1Program in Molecular and Cellular Biology and 2Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003; and 3Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908
Submitted 17 November 2003 ; accepted in final form 20 March 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
leukotriene B4; prostaglandin E2; spreading; migration; bridges
Wound healing in a fibroblast monolayer, in vitro, is similar to fibroblast cell adhesion to an ECM in that discrete transitional stages are observed. In the case of cell-ECM adhesion, sequential spreading and migration are initiated by the cell attachment stage (32), whereas in wound closure, the spreading and migration stages are initiated by wounding of the cell monolayer. Each stage of cell adhesion involves changes in overall morphology and cytoskeletal structure that are regulated inter alia by the oxidative enzymes of the AA cascade: LOX and COX. LOX and COX compete for AA released from membranes by phospholipase A2 (PLA2) to convert it to either leukotriene (LT) or prostaglandin (PG), respectively (1, 6, 33). More specifically, the conversion of AA to LTB4 via 5-LOX initiates a cascade of second messengers leading to actin polymerization and thereby activating cell spreading (4) (Stockton RA, Katsumi A, Dixon DA, Green JA, Roberts LA, and Jacobson BS, unpublished observations). Subsequent migration of spread cells requires redistribution and peripheral bundling of F-actin mediated by prostaglandins synthesized by the COX family of enzymes (9). Because of some apparent similarities between cell-ECM adhesion and wound healing stages, we investigated whether the LOX and COX oxidations of AA that regulate cell spreading and migration in adhesion also regulate cell spreading and migration during wound closure.
The present studies examine the roles of the respective activities of these arachidonate oxidative enzymes in cell spreading and motility associated with fibroblast wound closure. NIH/3T3 cell monolayers subjected to narrow scratch wounds were tested for COX-LOX activity over time postwounding vs. wound morphology, marginal cell spreading, and directed cell motility into wound gaps. Observation of the wound healing process as indicated by changes in wound margin cells and overall wound closure revealed phenotypically discernible events. The overall rate of closure of the wound appeared to be biphasic, as determined by measuring both the surface area of cells at wound margins and the area occupied by the entire wound. The use of specific pharmacological inhibitors indicated that LTB4 regulates the initial phase of wound closure by enhancing wound margin cell spreading, whereas prostaglandins regulate subsequent directed intrawound cell migration. In addition, a novel early postwounding organization of bridge cells acting as foci for later directed migration was noted, occurring at regular intervals perpendicularly to the wound axis, and was also examined with respect to arachidonate regulation. Wound closure was similarly studied with NIH/3T3 fibroblasts constitutively over- or underexpressing the 5-LOX or COX-2 enzymes to verify the results obtained by pharmacological knockout and rescue. The evidence presented supports the possibility that sequential activities of both 5-LOX and COX are essential for wound closure in fibroblasts.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
5-LOX and COX-2 overexpression and antisense constructs and transfections. NIH/3T3 cell lines either stably overexpressing or having antisense suppression of 5-LOX or COX-2 synthesis were previously described and characterized (Stockton RA, Dixon DA, Green JA, Roberts LA, and Jacobson BS, unpublished observations). In brief, a 2.2-kb fragment of human 5-LOX cDNA inserted in the sense orientation of pEGFP-C1 to promote synthesis of a 5-LOX-green fluorescent protein (GFP) fusion protein (provided by Dr. Colin Funk and described initially in Ref. 3), a 5-LOX antisense construct consisting of a 2.0-kb fragment of 5-LOX cDNA (courtesy of Dr. Jilly Evans) ligated in reverse orientation into pEGFP-C1, and human COX-2 cDNA with deleted 3'-untranslated region (7) cloned into the EcoRI site of pEGFP-C1 in frame in both sense and antisense orientations were transfected into wild-type NIH/3T3 cells with the use of Lipofectamine (GIBCO BRL, Grand Island, NY) according to the manufacturer's instructions and then selected for geneticin resistance. Resulting single-cell clones were isolated and tested for leukotriene or prostaglandin production, generating stable cell lines maintained with 1.0 mg/ml G418. "3T3-5LOX" (5-LOX overexpressing) cells produce fourfold more LTB4 than control cells do, whereas "3T3-XOL5" (5-LOX antisense) cells produce 70% less; "3T3-COX2" (COX-2 overexpressing) cells produce 10-fold more PGE2 than control cells do, whereas "3T3-2XOC" (COX-2 antisense) cells produce 50% less (Stockton RA, et al., unpublished manuscript).
