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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:794.)
© 2006 American Heart Association, Inc.

Vascular Biology

High Glucose Activates Nuclear Factor of Activated T Cells in Native Vascular Smooth Muscle

Jenny Nilsson; Lisa M. Nilsson; Yung-Wu Chen; Jeffery D. Molkentin; David Erlinge; Maria F. Gomez

From the Departments of Experimental Medical Science (J.N., L.M.N., M.F.G.) and Clinical Sciences (D.E.), Lund University, Sweden; Global Pharmaceutical Research and Development (Y.-W.C.), Abbott Laboratories, Abbott Park, Ill; and Department of Pediatrics (J.D.M.), Children’s Hospital Medical Center, Cincinnati, Ohio.

Correspondence to Maria F. Gomez, Department of Experimental Medical Science, BMC F12, SE-221 84 Lund, Sweden. E-mail maria.gomez{at}med.lu.se

	Abstract

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Objective— Hyperglycemia has been suggested to play a role in the development of vascular disease associated with diabetes. Atypical Ca²⁺ signaling and gene expression are characteristic of vascular dysfunction; however, little is known regarding the effects of high glucose on Ca²⁺-dependent transcription in the vascular wall.

Methods and Results— Using confocal immunofluorescence, we show that modest elevation of extracellular glucose (ie, from 2 to 11.5 mmol/L) increased [Ca²⁺]_i, leading to nuclear accumulation of nuclear factor of activated T cells (NFAT) in intact cerebral arteries from mouse. This was accompanied by increased NFAT-dependent transcriptional activity. Both the increase in Ca²⁺ and NFAT activation were prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. We provide evidence that the potent vasoconstrictors and growth stimulators UTP and UDP mediate glucose-induced NFAT activation via P2Y receptors. NFAT nuclear accumulation was inhibited by the voltage-dependent Ca²⁺ channel blockers verapamil and nifedipine, the calcineurin inhibitor cyclosporine A, and the novel NFAT blocker A-285222. High glucose also regulated glycogen synthase kinase 3ß and c-Jun N-terminal kinase activity, yielding decreased kinase activity and reduced export of NFAT from the nucleus, providing additional mechanisms underlying the glucose-induced NFAT activation.

Conclusions— Our results identify the calcineurin/NFAT signaling pathway as a potential metabolic sensor for the arterial smooth muscle response to high glucose.

Key Words: NFAT • high glucose • vascular smooth muscle • extracellular nucleotides • GSK-3

	Introduction

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Glucose has been shown to activate nuclear factor of activated T cells (NFAT) in pancreatic ß-cells, promoting insulin gene transcription.¹ Previous studies have shown that this transcription factor is expressed in native vascular smooth muscle and can be activated in response to vasoconstrictor agonist stimulation.² Furthermore, NFAT inhibition has been shown recently to reduce balloon injury–induced neointima formation in rat carotid artery.³ However, it is unknown whether glucose can activate NFAT in the vasculature.

There are 4 well-characterized members of the NFAT family, all of which depend on dephosphorylation by calcineurin to translocate to the nucleus.⁴ In immune cells, activation of this signaling pathway leads to production of cytokines and T-cell proliferation.⁴ Inhibition of NFAT nuclear translocation is largely responsible for the immunosuppressive actions of cyclosporine A and tacrolimus (FK506), which specifically block calcineurin.⁵ Although originally thought to be restricted to T cells, NFAT has since been shown to regulate heart valve development,⁶ skeletal muscle differentiation,⁷ and vascular development during embryogenesis.⁸ NFAT has also been implicated in the pathogenesis of cardiac⁹ and skeletal muscle hypertrophy¹⁰ and might be predicted to play a role in smooth muscle hypertrophy associated with, for example, hypertension and atherosclerosis. Indeed, Amberg et al¹¹ recently demonstrated that sustained angiotensin II stimulation, which in vivo results in hypertension, leads to NFAT activation in arterial smooth muscle. This activation causes decreased expression and function of voltage-dependent K⁺ (Kv) channels, resulting in enhanced vascular excitability.

