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RECEPTORS AND SIGNAL TRANSDUCTION
on the healing process of corneal alkali burn in miceDepartment of 1Ophthalmology, Wakayama Medical University, Wakayama, Japan; 2Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland; 3Department of Anatomy, Graduate School of Medicine, Osaka City University, Abeno, Osaka, Japan; and 4Department of Ophthalmology, University of Cincinnati Medical Center, Cincinnati, Ohio
Submitted 18 June 2006 ; accepted in final form 30 October 2006
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ABSTRACT |
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(PPAR) inhibits activation of ocular fibroblasts and macrophages in vitro and also induced anti-inflammatory and anti-fibrogenic responses in an alkali-burned mouse cornea. PPAR overexpression suppressed upregulation of inflammation/scarring-related growth factors and matrix metalloproteinases (MMPs) in macrophages. It also suppressed expression of such growth factors and collagen I
2 and myofibroblast generation upon exposure to TGF
1. Exogenous PPAR did not alter phosphorylation of Smad2, but inhibited its nuclear translocation. PPAR overexpression enhanced proliferation of corneal epithelial cells, but not of fibroblasts in vitro. Epithelial cell expression of MMP-2/-9 and TGF1 and its migration were suppressed by PPAR overexpression. In vivo experiments showed that PPAR gene introduction suppressed monocytes/macrophages invasion and suppressed the generation of myofibroblasts, as well as upregulation of cytokines/growth factors and MMPs in a healing cornea. In vivo re-epitheliazation with basement membrane reconstruction in the healing, burned, cornea was accelerated by PPAR-Ad expression, although PPAR overexpression was considered to be unfavorable for cell migration. Together, these data suggest that overexpression of PPAR may represent an effective new strategy for treatment of ocular surface burns.
peroxisome proliferator-activated receptor-; gene therapy; macrophage; fibroblast; Smad
A wound healing reaction is orchestrated by a variety of signals derived form endogenous soluble factors. It is widely accepted that transforming growth factor- (TGF) is one of the most critical growth factors regulating cellular responses in the process of wound healing or tissue inflammation (46, 49). For a mouse corneal alkali burn, we have shown that Smad3 gene ablation or Smad7 gene introduction suppresses the unfavorable pathogenic response (inflammation, neovascularization, and scarring) of corneal tissue as seen in other tissues, i.e., skin, lung, liver, kidney, or eye lens in mice (2, 13, 16, 17, 31, 37, 45, 47, 48, 50, 52). However, the entire mechanism regulating such signals, i.e., cross-talk between pathways or as-yet unidentified pathways, needs to be investigated.
The peroxisome proliferator-activated receptor (PPAR) family consists of three members; PPAR, -
or -, which are involved in modulation of adipose metabolism, and inflammatory cell function, as well as behaviors of noninflammatory cells, i.e., fibrogenic reaction or cell proliferation during wound healing (5, 6, 8, 11, 20, 33). PPAR is a nuclear receptor for ligands of 15-deoxy-12, 14-prostaglandin J2 (15d-PGJ2), thiazolidinedione, etc., although 15d-PGJ2 also has PPAR-independent actions. Although all of the signaling networks linked to PPAR are not fully described, stimulation of this receptor by ligand application results in reduction of the fibrogenic response and myofibroblast generation in cultures of hepatic stellate cells (22, 33, 36, 62) and lung fibroblasts (4, 65), suggesting potential efficacy in preventing/treating liver fibrosis or other fibrotic disorders. PPAR signal suppresses the inflammatory reaction by immune cells or macrophages in vitro (7, 9, 28, 38, 43, 44, 67), and also has therapeutic effects on inflammatory disease models (30, 32, 58). Indeed, adenoviral gene transfer of PPAR exhibits a therapeutic effect in experimental model of colitis and liver fibrosis (29, 63), although it was not shown which specific in vivo signal(s) was (were) the target(s) of cross-talk from PPAR signal.
