CLINICAL PERSPECTIVE

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(Circulation. 2005;112:1316-1322.)
© 2005 American Heart Association, Inc.

Molecular Cardiology

Glycogen Synthase Kinase-3 Mediates Endothelial Cell Activation by Tumor Necrosis Factor-

Masato Eto, MD, PhD; Alexei Kouroedov, MD, PhD; Francesco Cosentino, MD, PhD; Thomas F. Lüscher, MD

From the Department of Cardiology, Cardiovascular Center, University Hospital, and Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland (M.E., A.K., F.C., T.F.L.), and Division of Cardiology, 2nd Faculty of Medicine, University "La Sapienza," Rome, Italy (F.C.).

Correspondence to Thomas F Lüscher, MD, FRCP, Department of Cardiology, University Hospital, Ramistrasse 100, CH-8091 Zurich, Switzerland. E-mail cardiotfl{at}gmx.ch

Received March 21, 2004; revision received May 23, 2005; accepted May 24, 2005.

	Abstract

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Background— Endothelial cell transformation to a thrombogenic and inflammatory phenotype plays an important role in the pathogenesis of atherothrombosis, but the responsible signaling pathways remain to be elucidated. This study was designed to investigate the regulatory role of glycogen synthase kinase-3 (GSK-3) in tissue factor (TF) and vascular cell adhesion molecule (VCAM)-1 expression in tumor necrosis factor (TNF)-–stimulated endothelial cells.

Methods and Results— In human endothelial cells, TNF- as well as thrombin induced rapid and transient dephosphorylation and hence, activation of GSK-3. A GSK-3 inhibitor, LiCl, suppressed TNF-– and thrombin-induced TF and VCAM-1 expression, whereas NaCl had no effect. A specific GSK-3 inhibitor, TDZD-8, mimicked the inhibitory effects of lithium. GSK-3 inhibition also significantly suppressed the TNF-–induced increase in TF activity and VCAM-1 cell-surface expression. The luciferase reporter system demonstrated that regulation of TF and VCAM-1 expression by GSK-3 was mediated at the transcriptional level. The TNF-–induced increase in nuclear factor (NF)-B DNA-binding activity was significantly suppressed by TDZD-8. TDZD-8 completely prevented the TNF-–induced inhibitor of NF-B (IB)- degradation but had no effect on IB-kinase-ß phosphorylation.

Conclusions— GSK-3 regulates TNF-–induced IB- degradation and NF-B activation independent of IB-kinase-ß and subsequent induction of TF and VCAM-1 expression in human endothelial cells. This study provides the experimental basis for a novel strategy of using GSK-3 inhibition to treat atherothrombotic vascular disease.

Key Words: endothelium • inflammation • signal transduction

	Introduction

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The vascular endothelium is a highly dynamic tissue, constantly sensing and responding to changes in the local extracellular environment to modulate vascular tone, hemostatic balance, vascular permeability, and adhesion and transmigration of leukocytes.¹ Under normal conditions, endothelial cells show anticoagulant and antiinflammatory properties; however, on activation, they are transformed to a procoagulant and proinflammatory phenotype. This transformation is mainly mediated by gene expression of procoagulant molecules, such as tissue factor (TF), and adhesion molecules, such as vascular cell adhesion molecule (VCAM)-1.^2–6 In fact, neither TF nor VCAM-1 is detectable in unstimulated endothelial cells, but proinflammatory cytokines such as tumor necrosis factor (TNF)- markedly induce the expression of these proteins.^2,4

Endothelial cell activation plays a major role in the pathogenesis of several critical disorders, such as acute coronary syndromes, ischemic stroke, and sepsis.^1,7 In fact, recent morphological studies have clearly shown that both TF and VCAM-1 are expressed in endothelial cells of human coronary atherosclerotic lesions.^8–10 Levels of TF antigen and activity in coronary atherosclerotic plaques are increased in patients with acute coronary syndromes.¹¹ Moreover, VCAM-1 expression is more prevalent in atherosclerotic plaques than in nonatherosclerotic segments. In addition, increased intimal macrophage accumulation is strongly associated with VCAM-1 expression in plaques.^9,10 These data suggest the important role of endothelial cell transformation to a thrombogenic and inflammatory phenotype in atherothrombosis.