Wound closure assay.
Cells were detached from culture vessels with 0.01% trypsin-EDTA, washed with Hanks' balanced salt solution, and resuspended in fresh DMEM with 2% calf serum. Cells were plated on 35-mm polystyrene dishes at 6 x 105 cells/plate in DMEM with 2% calf serum overnight to permit formation of monolayers just reaching early confluence by 1624 h. Cells were washed and incubated for 2 h in DMEM with carrier solvent (control) or in AA861, indomethacin, LTB4, or PGE2 at concentrations indicated in figure legends. After incubation, medium was aspirated and replaced with fresh medium with or without additions, and then prewounding medium aliquots were removed for comparison with postwounding samples at later times. The monolayer was then immediately wounded by being scraped with the corner of a piece of Mylar film (used in copy machines) to produce a wound 300 µm (34 cell widths) wide across the entire length of the 35-mm plate monolayer. Cells were visualized by phase-contrast microscopy (Nikon Diaphot-TMD or Nikon Eclipse TE-300 inverted microscopes) and photographed at times indicated in figure legends with the use of a CCD100 video camera and Scion LG3-01 frame grabber or a Nikon N6000 camera and Kodak TMAX-100 professional film. Images were processed using Scion Image or Adobe Photoshop software.
To measure marginal cell surface area, cell bridges, and gap surface area, we photographed the wound at multiple random sites for each time point. To determine marginal cell surface area, we analyzed 50 cells per time point for each treatment. At each time point, NIH Image J software was used to quantify the cell surface area of those cells exposed to the wound gap. The mean area of treated cells for each time point and each treatment was normalized to the surface area of control cells at time 0 and expressed as a percentage of control cell surface area. Data from all experiments were pooled and subjected to ANOVA.
To evaluate cell bridges, we counted the number of bridges that completely spanned the wound in the entire photographic field (1 mm in length) for each treatment at 2 h postwounding. The result for each treatment was normalized to that for control untreated cells. Data from multiple experiments were then pooled and subjected to ANOVA.
To quantify gap surface area, we measured the gap at each time point for each treatment using NIH Image J analysis software. The results for each time point per treatment were normalized as percentages of control cell surface area at time 0. Data from all experiments were then pooled and subjected to ANOVA. All of the above experiments were graphed, with data points shown as means ± SE.
LTB4 and PGE2 enzyme immunoassays. Cells were seeded as described in Wound closure assay in 35-mm polystyrene plates and grown to early confluence. Cells on triplicate plates for each treatment were wounded after a 2-h incubation in medium containing 100 µM AA861, 75 µM indomethacin, 100 nM LTB4, 100 nM PGE2, or indicated combinations of these additions. Incubation medium was replaced with fresh medium plus additions immediately before wounding, and aliquots of the fresh medium were removed to serve as indicators of time 0 prewounding comparison control levels of LTB4 or PGE2. Control cell monolayers for each treatment were left unwounded. At indicated times postwounding, cells and medium in plates were lysed with cold enzyme immunoassay buffer (Cayman), in an amount equivalent to the medium volume, on ice. Cells were scraped, and then lysed cells plus medium were incubated on ice for 15 min, ruptured by sonication, and immediately transferred to 70°C. Colorimetric enzyme immunoassay to determine amounts of either LTB4 or PGE2 in the cell-medium lysate was performed according to the manufacturer's directions. Amounts of both eicosanoids were normalized to percentages of prewounding control levels to indicate relative changes over time.
Scanning electron microscopy. NIH/3T3 fibroblast monolayers were plated on surfaces and wounded as described. The monolayers were then rinsed twice with serum-free DMEM maintained at 37°C. The cells were then fixed with 0.5% glutaraldehyde in serum-free DMEM, also maintained at 37°C for 30 min. The cells were then rinsed twice for 5 min each time with DMEM and postfixed with 1% osmium tetroxide for 30 min. The cells were rinsed three times for 10 min each in serum-free DMEM. After postfixation, the cells were dehydrated by immersion in the following concentrations of ethanol for 2 min each: 20, 30, 40, 50, 60, 80, 90, 96, and 100%. The cells were then dried by critical point drying. Finally, the samples were mounted and coated with gold-palladium and imaged using a JEOL JSM-5400 scanning electron microscope.