In arterial smooth muscle, the extracellular nucleotide UTP and its degradation product uridine diphosphate UDP, acting on membrane P2Y₂ and P2Y₆ receptors, are potent growth factors stimulating cell cycle progression, cell division, and cellular hypertrophy.^12,13 UTP raises intracellular Ca²⁺ and engages multiple signaling pathways, resulting in increased NFATc3 nuclear accumulation, which is the predominant isoform in this tissue.² This is dependent on inositol triphosphate receptor-mediated release of Ca²⁺ from intracellular stores, extracellular Ca²⁺ influx from voltage-dependent Ca²⁺ channels (VDCCs), and calcineurin activity.

UTP effectively promotes NFATc3 nuclear accumulation not only by appropriate tuning of the Ca²⁺ signal but also by suppressing the activity of c-Jun N-terminal kinase (JNK), which phosphorylates and promotes the export of NFATc3 from the nucleus.¹⁴ The subcellular localization of NFATc3 therefore reflects the dynamic interplay between the cytosolic phosphatase activity of calcineurin, promoting NFAT nuclear import, and the activity of this serine-threonine kinase. Among other kinases, evidence obtained in transfection systems suggests that the glycogen synthase kinase 3 (GSK-3) can counteract NFAT nuclear accumulation.¹⁵ Both in cardiac and skeletal muscle, activation of GSK-3 suppresses hypertrophy via inhibition of NFAT signaling.^10,16

Apart from promoting NFAT export, GSK-3 inactivates glycogen synthase, the last enzyme in glycogen biosynthesis.¹⁷ Normally, when the glucose availability is high, GSK-3 activity is inhibited and glycogen can be stored. Insulin and growth factors, via phosphatidylinositol 3-kinase, inhibit GSK-3 acutely, leading to increased glycogen and protein synthesis. Interestingly, in vascular smooth muscle, phosphatidylinositol 3-kinase has been suggested to increase NFAT activation in response to very low-density lipoproteins via phosphorylation and thus inactivation of GSK-3ß.¹⁸ This kinase is also a downstream target of mammalian target of rapamycin (mTor), the substrate of the immunosuppressant rapamycin.

In this study, we test the hypothesis that changes in extracellular glucose levels may activate NFAT in vascular smooth muscle.

	Methods

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Tissue Samples
Aortas and cerebral arteries from Naval Medical Research Institute (Harlan, Scandinavia) mice and aortas and portal veins from NFAT-luciferase transgenic mice were used. Mice were euthanized by cervical dislocation in accordance with approved local ethical guidelines.

Immunofluorescence
Experiments were performed as described previously.^2,14 Primary antibody, rabbit anti-NFATc3 (Santa Cruz Biotechnology), and Cy5-labeled secondary antibody (Jackson ImmunoResearch Laboratories) were used. Nuclei were stained with the nucleic acid dye SYTOX Green (Molecular Probes). NFATc3 and nuclear regions were detected by monitoring Cy5 and green fluorescence on a Zeiss LSM 510 laser scanning confocal microscope. For scoring of NFATc3-positive nuclei, 3 to 5 fields for each vessel were imaged, and an average of 277 cells per field was counted under blind conditions. A cell was considered positive if colocalization (white) was observed in the nucleus and negative if no colocalization (green only) was visualized.

Luciferase Reporter Assay
Luciferase enzymatic activity in arteries from phenotypically normal NFAT-luciferase transgenic mice¹⁹ was determined using a commercial kit (Promega) according to the supplier indications. Optical density was measured (VICTOR 3 multilabel counter; Perkin–Elmer) and expressed as relative luciferase units normalized to protein concentration.

Confocal Ca²⁺ Measurements
Experiments were performed as described previously.² Arteries were loaded with fluo-4-AM and imaged using a Zeiss LSM 510 laser-scanning confocal microscope. Images were acquired every 1.56 s, before and after each treatment, and changes in global fluorescence (F/F₀) were calculated.

Western Blot Analysis
The following antibodies were used: goat polyclonal anti–GSK-3ß, anti–p-GSK-3ß (Santa Cruz Biotechnology), and rabbit polyclonal anti-JNK1&2[pTpY^183/185] (BioSource International, Inc.) on cerebral arteries and aortas.

Chemicals
Fluo-4 and pluronic acid were from Molecular Probes, Inc., GSK-3ß inhibitor was from Calbiochem, MRS2578 was a gift from Dr K.A. Jacobson (National Institutes of Health; Bethesda, Md), and A-285222 was provided by Abbott Laboratories. All other drugs were from Sigma.

