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Keratinocyte-Derived Vascular Endothelial Growth Factor Biosynthesis Represents a Pleiotropic Side Effect of Peroxisome Proliferator-Activated Receptor- Agonist Troglitazone but Not Rosiglitazone and Involves Activation of p38 Mitogen-Activated Protein Kinase: Implications for Diabetes-Impaired Skin Repair

Pharmazentrum Frankfurt/ZAFES (D.S., O.S., I.G., J.P.D., H.S., M.B., G.G., J.P., S.F.) and Zentrum der Chirurgie (R.S.), Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

Received for publication June 2, 2008.

Accepted for publication July 1, 2008.

	Abstract

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The peroxisome proliferator-activated receptors (PPARs) represent pharmacological target molecules to improve insulin resistance in type 2 diabetes mellitus. Here we assessed a functional connection between pharmacological activation of PPAR and vascular endothelial growth factor (VEGF) expression in keratinocytes and during diabetes-impaired acute skin repair in obese/obese (ob/ob) mice. PPARβ/ agonist 4-[3-[4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid (L165,041) and PPAR agonists ciglitazone and troglitazone, but not rosiglitazone, potently induced VEGF mRNA and protein expression from cultured keratinocytes. Inhibitor studies revealed a strong functional dependence of troglitazone- and L165,041-induced VEGF expression on p38 and p42/44 mitogen-activated protein kinase (MAPK) activation in keratinocytes. Rosiglitazone also induced activation of p38 MAPK but failed to mediate the activation of p42/44 MAPK in the cells. Functional ablation of PPARβ/ and PPAR from keratinocytes by small interfering RNA did not abrogate L165,041- and troglitazone-induced VEGF biosynthesis and suggested VEGF induction as a pleiotropic, PPAR-independent effect of both drugs in the cells. In accordance with the in vitro situation, we found activated p38 MAPK in wound keratinocytes from acute wounds of rosiglitazone- and troglitazone-treated diabetic obese/obese mice, whereas keratinocyte-specific VEGF protein signals were only prominent upon troglitazone treatment. In summary, our data from cell culture and wound healing experiments suggested p38 MAPK activation as a side effect of thiazolidinediones; however, only troglitazone, but not rosiglitazone, seemed to translate p38 MAPK activation into a PPAR-independent induction of VEGF from keratinocytes.

Thiazolidinediones (TZDs) represent a class of antidiabetic drugs that are capable of improving insulin resistance in target tissues such as muscle and liver (Henry, 1997). In 1995, it became clear that TZDs act through activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)- (Lehmann et al., 1995). The PPAR family of transcription factors consists of two additional members: PPAR and PPARβ/, and all members heterodimerize with 9-cis-retinoic acid retinoid X receptors (Kliewer et al., 1992, 1994). Insulin resistance now seems to be a consequence of the release of cytokines and adipokines from growing adipose tissue under conditions of obesity (Hotamisligil, 2006; Tilg and Moschen, 2006). TZDs interfere with the endocrine signaling process of adipocytes to muscle and liver and enhance insulin action in these organs through down-regulation of tumor necrosis factor (TNF)- and IL-6 and induction of the insulin-sensitizing hormone adiponectin in adipose tissue (Rangwala and Lazar, 2004). It is interesting that hyperlipidemia, representing an additional obesity-associated risk factor, can be improved by fibrates that activate PPAR (Staels and Fruchart, 2005).

Diabetes-associated severe ulcerations of the skin represent a serious problem of growing clinical importance. Diabetic ulcers still have a poor prognosis with high reulceration rates and a high mortality after limb amputations (Faglia et al., 2001). Besides their metabolic functions, it is interesting that PPAR,-β/, and - have been shown also to be involved in the homeostatic regulation of normal and injured skin (Michalik and Wahli, 2007). Epidermal keratinocytes express all three PPAR isoforms (Rivier et al., 1998). In these cells, PPARβ/ is the major isoform, whereas PPAR and PPAR are increased and functionally connected to keratinocyte differentiation in humans and mice (Rivier et al., 1998; Kömüves et al., 2000; Mao-Qiang et al., 2004). In contrast to PPAR, which remains hardly detectable upon skin wounding, PPAR and -β/ are reactivated in wound margin keratinocytes during acute healing in mice (Michalik et al., 2001). Keratinocytes represent a major source of vascular endothelial growth factor (VEGF) in skin wounds (Brown et al., 1992), and VEGF expression is induced by cytokines and growth factors in the cells (Frank et al., 1995). In this study, we investigated the potency of PPAR agonists to interfere with keratinocyte-derived VEGF expression. The PPAR agonist troglitazone, but not rosiglitazone, potently induced keratinocyte VEGF expression; however, our data constitute evidence that troglitazone-mediated induction of VEGF expression was independent from PPAR but functionally connected to p38 MAPK activation in cultured keratinocytes.