It is conceivable that application of PPAR ligands or introduction of the PPAR gene might have a therapeutic effect in an alkali-burned cornea because an alkali exposure causes tissue inflammation in the earlier phase and later causes stromal fibrosis/scarring, both much dependent on TGF/Smad signal. However, previous investigations of the effects of PPAR overexpression have been performed in internal organs, e.g., the liver and intestine (29, 63), and its effects on the skin or cornea have never been studied. The present study was undertaken to explore this possibility using a mouse model of an alkali ocular surface burn. We also employed adenoviral-mediated PPAR cDNA transfer instead of administration of a PPAR ligand, which might be easily washed out by blinking. Moreover, it has been reported that overexpression of the PPAR gene by transfection drives PPAR-related gene expression to such a high level that it was not reversed by administration of PPAR antagonist (19, 26), indicating that overexpressed PPAR activates its signal(s) independently on its ligands. First, the effects of PPAR overexpression on the behaviors of each cellular component of a burned cornea, e.g., epithelial and stromal cells and macrophages, and then evaluated the therapeutic efficacy of adenoviral-mediated PPAR gene transfer to the alkali-burned mouse cornea. Evaluation of epithelial healing, stromal repair, influx of inflammatory cells, and patterns of cytokine expression, all suggest that gene transfer of PPAR improves the healing of the injured tissue, and reduces scarring and neovascularization.
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MATERIALS AND METHODS |
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Adenovirus vector construction and virus purification.
We used the Adenovirus Cre/LoxP-Regulated Expression Vector Set (no. 6151; Takara, Tokyo, Japan) to make recombinant adenovirus expressing mouse PPAR as previously reported (47). A mixture of recombinant adenoviruses carrying CAG (cytomegalovirus enhancer, chicken -actin promoter plus a part of 3' untranslated region of rabbit -globin) promoter-driven Cre (Cre-Ad) and LNL-mouse PPAR cDNA (PPAR-Ad) was applied to the targets to express PPAR protein. Efficacy of gene transfer was confirmed as previously reported by using green fluorescent protein-carrying adenovirus and the observation that almost all the cells were positive (data not shown).
Effects of PPAR gene transfer on ocular fibroblast culture.
Because epithelial cells, keratocytes (corneal fibroblasts/myofibroblasts), and inflammatory cells are all involved in wound healing response to alkali exposure, we examined the effects of PPAR gene transfer on the behaviors of these cell types in vitro.
Mouse ocular fibroblasts were obtained from primary outgrowth of the cells from eye globes of postnatal day 1 mice. The confluent cells in 8-well chamber slides or 60-mm culture dishes were then subjected to infection with Cre-Ad and PPAR-Ad, while control cultures were infected with Cre-Ad only. The concentration of viral vector used was as previously described (25). Two days later, the fibroblasts were incubated in medium containing 1.0% fetal calf serum for 6 h. The cells in 60-mm dishes were cultured in the presence or absence of TGF1 (2.5 ng/ml) for 24 h for total RNA extraction for RT-PCR or real-time RT-PCR, as described below. The cells in 60-mm dishes, which were incubated for 48 h, were processed for Western blot analysis for -smooth muscle actin (SMA). The cells in wells of 8-well slides were incubated in the presence or absence of TGF1 (2.5 ng/ml) for 0.5, 1, 2, 4, or 48 h. These cells were processed for immunocytochemistry for PPAR, SMA, and collagens (samples after 48 h of incubation) or for phospho-Smad2 (other samples), as described below.
To further investigate whether PPAR overexpression affects phosphorylation of Smad2 in fibroblast cultures, we performed Western blot analysis. Mouse ocular fibroblasts were cultured until reaching confluence in 60 mm culture dishes and were then transfected with the PPAR adenoviral vector. After 48 h, the cells were then treated with culture medium supplemented with 3% fetal calf serum for 6 h. The cells were then treated with 2.5 ng/ml of TGF1 (R&D Systems) for 1, 3, and 5 h. The cell lysate was prepared by using cell lysis buffer as previously reported (47, 48). The cells were processed for SDS-PAGE and Western blot analysis for PPAR and phospho-Smad2 as previously reported (47, 48).