Endothelial expression of both TF and VCAM-1 is mainly regulated at the transcriptional level, and a transcription factor, nuclear factor (NF)-B, appears to play an important role in both cases.^2,4–6 However, the responsible upstream signaling pathways for gene expression remain to be clarified. Recently, we reported that in human endothelial cells, a phosphatidyl inositol 3-kinase inhibitor, wortmannin, potentiates thrombin-induced TF expression as well as Akt dephosphorylation.³ There is an inverse correlation between levels of TF and those of Akt phosphorylation, suggesting a negative regulatory role for Akt in TF expression. However, the downstream mechanisms underlying the pathways whereby Akt negatively regulates TF expression are unclear. Akt has several direct targets for mediating different cellular functions.¹² Among them, we focused on glycogen synthase kinase (GSK)-3 as a downstream effector in this context.

GSK-3 is a serine/threonine kinase that was originally identified as an enzyme phosphorylating and inactivating glycogen synthase.¹³ Its activity is inversely regulated by phosphorylation at a specific serine residue, and Akt, as a major upstream kinase, therefore serves as an inhibitor.¹⁴ Several lines of evidence have accumulated that GSK-3 regulates a wide range of cellular functions, including metabolism, cytoskeletal integrity, gene expression, cell cycle regulation, and cell survival.^15–22 GSK-3 is also involved in a variety of pathological processes, such as diabetes, cancer, Alzheimer’s disease, neovascularization, and ischemia/reperfusion injury in the heart and brain.^{13,14,23–30} However, the role of GSK-3 in cytokine-mediated endothelial cell activation remains to be elucidated.

This study was designed to investigate the regulatory role of GSK-3 in TF and VCAM-1 expression in TNF-–stimulated human endothelial cells. In addition, we also investigated the interaction between GSK-3 and the NF-B pathway.

	Methods

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Chemicals and Materials
Human recombinant TNF-, thrombin, and LiCl were purchased from Sigma. The Akt inhibitor SH-5 and the GSK-3 inhibitor 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione-8 (TDZD-8) were purchased from Calbiochem. Antibodies against Akt, phospho-Akt (serine 473), GSK-3ß, phospho-GSK-3/ß (serine 21/9), phospho–inhibitory protein (I)-B-kinase ß (IKKß) (serine 181), and IB- were obtained from Cell Signaling. TF antibody was obtained from American Diagnostica (American Diagnostica). VCAM-1 antibody was obtained from R&D Systems, and fluorescein isothiocyanate–conjugated VCAM-1 antibody was obtained from Serotec.

Cell Culture
Endothelial cells were isolated from human umbilical veins (ie, HUVECs) as described previously.³¹ Human aortic endothelial cells (HAECs) were obtained from Clonetics. In brief, fresh blood vessels were harvested in cold endothelial basal medium (EBM)-2 (Clonetics) with antibiotics. The vessels were cleaned of connective tissue and adventitia and then incubated with collagenase. Cell pellets were then collected by centrifugation, seeded into culture dishes coated with fibronectin, and cultured in EBM2 supplemented with 10% fetal calf serum in a humidified atmosphere. Endothelial cells were characterized by their typical cobblestone and nonoverlapping appearance and immunofluorescence stains with an antibody against von Willebrand factor. After reaching confluence, the cells were rendered quiescent by incubation in the medium with 0.5% fetal calf serum for 24 hours and then stimulated with TNF- or thrombin. Inhibitors were pretreated 1 hour before stimulation. Cells of the third to the sixth passage were used for experiments.

Western Blotting
HUVECs were harvested in lysis buffer for Western blotting.³¹ The samples (30 µg) were treated with 5x sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer,³¹ followed by heating at 95°C for 3 minutes, and were then subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred to Immobilon-P membranes (Millipore AG). The membranes were blocked with 5% milk and incubated with the primary antibodies. Immunoreactive bands were detected by a chemiluminescence system (ECLplus, Amersham).

TF Activity
TF activity was determined with use of the Actichrome kit (American Diagnostica), as described previously.³ After stimulation, cells were incubated with human factor VIIa (10 nmol/L, American Diagnostica) and the synthetic substrate Spectroenzyme (500 µmol/L, American Diagnostica) for 30 minutes in phenol red–free Dulbecco’s modified Eagle’s medium at 37°C. Afterward, the medium was removed, and optical density was monitored spectrophotometrically at 405 nm. Recombinant TF (American Diagnostica) was used for calibration.

Fluorescence-Activated Cell Sorting (FACS) Analysis
The cells were harvested by trypsinization, fixed (4% paraformaldehyde, 15 minutes), and incubated with fluorescein isothiocyanate–conjugated VCAM-1 antibody on ice for 1 hour. After immunofluorescence labeling, cells were washed and analyzed in a BD PharMingen FACScan flow cytometer.