	Materials and Methods

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Animals. Female C57BL/6J-ob/ob mice were obtained from Charles River WIGA (Sulzfeld, Germany) and were maintained under a 12-h light/dark cycle at 22°C until they were 10 weeks of age. At this time, they were caged individually, monitored for body weight, and wounded as described below.

Treatment of Mice. Mice were treated with rosiglitazone (Avandia, 5 mg/kg/day) or troglitazone (5 mg/kg/day) twice a day (6:00 AM and 6:00 PM) by gastrogavage for the indicated time points. The drug was freshly homogenized in 0.5% methylcellulose (Fluka; Sigma-Aldrich, Seelze, Germany) before oral administration. Treatment of mice with 0.5% methylcelluose alone served as a control. Rosiglitazone (Avandia) was from GlaxoSmithKline Consumer Healthcare (Bühl, Germany), and troglitazone was from Axxora (Lörrach, Germany).

Wounding of Mice. Wounding of mice was performed as described previously (Frank et al., 1999; Stallmeyer et al., 1999). In brief, mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg body weight)/xylazine (10 mg/kg body weight). Six full-thickness wounds (5 mm in diameter, 3-4 mm apart) were made on the back of each mouse by excising the skin and the underlying panniculus carnosus. An area measuring 7 to 8 mm in diameter, which included the granulation tissue and the complete epithelial margins, was excised at the indicated time points for analysis. As a control, a similar amount of skin was taken from the backs of nonwounded mice. For each experimental time point, tissue from four wounds each from three animals (n = 12 wounds, RNA analysis) and from two wounds each from three animals (n = 6 wounds, protein analysis) were combined and used for RNA and protein preparation. All animal experiments were carried out according to the guidelines and were approved by the local ethics animal review board.

RNA Isolation and RNase Protection Analysis. RNA isolation and RNase protection assays were carried out as described previously (Chomczynski and Sacchi, 1987; Frank et al., 1999). The cDNA probes were cloned using reverse transcription-polymerase chain reaction. The murine probes corresponded to nt 139 to 585 (VEGF, S38083 [GenBank] ), nt 1499 to 1779 (for PPARβ/, NM_006238 [GenBank] ), and nt 163 to 317 (for GAPDH, NM_002046 [GenBank] ). The human probes corresponded to nt 1362 to 1516 (for VEGF, NM_001025370.1) and nt 961 to 1070 (for GAPDH, M33197 [GenBank] ), respectively.

Immunohistochemistry. Mice were wounded as described above. Animals were sacrificed at day 5 after injury. Complete wounds were isolated from the back and fixed in formalin. Bisected wounds were embedded in paraffin. Immunohistochemistry from 4-µm deparaffinized serial sections was performed as described previously (Stallmeyer et al., 1999). Sections were stained with the avidin-biotin-peroxidase complex system using 3,3-diaminobenzidine-tetrahydrochloride as a chromogenic substrate. Nuclei were counterstained with hematoxylin. The antibody directed against murine VEGF was from Santa Cruz (Heidelberg, Germany). The phosphospecific anti-p38 MAPK antibody was obtained from Cell Signaling Technology (Frankfurt, Germany).

Cell Culture. Human HaCaT epidermal keratinocytes (Boukamp et al., 1988) were grown to confluence in Dulbecco's modified Eagles's medium (Invitrogen AG, Karlsruhe, Germany). Human primary keratinocytes were isolated according to a protocol from PromoCell (available at http://www.promocell-academy.com) and cultured in keratinocyte growth medium 2 (PromoCell, Heidelberg, Germany). The murine macrophage cell line RAW264.7 was cultured in RPMI medium (Invitrogen AG). Confluent keratinocytes were subsequently treated with epidermal growth factor (EGF; 10 ng/ml), ciglitazone (0.1-25 µM), troglitazone (0.1-25 µM), rosiglitazone (0.1-50 µM), L165,041 (0.1-50 µM), WY14643 (50 µM), clofibrate (500 µM) in the presence or absence of 200 nM wortmannin (WTM), 10 µM U0126, 5 µM SB203580, or 10 µM actinomycin D (Act D) for the indicated periods of time. RAW264.7 cells were stimulated with lipopolysaccharide (LPS; 200 ng/ml) and interferon- (IFN-; 2 ng/ml). EGF and IFN- were purchased from Roche Biochemicals (Mannheim, Germany). WTM and SB203580 were obtained from Calbiochem (Darmstadt, Germany), and U0126 was from Alexis (San Diego, CA). Ciglitazone, troglitazone, L165,041, WY14643, and clofibrate were obtained from Merck (Darmstadt, Germany). Rosiglitazone was from Axxora (Lörrach, Germany). LPS was from Sigma (Taufkirchen, Germany).

