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(Hypertension. 2002;40:748.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Leptin Induces Endothelial Cell Migration Through Akt, Which Is Inhibited by PPAR-Ligands

Stephan Goetze; Anne Bungenstock; Cornelia Czupalla; Friedrich Eilers; Philipp Stawowy; Ulrich Kintscher; Chantel Spencer-Hänsch; Kristof Graf; Bernd Nürnberg; Ronald E. Law; Eckart Fleck; Michael Gräfe

From the Department of Medicine/Cardiology, German Heart Institute Berlin (S.G., A.B., F.E., P.S., U.K., C.S.-H., K.G., E.F., M.G.); Department of Pharmacology, Free University Berlin (C.C., B.N.); Institut für Physiologische Chemie II, Universität Düsseldorf, Germany (C.C., B.N.); and Division of Endocrinology, Diabetes, and Hypertension, University of California Los Angeles School of Medicine (R.E.L.), Los Angeles, Calif.

Correspondence to Stephan Goetze, MD, Cardiology Fellow at the Department of Medicine/Cardiology, German Heart Institute Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail goetze{at}dhzb.de

	Abstract

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Migration of endothelial cells (EC) is a key event in angiogenesis that contributes to neovascularization in diabetic vasculopathy. Leptin induces angiogenesis and is elevated in obesity and hyperinsulinemia. The antidiabetic thiazolidinediones (TZD) inhibit leptin gene expression and vascular smooth muscle cell migration through activation of the peroxisome proliferator–activated receptor- (PPAR). This study investigates the role of leptin in EC migration, the chemotactic signaling pathways involved, and the effects of the TZD-PPAR ligands troglitazone (TRO) and ciglitazone (CIG) on EC migration. We demonstrate that leptin induces EC migration. Because activation of two signaling pathways, the phosphatidylinositol-3 kinase (PI3K)AkteNOS and the ERK1/2 MAPK pathway, is known to be involved in cell migration, we used the pharmacological inhibitors wortmannin and PD98059 to determine if chemotactic signaling by leptin involves Akt or ERK1/2, respectively. Both wortmannin and PD98059 significantly inhibited leptin-induced migration. Treatment with the TZD-PPAR-ligands TRO and CIG significantly inhibited the chemotactic response toward leptin. Both PPAR-ligands inhibited leptin-stimulated Akt and eNOS phosphorylation, but neither attenuated ERK 1/2 activation in response to leptin. The inhibition of Akt-phosphorylation was accompanied by a PPAR-ligand–mediated upregulation of PTEN, a phosphatase that functions as a negative regulator of PI3KAkt signaling. These experiments provide the first evidence that activation of Akt and ERK 1/2 are crucial events in leptin-mediated signal transduction leading to EC migration. Moreover, inhibition of leptin-directed migration by the PPAR-ligands TRO and CIG through inhibition of Akt underscores their potential in the prevention of diabetes-associated complications.

Key Words: signal transduction • endothelium • kinase • diabetes mellitus • vasculature • phosphorylation

	Introduction

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Angiogenesis, the formation and organization of new blood vessels from the preexisting vasculature, contributes to physiological and pathological changes in vascular structure and is tightly controlled by a variety of angiogenic and antiangiogenic factors.¹ Depending on the physiological or pathological context, angiogenesis can be beneficial, such as in collateral formation in ischemic disease states, or detrimental, as in diabetic retinopathy. One of the factors that potentially contributes to the development and progression of diabetic vascular alterations is leptin, the product of the ob gene. Leptin is a pleiotropic molecule that regulates food intake and metabolic and endocrine responses and plays a regulatory role in hematopoiesis, inflammation, and immunity.^2,3 Elevated leptin levels strongly correlate with obesity, hyperinsulinemia, and insulin resistance—conditions found in most patients with non–insulin-dependent diabetes mellitus (NIDDM).⁴ In the vasculature, leptin functions as a potent inducer of angiogenesis, where it stimulates endothelial cell (EC) proliferation and cell survival through activation of the endothelial ob receptor.^5,6 Other important components of angiogenesis include cell invasion and EC migration¹; however, little is known about mechanisms of leptin-induced EC migration and the chemotactic signaling pathways involved.