Statistical Analysis
Results are expressed as means±SEM. Statistical significance was determined using 1-way ANOVA followed by Bonferroni or Tukey–Kramer tests (for comparisons between <5 and >6 groups, respectively).

For a detailed version of the methods, please see the online supplement, available at http://atvb.ahajournals.org.

	Results

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Glucose Acutely Induces NFATc3 Activation
In intact cerebral arteries, raising the extracellular glucose concentration from 11.5 mmol/L (control) to 20 mmol/L (high glucose [HG]) for 30 minutes significantly increases NFATc3 nuclear accumulation (Figure 1A). The number of NFATc3-positive nuclei increases from 19% in control vessels to 49% in arteries exposed to HG (Figure 1B). This was prevented by the calcineurin inhibitor cyclosporin A (1 µmol/L) and by the novel NFAT blocker A-285222^20,21 (1 µmol/L; Figure 1B). Mannitol had no effect on NFATc3 nuclear accumulation, ruling out a possible osmotic effect of glucose; and L-glucose, which cannot be metabolized by the cell, had no effect either (Figure 1B).

View larger version (22K): [in this window] [in a new window]   Figure 1. HG activates NFAT in vascular smooth muscle. A, Representative images showing cytosolic localization of NFATc3 in control condition (11.5 mmol/L glucose) and nuclear localization on 30-minute HG (20 mmol/L) in cerebral arteries, with or without the NFAT inhibitor A-285222 (1 µmol/L). White indicates nuclear colocalization of NFATc3 (red) and the DNA-binding dye SYTOX (green). Bars=20 µm. B, Percentage of cells exhibiting NFATc3 nuclear accumulation in cerebral vessels treated with HG for 30 minutes with or without cyclosporin A (1 µmol/L; cyclosporine A [CsA]) or A-285222 (1 µmol/L), mannitol, and L-glucose (both 20 mmol/L; 30 minutes). Experiments were performed at room temperature. ***P<0.001, HG vs all other bars (m indicates No. of mice; v, No. of vessels). C, NFAT-dependent transcriptional activity in aortas and portal veins from transgenic mice. Vessels were exposed to HG with and without apyrase (0.32 U/mL) or to UTP (100 µmol/L) for 30 minutes and collected 6 hours later. Data expressed as optical density normalized to protein concentration (relative luciferase units/µg protein). Values from nontransgenic FVBN mice (WT) are shown as negative controls. Experiments are performed in triplicate (m=8; ***P<0.001, **P<0.01, *P<0.05).

Exposure to HG for 30 minutes also resulted in increased NFAT-dependent luciferase activity in aorta and portal vein from mouse (Figure 1C), indicating that nuclear accumulation of NFAT is accompanied by enhanced transcriptional activity. Corresponding experiments on cerebral arteries were hampered by low tissue yield.

Time and Dose Dependency of Glucose-Induced NFAT Activation
Time- and dose-response experiments reveal significantly increased NFATc3 nuclear accumulation after 8-minute exposure to HG and a stepwise response when raising the extracellular glucose concentration >11.5 mmol/L (Figure 2A and 2B). A 3.5-mmol/L increase (to 15 mmol/L) was sufficient to achieve significant NFATc3 nuclear accumulation, whereas lowering glucose to 8 or 5 mmol/L had no effect (Figure 2B).

View larger version (26K): [in this window] [in a new window]   Figure 2. Time and dose response of glucose-induced NFATc3 nuclear accumulation. Summarized data showing NFATc3 nuclear accumulation A, After treatment with HG for 3, 5, 8, 10, 20, and 30 minutes. B, In response to varying doses of glucose (5, 8, 11.5, 13, 14, 15, 20, and 25 mmol/L) for 30 minutes. ***P<0.001, **P<0.01, *P<0.05, vs control (m indicates No. of mice; v, No. of vessels).