Luciferase Assay
TF luciferase plasmids³² and VCAM-1 luciferase plasmids⁵ were kind gifts from Dr Nigel Mackman (Scripps Research Institute, La Jolla, Calif) and Dr William C. Aird (Beth Israel–Deaconess Medical Center, Boston, Mass). For transient transfer, 3 µg of cDNA plasmids was mixed with 15 µL of Superfect (Qiagen) for 10 minutes for complex formation. The complex was added to the cells for 3 hours and then removed. The cells were incubated in growth medium for 24 hours, and then medium was changed to EBM2 with 0.5% fetal calf serum 24 hours before stimulation. Cell lysates were assayed for firefly luciferase activity, as described in the manufacturer’s protocol (Promega), with use of a luminometer. Cells were cotransferred with pRL-CMV, which expresses Renilla luciferase (Promega). Renilla luciferase activity was used to normalize the activity of firefly luciferase.

NF-B DNA-Binding Assay
Nuclear protein was obtained with use of a nuclear extract kit (Active Motif). Cells were harvested in hypotonic buffer (Active Motif). The homogenate was centrifuged to obtain a pellet of nuclei. Isolated nuclei were resuspended in hypertonic buffer (Active Motif), and nuclear protein was extracted by incubation on ice for 20 minutes. The supernatant containing the nuclear protein was collected after centrifugation. The DNA binding reaction was carried out with 8 µg of nuclear protein in a 96-well plate coated with oligonucleotides (GGGACTTTCC) to consensus sequences for NF-B (Active Motif) for 1 hour at room temperature. Then, after washing, p65 antibody (Active Motif) was added and incubated for 1 hour. A horseradish peroxidase–conjugated secondary antibody (Active Motif) was then added and incubated for 1 hour. Finally, DNA-binding NF-B protein was assessed spectrophotometrically at 450 nm.

Statistics
Data are given as mean±SEM. For statistical analysis, nonparametric methods (Kruskal-Wallis test) were used, followed by Fisher’s protected least significant difference test for comparison between groups. A value of P<0.05 was considered statistically significant.

	Results

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Akt–GSK-3 Pathway and TF Expression
Similar to a previous finding reported by another group,³³ TNF- (10 ng/mL) induced rapid and transient dephosphorylation of Akt in HUVECs (Figure 1A). Consistent with the response of Akt, GSK-3 was also dephosphorylated and thus, activated by TNF- (Figure 1B), as previously reported in another type of cell.³⁴ Thrombin (4 U/mL) also induced rapid and transient dephosphorylation of GSK-3 (Figure 1C).

View larger version (33K): [in this window] [in a new window]   Figure 1. A, Effect of TNF- on the levels of phosphorylated Akt (active form). Phosphorylation levels of Akt were determined by Western blotting. Similar results were obtained in 3 independent experiments. B, Effect of TNF- on the levels of phosphorylated GSK-3 (inactive form). Phosphorylation levels of GSK-3 were determined by Western blotting. Similar results were obtained in 3 independent experiments. C, Effect of thrombin on the levels of phosphorylated GSK-3. Phosphorylation levels of GSK-3 were determined by Western blotting. Similar results were obtained in 3 independent experiments.

TF protein was not detectable by Western blotting in unstimulated HUVECs, but TNF- (10 ng/mL, 5 hours) markedly induced TF expression (Figure 2A-D). SH-5 (10^–5 mol/L), which is a specific Akt inhibitor,³⁴ potentiated TF expression induced by TNF- (Figure 2A). LiCl (5x10^–2 mol/L), which is a potent GSK-3 inhibitor with an IC₅₀ value of 1 to 2x10^–3,^35,36 prevented TNF-–induced TF expression, but NaCl (5x10^–2 mol/L), which served as an osmotic control, failed to prevent it (Figure 2B). This inhibitory effect of lithium was concentration dependent, and even a clinically relevant concentration (1 mmol/L) of lithium³⁷ significantly suppressed TF expression (67.1±6.8% of positive control, n=3, P<0.01; Figure 2C). Lithium also prevented TNF-–induced TF expression in HAECs (Figure 2D). Moreover, TDZD-8 (2x10^–7 to 2x10^–5 mol/L), which is a specific GSK-3 inhibitor with an IC₅₀ value of 2x10^–6,^36,38 prevented TNF-–induced TF expression in a concentration-dependent manner (Figure 2E). Thrombin is also known to induce TF expression in endothelial cells.³ Lithium also prevented thrombin-induced TF expression in HUVECs (Figure 2F).