Recent research has identified the peroxisome proliferator–activated receptor- (PPAR) as a potential target for therapeutic inhibition of pathological neovascularization.^7,8 PPAR is a member of the steroid receptor superfamily and, as such, a ligand-activated transcription factor.⁹ Activators of PPAR include high-affinity synthetic ligands, such as the antidiabetic thiazolidinediones, which are in clinical use for the treatment of NIDDM.⁹ Previous studies have demonstrated expression and functional relevance of PPAR in ECs,^7,10,11 where its activation by PPAR ligands inhibits VEGF-induced angiogenesis and EC proliferation and migration.^7,8 PPAR activation by thiazolidinediones has also been shown to inhibit proteolysis required for angiogenesis by inhibiting urokinase plasminogen activator expression and by increasing plasminogen activator inhibitor type-1 in ECs.^7,11 In vascular smooth muscle cells and monocytes, activated PPAR inhibits directed migration and targets the expression of matrix metalloproteinases downstream of the extracellular signal–regulated kinases 1 and 2 (ERK 1/2).^12,13 In addition, activated PPAR has a number of other effects in the vasculature, including the modulation of adhesion molecule expression and the regulation of atherogenic and inflammatory processes (for review see Neve et al¹⁴).

Leptin levels are elevated in obesity and hyperinsulinemia, and leptin has been reported to upregulate PPAR expression.¹⁵ Interestingly, PPAR activation by thiazolidinediones inhibits leptin gene expression in vivo and in vitro.^16,17 Thus, regulation and function of leptin and PPAR are possibly interrelated and relevant in patients with NIDDM. The present study investigates the role of leptin in EC migration, the chemotactic signaling pathways involved, and the effects of the TZD-PPAR ligands troglitazone (TRO) and ciglitazone (CIG) on EC migration.

	Methods

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Materials
Medium 199 Earle and fetal calf serum (FCS) were purchased from Seromed. Leptin, VEGF, L-glutamine, antibiotics, HEPES, DMSO, gelatin, L--phosphatidylethanolamine, L--phosphatidylcholine, L--phosphatidyl-L-serine, sphingomyelin, and L--phosphatidylinositol-4,5-diphosphate were from Sigma. The F-actin antibody was from Abcam, UK. Tumor necrosis factor- was from Pepro Tech, Hybond ECL nitrocellulose membrane, horseradish peroxidase–linked anti-rabbit antibody, as well as ECL Western blotting detection reagents were from Amersham Life Sciences. Ciglitazone was purchased from Biomol. Troglitazone was kindly provided by Parke Davis. Culture plastic ware was from Falcon. The MAPK-ERK Kinase (MEK) inhibitor PD98059 and antibodies against Akt, Phospho-Akt (Ser 473), Phospho-eNOS (Ser 1177), ERK 1/2 and phospho-ERK 1/2 (Thr 202/Tyr 204) were purchased from New England BioLabs. Antibodies against eNOS, p85 PI3K, and PTEN were obtained from Santa Cruz Biotechnology; the JAK2 antibody was from Upstate Biotechnology.

Cell Culture
Human umbilical vein ECs (HUVEC) were prepared as described previously.¹⁸ Cells were cultured in medium 199 Earle containing 20% FCS, 150 mmol/L HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L glutamine, 10 ng/mL aFGF and 5 U/mL heparin. Western blot and migration experiments were performed with medium 199 Earle containing 5% FCS (heat inactivated), 150 mmol/L HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. Purity of EC cultures was verified by immunostaining with an antibody against CD31. For all data shown, each individual experiment represented in the n value was performed with an independent preparation of HUVEC. DMSO was used as a solvent for TRO, CIG, wortmannin, and PD98059, whereas leptin and VEGF were dissolved in phosphate-buffered saline. DMSO was present at equal concentrations in all groups, including the control.