For this study, 11.5 mmol/L was considered as control glucose concentration because all our previous NFAT data in cerebral arteries were obtained using physiological saline solution containing this level of glucose. This level is higher than the levels the arteries may face in vivo, but it constitutes the only energy source during in vitro experiments and was therefore chosen as "control" condition. Because the lack of response at lower glucose concentrations (5 and 8 mmol/L) could be attributed to the elevated basal level used as control, we used a different experimental paradigm in which cerebral vessels were allowed to equilibrate in media containing 2 mmol/L glucose overnight and were then stimulated for 30 minutes with 11.5 mmol/L or 20 mmol/L glucose. This resulted in a dose-dependent and significant increase in NFATc3 nuclear accumulation (Figure 3A). Similar experiments using aortas from NFAT-luciferase mice show that stimulation with media containing 7, 11.5, or 20 mmol/L glucose after overnight equilibration in 2 mmol/L glucose yields enhanced NFAT-dependent transcriptional activity (Figure 3B). Interestingly, the level of NFATc3 nuclear accumulation or NFAT-dependent transcriptional activity after overnight incubation with 2 mmol/L was higher than the levels measured in noncultured arteries, suggesting that culture per se may result in NFAT activation (Figure 1A and 1B versus 1C).

View larger version (19K): [in this window] [in a new window]   Figure 3. Dose response after overnight equilibration in low-glucose media. A, Summarized NFATc3 nuclear accumulation in arteries equilibrated overnight in 2 mmol/L glucose media and subsequently stimulated for 30 minutes with 11.5 or 20 mmol/L glucose. B, NFAT-dependent transcriptional activity in aortas equilibrated overnight as in A and then incubated for 6 hours in media containing 7, 11.5, or 20 mmol/L glucose. Data expressed as optical density normalized to protein concentration (relative luciferase units/µg protein). ***P<0.001, **P<0.01, *P<0.05, vs 2 mmol/L (m indicates No. of mice; v, No. of vessels).

Release of Extracellular Nucleotides Mediates Glucose-Induced NFATc3 Nuclear Accumulation
The ectonucleotidase apyrase (0.32 U/mL) prevents HG-induced NFATc3 nuclear accumulation (Figure 4A) and NFAT-dependent luciferase activity (Figure 1C), suggesting enhanced release of extracellular nucleotides on HG exposure. MRS2578, a selective antagonist of the UDP receptor P2Y₆ significantly decreased glucose-induced NFATc3 nuclear accumulation (Figure 4A), providing further evidence for the involvement of extracellular nucleotides in this response.

View larger version (27K): [in this window] [in a new window]   Figure 4. Glucose-induced NFATc3 nuclear accumulation involves extracellular nucleotides. Summary data showing A, the effect of 30-minute treatment with HG with or without apyrase (0.32 U/mL), apyrase+2-theophylline (theo; 10 µmol/L), or the P2Y6 receptor antagonist MRS2578 (10 µmol/L). ***P<0.001, HG vs all groups, *P<0.05, vs control, and B, the effect of 30-minute exposure to UDPßs, UTPs, or UTP (both 10 µmol/L) on NFATc3 nuclear accumulation. ***P<0.001 and *P<0.05 vs controls. (m indicates No. of mice; v, No. of vessels).

UTP was slightly more effective than the stable pyrimidines UDPßs (selective for P2Y₆ receptors) and UTPs (selective for P2Y_2/4 receptors), in stimulating NFATc3 nuclear accumulation (Figure 4B). It is possible that the less stable UTP may act both on P2Y_2/4 receptors and, after degradation to UDP, on P2Y₆ receptors. As shown in Figure 1C, UTP also effectively increased NFAT-dependent luciferase activity.

Hydrolysis of extracellular nucleotides by apyrase can lead to increased levels of adenosine and, consequently, to activation of K_ATP channels, hyperpolarization of the cell membrane, and reduced influx of Ca²⁺ via VDCCs.²² The K_ATP channel opener pinacidil has been shown previously to inhibit NFATc3 nuclear accumulation in these arteries.² Hence, adenosine could be predicted to decrease glucose-induced NFATc3 nuclear accumulation by virtue of its effects on K_ATP channels. To test this, we tried apyrase in the presence of the adenosine inhibitor 2-theophylline (10 µmol/L). Blockade of adenosine failed to affect the inhibition of glucose-induced NFATc3 nuclear accumulation achieved by apyrase, excluding an indirect effect of apyrase other than reduction of available extracellular nucleotides (Figure 4).