Statistical analysis. Data are presented as means ± SE of minimally three separate experiments. Statistical significance was determined using SigmaPlot and SigmaStat software (Jandel Scientific). To determine statistical significance for differences between treatments and controls in kinetic assays, we compared all time point values as a whole for a particular treatment with control values as a whole, unless a particular treatment intersected or merged with control points for a part of the assay. In that case, those distinct sections of the kinetic course were evaluated both overall and as separate parts. P values derived from ANOVA are indicated in figure legends.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Figure 4A indicates that during the first 120 min postwounding in wild-type cells, there was a quantitative increase in the cell surface area of the cells at the edge of the wound; this unidirectional spreading of the marginal cells increased over the course of wound closure. This marginal cell spreading can be further correlated with the kinetics of the LTB4 increase shown in Fig. 3. The increase in marginal spread cell surface area was inhibited by the specific 5-LOX inhibitor AA861; this spreading inhibition was completely reversed with the addition of LTB4, but reversal of inhibition was not seen with coaddition of AA861 and PGE2 (Fig. 4A, top). Overcoming AA861 inhibition of LOX by the exogenous addition of LTB4 with regard to restoration of cell spreading also confirmed that the effect of the LOX inhibitor was not due to nonspecific general cytotoxicity (Fig. 4A, top).
|
Additional studies of the role of LOX and COX on wound closure in NIH/3T3 fibroblasts were done with fibroblasts transfected such that they overexpressed or underexpressed either 5-LOX or COX-2. Overexpression of 5-LOX (5LOX in Fig. 4B, top) or underexpression of COX-2 (2XOC in Fig. 4B, bottom) led to an increase in the marginal cell surface area during the course of wound closure, whereas underexpression of 5-LOX (XOL5 in Fig. 4B, top) or overexpression of COX-2 (COX2 in Fig. 4B, bottom) decreased the extent of marginal cell spreading during wound closure. These results confirm and complement the biochemical and pharmacological experiments described above.
After the marginal cell spreading and elongation into the denuded area in wild-type cells, some of the spread, extended cells make contact with a counterpart on the opposite side, eventually forming a perpendicular cell-cell bridge across the wound space (Fig. 2A). The formation of these bridge structures appears to be critical in the shift from directional marginal cell spreading to directional peripheral cell migration. Because the onset of bridging occurs during the initial cell-spreading phase of wound gap closure, cells were treated with the 5-LOX inhibitor AA861 to determine whether the role played by leukotrienes in signaling marginal cell spreading also extends to this more selected and limited type of unidirectional spreading. The addition of the 5LOX inhibitor to wild-type cells produced a significant reduction in the frequency of bridge formation to <30% of that in untreated control cells (Fig. 5A, top). Conversely, addition of exogenous LTB4 enhanced the formation of bridges 1.5-fold (Fig. 5A, top). Furthermore, addition of the COX2 inhibitor indomethacin resulted in enhanced bridge formation that corresponds to the increase in LTB4 levels shown in Fig. 3A. Addition of exogenous PGE2 also produced a reduction in the number of bridges (Fig. 5A, bottom).
|
To further corroborate these findings, we examined the formation of the cell bridges with the stably transfected 5-LOX- and COX-2-over- and -underexpressing fibroblasts at 120 min postwounding. Either 5-LOX overexpression (3T3-5LOX in Fig. 5B, top) or COX-2 underexpression (3T3-2XOC in Fig. 5B, bottom) enhanced bridge formation more than threefold compared with untreated wild-type cells, whereas either COX-2 overexpression (3T3-COX2 in Fig. 5B, bottom) or 5-LOX underexpression (3T3-XOL5 in Fig. 5B, top) decreased the extent of cell bridging to less than 20 and 35%, respectively, of that in control wild-type cells during wound closure.