Western Blot Analysis
For protein analysis, early passaged (3 or less) HUVEC were grown to 60% to 70% confluency and then starved for 16 hours in 5% FCS/medium 199. For inhibitor studies, cells were pretreated for 30 minutes with PD98059 (10 to 30 µmol/L), wortmannin (50 to 100 nmol/L), TRO (10 to 20 µmol/L), CIG (10 to 20 µmol/L), or vehicle (5% FCS/medium 199) alone, followed by the addition of Leptin (50 ng/mL). Western blot analysis was performed as described previously.¹³ For immunoblotting, antibodies were used that recognize either (a) Akt, which is phosphorylated on serine 473, that is, "phospho Akt," (b) eNOS, when phosphorylated on serine 1177, that is, "phospho eNOS," (c) ERK1 or ERK2, which are phosphorylated on threonine 202 and tyrosine 204, that is, "phospho ERK1/2 MAPK," (d) all Akt protein, independent of its phosphorylation state, that is, "total Akt," (e) all eNOS protein, that is, "total eNOS," or (f) all ERK1 and ERK2 proteins, that is, "total ERK1/2 MAPK." To control for equal protein concentrations, two gels of each group were loaded in parallel with the same protein samples and blotted for activated, phosphorylated proteins or total Akt, eNOS, or ERK1/2. The phosphorylation state of these signaling proteins in the presence of 5% FCS was not affected by any of the pharmacological inhibitors (data not shown).

PI-3–Kinase Assay
Cultured HUVEC were starved for 16 hours in 5% FCS/medium 199. After treatment with leptin (50 ng/mL, 5 minutes) or vehicle only, cell lysates were prepared as described previously.¹⁹ Lysates were centrifuged at 15 000g for 10 minutes at 4°C, and aliquots of the supernatants containing equal amounts of protein were incubated with an anti-JAK2 antibody for 1 hour at 4°C, followed by addition of protein A-sepharose for 2 hours at 4°C. Precipitates were washed once with lysis buffer, twice with 1 mol/L LiCl in 100 mmol/L Tris/HCl (pH 7.5), and once with PI-3–kinase buffer containing 40 mmol/L HEPES (pH 7.5), 100 mmol/L NaCl, 7 mmol/L MgCl₂, 1 mmol/L EGTA, 200 µmol/L EDTA, 1 mmol/L dithiothreitol, and 1 mmol/L ß-glycerophosphate.

Lipid kinase activity of immunoprecipitates was determined as described previously.¹⁹ In parallel, to determine the quantity of precipitated PI-3–kinase, aliquots of the beads were subjected to SDS-PAGE and Western blot analysis with an anti-p85 antibody as described elsewhere.²⁰

Migration
HUVEC migration was examined in transwell cell culture chambers with gelatin-coated polycarbonate membranes as described previously.¹³ Cells were pretreated with PD98059 (30 µmol/L), wortmannin (100 nmol/L), TRO (10 to 20 µmol/L), CIG (10 to 20 µmol/L), or vehicle (5% FCS/medium 199) for 30 minutes at 37°C. Inhibitors were added to both the upper and the lower compartments and were present throughout the duration of the experiment. Migration was induced by addition of leptin (0.1 to 50 ng/mL) for 5 hours to the lower compartment. Experiments were performed in duplicate or triplicate and were repeated at least 3 times. A modified checker-board analysis revealed that leptin is chemotactic for HUVEC (data not shown).

Statistics
Densitometry of Western blots was done with the NIH Image program 1.60 for Macintosh (data not shown), and ANOVA was performed to compare for differences between the groups. Probability values <0.05 were considered to be statistically significant.