Effect of PPAR gene introduction on fibroblast proliferation stimulated by platelet-derived growth factor-BB (PDGF-BB) was also examined. The cells (5.1 x 104/100 ml/well) were seeded into the wells of a 96-well culture plate and then cultured for 24 h. The cells were processed for adenoviral PPAR gene introduction. After 36 h, the cells were further incubated for 36 h in the presence or absence of PDGF-BB (5.0 ng/ml; R&D Systems). The cells were then processed for Alamar Blue cell proliferation assay according to the manufacturer's instruction (21).
PPAR gene transfer in macrophage culture.
Macrophages were obtained from the mouse peritoneal space by irrigation 4 days after irritation by intraperitoneal injection of 5% oyster glycogen in sterile saline. The macrophages were seeded in 60 mm culture dishes, and were then incubated for 24 h. The cells were then infected with Cre-Ad and PPAR-Ad. Control cultures received Cre-Ad only. The concentration of the viral vectors used was as previously described (25). After 2 days, the macrophages were then incubated in serum-free medium for 6 h and then were further cultured in the presence or absence of TGF1 (2.5 ng/ml) for 24 h for total RNA extraction for real-time RT-PCR. Macrophages were also seeded in wells of 8-well chamber slides and processed for gene introduction as described above. The macrophages were then incubated in the presence of TGF1 (2.5 ng/ml) for 0.5, 1, 2, 4, and 7 h and processed for phospho-Smad2 detection by immunocytochemistry with diaminobenzidine.
Effect of PPAR gene transfer of epithelial cell proliferation, migration, and expression of wound healing-related genes.
An SV40-immortalized human corneal epithelial cell line was kindly provided by Dr. Kaoru Araki-Sasaki (Kumamoto University School of Medicine, Kumamoto, Japan) (1). The effect of PPAR overexpression on epithelial cell migration was evaluated using an in vitro wound closure model. A monolayer cell sheet of Araki-Sasaki corneal epithelial cells were treated with PPAR or Cre-Ad only gene transfer. After 48 h, a linear defect was produced in the monolayers with the use of a silicone rubber needle, and the rate of closure was monitored in medium containing 1.0% fetal calf serum. This was done by staining with hematoxylin and eosin and observed with light microcopy.
The cells (1 x 103/100 µl/well) were seeded in to the wells of a 96-well culture plate and incubated for 24 h. The cells were then processed for adenoviral gene transfer of PPAR cDNA and further incubated for 48 h. Control cells were treated with Cre-Ad. Cell proliferation in medium containing 1.0% fetal calf serum was assayed by using Alamar blue (Trek Diagnostic Systems, UK) according to the manufacturer's protocol (21).
Two days after gene transfer the cells were incubated in medium containing 1.0% fetal calf serum for 6 h. The cells in 60-mm dishes were cultured in the presence or absence of TGF1 (2.5 ng/ml) for 24 h for total RNA extraction for real-time RT-PCR as described below.
Alkali burn in a cornea of C57BL/6 mice and treatment with PPAR gene transfer.
Three microliters of 1 N sodium hydroxide solution was applied to the right eye of adult C57BL/6 mice (n = 75) to produce an ocular surface alkali burn under both general and topical anesthesia (47, 48). A mixture of Cre-Ad and PPAR-Ad was administered at 24 h, days 5 and 10 after an alkali exposure (PPAR-Ad group). Each adenoviral vector was used at the concentration of 1.0 x 107 PFU/µl. The efficiency of gene transfer to a burned mouse cornea by the Cre/LoxP system was established by using adenoviral vector of green fluourescein protein (GFP) in our previous study (47, 48). In this report, by using the same alkali burn model, GFP was well detected in healing epithelium and cells in the affected stroma. Although GFP was minimally expressed in cells in an uninjured mouse cornea (47), expression of protein derived from an exogenous gene was observed as early as day 3 with a peak at day 5 (47). Our previous experiments (47, 48) showed that there was no obvious difference in the histology or in healing at the macroscopic level in an alkali-burned mouse eye with (Cre-Ad group) or without application of adenovirus carrying Cre (No vector group). Thus corneas of Cre-Ad group were used as controls. After the evaluation of the corneal surface, the eye globe was enucleated 2 h after being labeled with bromodeoxyuridine (BrdU) and processed for histological examination in either paraffin or cryosections at days 5, 10, and 20. The number of eyes used for paraffin sections was 5 and 5 (day 5), 11 and 10 (day 10), or 6 and 6 (day 20) in the Cre-Ad and PPAR-Ad groups, respectively. The number of eyes used for cryosections was 2 and 2 (day 5), 3 and 3 (day 10), or 2 and 2 (day 20) in Cre-Ad group and PPAR-Ad group, respectively. For gene expression analysis, five corneas each at day 10 or 20 were processed for total RNA extraction and RT-PCR or real-time RT-PCR as previously reported (47, 48).