View larger version (18K): [in this window] [in a new window]   Figure 2. Akt–GSK-3 pathway and TF expression in endothelial cells. A, Effect of an Akt inhibitor, SH-5, on TNF-–induced TF expression in HUVECs. TF expression was evaluated by Western blotting. Similar results were obtained in 3 independent experiments. B, Effect of LiCl on TF expression in HUVECs. NaCl was used as an osmotic control. Similar results were obtained in 3 independent experiments. C, Concentration-dependent effect of LiCl on TF expression. Similar results were obtained in 3 independent experiments. D, Effect of LiCl on TF expression in HAECs. Similar results were obtained in 3 independent experiments. E, Concentration-dependent effect of a GSK-3 inhibitor, TDZD-8, on TF expression in HUVECs. Similar results were obtained in 3 independent experiments. F, Effect of lithium on thrombin-induced TF expression in HUVECs. Similar results were obtained in 3 independent experiments. G, Effects of LiCl and TDZD-8 on TF activity in HUVECs. The cells were stimulated with TNF- in the presence or absence of LiCl or TDZD-8, and 5 hours after stimulation, TF activity was determined chromogenically.

Consistent with the protein expression in cell lysates, TF activity was significantly increased by TNF- (10 ng/mL, 5 hours) in HUVECs (Figure 2G). LiCl (5x10^–2 mol/L) as well as TDZD-8 (2x10^–5 mol/L) significantly suppressed the TNF-–induced increase in TF activity (Figure 2G).

GSK-3 and VCAM-1 Expression
VCAM-1 protein was not detectable by Western blotting in unstimulated HUVECs, and TNF- (10 ng/mL, 5 hours) markedly induced VCAM-1 expression (Figure 3A–3D). LiCl (5x10^–2 mol/L) prevented TNF-–induced VCAM-1 expression, but NaCl (5x10^–2 mol/L) had no effect (Figure 3A). This inhibitory effect of lithium was concentration dependent, and even a clinically relevant concentration (1 mmol/L) of lithium significantly suppressed VCAM-1 expression (56.6±8.2% of positive control, n=3, P<0.01; Figure 3B). Lithium also prevented TNF-–induced VCAM-1 expression in HAECs (Figure 3C). Moreover, TDZD-8 (5x10^–5 mol/L) also prevented TNF-–induced VCAM-1 expression (Figure 3D). In addition, lithium also prevented thrombin-induced VCAM-1 expression in HUVECs (Figure 3E).

View larger version (21K): [in this window] [in a new window]   Figure 3. The GSK-3 pathway and VCAM-1 expression in endothelial cells. A, Effect of LiCl on TNF-–induced VCAM-1 expression in HUVECs. VCAM-1 expression was evaluated by Western blotting. NaCl was used as an osmotic control. Similar results were obtained in 3 independent experiments. B, Concentration-dependent effect of LiCl on VCAM-1 expression. Similar results were obtained in 3 independent experiments. C, Effect of LiCl on VCAM-1 expression in HAECs. Similar results were obtained in 3 independent experiments. D, Effect of a GSK-3 inhibitor, TDZD-8, on VCAM-1 expression in HUVECs. Similar results were obtained in 3 independent experiments. E, Effect of LiCl on thrombin-induced VCAM-1 expression in HUVECs. Similar results were obtained in 3 independent experiments. F, Effects of LiCl on VCAM-1 cell-surface expression on HUVECs. The cells were stimulated with TNF- in the presence or absence of LiCl, and 5 hours after stimulation, VCAM-1 cell-surface expression was determined by FACS analysis. Similar results were obtained in 2 independent experiments.

Consistent with the protein expression in cell lysates, VCAM-1 cell-surface expression was significantly increased by TNF- (10 ng/mL, 5 hours) in HUVECs (Figure 3F). LiCl (5x10^–2 mol/L) almost completely suppressed the TNF-–induced increase in VCAM-1 cell-surface expression (Figure 3F).