	Results

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Leptin Stimulates EC Migration
To examine the directed migration responses of HUVEC toward leptin, experiments were performed with a transwell migration chamber assay. With increasing concentrations of leptin (0.1 to 50 ng/mL), we observed a dose-dependent increase in directed migration of cultured HUVEC (Figure 1). Although leptin did not induce a significant increase in EC migration at concentrations up to 10 ng/mL, a significantly augmented migration occurred at 20 ng/mL and 50 ng/mL leptin, leading to a 1.59-fold and 1.93-fold stimulation, respectively (both P<0.05 compared with control). Higher leptin concentrations would have been above the leptin levels found in humans²¹ and were therefore not tested in the migration experiments. To compare for the chemotactic potency of leptin, additional migration experiments were performed with the known potent chemoattractant VEGF. Migration responses toward VEGF (20 ng/mL) were 2.2-fold higher than the maximal induction of migration achieved with leptin (50 ng/mL; P<0.05).

View larger version (13K): [in this window] [in a new window]   Figure 1. Endothelial cell migration toward increasing concentrations of leptin (0.1 to 50 ng/mL). Migration of HUVEC was determined with a modified Boyden chamber assay as described in Methods and is shown in cell count per high powered field. Results represent at least 3 independent experiments done in duplicate or triplicate. Data are expressed as mean±SEM. *P<0.05 vs control.

Leptin-Induced Migration Is Akt- and ERK1/2-Dependent
The activation of Akt and its downstream target eNOS are known to be important steps in VEGF-directed migration of ECs.^22,23 In cancer cells, leptin-induced cancer cell invasion is inhibited by the phosphatidylinositol-3 kinase (PI3K)Akt pathway inhibitor wortmannin.²⁴ We therefore tested the effect of wortmannin to examine a possible involvement of the PI3KAkteNOS pathway in leptin-induced migration. Treatment with wortmannin (100 nmol/L) completely abolished leptin-stimulated migration of HUVEC (P<0.05) (Figure 2). We next examined whether ERK1/2, which are required for EC migration in response to basic fibroblast growth factor,²⁵ are also involved in leptin-induced migration. Blockade of the ERK-MAPK pathway with the pharmacological inhibitor PD98059 (30 µmol/L) resulted in a significant inhibition of EC migration toward leptin (-98±12%, P<0.05)(Figure 2). Neither wortmannin (100 nmol/L), nor PD98059 (30 µmol/L) significantly affected the migration of unstimulated ECs, which were kept in 5% FCS (data not shown). Also, none of the pharmacological inhibitors caused any cytotoxic effects at the concentrations used. There was no evidence of cell detachment or loss of plasma membrane integrity, as evidenced by the uptake of trypan blue.

View larger version (30K): [in this window] [in a new window]   Figure 2. Migration of HUVEC toward leptin is Akt- and ERK1/2-dependent. Cells were incubated with wortmannin (100 nmol/L) or PD98059 (30 µmol/L) for 30 minutes before leptin (50 ng/mL) was added. HUVEC migration is shown as x-fold induction over control. Experiments were repeated 3 times and were done in duplicate. Data are expressed as mean±SEM. *P<0.05 vs control; #P<0.05 vs leptin alone.

Leptin Transiently Activates Akt and ERK 1/2 in ECs
To corroborate our findings that the PI3K pathway inhibitor wortmannin inhibited leptin-induced chemotactic signaling in ECs, we examined the effect of leptin on the PI3KAkteNOS pathway. By using a radioactive PI3K activity assay, we observed a 2.7±0.34-fold upregulation of PI3K activity 5 minutes after stimulation with leptin (50 ng/mL; n=3) (Figure 3). The subsequent phosphorylation and activation of Akt and its downstream substrate eNOS was assessed by immunoblotting with phosphospecific Akt or eNOS antibodies. Unstimulated cells in the control groups exhibited low Akt and eNOS activity, as evidenced by the faint bands detected with the phosphospecific antibodies. Stimulation with leptin induced a transient activation of Akt with a maximal induction at 30 minutes (3.2-fold) and a return to baseline values within 60 minutes (Figure 4). The phosphorylation of eNOS in response to leptin followed a comparable time course, revealing a maximal 4.2-fold increase versus untreated control after 30 minutes (Figure 4). Leptin also induced the rapid and transient phosphorylation and activation of ERK 1/2 leading to a maximal 3.4-fold increase at 10 minutes (Figure 4). Leptin did not affect the amount of total Akt, eNOS, or ERK 1/2 protein during the investigated time courses.