In arterial smooth muscle, extracellular nucleotides (ie, ATP, ADP, UTP, and UDP) increase global [Ca²⁺]_i.^2,23,24 Here we show that HG increases global [Ca²⁺]_i in intact cerebral arteries (Figure 5A through 5C). If activation of P2Y receptors by a glucose-stimulated increase in extracellular nucleotides is responsible for the observed increase in [Ca²⁺]_i, then treatment with apyrase would be expected to prevent a raise in [Ca²⁺]_i. Indeed, no increase of Ca²⁺ was observed when cerebral arteries were exposed to HG in the presence of apyrase (Figure 5C). The L-type VDCC blockers verapamil (10 µmol/L) and nifedipine (100 nmol/L) partially inhibited HG-induced NFATc3 nuclear accumulation (supplemental Figure I, available online at http://atvb.ahajournals.org), indicating that Ca²⁺ influx via these channels participates in this response.

View larger version (83K): [in this window] [in a new window]   Figure 5. Dependence of glucose-induced NFATc3 nuclear accumulation on Ca2+. A, Selected confocal images of a cerebral artery segment loaded with fluo-4, showing fluorescence intensity before (0 s) and after application of HG (40, 80, 120, and 150 s). Five individual smooth muscle cells in the artery wall are highlighted at 150 s (pseudocolored with values ranging from 0 to 255). B, Mean pixel intensity within the 5 cells highlighted in A, before and after HG application. C, Summarized F/F0 data before and 1 minute after HG exposure with or without apyrase (0.32 U/mL). Five to 10 boxes of 10.24 µm2 were placed in each artery. ***P<0.001, HG vs control and HG+apyrase (m indicates No. of mice).

GSK-3ß Negatively Regulates NFATc3 Nuclear Accumulation
Intact cerebral arteries treated for 30 minutes with the cell-permeable GSK-3ß inhibitor Myr-N-GKEAPPAPPQSpP-NH₂ induced a robust NFATc3 nuclear accumulation, resulting in levels comparable to those observed after HG exposure (Figure 6A). This indicates that GSK-3ß is tonically active and contributes to NFATc3 export regulation in these arteries. Also, simultaneous incubation of vessels with the GSK-3ß inhibitor and HG failed to achieve higher levels of nuclear NFATc3 than those observed after HG alone, consistent with the effects of glucose on NFATc3 activation being, at least in part, mediated by inhibition of GSK-3ß export activity.

View larger version (18K): [in this window] [in a new window]   Figure 6. GSK-3ß is constitutively active in cerebral arteries from mice and is phosphorylated by HG. A, Effect of HG with or without the GSK-3ß inhibitor Myr-N-GKEAPPAPPQSpP-NH2 (GSKi; 120 µmol/L) on NFATc3 nuclear accumulation. The effect of GSKi alone is also shown. ***P<0.001, **P<0.01 vs control, *P<0.05 HG vs HG+GSKi (m indicates No. of mice; v, No. of vessels). B, Increased GSK-3ß phosphorylation on 30-minute HG stimulation. C, Summarized levels of phosphorylated GSK-3ß (P-GSK-ß), normalized to total GSK-3ß, expressed as percentage of control (3 separate experiments including cerebral arteries from 14 mice). The effect of insulin (100 nmol/L) is shown for comparison (value from a single experiment with pooled arteries from 10 mice).

Glucose Downregulates GSK-3ß and JNK Activity
Raising the glucose concentration from 11.5 to 20 mmol/L for 30 minutes increased GSK-3ß phosphorylation by 66% in intact cerebral arteries (Figure 6B and 6C). Insulin (100 nmol/L), which has proven to effectively increase GSK-3ß phosphorylation in human skeletal muscle,²⁵ yielded increased GSK-3ß phosphorylation by 55%. We have previously shown in cerebral vessels that UTP decreases the activity of JNK.¹⁴ If HG acts through nucleotides like UTP, it would be predicted that it should also inhibit JNK activity. Indeed, stimulation for 30 minutes with HG significantly decreased the levels of phosphorylated JNK in mouse aorta (supplemental Figure IIA and IIB).