Immunohistochemistry.
Deparaffinized sections (5 µm thick), cryosections (7 µm thick) fixed in cold acetone for 5 min, and fixed cultured cells were processed for indirect immunohistochemistry as previously reported (47, 48). The following antibodies were diluted in PBS; rabbit polyclonal anti-phospho-Smad2 antibody [(1:100; Chemicon, Temecula, CA)], mouse monoclonal anti-SMA antibody (1:100; Neomarker, Fremont, CA), goat polyclonal anti-type IV collagen antibody (1:100; Southern Biotechnology, Birmingham, AL), and rat monoclonal anti-CD31 (PECAM) antibody (1:100, Santa Cruz). TGF1 and TGF2 were immunostained as previously reported (18).
The rat monoclonal F4/80 anti-macrophage antigen antibody (clone A3-1, 1:400 dilution in PBS; BMA Biomedicals, Augst, Switzerland) was used to detect monocytes/macrophages. The number of labeled cells in the central cornea (200 µm length) was determined in four corneas in each treatment group. FITC-conjugated specific secondary antibodies were used for detection of the primary antibody and 4,6-diamidino-2-phenylindole was used for nuclear counterstaining. Cell proliferation in the healing epithelium was determined by immunostaining with an anti-BrdU antibody (1:11 in PBS; Roche Diagnostics, Mannheim, Germany) and diaminobenzidine reaction with hematoxylin counterstaining as previously reported (47, 48), followed by counting the number of labeled cells in the healing epithelia in the affected cornea. Specimens were treated with 2N HCl for 60 min at 37°C before antibody application. Deparaffinized sections were also stained with hematoxylin and eosin and observed under light microscopy.
RT-PCR for PPAR mRNA expression and real-time RT-PCR for mRNA expression of TGF1, TGF2, VEGF, monocyte/macrophage chemoattractant protein-1, MMP-2, or MMP-9.
mRNA expression of endogenous and exogenous PPAR in alkali-burned cornea samples and cultured cells were examined by RT-PCR. Total RNA from corneal tissue or from cultured cells was processed for TaqMan real-time RT-PCR for mRNAs of mouse TGF1, TGF2, VEGF, monocyte/macrophage chemoattractant protein-1 (MCP-1), MMP-2, or MMP-9 as previously reported (47, 48). Primers and TaqMan probes for mouse or human mRNAs were shown in Tables 1 and 2, respectively.
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Statistical analysis. Data were analyzed with ANOVA or unpaired t-test, if needed.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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gene introduction to mouse ocular fibroblasts in vitro.
Marked expression of exogenous PPAR mRNA (Fig. 1a) and protein (Fig. 1b) was detected in cells that received adenoviral PPAR gene introduction compared with nontreated cells that exhibited very faint expression. Exogenous PPAR protein was detected in the cell nuclei (Fig. 1c). Immunocytochemistry showed inhibition of nuclear translocation of phosphorylated Smad2 (Fig. 1, d and e) and suppression of nuclear translocation of Smad3 (Fig. 1g) with PPAR overexpression. Western blot analysis, however, showed that PPAR overexpression did not alter the phosphorylation level of the Smad2 C-terminus upon exposure to exogenous TGF1 (Fig. 1f).