View larger version (55K): [in this window] [in a new window]   Figure 3. Leptin stimulates PI3-kinase activity. HUVEC were treated with leptin (50 ng/mL) for 5 minutes, JAK2 was immunoprecipitated, and lipid kinase activity of the coprecipitated class I PI3-kinase was determined. Representative autoradiograph of the PI3-kinase assay (I) and corresponding Western blot of the gel-separated immunocomplex stained with PI3-kinase anti-p85 mAB (II) are shown. Three independent experiments were performed in duplicate.

View larger version (67K): [in this window] [in a new window]   Figure 4. Leptin stimulates activation of PI3KAkteNOS and ERK1/2 pathways. HUVEC were treated with leptin (50 ng/mL) for 10 to 60 minutes and protein samples were immunoblotted for (A) activated, phosphorylated Akt (I), total Akt (II), or (B) phosphorylated eNOS (I), or total eNOS (II), (C) activated, phosphorylated ERK1/2 (I), or total ERK 1/2 (II). Western blots shown are representative of 3 experiments with different cell preparations.

The PI3K pathway inhibitor wortmannin (50 to 100 nmol/L) potently inhibited leptin-stimulated Akt activation and downstream eNOS phosphorylation (Figure 5). Leptin-induced phosphorylation and activation of ERK 1/2 was significantly attenuated by PD98059 at 10 µmol/L and totally blocked at 30 µmol/L (Figure 6). Wortmannin had no effect on leptin-stimulated ERK 1/2 phosphorylation (data not shown).

View larger version (72K): [in this window] [in a new window]   Figure 5. Wortmannin inhibits leptin-induced Akt and eNOS phosphorylation. Cells were incubated with wortmannin (50 to 100 nmol/L) for 30 minutes before stimulation with leptin (50 ng/mL; 30 minutes). Then, cell lysates were immunoblotted with antibodies against (A) phosphorylated Akt (I), total Akt protein (II), or (B) phosphorylated eNOS (I), or total eNOS (II). The Western blots shown are representatives of 3 independently performed experiments in each group.

View larger version (39K): [in this window] [in a new window]   Figure 6. Leptin-stimulated ERK1/2-activation is inhibited by PD98059. After incubation with PD98059 (10 to 30 µmol/L) for 30 minutes cells were stimulated with leptin (50 ng/mL; 10 minutes). Immunoblotting was performed with antibodies against activated, phosphorylated ERK1/2 (I), or total ERK1/2 (II). Three experiments were performed in each group.

PPAR-Ligands Inhibit EC Migration Toward Leptin
The thiazolidinediones TRO and CIG are antidiabetic insulin sensitizers that function as ligands for PPAR.⁹ The present study examined the effects of the PPAR ligands TRO and CIG on leptin-induced migration. The data in Figure 7 show that migration of HUVEC toward leptin was significantly inhibited by treatment with 10 µmol/L and 20 µmol/L CIG by 66±12% and 89±5%, respectively (both P<0.05 versus leptin 50 ng/mL alone). An even more potent effect was observed for TRO that completely abolished migration toward leptin at 20 µmol/L (P<0.05).

View larger version (16K): [in this window] [in a new window]   Figure 7. Migration of HUVEC toward leptin is inhibited by the PPAR ligands CIG (10 to 20 µmol/L) and TRO (10 to 20 µmol/L) in a concentration-dependent manner. Migration responses are shown as percent of leptin-induced increase. Results represent 4 independent experiments that were done in duplicate. Data are expressed as mean±SEM. #P<0.05; *P<0.01 vs leptin alone.