Nitrite Determination in Cell Culture Supernatants. Nitrite, a stable nitric oxide (NO) oxidation product, was determined in cell culture supernatants using the Griess reaction (Green et al., 1982). In brief, cleared supernatants were mixed with 20 µl of sulfanilamide (dissolved in 1.2 M HCl) and 20 µl of N-naphthylethylenediamine dihydrochloride. After 5 min, the absorbance was measured at 540 nm.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Dose-dependent induction of VEGF expression in keratinocytes by PPAR agonists. Quiescent human HaCaT keratinocytes were stimulated with troglitazone (a), rosiglitazone (b), or L165,041 (c) for 24 h as indicated. EGF (10 ng/ml) was used as a control. After 24 h, VEGF mRNA expression of nonstimulated (ctrl) or stimulated keratinocytes was analyzed by RNase protection assay (top and middle). VEGF165 protein release from the cells was assessed by ELISA analyses of the corresponding conditioned cell culture supernatants (bottom). **, P < 0.01; *, P < 0.05; n.s., not significant compared with nonstimulated control cells (ctrl). Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

Enzyme-Linked Immunosorbent Assay. Quantification of human VEGF₁₆₅ protein from keratinocyte cell culture supernatants was performed using the human VEGF enzyme-linked immunosorbent assay (ELISA) kit (Biosource, Nivelles, Belgium).

Preparation of Protein Lysates and Immunoblot Analysis. Keratinocyte cell culture samples and murine skin were homogenized as described previously (Goren et al., 2006). Twenty to fifty micrograms of total protein lysate was separated using SDS-gel electrophoresis, and specific proteins were detected using antisera directed against total Akt, phospho-Akt (Ser473), phospho-p38 MAPK (Thr180, Tyr182), phospho-p42/44 MAPK (Thr202, Tyr204) (Cell Signaling, New England Biolabs), PPAR (Santa Cruz), and β-actin (Sigma). A secondary antibody coupled to horseradish peroxidase and the enhanced chemiluminescence detection system was used to visualize the proteins of interest. Phenylmethylsulfonyl fluoride, aprotinin, NaF, Na₂VO₄, and dithiothreitol were from Sigma. Ocadaic acid and leupeptin were from BioTrend (Köln, Germany), and the enhanced chemiluminescence detection system was obtained from GE Healthcare (Freiburg, Germany).

Silencing of PPAR and PPARβ/ Expression by Small Interfering RNA. HaCaT keratinocytes (2 x 10⁵) were grown in six-well plates to reach 40 to 60% confluence. Cells were subsequently transfected twice with the respective small interfering RNA (siRNA; 50 nM final concentration) using Oligofectamine (Invitrogen, Karlsruhe, Germany) and Opti-MEM (Invitrogen) as described by the manufacturer.

Determination of Rosiglitazone and Troglitazone in Mouse Plasma Samples by Liquid Chromatography/Mass Spectrometry/Mass Spectrometry. Aliquots of mouse plasma samples were extracted by methanol/water (50:50 v/v) precipitation. Pioglitazone and ciglitazone were used as internal standards for rosiglitazone and troglitazone, respectively. HPLC analysis was done under gradient conditions using a Luna C18(2) column (Phenomenex, Aschaffenburg, Germany). Mass spectrometry and tandem mass spectrometry analyses were performed on a 4000 Q TRAP triple-quadrupole mass spectrometer with a Turbo V source (Applied Biosystems, Darmstadt, Germany) in the negative ion mode. Precursor-to-product ion transitions of m/z 35642 for rosiglitazone, 44042 for troglitazone, 35542 for pioglitazone, and m/z 33242 for ciglitazone were used for the multiple reaction monitoring with a dwell time of 150 ms. Concentrations of the calibration standards, quality controls, and unknowns were evaluated by Analyst software (version 1.4; Applied Biosystems). Variations in accuracy and intraday and interday precision (n = 6 for each concentration, respectively) were <15% over the range of calibration.

Statistical Analysis. Data are shown as means ± S.D. Data analysis was carried out using the unpaired Student's t test with raw data. Statistical comparison between more than two groups was carried out by analysis of variance (ANOVA; Dunnett's method).