GSK-3 and TF and VCAM-1 Promoter Activity
Gene expression of TF and VCAM-1 is regulated mainly at the transcriptional level. Thus, the effects of GSK-3 inhibition on TF and VCAM-1 promoter activities were explored. TNF- (10 ng/mL, 5 hours) significantly increased TF promoter activity in HUVECs (Figure 4A). LiCl (5x10^–2 mol/L) as well as TDZD-8 (2x10^–5 mol/L) significantly prevented the increase in TF promoter activity mediated by TNF- (Figure 4A). TNF- (10 ng/mL, 5 hours) also significantly induced the increase in VCAM-1 promoter activity, which was significantly prevented by LiCl and TDZD-8 (Figure 4B).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of GSK-3 inhibition on the promoter activities of TF and VCAM-1 in HUVECs. The cells were transferred with TF luciferase plasmid or VCAM-1 luciferase plasmid and stimulated with TNF- in the presence or absence of LiCl or TDZD-8, and then 5 hours after stimulation, promoter activities were measured by luciferase assay.

GSK-3 and NF-B Pathway
An important transcription factor for TF and VCAM-1 gene transcription is NF-B. Thus, the effect of inhibition of GSK-3 on the NF-B pathway was investigated. TNF- (10 ng/mL, 1 hour) significantly increased NF-B DNA binding in HUVECs (Figure 5A). TDZD-8 (2x10^–5 mol/L) significantly prevented the TNF-–induced increase in NF-B DNA binding (Figure 5A). Phosphorylation of IKKß and subsequent degradation of IB- are important early steps in NF-B activation. Actually, TNF- (10 ng/mL) induced a rapid and transient phosphorylation of IKKß as well as degradation of IB- (Figure 5B). The GSK-3 inhibitor TDZD-8 (2x10^–5 mol/L) completely prevented the TNF-–induced IB- degradation but not the IKK-ß phosphorylation (Figure 5C).

View larger version (31K): [in this window] [in a new window]   Figure 5. Effects of GSK-3 inhibition on the NF-B pathway. A, Effect of a GSK-3 inhibitor, TDZD-8, on NF-kB DNA binding in HUVECs. The cells were stimulated with TNF- in the presence or absence of TDZD-8, and 1 hour after stimulation, the NF-B binding assay was performed. B, Effects of TNF- on IKKß phosphorylation and IB- expression. Phosphorylated IKKß and total IkB- expression were determined by Western blotting. Similar results were obtained in 3 independent experiments. C, Effects of TDZD-8 on TNF-–induced IKKß phosphorylation and IkB- degradation. Similar results were obtained in 3 independent experiments.

	Discussion

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In the present study, we have shown that GSK-3 is essential for TNF-–induced NF-B activation and subsequent induction of TF and VCAM-1 expression in human endothelial cells.

GSK-3 is a serine/threonine kinase that mediates multiple cellular functions, and its activity is negatively regulated by phosphorylation at a specific serine residue (serine 21 for the -isoform and serine-9 for the ß-isoform¹⁴). Interestingly, we observed that TNF- induced a rapid and transient dephosphorylation and thereby activation of GSK-3 in human endothelial cells. In general, phosphorylation levels of enzymes are determined by the balance between the activity of upstream kinases and that of phosphatases. In this study, GSK-3 dephosphorylation by TNF- was accompanied by a transient dephosphorylation of Akt. Because Akt is a major upstream kinase of GSK-3¹⁴ and Akt activity is positively regulated by phosphorylation¹² in contrast to GSK-3, TNF-–induced GSK-3 activation appears to be mediated by inactivation of Akt. However, the possibility cannot be ruled out that some serine/threonine phosphatase, such as protein phosphatase 1, which has been reported to dephosphorylate and thus activate GSK-3,³⁶ might be activated by TNF- in endothelial cells.

We discovered the essential role of GSK-3 in TF and VCAM-1 expression in TNF-–stimulated endothelial cells by using 2 different inhibitors. Lithium is known to reduce GSK-3 activity, with an IC₅₀ value of 2 mmol/L.³⁵ LiCl significantly suppressed TF expression and its cell-surface activity, as well as whole-cell and cell-surface expression of VCAM-1. Because NaCl had no effect on TF or VCAM-1 expression, we can rule out osmolality as an alternative explanation for the inhibitory effects of lithium. Because lithium is a potent but nonspecific GSK-3 inhibitor,³⁹ we also examined the effects of a highly specific GSK-3 inhibitor, TDZD-8,³⁸ to confirm the regulatory role of GSK-3 in this context. As expected, TDZD-8 mimicked the inhibitory effects of LiCl on TF and VCAM-1 expression. Taken together with the rapid and transient activation of GSK-3 by TNF-, we conclude that GSK-3 positively regulates endothelial induction of TF and VCAM-1 expression in response to TNF-.