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SMA expression is the hallmark of myofibroblast differentiation. Immunohistochemistry or Western blot analysis showed that PPAR gene introduction abolished SMA expression and reduced protein expression of collagen types I and III in the presence of TGF1 (Fig. 2, a and b). PPAR overexpression counteracted PDGF-BB-induced acceleration of cell proliferation of ocular fibroblasts (Fig. 2c). PPAR gene transfer also reduced mRNA expression of TGF1, TGF2, CTGF, and collagen I2 in the presence or absence of exogenous TGF1 (Fig. 2d).
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gene introduction to mouse macrophages in vitro.
Expression level of PPAR mRNA and protein was higher in cultured macrophages treated with PPAR-Ad (Fig. 3a). Immunocytochemistry detected macrophages with nuclear PPAR immunoreactivity in both Cre-Ad- or PPAR-Ad-treated specimens, although immunoreactivity seemed more marked in specimens with PPAR-Ad compared with Cre-Ad (Fig. 3b). Immunocytochemistry showed upregulation and nuclear accumulation of COOH-terminallly phospho-Smad2 at 1 and 2 h post-TGF1 addition in Cre-Ad-treated macrophages, but such phospho-Smad2 upregulation was not observed in PPAR-Ad-treated cells (Fig. 3, c and d).
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gene reduced expression levels of TGF1, TGF2, VEGF, and MCP-1 mRNAs in cultured macrophages (Fig. 3e). Expression of MMPs in macrophages in a healing tissue is reportedly involved in degradation and remodeling of extracellular matrix, including basement membrane. We therefore evaluated the effect of PPAR gene transfer on expression of MMP-2 and MMP-9 in cultured macrophages by using real-time RT-PCR, as previously reported. The results showed that the relative RNA expression level of MMP-2 was almost one-tenth that of MMP-9. PPAR gene transfer reduced expression level of MMP-2 and MMP-9 with more marked reduction in MMP-9 expression (Fig. 3f).
Effect of PPAR gene transfer of epithelial cell proliferation and migration.
We evaluated the effect of PPAR overexpression on proliferation of a corneal epithelial cell line in vitro. PPAR gene transfer delayed closure of the wound in monolayer sheet of the corneal epithelial cell line (Fig. 4a), but enhanced proliferation (Fig. 4b). As shown in Fig. 4, c–e, PPAR overexpression reduced mRNA expression of MMP-2, MMP-9, and TGF1-like in fibroblast and macrophages.
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in an alkali-burned cornea.
RT-PCR showed higher expression of PPAR mRNA in burned corneas compared with an uninjured cornea and that with Cre-Ad (Fig. 5a). Very faint PPAR protein expression was detected by immunohistochemistry in the corneal epithelium of an uninjured cornea (Fig. 5bA) and those burned with alkali and treated with Cre-Ad (Fig. 5, bB and C). PPAR protein was readily observed in regenerated corneal epithelium and stromal cells in burned corneas that had received PPAR-Ad (Fig. 5dD).
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show improved healing.
Since previous experiments showed no obvious differences in either the histology or the rate of healing of alkali-burned mouse corneas treated with either Cre-Ad alone or no vector (21, 22), we used corneas of the Cre-Ad group as controls (Fig. 6, a and b). At day 5, 100% of the corneas in Cre-Ad group showed epithelial defect(s), while 57% (4 of 7) in the PPAR-treated group had the defect. At day 10 post-injury, 67% (8 of 12) corneas of the Cre-Ad group still showed an epithelial defect or stromal ulceration, whereas 4 of 13 corneas treated with PPAR-Ad showed epithelial defects. At this same time point, corneas in the Cre-Ad group that remained free of epithelial defects (4/12) exhibited more marked stromal opacification, compared with the opacification in all of the corneas of the PPAR group. At day 20 post-injury, 50% (4/8) of the corneas in the Cre-Ad group still exhibited epithelial defects/ulceration, and the remaining corneas showed dense stromal opacification (Fig. 6b). In contrast, 87.5% (7/8) corneas in the PPAR-Ad group exhibited only minor opacification without epithelial defects.
