-to-mesenchyme transition in renal proximal tubular epithelial cells (37), which is essentially the opposite of the coordination of motility we observe by combining serum and EGF.

The abundance of a variety of molecules involved in cell adhesion and signaling was examined by immunoblotting in an attempt to identify serum-induced proteins. Among the candidate proteins tested, only {gamma}-catenin, integrin {alpha}5, and fibronectin varied with treatment. However, these responses were small and were related to EGF, and not to serum treatment, and thus do not help to explain how serum coordinates cellular motility. Disassembly of cell-cell adhesive complexes may be important for cell migration into the denuded area of the culture, but this is expected to be a local property of the wound margin and not one of the bulk culture. Reductions in tight and adherens junction complexes are typical of an epithelial-to-mesenchymal cell transition associated with tumor invasion and metastasis (48) and have been linked to tyrosine phosphorylation of E-cadherin and {beta}-catenin (2), so changes in phosphorylation status may be more relevant than the changes in mass measured in the present experiments. In any event, the morphological responses strongly indicate that one or more cell adhesion molecules are necessary for the epithelial behavior adopted by these cells.

In serum-free medium, EGF stimulated CV-1 cell motility but had only a modest effect on wound closure. This uncoupling of EGF activity and wound closure occurred because the EGF-stimulated CV-1 cell motility was not well coordinated. Although cells did move into the wound area, the cells adopted the typical morphology of migrating fibroblasts and did not cover the substrate effectively. Cells moved freely around and past one another, indicating that there was very little adhesion of cells to one another. However, by contrast to IMR-90 fibroblasts, CV-1 cells exhibit contact inhibition and do not grow on top of one another at high cell densities the way fibroblasts do. Human bronchiolar epithelial cells (16HBE cells) adhered to one another, and the motility of individual cells was constrained. Treatment of CV-1 cells with EGF+serum modified their behavior such that the monolayers were more reminiscent of epithelial cell sheets, although the cell-to-cell adhesions were not as extensive as they were for 16HBE cells.

Upon binding the EGF receptor, EGF activates a variety of processes leading to enhanced growth and motility. The pathways that mediate these responses diverge and involve both short-term (enzymatic) and long-term (transcriptional) mechanisms (6, 7, 50). Enzymes whose activation is necessary for EGF-induced cell motility in fibroblasts include phospholipase C{gamma} for mediating cytoskeletal reorganization (7), MEK for reducing adhesion to substrates (53), and calpain, necessary for detachment of the rear of the cells from the substrate (17, 38). It is postulated that these pathways should be negatively regulated to limit or terminate the wound-healing response and thereby minimize scarring. Indeed, the chemokine IP-10 has been shown to suppress the activation of calpain by EGF and thereby inhibit fibroblast motility (41). Negative regulation of these pathways is likely mediated by the same mechanisms that lead to contact inhibition of growth and to growth arrest at high cell density.

The present observation that wound closure rates accelerate 6–8 h after the initiation of EGF treatment is consistent with earlier observations that maximal fibroblast motility occurs 6–8 h after EGF stimulation (27). We reasoned that this period of time was sufficient for the induction of new protein expression. Indeed, transient treatment with either an inhibitor of transcription or translation blocked the ability of EGF to acutely enhance wound closure. Inhibitor concentrations were selected such that serum-stimulated wound closure was not blocked by transient treatment. Both inhibitors also blocked the sustained stimulation of wound healing by EGF. On the basis of these results, we conclude that EGF treatment promotes the expression of one or more new proteins that mediate enhanced motility. These proteins appear stable because their effects persist after the downregulation of receptors and removal of EGF. Thus a simple activation of enzymes by EGF receptors is not adequate for an enhanced wound closure response by CV-1 cells. However, enhancement of cell motility alone is not adequate to promote efficient wound healing.

To make the best use of the ability of EGF to promote wound healing, it is necessary to understand 1) the mechanisms by which EGF promotes motility of the individual cells, 2) how enhanced motility is channeled to promote the appropriate behavior of a sheet of cells (vectorial movement of cells essential for rapid wound closure), and 3) how the motility process is terminated once the wound is closed. The present study is most relevant to the second question and defines a clear and robust synergy between EGF and serum factors in enhancing the rate of wound healing in vitro. This interaction between EGF and an unidentified serum factor(s) is functionally relevant to reepithelialization of wounds. Identification of the factor and the molecular basis for its activity could prove important for the combinatorial optimization of therapeutic approaches for the treatment of impaired wound healing. Our findings also indicate that chronic administration of EGF (and possibly other growth factors) is not necessary in wound- healing therapies and may actually be counterproductive because of receptor downregulation.


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This work was supported by a University of Arkansas for Medical College (UAMS) College of Medicine Pilot Study Grant (to R. C. Kurten), the UAMS Dean's/CUMG Research Development Fund SG-030102-SJ (to S. M. Jones), and National Institutes of Health (NIH) Grants K23 AI-01818 (to S. M. Jones) and CA-75577 (to T. C. Chambers). Use of the facilities in the UAMS Digital and Confocal Microscopy Laboratory, supported by NIH Grants 1 P20 RR-16460 and PAR-98-092, 1-R24 CA-82899 is acknowledged. Funding was also provided by the Arkansas Biosciences Institute (to R. C. Kurten), a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas-Division of Agriculture, the University of Arkansas, Fayetteville, and the UAMS.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Kurten, Dept. of Physiology & Biophysics, Univ. of Arkansas for Medical Sciences, 4301 West Markham #750, Little Rock, AR 72205-0750 (E-mail: KurtenRichardC{at}uams.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.

1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00024.2003/DC1. Back


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