To determine differences in wound closure mediated by 5-LOX or COX oxidative products between marginal cell spreading as opposed to peripheral cell motility into the gap, we also measured the reduction of wound surface area over time by adding the same enzyme inhibitors and metabolites described above. Although marginal cell spreading was shown to be necessary for wound closure in the previously described assays, most of the total reduction in wound surface area occurred later than 120 min postwounding, which suggests that either directed cell migration (migration of cells resulting from early formation of bridges) or random cell migration (migration of cells without the early formation of bridges) into the gap after earlier marginal cell spreading is responsible for the majority of wound closure. Evaluation of wound surface area in wild-type cells at 300 min postwounding indicates that inhibitors of 5-LOX (AA861) and COX (indomethacin) both slow wound closure, with 5-LOX inhibition resulting in a slower closure overall (Fig. 6A, top and middle). Inhibition of either enzyme in wild-type cells could be reversed by addition of its own respective downstream metabolite but not by addition of PGE2 to 5-LOX-inhibited cells (Fig. 6A, top) or addition of LTB4 to COX-inhibited cells (Fig. 6A, middle), although wound closure was partially reversed in the latter case, likely due to increased marginal cell spreading, since motility is inhibited. Interestingly, addition of exogenous PGE2 alone did not inhibit overall wound closure at 300 min, even though it slowed initial cell spreading and bridge formation (Fig. 6A, bottom).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is important to study the kinetic and empirical stages of wound healing to better understand both how the process can go awry and how to develop drugs to accelerate wound closure. The goal of these studies was to evaluate whether LOX and COX metabolites might signal the unique directional cell spreading and motility required for wound closure in stable confluent monolayers where cells are already spread, as has been shown to be the case with suspended NIH/3T3 cells plated onto a fibronectin (FN) surface (46, 32, 33).
In previous studies, we (32) showed that the production of LTB4 and PGE2 in these cells during the course of cell spreading and migration is dependent on the production of AA via cytosolic PLA2 activation. Our preliminary work using pan and specific inhibitors of PLA2 also suggested that calcium-dependent secretory PLA2 may be required for wound healing (unpublished observations).
We (32) and others (23) also showed that when cells are stimulated by either mechanical wounding and/or adhesion to the ECM, AA production is also stimulated. We can therefore infer that in this case, wounding, and more specifically, wounded marginal cells, are responsible for the LTB4 and PGE2 production described in these assays, because little activity was observed in the absence of wounding (Fig. 3B). Whether an AA release providing substrate for the various oxidative enzymes is stimulated by mechanical forces from wounding or by exposure of marginal cells to an increased area of available FN surface cannot be determined from these data.
Although marginal cell spreading into a wound gap is unidirectional compared with the spreading of newly plated detached cells, wound marginal cell spreading was similarly shown to be regulated by 5-LOX metabolites (Fig. 4, A and B) and can be correlated to 5-LOX activity as measured by increases in LTB4 levels stimulated rapidly after monolayer wounding. LTB4 increased 2.5-fold over basal levels by 60 min postwounding in untreated wild-type cells, whereas no increases were seen in unwounded monolayers (Fig. 3). This 5-LOX-mediated LTB4 rise in wild-type cells also corresponded to development of periodic cell bridges across the wound gap, which were required for later directed motility (Fig. 5A). Cells constitutively expressing 5-LOX demonstrated a proportional increase in cell bridge formation (Fig. 5B). This finding suggests that initial bridge extensions are a later developing part of the process of marginal cell spreading and that both are regulated by LTB4. Both marginal cell spreading and bridge formation were required for later directed cell motility into wound gaps but not for motility stimulated by prostaglandin release, which seems to occur without much additional spreading (Figs. 4 and 5). Increased spreading and bridge cell formation were also enhanced by indomethacin inhibition of COX, likely due to increased LTB4 production accompanying COX inhibition as shown in Fig. 3. Interestingly, even though 5-LOX overexpression or COX-2 antisense enhanced initial marginal spreading and bridge formation compared with wild-type cells, overall wound closure was inhibited. This is likely due to the observation that those particular cells have impaired motility following the spreading stage of cell adhesion to FN (Stockton RA and Jacobson BS, unpublished observation).
Conversely, COX activity as indicated by PGE2 levels was unchanged in wild-type cells for the first 60120 min postwounding but then rose continuously over the remaining time course of wound closure (Fig. 3). This increase also corresponded to increased cell migration into the wound gap, as measured by a decrease in wound surface area over time (Fig. 6).