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-Ad groups showed epithelial defects and stromal inflammation at day 5 and 10, corneas in the Cre-Ad group were characterized by a less organized epithelium with invasion into the underlying stroma (Fig. 6, cA–D). Our previous study showed that the regenerated epithelium in this alkali burn model is of conjunctival origin (data not shown). At day 20, the affected corneal stroma seemed better organized in the PPAR-Ad treatment group, compared with the Cre-Ad group (Fig. 6, cE and F).
Invasion of monocytes/macrophages in the affected stroma of PPAR-Ad-treated corneas and control corenas and expression of growth factors in burned cornea.
Since invasion of monocytes/macrophages plays an important role in tissue damage following injury, including alkali burning in the cornea, we used the F4/80 antibody to quantify the number of monocytes/macrophages in the central stroma of alkali-injured corneas (Fig. 7, a and b). At day 10, there was a significant reduction in the number of F4/80-positive cells in corneas of PPAR-Ad-treated mice compared with their respective Cre-Ad controls.
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-Ad treatment on expression of growth factors implicated in the tissue destruction in alkali-burned corneas. Real-time RT-PCR showed an upregulation of TGF1, VEGF, and MCP-1 mRNAs, at day 10 post-alkali burn. PPAR gene transfer counteracted upregulation of these components (Fig. 7c). Reduction of expression levels of TGF1 and MCP-1 was considered to contribute to the reduction of macrophage invasion. Immunohistochemical results coincided with the findings obtained by real-time RT-PCR. Immunoreactivity for TGF1 and 2 was detected in the epithelium of the uninjured cornea as previously reported (data not shown). The levels of extracellular TGF1 were markedly elevated at day 10 (not shown) and persisted at day 20 post-injury in Cre-Ad-treated eyes (Fig. 7dA and B). A similar pattern was observed for TGF2, with stromal expression of TGF2 being particularly intense at day 20 (Fig. 7, dC and D). This upregulation of TGF1 and 2 was suppressed by PPAR-Ad treatment at day 10 (not shown) and day 20.
Neovascularization and scar formation are suppressed by PPAR gene introduction.
One of the hallmarks of corneal stromal scarring is the development of neovascularization. Such neovascularization of the corneal stroma likely contributes to stromal opacification and is associated with inflammation. Examination of stromal neovascularization using an antibody to CD31 (PECAM) showed marked staining in Cre-Ad-treated corneas at all time points examined which was substantially reduced in eyes treated with PPAR-Ad (Fig. 8a). This effect might be attributed to the reduction of expression of TGF1 and VEGF in tissue treated with PPAR-Ad.
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SMA-positive myofibroblasts (58–61). Examination of the expression pattern of SMA in injured corneas showed that, at day 10 post-injury, many stromal cells in injured corneas of Cre-Ad-treated mice expressed SMA, but that this was substantially reduced in PPAR-Ad-treated mice (Fig. 8, b and c). These differences persisted at day 20 post-wounding (Fig. 8, b and c).
mRNA expression of CTGF was also assayed to evaluate tissue fibrogenic reaction by stromal cells. Expression of these components was markedly upregulated at day 20 in Cre-Ad-treated corneas, but was suppressed in PPAR-treated corneas at day 20 (Fig. 8d).
Degradation of basement membrane and expression of MMPs.
Development of corneal ulceration post-alkali burn is characterized by degradation of the epithelial basement membrane by MMPs expressed by inflammatory cells, i.e., macrophages, as well as by healing resident corneal cells. In situ zymography showed that matrix degradation activity of MMP was more marked in control corneas than in corneas treated with PPAR gene transfer at day 10 (Fig. 9a). The activity was abolished by treating the membrane with the synthetic MMP inhibitor that included in the kit, indicating that the activity was derived from MMP, but not from proteinases other than MMP (data not shown). Upregulation of MMPs in inflammatory cells, i.e., macrophages, is reportedly involved in the degradation of epithelial basement membrane in a healing cornea. Real-time RT-PCR showed that both MMP-2 and MMP-9 were upregulated in healing burned corneas on days 10 and 20. PPAR gene transfer suppressed such upregulation slightly at day 10 and obviously at day 20 (Fig. 9b).