	Discussion

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This study shows that changes in the extracellular glucose concentration are readily detected by NFATc3 in native cerebral artery smooth muscle, leading to increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity. We therefore propose a role for NFAT as a metabolic sensor in the vascular wall of potential relevance for vascular dysfunction in diabetes. The effect of glucose on NFATc3 activation involves the release of extracellular nucleotides acting on P2Y receptors, leading to increased [Ca²⁺]_i and subsequent activation of calcineurin and NFATc3. Normally, the subcellular localization and transcriptional activity of NFAT reflects a dynamic balance between stimuli promoting and opposing nuclear accumulation of NFAT. Here we show that HG not only engages influences promoting NFAT nuclear translocation but also decreases the export of NFAT from the nucleus by inhibiting the otherwise constitutively elevated kinase activity of GSK-3ß and JNK.

HG-induced nuclear accumulation of NFATc3 is robust and comparable to activation levels obtained after agonist stimulation with UTP or endothelin-1². The time course of this response is consistent with previous data reported for UTP-induced NFATc3 activation in these arteries.¹⁴ HG-induced NFATc3 nuclear accumulation is prevented by A-285222, which is a 3,5-bis(trifluoromethyl)pyrazole derivative recently identified as NFAT blocker in immune cells.^20,21,26 Traditionally, the NFAT pathway has been studied using calcineurin blockers, such as cyclosporin A, and FK506. Because calcineurin not only interacts with NFAT but also with other substrates (ie, the type II regulatory subunit of protein kinase A,²¹), these drugs are ambiguous tools for dissecting the NFAT-signaling pathway. Instead, the A-285222 compound has been shown to maintain NFAT in a phosphorylated state by a mechanism independent of calcineurin activity.

Regarding the mechanism of action underlying HG-induced NFAT activation, a role for extracellular nucleotides is strongly suggested because the response was blocked by apyrase. This is consistent with studies in endothelial and pancreatic ß-cells showing that extracellular nucleotides such as UTP and ATP are released on mechanical stress and increased extracellular glucose concentration.^27–29

In the vasculature, autocrine or paracrine release of nucleotides can lead to both vasoconstriction and growth stimulation depending on the activation of specific nucleotide receptor subtype.²⁴ In human and rat cerebral arteries, the UDP receptor P2Y₆ has been shown to play a prominent role in the regulation of vascular tone.^30,31 The observation that the P2Y₆ receptor antagonist MRS2578 partially decreased HG-induced NFAT nuclear accumulation provides further evidence for nucleotide release on HG stimulation and highlights the possible engagement of multiple purinergic receptors. A detailed characterization of the nature of the nucleotides released and receptors involved is beyond the scope of this study, but the experiments with the stable analogs UDPßs and UTPs, suggest that UTP acting on P2Y_2/4 receptors and UDP acting on P2Y₆ receptors are strong candidates.

Consistent with previous data,³² an increase in global [Ca²⁺]_i was observed on exposure to HG in cerebral artery smooth muscle. This was prevented by apyrase, suggesting that the increase in Ca²⁺ is attributable to autocrine/paracrine activation of P2Y purinergic receptors by released extracellular nucleotides. It was shown previously that UTP-induced NFATc3 nuclear accumulation requires both the release of Ca²⁺ from intracellular stores and influx of Ca²⁺ via VDCCs because inhibition of either pathway completely abrogates the response.² Interestingly, HG-induced NFATc3 activation was only partially reduced by inhibition of VDCCs, as shown by experiments using verapamil and nifedipine, highlighting potential differences in the Ca²⁺ signaling pathways engaged in response to glucose with those of UTP.

UTP not only acts through multiple mechanisms to increase [Ca²⁺]_i but also by suppressing relatively elevated basal JNK2 activity.¹⁴ A model describing a dual signal mechanism for induction of NFATc3 in arterial smooth muscle was proposed, requiring both a calcineurin-activating Ca²⁺ signal and engagement of pathways that downregulate NFAT nuclear export. The data presented here are supportive of this model and describe for the first time in native arterial smooth muscle the involvement of another kinase in NFATc3 export regulation: GSK-3ß. This kinase is constitutively active in this tissue because pharmacological inhibition of GSK-3ß resulted in enhanced NFATc3 nuclear accumulation. The fact that HG also decreases the levels of phosphorylated JNK further supports the concept of HG acting through nucleotides, even if a direct effect of HG on JNK cannot be ruled out.