Cytokine-mediated gene expression of TF and VCAM-1 is mainly mediated at the transcriptional level.^2,4 In this study, a luciferase assay clearly demonstrated that the increase in TF and VCAM-1 promoter activities by TNF- was significantly decreased by GSK-3 inhibition, suggesting a significant role for GSK-3 in the regulation of gene transcription. NF-B is a common important transcription factor for both genes.^2,4 Signaling pathways leading to NF-B activation, especially in cytokine-stimulated cells, have been extensively explored; IKKß-dependent phosphorylation and subsequent degradation of IB play a central role.^40,41 Under unstimulated conditions, NF-B binds to IB in the cytosol. On stimulation, IKKß is phosphorylated and activated and in turn phosphorylates IB. Phosphorylation of IB induces rapid IB degradation via the ubiquitin-proteosome system, which allows NF-B to travel into the nucleus and to bind the promoter regions of target genes, including those for TF and VCAM-1. However, the relation between GSK-3 and the NF-B pathway has not been fully characterized. Previously, it was reported that TNF-–induced NF-B activation was blunted in fibroblasts obtained from GSK-3ß–deficient mice,²² but that study did not clarify the mechanism underlying the regulation of NF-B by GSK-3. In this study, we observed that the increase in NF-B DNA binding activity induced by TNF- was significantly suppressed by GSK-3 inhibition. In addition, TDZD-8 completely blocked IB- degradation by TNF-, without affecting phosphorylation of IKKß. Because both IKKß-dependent phosphorylation and subsequent ubiquitination are necessary for NF-B activation, it appears that GSK-3 regulates NF-B activity through the ubiquitin-proteosome system, not through the IKKß pathway. In fact, it has been reported that GSK-3 facilitates the ubiquitin-proteosome system to induce proteolysis of ß-catenin,⁴² and IB- and ß-catenin have a common motif, which associates with the specific E3 ubiquitin ligase complex.^43–46

Lithium has been widely used as an effective drug for patients with bipolar mood disorders.³⁷ More recently, in animal and cellular models of Alzheimer’s disease, beneficial effects of lithium have been demonstrated.^25–27 In our study, even a relatively low concentration of lithium (1 mmol/L), which is close to plasma levels seen in patients receiving this medication,³⁷ significantly reduced both TF and VCAM-1 induction by TNF-, suggesting clinical relevance for these beneficial effects of lithium. In line with this observation, lithium or another specific GSK-3 inhibitor, SB216763, has been reported to reduce infarct size after ischemia/reperfusion in isolated rat hearts and in a rat stroke model.^29,30 Based on these findings, GSK-3 blockade by lithium or newly developed specific inhibitors, including TDZD-8, could be used as a novel therapeutic strategy for atherothrombotic vascular diseases such as acute coronary syndromes and ischemic stroke through reduction of thrombus formation, vascular inflammation, and ischemic organ damage, although this strategy needs to be tested in animal experiments and clinical trials.

In summary, in human endothelial cells, GSK-3 regulates TNF-–induced IkB- degradation and NF-B activation, independent of IKKß and subsequent induction of TF and VCAM-1 expression. These results therefore demonstrate a novel, essential signaling pathway leading to endothelial cell activation in response to proinflammatory cytokines. Hence, this study provides the experimental basis for a novel target in the treatment of atherothrombotic vascular disease.

	Acknowledgments

 
This study was supported by the Swiss National Research Foundation (32-67202.01) and the Swiss Heart Foundation.

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CLINICAL PERSPECTIVE

Endothelial cell transformation to a thrombogenic and inflammatory phenotype plays an important role in the pathogenesis of atherothrombosis, such as acute coronary syndromes and ischemic stroke. This study was designed to investigate the role of GSK-3 in TF and VCAM-1 expression in endothelial cells, because endothelial induction of these proteins is the hallmark of thrombosis and vascular inflammation. In human endothelial cells, TNF- activated GSK-3. The GSK-3 inhibitors LiCl and TDZD-8 suppressed TNF-–induced TF and VCAM-1 expression. The TNF-–induced increase in NF-B activity was significantly suppressed by TDZD-8, which also completely prevented TNF-–induced IB- degradation, but had no effect on IKKß phosphorylation. GSK-3 thus regulates TNF-–induced NF-B activation and subsequent induction of TF and VCAM-1 expression in human endothelial cells. These results suggest that inhibition of GSK-3 by lithium, which is widely used in clinical practice for patients with bipolar disorder, or other new GSK-3 inhibitors is a potential therapeutic strategy for atherothrombotic vascular disease.

	Footnotes

 
Guest Editor for this article was James T. Willerson, MD.

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