Immunostaining of healing corneas in the Cre-Ad control group for type IV collagen showed degradation of basement membrane and loss of epithelial integrity beginning at day 5 through 20 (Fig. 9, cA, C, and E). In contrast, corneas in the PPAR-Ad group showed a linear pattern of subepithelial immunoreactivity indicating the preservation of the basement membrane (Fig. 9cD and F).
PPAR expression alters the proliferation profile of the healing epithelium.
Proliferation and migration of regenerated epithelium are critical to corneal wound healing. Although migration and proliferation of epithelial cells are affected by various growth factors including TGF signaling, such behaviors are considered to be regulated in a complex way by both autocrine and paracrine signaling from other cell types in the local tissue. We examined the effect of PPAR gene introduction to burned cornea on proliferation activity in the healing corneal epithelium. Although our in vitro experiment showed that PPAR gene transfer enhanced cell proliferation of corneal epithelial cell line, the incidence of BrdU-labled epithelial cells was similar between Cre-Ad group and PPAR-Ad group (data not shown).
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DISCUSSION |
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fibrogenic behaviors both in ocular fibroblasts and in macrophages in vitro. PPAR gene transfer suppressed profibrogenic myofibroblast generation from ocular fibroblasts in the presence of TGF1 and expression of fibrogenic cytokines, i.e., TGF1, TGF2, and CTGF, as well as of collagen I2. Exogenous PPAR also countereacted acceleration of ocular fibroblast proliferation promoted by PDGF. Similar effect of PPAR signal on behaviors of mesenchymal cell types have been reported (4, 33, 62, 65). In corneal epithelial cell line, expression of MMP-2/9 and TGF1 was suppressed. These all findings prompted us to hypothesize that PPAR gene transfer might have a therapeutic effect on alkali-burned cornea, because these cellular components all participate in the unfavorable inflammatory/fibrogenic process of healing in this condition. Next, we observed such beneficial effect by using mouse model of corneal alkali burn. Although similar therapeutic potential of PPAR gene introduction via adenoviral vector has been reported in experimental colitis and liver fibrosis model (29, 63), the present study showed this is the case also in a body surface tissue (cornea) composed of stratified epithelium and mesenchymal tissue. As previously reported (19, 26), overexpression of PPAR gene exhibited its effects without further addition of its ligands in a burned cornea.
Modulation of cytoplasmic signaling by PPAR is not fully understood and requires further investigation. Nevertheless, cross-talk between PPAR-linked signaling and other signal(s) derived from various ligands/receptors have been in part clarified. For example, PPAR receptor-derived signal activates AP-1, MAP kinase/Erk, p38 MAP kinase, etc., which in turn exhibit complex cross-talks (4–6, 8, 11, 20, 22, 33, 36, 41, 62, 65). On the other hand, we previously reported that TGF/Smad signaling is critical in the progression of inflammation and fibrosis in an alkali-burned cornea by showing that adenoviral gene transfer of Smad7, an inhibitory Smad, almost completely rescued the corneal transparency in a healing mouse cornea post-alkali burn (47). We therefore focused on the effects of PPAR signaling on the Smad pathway (34, 59). Our present study shows that PPAR overexpression inhibits nuclear translocation of phospho-Smad2 and Smad3, but does not alter the COOH-terminal phosphorylation of Smad2 in fibroblasts or macrophages in vitro. The mechanism of inhibition of nuclear import of phospho-Smad2 and Smad3 by PPAR signal must be further investigated. Suppression of TGF1-dependent nuclear translocation of phospho-Smad2 and Smad3 might be included in the mechanisms of PPAR's anti-fibrogenic effects in the cultured fibroblasts. It is known that expression of SMA depends on Smad2 signaling and that of collagen on Smad3 (14, 24, 39, 53, 64). On the other hand, nuclear factor-
B signaling is also involved in the pathogenesis of excess inflammation and late scarring in an alkali-burned cornea (51). However, PPAR gene transfer did not affect the phosphorylation of RelA in cultured mouse ocular fibroblasts (data not shown).