At all concentrations of the PPAR ligands CIG or TRO (each 20 µmol/L) used in migration assays, we observed no cytotoxic effects, as evidenced by the lack of cell detachment or the absence of a significant number of cells staining positively for trypan blue.

PPAR Ligands Inhibit Leptin-Induced Akt Activation and eNOS Phosphorylation
To determine whether the PPAR ligands CIG and TRO inhibited migration by targeting the PI3KAkteNOS or the ERK1/2 MAPK pathway, we investigated their effects on leptin-induced Akt/eNOS and ERK1/2 activation. Leptin-induced phosphorylation of Akt was dramatically attenuated in cells that were treated with CIG or TRO, and complete inhibition of Akt phosphorylation was observed for both PPAR ligands at concentrations of 20 µmol/L (Figure 8). Leptin-induced eNOS phosphorylation downstream of Akt was also profoundly inhibited after treatment with the two PPAR ligands (Figure 8). In contrast, neither CIG nor TRO affected ERK1/2 activation in response to leptin (Figure 8).

View larger version (90K): [in this window] [in a new window]   Figure 8. PPAR ligands inhibit leptin-induced Akt and eNOS phosphorylation but do not affect ERK 1/2 activation. After a 30-minute preincubation with CIG (10 to 20 µmol/L) or TRO (10 to 20 µmol/L) HUVEC were stimulated with leptin (50 ng/mL) for 30 (A, B), or 10 minutes (C). Immunoblotting was performed as described in Figure 4 [A, I phospho–Akt II, total Akt, (B) phosphorylated eNOS (I), or total eNOS (II), or (C) phosphorylated ERK1/2 (I) or total ERK1/2 (II)]. Western blots shown are representatives of 3 independently performed experiments.

In vascular smooth muscle cells, PPAR ligands inhibit migration and proliferation downstream of activated, phosphorylated ERK 1/2.¹³ The present study shows that EC migration is Akt-dependent and ERK 1/2–dependent and inhibited by PPAR ligands. To investigate the possibility that the antimigratory actions of PPAR ligands might be due to an inhibition of chemotactic signaling from upstream ERK1/2 to downstream Akt, we next tested the effect of PD98059 on leptin-induced Akt phosphorylation. We find that leptin-stimulated Akt phosphorylation was not affected by PD98059, indicating that the PI3KAkteNOS and the ERK1/2 MAPK pathway are two distinct signaling pathways that independently transmit chemotactic signals in response to leptin (Figure 9).

View larger version (43K): [in this window] [in a new window]   Figure 9. Leptin-stimulated Akt phosphorylation is independent of ERK1/2. Cells were incubated with the MEK inhibitor PD98059 (30 µmol/L) for 30 minutes before leptin (50 ng/mL) was added. After stimulation with leptin for 30 minutes, cell lysates were immunoblotted for activated, phosphorylated Akt (I) or total Akt (II). Western blots shown are representative of 2 experiments with different cell preparations.

Expression of PTEN Is Upregulated by PPAR Ligands
PTEN is a lipid phosphatase that antagonizes PI3K-mediated phosphorylation of Akt by dephosphorylating the D3 position of phosphatidylinositol (3,4,5) trisphosphate, which functions as a second messenger to activate Akt.²⁶ In accordance with that, a recent study reported the inhibition of VEGF-induced EC migration and angiogenesis by PTEN through blockade of the inducible phosphorylation of Akt.²⁷Interestingly, PPAR ligands have been shown to upregulate PTEN mRNA and protein expression in macrophages and different tumor cell lines, which was prevented when cells had been exposed to PPAR antisense oligonucleotides.²⁸ These findings prompted us to investigate the effects of the PPAR ligands TRO and CIG on PTEN levels in ECs. We observed that stimulation of quiescent EC with both PPAR ligands caused a rapid and profound upregulation of PTEN expression in these cells, leading to a 3.6-fold or 3.2-fold increase in PTEN protein levels after incubation for 45 minutes with TRO or CIG, respectively (Figure 10). A further increase in PTEN protein occurred after 90 minutes of PPAR ligand treatment (TRO 5.8-fold, CIG 5.5-fold). The observed time course for PPAR-mediated PTEN upregulation corresponds to the exposure time of ECs to PPAR ligands before the inhibition of leptin-induced Akt phosphorylation (30 minutes PPAR ligand pretreatment plus 30 minutes costimulation during leptin-stimulated phosphorylation of Akt) and may therefore constitute a potential mechanism by which PPAR ligands inhibit Akt phosphorylation and EC migration.