The role of NFAT in vascular smooth muscle is still unclear; however, NFATc3 may be predicted to face considerable levels of [Ca²⁺]_i and calcineurin activation under pressurized conditions. Indeed, recent work by Gonzalez Bosc et al demonstrates that acute increases in intraluminal pressure stimulate NFATc3 nuclear accumulation in mouse cerebral arteries.³³ Thus, the presence of 2 constitutively active kinases able to suppress NFAT activity provides additional levels of regulation of potential interest in response to metabolic changes or agonist stimulation. The mechanism connecting increases in glucose to the inactivation of GSK-3ß is currently unknown, but enhanced GSK-3ß phosphorylation in vascular smooth muscle cells (VSMCs) during neointima formation has been reported as a consequence of upregulated glucose transport and metabolism.³⁴

The described link between HG levels and NFAT activation mediated by activation of P2Y receptors by released extracellular nucleotides is interesting from the clinical perspective. Diabetic patients experience microvascular disease characterized by increased wall-lumen ratio mainly because of increased amounts of vascular smooth muscle cells. Our findings, combined with the potent growth stimulatory effects of both UTP and UDP,^12,13 could represent a new mechanism contributing to diabetic microvascular disease. The described NFAT-dependent regulation of Kv channels, leading to enhanced excitability of arterial smooth muscle,¹¹ may represent a contributing factor to vascular dysfunction in the diabetic situation.

Diabetic patients have higher rates of restenosis after coronary angioplasty, resulting in increased morbidity and mortality. The mechanism of restenosis is excessive growth of VSMCs, creating a neointima. UTP has been shown to contribute to neointimal development.³⁵ In diabetic patients, drug-eluting stents coated with sirolimus (rapamycin) or paclitaxel have been valuable to avoid restenosis.³⁶ Interestingly, sirolimus is closely related to the NFAT inhibitors tacrolimus and cyclosporine and reduces GSK-3 activity via inhibition of mTor (see Introduction). It is possible that our results provide a link between diabetes and increase restenosis rates, involving HG-induced release of extracellular nucleotides acting on P2Y receptors and concomitant GSK-3ß inhibition to stimulate VSMC growth via NFAT activation.

Although a basal glucose level of 11.5 is typical for in vitro experiments, it would be considered hyperglycemic in vivo. Nevertheless, the narrow concentration range of the response (from 11.5 to 15 mmol/L) is interesting because plasma glucose concentrations >11.1 mmol/L are considered in clinical practice as indicative for diabetes.³⁷ The experiments using 2 mmol/L glucose as basal level provide further evidence for a glucose-responsive NFAT pathway at physiologically relevant hyperglycemic levels. The vessels are able to adjust to the glucose environment they are exposed to (11.5 or 2 mmol/L) by equilibrating at relatively low levels of NFAT nuclear accumulation, allowing the system to sense further changes in extracellular glucose and responding with increased nuclear accumulation and transcriptional activity. This may be important in the clinical situation because modest fluctuations (of a few mmol/L) in plasma glucose or hyperglycemic peaks may be sufficient for NFAT activation.

In conclusion, modest elevations in extracellular glucose lead to increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in arterial smooth muscle. We therefore propose a role for NFAT as a metabolic sensor in the vascular wall of potential relevance for vascular dysfunction in diabetes. The effect of glucose on NFATc3 nuclear activation involves the release of extracellular nucleotides acting on P2Y receptors, leading to increased intracellular Ca²⁺ levels and subsequent activation of calcineurin, combined with inhibition of GSK-3b and JNK, leading to reduced nuclear export of NFATc3 (for diagram of proposed mechanisms, please see the online supplement).

	Acknowledgments

 
This work was supported by grants from the Crafoord, Bergvall, Zoéga, Söderberg, Wiberg and Swedish Heart-Lung Foundations, from the Royal Physiographic Society, the Vascular Wall Programme at the Medical Faculty in Lund, and the Swedish Research Medical Council (13130). We thank Ina Nordström for skillful technical assistance.

	Footnotes

 
Y.-W.C. was employed by Abbott Laboratories at the time this work was conducted. Abbott Laboratories own the 3,5-bis(trifluoromethyl)pyrazole compounds.

Received October 22, 2005; accepted January 18, 2006.

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