Macrophages also have important roles in the development of ulceration, excess scarring and neovascularization in an injured tissue including an alkali-burned cornea during wound healing process (40, 56, 60). It is believed that invasive macrophages are one of the main sources of cytokines, including TGF, that activate keratocytes and induce their transformation to myofibroblasts. PPAR gene transfer reduced mRNA expression of TGF1, TGF2, VEGF, and MCP-1, all of which are chemotactic factors for monocytes/macrophages. Moreover, PPAR overexpression decreased mRNA expression of MMP-2/-9 that is involved in degradation of basement membrane in the healing cornea. As seen in fibroblasts, PPAR signal counteracted TGF/Smad2 signal in macrophages. Similar deactivation of macrophages by stimulating PPAR signal has been extensively investigated using specific PPAR ligands (5, 6, 8, 11, 20, 33, 41).
These in vitro results strongly suggested that PPAR gene introduction might be effective in suppression of the fibrogenic, or scarring reaction in an alkali-burned cornea in vivo. We showed here that PPAR gene introduction prevents tissue destruction in an alkali-burned mouse cornea. Undesirable pathogenic processes of corneal healing, i.e., delayed resurfacing (breakdown of the epithelial basement membrane), scarring and neovascularization were all less severe in PPAR-Ad treated burned corneas compared with those with control, Cre-Ad. PPAR's favorable effects on corneal fibroblasts and inflammation might overcome its negative effect on epithelial cell migration, although PPAR gene transfer retarded epithelial cell migration. Conversion of keratocytes to myofibroblasts (12, 27, 54, 61), as characterized by expression of SMA, is a very important element in scarring of the stroma in a healing cornea following exposure to alkali and is associated with upregulation of matrix components involved in stromal scarring. In the present study, immunohistochemistry showed that treatment with PPAR-Ad suppressed myofibroblast generation in the burned stroma. Real-time RT-PCR further showed that PPAR overexpression suppressed CTGF, TGFb1, and TGFb2 mRNA expression at day 20, further confirming the anti-fibrogenic effect of PPAR signal. Although myofibroblast generation is reportedly regulated by extracellular matrix, i.e., fibronectin ED-A (12, 27, 54, 61), TGF/Smad2 signaling is essential for it (14, 24, 39, 64). Moreover, Smad3 gene ablation or Smad7 gene introduction suppresses appearance of myofibroblasts in cutanenous wounds or in alkali-burned corneal stroma, clearly indicating that the Smad signal is a key in injury-induced fibroblast-myofibroblast conversion in vivo.
PPAR-Ad treatment also suppressed recruitment of monocytes/macrophages to the healing stroma post-alkali burn. TGF1, VEGF, and MCP-1, all abundantly expressed by macrophages, were dramatically upregulated in a burned cornea. Suppression of expression levels of these factors in tissue might result in reduction of further invasion of monocytes/macrophages that express inflammatory growth factors and cytokines, and that of VEGF in reduction of injury-induced unfavorable neovascularization.
Epithelial healing in vivo depends on epithelial cell migration and proliferation, as well as the integrity of the epithelial basement membrane. Expression of MMP-2/-9 and TGF1 in the corneal epithelial cell line was suppressed by PPAR gene transfer, which might account for retarded migration of the PPAR-Ad-treated epithelial cells in vitro. Since TGF normally inhibits growth of epithelial cells, its suppression by PPAR should increase proliferation of these cells contributing to the enhanced re-epithelialization seen in vivo. MMPs expressed by corneal cells and inflammatory cells are involved in degradation of the epithelial basement membrane. The present experiments show that PPAR gene introduction suppressed expression of MMP-9 dramatically in macrophages in vitro and also reduced expression of MMP mRNAs and activity in healing corneas in vivo. The ability of PPAR to inhibit these MMPs likely contributes to the rapid restoration of the basement membrane and thus to the promotion of epithelial healing seen in vivo post-injury.
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FOOTNOTES |
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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.
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