Overall wound closure appeared to be biphasic, in that initial reductions in wound surface area were associated with marginal cell spreading signaled by LTB4 over the first 60120 min postwounding, whereas subsequent full gap closure was associated with directed motility of reorganized peripheral cells into the wound gap over and adjacent to the established bridges (Figs. 1, 5, and 6). These leukotriene-mediated early bridge structures, to our knowledge, appear to represent a novel and previously unreported stage of wound healing in vitro.
The overall effects of leukotrienes as opposed to prostaglandins in all stages of wound closure can be argued to be largely dominated by the relative ratios of oxidative products over time. Although exogenous PGE2 enhanced overall wound closure in untransfected cells at 5 h postwounding, it inhibited initial LTB4 production and concomitant marginal spreading and bridge formation. This is likely, because LTB4 production in cells treated with PGE2 returns to control levels after 2 h. COX-2 overexpression, like addition of PGE2, caused a delay in the onset of wound healing, presumably because of decreased spreading and bridge formation (Fig. 4 and 5). The effect of COX-2 on overall wound healing can be better observed with the COX-2-overexpressing cells in which gap closure was delayed up until 4 h postwounding. In these cells, no increase in spreading could be observed during the entire period studied; therefore, any movement of the cells occurred by random motility, according to our definition of random motility as that occurring in the absence of any increase in spreading or bridge formation and not specifically wound gap-directed in overall direction. In wild-type cells, exogenous PGE2 enhanced general motility of cells into the wound while reducing initial marginal cell spreading, as stated previously. After 120 min, spreading as well as LTB4 levels catch up to control levels to produce an accelerated rate of wound closure. This is further evidence that both LOX and COX activity are required for optimal closure.
The results seen with the COX-2-overexpressing cells can be compared with untransfected cells treated with both AA861 and PGE2, which also show no increase in spreading during the period studied, indicated by the fact that both have delayed wound closure. Cells treated with both AA861 and PGE2, however, take much longer than the 5-h period shown in this study to close (data not shown). Although we have shown that enhanced PGE2 production inhibits initial LTB4 production, it is likely that 5-LOX activity is not completely inhibited in COX-2 overexpressers but that the mass ratio of products has heavily shifted toward prostaglandin signaling. We cannot rule out, however, other indirect effects that PGE2 might have on LTB4 production such as the inhibition of 5-LOX-activating protein (FLAP) via an IL-10-dependent mechanism as was seen in dendritic cells (12).
Leukotrienes are known to play an important role in the initiation of inflammatory bowel disease (IBD) in rats. Interestingly, IBD is only inhibited when administration of an inhibitor of leukotriene synthesis is begun before the inflammatory stimulus is applied and not after (20). This suggests that leukotrienes may play a role early in the inflammatory process such that inhibition after inflammation has already occurred would be futile. This requirement for preaddition of wound-associated inhibitors was also observed in corneal endothelial cells treated with indomethacin such that indomethacin added before, but not after, wounding promoted enhanced spreading of the confluent monolayer into the wound (17).
Other recent work showed that inhibition of 5-LOX did not impair healing of 3T6 fibroblast wounds (22); however, in those experiments wound closure was evaluated at more than 5 h postwounding, by which time random migration rather than bridge-directed migration of COX-activated cells would have produced a considerable degree of closure. Our data indicate that 5-LOX inhibition slows the initial stages of gap closure and prevents later directed motility dependent on bridge formation but that it has little effect on random peripheral cell motility. In contrast, cells treated with PGE2 had inhibited LTB4 production and consequential bridge formation, but the later stages of wound closure were not affected (Fig. 6A). Wounds treated with both a 5-LOX inhibitor and exogenous PGE2 showed an initial lag in closure compared with control cell monolayers but did eventually close at later times (data not shown), possibly due to eventual prostaglandin-stimulated random movement into the wound (Figs. 3 and 4). It is also interesting to note that AA861 has a broad inhibitory effect on both LTB4 and PGE2 production. The observation that exogenous LTB4 alone can reverse the inhibitory effects of AA861, however, strongly suggests that 5-LOX activation and COX activation are sequential during wound healing. The overall stages of wound healing and their proposed regulatory sites by LTB4 and PGE2 are schematized in Fig. 7.
|
This wound closure regulation by arachidonate pathway switching modulates activities of both branch enzymes, and we hypothesize that in addition to limiting undirected cell motility from prostaglandin effects, this also serves to reduce postwounding localized fibrosis, which may be associated with sustained increases in leukotriene syntheses. Elevated leukotriene synthesis is seen systemically in scleroderma (31) and other conditions of systemic fibrosis (19, 21) and specifically in keloid scar formation (2). These data suggest that sequential and coordinated activities of first LOX and then COX form a system of tight regulation of early inflammation-stimulated cell motility, both to optimize wound healing and, possibly, to reduce potentially dangerous increases in fibroblast and epithelial cell random motility associated with increased prostaglandin levels.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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.