View larger version (49K): [in this window] [in a new window]   Figure 10. PPAR ligands stimulate endothelial PTEN expression. Quiescent ECs were incubated with PPAR ligands TRO (20 µmol/L) or CIG (20 µmol/L) for 45 or 90 minutes, and cell lysates were immunoblotted for PTEN protein. The same protein samples were also immunoblotted against F-actin, which served as control. Western blots shown are representative of 3 experiments with different cell preparations.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present investigation demonstrates that leptin induces EC migration through activation of the PI3KAkteNOS and the ERK 1/2 signaling pathways. In addition, we found that activation of PPAR with the antidiabetic PPAR ligands TRO and CIG blocks leptin-stimulated EC migration by inhibition of Akt and eNOS.

Leptin is a pluripotent hormone that exerts several effects in the vasculature, where it stimulates EC proliferation, inhibits apoptotic cell death, and promotes angiogenesis.^3,5,6 Other vascular effects of leptin include the induction of reactive oxygen species and MCP-1 in ECs, actions that have been implicated with a potential atherogenic role of the hormone.²⁹ Both angiogenic and proatherosclerotic actions of leptin may be of clinical importance in disease states that correlate with pathological alterations in vascular architecture and elevated leptin levels, such as NIDDM.

The mechanisms and signaling pathways leading to EC migration are not completely understood. Migration itself is a complex vascular response that involves chemotaxis, locomotion, and invasion, cellular functions that are regulated by cytosolic and nuclear signaling events. Among the cytosolic signaling steps that participate in the migration process, recent studies have recognized the AkteNOS pathway as an important signaling cascade in VEGF-mediated EC migration.^22,23 The chemoattractant-induced activation of Akt has been shown to induce reorganization of the actin/myosin cytoskeleton and subsequent cell movement.²³ In addition, Akt has been reported to phosphorylate and activate endothelial nitric oxide synthase,²² which probably contributes to angiogenesis through endothelial nitric oxide production.³⁰ In combination, activation of the protein kinase Akt by VEGF orchestrates several signaling events that contribute to EC migration and angiogenesis.

Leptin promotes angiogenesis and has been reported to activate Akt in endothelial and nonvascular cells.^31,32 We identified that activation of the PI3KAkteNOS signaling pathway is a critical signaling step in leptin-induced EC migration. These results are in line with the previous observations on the role of Akt and eNOS in VEGF-stimulated migration,^22,23 indicating that Akt functions as an essential signaling kinase in the migratory response of ECs.

Our results on leptin-induced phosphorylation and activation of Akt and eNOS are in line with a recently published study by Vecchione et al, who made comparable observations in human aortic ECs.³² However, Vecchione and colleagues report a PI3-kinase–independent phosphorylation of Akt and eNOS in their study cells, which is different from our observations. Our results support a role of PI3 kinase in leptin-induced Akt and eNOS phosphorylation, since we find an activation of PI3 kinase in leptin-stimulated ECs and an inhibition of the subsequent Akt and eNOS phosphorylation by a pharmacological PI3 kinase inhibitor. From the currently available data, we have no explanation for these different findings, and future studies might help to fully understand the role of PI3 kinase in signaling by leptin in ECs.