	Results

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Troglitazone and L165,041 but Not Rosiglitazone Potently Induce VEGF Expression in Keratinocytes. Epidermal keratinocytes express all three PPAR subtypes (Rivier et al., 1998). Because angiogenesis is central to skin repair (Singer and Clark, 1999; Eming and Krieg, 2006) and keratinocytes are well known producers of VEGF at the wound site (Brown et al., 1992) and in vitro (Frank et al., 1995), we tested the potency of diverse PPAR agonists to drive VEGF expression in keratinocytes. The PPAR agonists troglitazone (Fig. 1a), ciglitazone (Supplemental Figure S1), and the PPARβ/ agonist L165,041 (Fig. 1c) are potent inducers of VEGF mRNA (top and middle) and protein (bottom) expression in cultured human HaCaT keratinocytes. By contrast, the PPAR agonist rosiglitazone failed to induce VEGF expression in the cells at low (0.1-25 µM; data not shown) and even at highest concentrations (Fig. 1b). It is interesting that the dose-response experiment revealed that the PPAR agonists troglitazone and ciglitazone or the β/ agonist L165,041 mediated an "on/off" phenomenon with respect to VEGF expression at higher concentrations (glitazones, 25 µM; L165,041, 50 µM) (Fig. 1, a and c; Supplemental Figure S1). By contrast, PPAR agonists WY14643 (50 µM) and clofibrate (500 µM) did not induce VEGF mRNA and protein expression in keratinocytes even at the highest concentrations (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Time-dependent induction of VEGF expression in keratinocytes by PPAR agonists. Confluent human HaCaT keratinocytes were stimulated with 25 µM troglitazone (a) or 50 µM L165,041 (b) for the indicated time points. At the indicated time points of stimulation, VEGF mRNA expression of nonstimulated (ctrl, 0 h) or stimulated keratinocytes was analyzed by RNase protection assay (top and middle). VEGF165 protein release from the cells was assessed by ELISA analyses of the corresponding conditioned cell culture supernatants (bottom). **, P < 0.01; *, P < 0.05 compared with nonstimulated control cells (ctrl). Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

To test the bioactivity of rosiglitazone, we assessed the ability of this drug to suppress the proinflammatory stimulation of murine macrophages in vitro. LPS and IFN-, which characteristically trigger macrophage inflammatory activity (Gordon, 2003), potently mediated the accumulation of nitrite in cell culture supernatants of stimulated RAW264.7 macrophages. Nitrite represents a reliable readout for inducible nitric-oxide synthase activity. Rosiglitazone (50 µM) turned out to be effective, because the drug significantly reduced the production of nitrite from activated RAW264.7 macrophages (Fig. 3).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Rosiglitazone and troglitazone are functionally active. Quiescent murine RAW264.7 macrophages were activated using LPS (200 ng/ml) and IFN (2 ng/ml) for 24 h in the presence or absence of 50 µM rosiglitazone (a) or 25 µM troglitazone (b). Nitrite accumulation as an assessment of inducible NOS activity and thus macrophage activation was assessed using the Griess reaction. **, P < 0.01; *, P < 0.05 as indicated by the bracket. Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Induction of VEGF expression by PPARβ/ and PPAR agonists in human primary keratinocytes. Quiescent human primary keratinocytes were stimulated with EGF (10 ng/ml), troglitazone (25 µM), or L165,041 (50 µM) for 24 h. After 24 h, VEGF mRNA expression of nonstimulated (ctrl) or stimulated primary keratinocytes was analyzed by RNase protection assay (a). **, P < 0.01 compared with nonstimulated control cells (ctrl). Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3). VEGF165 protein release from the cells was assessed by ELISA analyses of conditioned cell culture supernatants (b). VEGF165 protein expression from one representative experiment is shown.

View larger version (38K): [in this window] [in a new window]   Fig. 5. PPARβ/ and PPAR agonists do not stabilize VEGF mRNA. Quiescent HaCaT keratinocytes were stimulated with EGF (10 ng/ml), troglitazone (25 µM), or L165,041 (50 µM) for 16 h. After 16 h, Act D (10 µM) was added to the cells. Keratinocyte VEGF mRNA levels were determined at the indicated time points of treatment by RNase protection assay. Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3). A RNase protection assay demonstrating VEGF mRNA in stimulated keratinocytes in the absence of Act D (16 and 24 h as indicated) is shown at the bottom.

Troglitazone- and L165,041-Induced VEGF Expression Is Independent from PPAR Activation. As given in Fig. 6, inhibitor experiments strongly argue for MAPK activation as the functional basis of troglitazone-mediated VEGF expression in keratinocytes. It is noteworthy that inhibition of either p42/44 (by U0126) or p38 (by SB203580) MAPK nearly completely abrogated troglitazone-induced the expression of VEGF mRNA in keratinocytes, whereas inhibition of PI3K by wortmannin had no effect (Fig. 6a, top). As a consequence, we observed a marked reduction of troglitazone-induced VEGF protein upon inhibition of p42/44 and p38 MAPK at the early 16-h experimental time point, and inhibition of VEGF protein expression remained significantly reduced even after 24 h of stimulation (Fig. 6a, bottom). In addition, induction effects of the PPARβ/ agonist L165,041 on keratinocyte VEGF mRNA (Fig. 6b, top) and protein (Fig. 6b, bottom) expression seemed to be sensitive to inhibition of PI3K or p42/44 and p38 MAPK activation.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Troglitazone- and L165,041-mediated VEGF expression is dependent on MAPK activation. Quiescent human HaCaT keratinocytes were stimulated with troglitazone (a) or L165,041 (b) for 16 and 24 h in the absence or presence of WTM (200 nM), SB203580 (5 µM), or U0126 (10 µM) as indicated and subsequently analyzed for VEGF mRNA expression by RNase protection assay (top and middle). VEGF165 protein release from the cells was assessed by ELISA analyses of the corresponding conditioned cell culture supernatants (bottom). **, P < 0.01; *, P < 0.05 compared with stimulated keratinocytes in the absence of the respective inhibitor. Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