* J. A. Green and R. A. Stockton contributed equally to this work.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Brissett A and Sherris D. Scar contractures, hypertrophic scars, and keloids. Facial Plast Surg 17: 263272, 2001.[CrossRef][Medline]
3. Chen XS, Zhang YY, and Funk CD. Determinants of 5-lipoxygenase nuclear localization using green fluorescent protein/5-lipoxygenase fusion proteins. J Biol Chem 273: 3123731244, 1998.
4. Chun J, Auer KA, and Jacobson BS. Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of 1 integrin. J Cell Physiol 173: 361370, 1997.[CrossRef][ISI][Medline]
5. Chun J and Jacobson B. Requirement for diacylglycerol and protein kinase C in HeLa cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading. Mol Biol Cell 4: 271281, 1993.[Abstract]
6. Chun JS and Jacobson BS. Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering. Mol Biol Cell 3: 481492, 1992.[Abstract]
7. Dixon DA, Kaplan CD, McIntyre TM, Zimmerman GA, and Prescott SM. Post-transcriptional control of cyclooxygenase-2 gene expression. The role of the 3'-untranslated region. J Biol Chem 275: 1175011757, 2000.
8. Gilroy DW, Saunders MA, and Wu KK. COX-2 expression and cell cycle progression in human fibroblasts. Am J Physiol Cell Physiol 281: C188C194, 2001.
9. Glenn H and Jacobson B. Cyclooxygenase and cAMP-dependent protein kinase reorganize the actin cytoskeleton for motility in HeLa cells. Cell Motil Cytoskeleton 55: 265277, 2003.[CrossRef][ISI][Medline]
10. Gotlieb A, Wong M, Boden P, and Fone A. The role of the cytoskeleton in endothelial repair. Scanning Microsc 1: 17151726, 1987.[ISI][Medline]
11. Gupta AG, Hirakata A, and Proia AD. Effect of inhibitors of arachidonic acid metabolism on corneal re-epithelialization in the rat. Exp Eye Res 56: 701708, 1993.[CrossRef][ISI][Medline]
12. Harizi H, Juzan M, Moreau JF, and Gualde N. Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism. J Immunol 170: 139146, 2003.
13. Hofbauer R, Moser D, Kaye AD, Knapp S, Gmeiner B, Kapiotis S, Wagner O, and Frass M. Prostaglandin E1 is able to increase migration of leukocytes through endothelial cell monolayers. Microvasc Res 59: 354360, 2000.[CrossRef][ISI][Medline]
14. Jovanovic D, Fernandes J, Martel-Pelletier J, Jolicoeur F, Reboul P, Laufer S, Tries S, and Pelletier J. In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML-3000 reduces the progression of experimental osteoarthritis: suppression of collagenase 1 and interleukin-1beta synthesis. Arthritis Rheum 44: 23202330, 2001.[CrossRef][ISI][Medline]
15. Joyce NC, Joyce SJ, Powell SM, and Meklir B. EGF and PGE2: effects on corneal endothelial cell migration and monolayer spreading during wound repair in vitro. Curr Eye Res 14: 601609, 1995.[ISI][Medline]
16. Joyce NC and Meklir B. PGE2: a mediator of corneal endothelial wound repair in vitro. Am J Physiol Cell Physiol 266: C269C275, 1994.
17. Joyce NC, Meklir B, and Neufeld AH. In vitro pharmacologic separation of corneal endothelial migration and spreading responses. Invest Ophthalmol Vis Sci 31: 18161826, 1990.[Abstract]
18. Kohyama T, Ertl RF, Valenti V, Spurzem J, Kawamoto M, Nakamura Y, Veys T, Allegra L, Romberger D, and Rennard SI. Prostaglandin E2 inhibits fibroblast chemotaxis. Am J Physiol Lung Cell Mol Physiol 281: L1257L1263, 2001.