The mitogen-activated protein kinases ERK1/2 represent other critical signaling molecules that mediate migration of vascular and nonvascular cells by regulating cytosolic and nuclear signaling events.^13,33–35 In the present study, we demonstrated that blockade of the ERK1/2 pathway leads to inhibition of EC migration in response to leptin. ERK 1/2 have been shown to modulate cell motility through the downstream phosphorylation and activation of myosin light chain kinase.³⁴ In addition to these cytosolic events, several studies reveal that cell migration requires ERK 1/2–dependent expression and release of matrix metalloproteinases, suggesting an additional involvement of nuclear signaling events.^33,36–38

Previous studies have shown that activation of PPAR by its synthetic ligands inhibits chemoattractant-inducible migration of vascular smooth muscle cells and monocytes.^12,13,33 Activated PPAR is also known to inhibit the expression of MMPs in these cells, which probably accounts for some of the antimigratory effects of PPAR.^12,33 Because PPAR ligands do not affect the cytosolic activation of ERK 1/2, it is conceivable that PPAR inhibits the induction of MMPs at a transcriptional level by interfering with regulatory transcription factors. This hypothesis is supported by several studies that report the inhibition of target gene expression by PPAR through negative interference with several transcription factor pathways including NFB, STAT-1, and AP-1 (for review, see Neve et al¹⁴).

Inhibition of cell invasion by PPAR through blockade of MMP production is a process that is likely to occur after hours or days when PPAR ligands are administered. Yet, the fact that cell migration is completely abrogated by PPAR ligands within <5 hours prompted us to examine additional chemotactic signaling steps that might be affected by PPAR. Cell migration requires not only invasion but also locomotion, which is regulated by different cytosolic signaling events.¹ The protein kinase Akt is known to facilitate cell movement by reorganizing the actin/myosin cytoskeleton in ECs.²³ Here we show that the PPAR ligands TRO and CIG inhibit EC migration by blocking leptin-induced activation of Akt and eNOS. In contrast to Akt and eNOS is the ERK 1/2–dependent activation of myosin light chain kinase not affected by PPAR.³⁹ Thus, inhibition of Akt and eNOS by PPAR might constitute the cytosolic signaling steps involved in locomotion, which are responsible for the immediate antimigratory effect of PPAR ligands.

What is the underlying mechanism for PPAR-mediated inhibition of the cytosolic protein kinase Akt? Many studies have demonstrated the presence and function of PPAR in an increasing number of cell types, where its localization has been predominantly defined to the nucleus.^9,14 Most of the known actions of PPAR are mediated by signaling events in the nucleus, where PPAR heterodimerizes with retinoid X receptors, binds to specific PPAR response elements in the promotor of target genes, and thereby regulates their transcription.^9,14

A recent study in macrophages and different tumor cell lines identified a PPAR-mediated increase in the gene expression of the lipid phosphatase PTEN, which functions as a negative regulator of the PI3KAkt signaling pathway.^26,28 In ECs, it has been reported that PTEN inhibits VEGF-induced angiogenesis and migration through blockade of inducible Akt phosphorylation.²⁷ In line with these studies, we observed an upregulation in endothelial PTEN levels after treatment with the PPAR ligands TRO and CIG, which suggests a potential role for PTEN in the antimigratory actions of PPAR ligands in ECs.

In conclusion, we were able to identify Akt as an important signaling step in leptin-induced EC migration that is inhibited by PPAR ligands. These results provide a novel target of antidiabetic PPAR activators in ECs and underscore their potential as useful therapeutic agents in the treatment of diabetes-associated vasculoproliferative disorders.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG GO 800/2-1 and DFG NU 53/6-1) and Fonds der Chemischen Industrie. The authors thank Verena Fromm for assistance in preparing the manuscript and Jürgen Malkewitz for technical assistance.

Received May 9, 2002; first decision June 10, 2002; accepted August 16, 2002.

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