In accordance with wortmannin-insensitive VEGF induction by troglitazone, we observed no increase in phosphorylation of protein kinase B/Akt, which represents a target of active PI3K (Franke et al., 1995) (data not shown). In contrast, troglitazone mediated a strong phosphorylation and thus activation of both p38 and p42/44 MAPK (Fig. 7a). The PPAR agonist rosiglitazone did not induce VEGF expression in keratinocytes (Fig. 1b); however, the drug shared its capability to potently induce p38 MAPK activation with troglitazone. Rosiglitazone failed to mediate p42/44 MAPK activation (Fig. 7a). Activation of the MAPKs by the assessed drugs in keratinocytes seemed to be independent from a cellular response toward stress or toxicity, because a cell viability assay showed only moderate changes in cell survival upon drug exposure (Fig. 7b).

View larger version (17K): [in this window] [in a new window]   Fig. 7. Differential activation of p38 and p42/44 MAPK by PPAR agonists. a, quiescent HaCaT keratinocytes were treated with L165,041 (50 µM), troglitazone (25 µM), or rosiglitazone (50 µM) for the indicated time periods. Nonstimulated cells (ctrl 0', ctrl 90') served as controls. Total cell lysates were analyzed for the presence phosphorylated p38 MAPK (p-p38 Thr180, Tyr182) or phosphorylated p42/44 MAPK (p-p42/44 Thr202, Tyr204) by immunoblot. Loading of gels was controlled by the analysis of β-actin expression as indicated. b, keratinocyte viability as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay in the presence or absence of troglitazone (25 µM), rosiglitazone (50 µM), or L165,041 (50 µM) as indicated. **, P < 0.01; *, P < 0.05; n.s., not significant compared with nonstimulated control cells (ctrl). Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

View larger version (34K): [in this window] [in a new window]   Fig. 8. Troglitazone- and L165,041-induced VEGF expression are independent of PPAR expression. Cultured HaCaT keratinocytes were treated with Oligofectamine alone (ctrl) and in combination with a nonspecific (scrambled) siRNA, a PPAR-specific siRNA (si-PPAR) (a) or a PPARβ/-specific siRNA (si-PPARβ/) (b) as indicated. Abrogation of the respective PPAR in keratinocytes was controlled by immunoblot (a, left) or RNase protection assay (b, left) as indicated. Analysis of β-actin (a) or GAPDH (b) was used to control an equal loading. VEGF165 protein release from control-(ctrl), scrambled RNA (scr)- or PPAR-specific siRNA (si-P; si-Pβ/)-treated keratinocytes in the absence (w/o) or presence (±) of troglitazone (25 µM) or L165,041 (50 µM) as indicated. **, P < 0.01 compared with conditions without drug. ##, P < 0.01; n.s., not significant as indicated by brackets. Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

Opposite Effects of PPAR Agonists Rosiglitazone and Troglitazone on Wound Keratinocytes in Diabetes-Impaired Skin Wounds. PPAR is only hardly detectable in acute wound tissue and seems to be of minor importance for tissue repair (Michalik et al., 2001). However, because we had demonstrated the robust VEGF induction to be independent from PPAR in keratinocytes (Figs. 6 and 8), it was interesting to assess a potential therapeutic action of PPAR agonists to improve the strongly impaired angiogenic process in diabetic wound tissue (Frank et al., 1995; Kämpfer et al., 2001; Stallmeyer et al., 2001; Eming and Krieg, 2006) by stimulating VEGF release from wound keratinocytes. Figure 9 demonstrates the marked loss of VEGF protein expression in acute wound tissue of diabetic ob/ob mice. To improve these disturbed conditions, we treated wounded diabetic ob/ob mice orally with the PPAR agonists rosiglitazone or troglitazone, respectively. Liquid chromatography/mass spectrometry/mass spectrometry analysis of blood serum from treated animals revealed the high bioavailability of both drugs: oral administration of 5 mg/kg/day rosiglitazone resulted in average blood serum levels of 12.5 ± 2.5 µg rosiglitazone; oral administration of 5 mg/kg/day troglitazone led to average blood serum levels of 0.9 ± 0.3 µg/ml troglitazone./ml

View larger version (17K): [in this window] [in a new window]   Fig. 9. Impaired VEGF protein expression in acute wounds of diabetic mice. VEGF ELISA analyses from lysates of nonwounded skin (ctrl) and lysates of wound tissue isolated from wild-type (C57BL/6J) and diabetic ob/ob mice. VEGF protein is expressed as picograms per 50 µg of skin or wound lysate. **, P < 0.01; *, P < 0.05; n.s., not significant (unpaired Student's t test). Bars indicate the mean ± S.D. obtained from wound lysates from 12 individual mice (n = 12).

View larger version (51K): [in this window] [in a new window]   Fig. 10. Rosiglitazone impairs VEGF expression from keratinocytes in acute wounds of ob/ob mice. a, VEGF mRNA expression in total wound tissue of nontreated (mock) or rosiglitazone (rosi)-administered ob/ob mice was determined 1 day (1 d wound tissue) or 5 days (5 d wound tissue) upon injury by RNase protection assay. Expression levels of VEGF mRNA are expressed as arbitrary PhosphorImager PSL units. *, P < 0.05 compared with nonwounded control skin (ctrl) (ANOVA, Dunett's method). Bars indicate the mean ± S.D. from four wounds (n = 4) from three individual mice (n = 3). b, overview to show tissue sampling for inner wound (iw) and wound margin (wm) compartments (left). RNase protection analysis of VEGF mRNA levels in wm and iw tissue compartments isolated from mock (mock)- or rosiglitazone (rosi)-treated ob/ob mice 1 day (1 d wd) and 5 days (5 d wd) upon injury. Ctrl skin refers to back skin biopsies of nonwounded mice. A tRNA was used to control the specificity of the VEGF antisense RNA. A simultaneous hybridization of GAPDH within the same samples served to control an equal loading. Every experimental time point depicts isolated wm and iw compartments from two wounds each (n = 2) obtained from three individual mice (n = 3), which have been pooled before analysis. c, paraffin-fixed sections from mouse wounds (day 5 after wounding) were incubated with a polyclonal goat antiserum directed against murine VEGF. Immunopositive signals within the sections are indicated with arrows. Wound margin epithelia are indicated by a red line. ac, adipocytes; gt, granulation tissue; he, hyperproliferative epithelium; sc, scab.

View larger version (151K): [in this window] [in a new window]   Fig. 12. Differential effects of PPAR agonists in diabetes-impaired wound tissue: histology. Paraffin-fixed sections from mouse wounds (day 5 after wounding) were incubated with antisera directed against murine VEGF (top) or murine phosphorylated p38 MAPK (2-4). Immunopositive signals within the sections are indicated with arrows. Wound margin epithelia are indicated by a red line. ac, adipocytes; gt, granulation tissue; he, hyperproliferative epithelium; sc, scab. Scale bar, 100 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 11. Differential effects of PPAR agonists in diabetes-impaired wound tissue: MAPK activation. Total wound tissue of nontreated (mock), or rosiglitazone-(rosi)-, and troglitazone (trogli)-administered ob/ob mice was analyzed for the presence of phosphorylated p38 MAPK (p-p38, Thr180, Tyr182) or phosphorylated p42/44 MAPK (p-p42/44, Thr202, Tyr204) by immunoblot. Mouse 1, 2, or 3 represent individual mice. Every experimental time point depicts two wounds (n = 2) obtained from an individual mouse, which have been pooled before analysis. β-Actin was used to control loading.

Here, we further analyzed wound granulation tissue and scab for activated p38. We did so to explain the different signals for activated p38 in immunoblots and histologies: rosiglitazone-treated ob/ob mice showed an overall reduction, and troglitazone-treated mice showed any change in p38 activation in wound lysates (Fig. 11) in the presence of a significant phospho-p38 staining in keratinocytes (Fig. 12, second panel). Lower levels of activated p38 in immunoblots of total wound tissue upon rosiglitazone treatment seemed to reflect an overall reduction of phosphorylated p38 in wound macrophages (Fig. 12, third panel). According to the immunoblot (Fig. 11), troglitazone did not reduce activated p38 in wound macrophages compared with mock-treated mice (Fig. 12, third panel). Polymorphonuclear neutrophils did not contribute to this regulation, because this cell type did not express activated p38 (Fig. 12, bottom). In summary, the action of both PPAR agonists on wound macrophages might explain, at least partially, the observed status of p38 activation in total wound tissues of TZD-treated mice.

PPAR Agonists Do Not Interfere with Cytokine- and Growth Factor-Induced VEGF Expression in Keratinocytes. Finally, we tested the potency of rosiglitazone and troglitazone to interfere with keratinocyte responses toward wound-derived mediators such as cytokines and EGF. This was important, because VEGF expression from wound keratinocytes in rosiglitazone (reduced VEGF expression)- and troglitazone (robust VEGF expression)-treated ob/ob mice might not only be a result of the observed PPAR-independent effects but might also be based on a functional interaction with responses of wound keratinocytes toward wound-derived mediators. To this end, we stimulated keratinocytes with submaximal concentrations of cytokines and EGF to enable a modulation of induced VEGF expression by the drugs. We determined the EC₅₀ value for cytokine (combination of TNF, 0.25 ng/ml; IL-1β, 0.5 ng/ml; IFN, 0.1 ng/ml) and EGF (2 ng/ml) stimulation (Fig. 13, top). Next, we stimulated keratinocytes with the assessed EC₅₀ value of cytokines and EGF in the presence of increasing amounts of rosiglitazone and troglitazone, respectively. We did not observe any changes in cytokine- and EGF-induced VEGF expression in the presence of the respective PPAR agonist (Fig. 13, bottom).

View larger version (29K): [in this window] [in a new window]   Fig. 13. PPAR agonists do not interfere with cytokine- and EGF-induced VEGF expression in keratinocytes. a, quiescent human HaCaT keratinocytes were stimulated with increasing concentrations of cytokines (1 = TNF, 25 ng/ml; IL-1β, 50 ng/ml; IFN, 10 ng/ml) (a) or EGF (0.1-10 ng/ml) (b) as indicated to determine the EC50 value. After 24 h, VEGF165 protein release from the cells was assessed by ELISA analyses of the corresponding conditioned cell culture supernatants (top). Cells were subsequently stimulated with EC50 doses of cytokines (a, bottom) or EGF (b, bottom) in the presence or absence of troglitazone or rosiglitazone as indicated. **, P < 0.01 compared with nonstimulated control cells (ctrl). Bars indicate the mean ± S.D. from three independent cell culture experiments (n = 3).

	Discussion

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Diabetic conditions severely impair the healing process of cutaneous wounds in humans (Falanga, 2005) and in mice (Frank et al., 2000; Goren et al., 2003, 2006). In this context, it was tempting to argue for a possible function of therapeutically mediated PPAR activation to improve tissue movements during skin repair. In this study, we focused on the expression of VEGF for the following reasons: the expression of VEGF is markedly reduced in diabetes-impaired wounds (Frank et al., 1995), and keratinocytes represent the principal source of VEGF during skin repair (Brown et al., 1992) and express PPAR, -β/, and - isoforms (Michalik and Wahli, 2007).

In fact, both PPAR agonists, clofibrate and WY14643, completely failed to induce VEGF in HaCaT keratinocytes. Our observation is supported by findings from an epithelial colon carcinoma cell line, in which clofibrate and WY14643 were even potent suppressors of phorbolester-induced VEGF and Cox-2 expression. It is interesting that both substances inhibited DNA binding of activator protein-1 and thus transcriptional activation of VEGF and Cox-2 in a PPAR-dependent manner (Grau et al., 2006). By contrast, both the PPARβ/ agonist L165,041 and the PPAR agonists troglitazone and ciglitazone mediated a robust induction of VEGF mRNA and protein expression from cultured keratinocytes. This observation is in good accordance with recent findings in diverse cellular systems. Myofibroblasts exhibit a PPAR-dependent biosynthesis of VEGF upon treatment with troglitazone and 15-deoxy-prostaglandin J2. In these cells, PPAR-induced VEGF increase was functionally coupled to a decrease of nuclear factor-B activity (Chintalgattu et al., 2007). In addition, bovine articular chondrocytes have been shown to up-regulate VEGF via oxidized low-density lipoprotein-mediated PPAR activation (Kanata et al., 2006). However, the specific PPAR inhibitor GW9662 suppressed oxidized low-density lipoprotein-induced VEGF expression in chondrocytes, providing evidence that PPAR activation was, in contrast to keratinocytes, pivotal to VEGF expression. Nevertheless, the PPAR agonist rosiglitazone failed to induce VEGF expression in keratinocytes, suggesting a different profile of pleiotropic actions for this member of the TZD family of drugs. It is interesting that it has been reported for several years that especially rosiglitazone provides antiangiogenic effects in endothelial and epithelial cells. In accordance to the failure of rosiglitazone to stimulate VEGF expression in keratinocytes (this study), it is noteworthy that this drug exerted antiangiogenic properties in particular by a marked reduction of VEGF biosynthesis during physiological processes such as endometrium-driven uterine angiogenesis (Peeters et al., 2005) and during pathophysiological processes such as angiogenesis in tumor growth and metastasis (Panigrahy et al., 2002).

There is growing evidence that activation of PPARβ/ stimulates VEGF release in close functional connection to inhibition of apoptosis and stimulation of proliferation in human endothelial cells (Piqueras et al., 2007), colon carcinoma cells (Wang et al., 2006), and keratinocytes (Di-Poï et al., 2002). The potential of PPARβ/ to trigger VEGF release in close functional connection to cell survival in a colon cancer cell line must be critically evaluated in the context of cancer development. By contrast, the ability of activated PPARβ/ to drive VEGF expression (this study) and cell survival in keratinocytes by activation of protein kinase B/Akt1 (Di-Poï et al., 2002) might be of functional importance in skin repair, where keratinocytes drive angiogenic processes via VEGF release (Brown et al., 1992; Frank et al., 1995) and cover the wound site from proliferative epithelia located at the margins of the wound (Singer and Clark, 1999).

In addition, it was reasonable and interesting to assess whether the observed unequal VEGF-stimulating capabilities of individual TZDs in cultured keratinocytes, even despite the observed independence of this process from binding to PPAR, might also be transferred into diabetes-impaired wound conditions associated with disturbed angiogenic processes (Frank et al., 1995; Kämpfer et al., 2001; Eming and Krieg, 2006). Unfortunately, a functional role for PPAR in skin repair still remains unresolved, especially because this receptor isoform was only hardly detectable in skin tissue upon injury (Michalik et al., 2001). Moreover, the role of PPAR in the control of keratinocyte proliferation and differentiation still remains complex (Michalik and Wahli, 2007). The TZD ciglitazone mediated differentiation in human keratinocytes in vitro, as assessed by the induction of the typical keratinocyte differentiation markers involucrin and trans-glutaminase 1. A PPAR-mediated process of differentiation could also be induced in murine epidermis, because ciglitazone induced the expression of keratinocyte differentiation markers loricrin and filaggrin in wild-type but not in PPAR-null keratinocytes in mice (Mao-Qiang et al., 2004). In addition, PPAR-independent effects seem to contribute to the TZD-mediated cellular decisions toward differentiation, because troglitazone caused a PPAR-independent inhibition of keratinocyte proliferation by abrogation of cyclin D1 expression (He et al., 2004). In accordance to other reported PPAR-independent mechanisms of TZDs in keratinocytes, we found that inhibition of p38 and p42/44 MAPK and not a functional ablation of PPAR by siRNA abrogated TZD-stimulated VEGF expression in the cells. This observation confirms the findings of a most recent report on keratinocytes showing that troglitazone-induced Cox-2 expression did not involve PPAR but p42/44 activation (He et al., 2006).

It is interesting that our experiments on TZD actions in acute wound healing in diabetic ob/ob mice basically support our major findings obtained from cultured keratinocytes: troglitazone and rosiglitazone both activated p38 MAPK in the cells, whereas the failure to induce VEGF expression was restricted to rosiglitazone only. This observation was quite interesting, because it again demonstrated potent differences in pleiotropic actions associated with drugs belonging to the same class of substances. In particular, those differences between troglitazone, which was withdrawn from the market in 2000 because of its drug-induced side effects, and rosiglitazone, which is still considered safe and of current therapeutic use, might contribute to the overall safety of individual members of the TZD family of drugs. In addition, our data from diabetes-impaired wound tissue in vivo suggested a rosiglitazone-mediated inhibition of wound keratinocyte proliferation, probably via activation of PPAR (Mao-Qiang et al., 2004). Thus, it is reasonable to argue that the reduced VEGF expression from wound margin epithelia upon rosiglitazone treatment might be partially explained by decreased keratinocyte cell numbers. By contrast, troglitazone did not interfere with the size of wound margin epithelia, revealing an additional difference between both TZD drugs. It is remarkable that the functional connection between troglitazone and VEGF expression via activation of p38 and p42/44 MAPK in cultured keratinocytes seemed to be a key regulatory mechanism in VEGF biosynthesis and in wound keratinocytes in vivo, because we could observe a ubiquitous appearance of activated p38 MAPK in VEGF-expressing wound margin keratinocytes. In summary, our data from cell culture and wound healing experiments suggested p38 MAPK activation as a side effect of TZDs; however, only troglitazone, but not rosiglitazone, seemed to translate p38 MAPK activation into a PPAR-independent induction of VEGF from keratinocytes.

	Acknowledgements

 
We are grateful to Dr. Elke Müller for determination of VEGF protein levels from mouse wound tissue.

	Footnotes

 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 553, grant FR 1540/1-2, GRK 1172).

D.S. and O.S. contributed equally to this work.

ABBREVIATIONS: TZD, thiazolidinedione; PPAR, peroxisome proliferator-activated receptor; VEGF, vascular endothelial growth factor; L165,041, 4-[3-[4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA; TNF, tumor necrosis factor; IL, interleukin; nt, nucleotide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor; WTM, wortmannin; Act D, actinomycin D; LPS, lipopolysaccharide; IFN-, interferon-; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance; PI3K, phosphoinositide 3-kinase; wm, wound margin; iw, inner wound; WY14643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-)thioacetic acid; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; GW9662, 2-chloro-5-nitrobenzanilide.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Stefan Frank, Pharmazentrum Frankfurt/ZAFES, Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der JW Goethe-Universität Frankfurt/M., Theodor-Stern-Kai 7, D-60590 Frankfurt/M., Germany. E-mail: s.frank{at}em.uni-frankfurt.de

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