19. Kowal-Bielecka O, Distler O, Neidhart M, Kunzler P, Rethage J, Nawrath M, Carossino A, Pap T, Muller-Ladner U, Michel BA, Sierakowski S, Matucci-Cerinic M, Gay RE, and Gay S. Evidence of 5-lipoxygenase overexpression in the skin of patients with systemic sclerosis: a newly identified pathway to skin inflammation in systemic sclerosis. Arthritis Rheum 44: 18651875, 2001.[CrossRef][ISI][Medline]
20. LeDuc L, Su K, Guth E, Reedy T, and Guth P. Effects of cyclooxygenase and lipoxygenase inhibition on eicosanoids and healing of acetic acid colitis in rats. Dig Dis Sci 38: 289294, 1993.[ISI][Medline]
21. Marra F. Chemokines in liver inflammation and fibrosis. Front Biosci 7: d1899d1914, 2002.[ISI][Medline]
22. Moreno J. Cyclooxygenase and cytochrome P-450 pathways induced by fetal calf serum regulate wound closure in 3T6 fibroblast cultures through the effect of prostaglandin E2 and 12 and 20 hydroxyeicosatetraenoic acids. J Cell Physiol 195: 9298, 2003.[CrossRef][ISI][Medline]
23. Moreno J. Regulation of arachidonic acid release and prostaglandin formation by cell-cell adhesive interactions in wound repair. Pflügers Arch 433: 351356, 1997.[ISI][Medline]
24. Ribardo DA, Crowe SE, Kuhl KR, Peterson JW, and Chopra AK. Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D. J Biol Chem 276: 54675475, 2001.
25. Rieger G, Hein R, Adelmann-Grill B, Ruzicka T, and Krieg T. Influence of eicosanoids on fibroblast chemotaxis and protein synthesis in vitro. J Dermatol Sci 1: 347354, 1990.[Medline]
26. Saika S, Ohnishi Y, Ooshima A, Liu C, and Kao W. Epithelial repair: roles of extracellular matrix. Cornea 21: S23S29, 2002.[CrossRef][Medline]
27. Savla U, Appel HJ, Sporn PH, and Waters CM. Prostaglandin E2 regulates wound closure in airway epithelium. Am J Physiol Lung Cell Mol Physiol 280: L421L431, 2001.
28. Serhan CN, Clish CB, Brannon J, Colgan SP, Gronert K, and Chiang N. Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions. J Physiol Pharmacol 51: 643654, 2000.[ISI][Medline]
29. Sheng H, Shao J, Washington MK, and DuBois RN. Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 276: 1807518081, 2001.
30. Sherrard E. The corneal endothelium in vivo: its response to mild trauma. Exp Eye Res 22: 347357, 1976.[CrossRef][ISI][Medline]
31. Steen VD. Treatment of systemic sclerosis. Am J Clin Dermatol 2: 315325, 2001.[Medline]
32. Stockton RA and Jacobson BS. Modulation of cell-substrate adhesion by arachidonic acid: lipoxygenase regulates cell spreading and ERK1/2-inducible cyclooxygenase regulates cell migration in NIH-3T3 fibroblasts. Mol Biol Cell 12: 19371956, 2001.
33. Whitfield RA and Jacobson BS. The 1 integrin cytosolic domain optimizes phospholipase A2-mediated arachidonic acid release required for NIH-3T3 cell spreading. Biochem Biophys Res Commun 258: 306312, 1999.[CrossRef][ISI][Medline]
34. Yamaguchi Y and Yoshikawa K. Cutaneous wound healing: an update. J Dermatol 28: 521534, 2001.[ISI][Medline]
35. Zha S, Gage WR, Sauvageot J, Saria EA, Putzi MJ, Ewing CM, Faith DA, Nelson WG, De Marzo AM, and Isaacs WB. Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. Cancer Res 61: 86178623, 2001.
36. Zhu YK, Liu XD, Skold MC, Umino T, Wang H, Romberger DJ, Spurzem JR, Kohyama T, Wen FQ, and Rennard SI. Cytokine inhibition of fibroblast-induced gel contraction is mediated by PGE2 and NO acting through separate parallel pathways. Am J Respir Cell Mol Biol 25: 245253, 2001.
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |