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(Circulation. 2004;110:91-96.)
© 2004 American Heart Association, Inc.

Original Articles

Selective Inhibition of Protein Kinase Cß₂ Prevents Acute Effects of High Glucose on Vascular Cell Adhesion Molecule-1 Expression in Human Endothelial Cells

Alexei Kouroedov, MD; Masato Eto, MD, PhD; Hana Joch; Massimo Volpe, MD; Thomas F. Lüscher, MD; Francesco Cosentino, MD, PhD

From Cardiovascular Research, Institute of Physiology, University of Zürich and Cardiovascular Center, University Hospital, Zürich, Switzerland (A.K., M.E., H.J., T.F.L., F.C.); and the Division of Cardiology, 2nd Faculty of Medicine, University La Sapienza, Rome, and IRCCS Neuromed, Pozzilli, Italy (M.V., F.C.).

Correspondence to Francesco Cosentino, MD, PhD, Cardiovascular Research, Institute of Physiology, University of Zürich-Irchel, CH-8057 Zürich, Switzerland. E-mail f_cosentino{at}hotmail.com

Received December 11, 2003; revision received March 30, 2004; accepted April 2, 2004.

	Abstract

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Background— Enhanced expression of adhesion molecules by the endothelium may account for vascular damage in diabetics and nondiabetic patients who develop stress hyperglycemia during acute myocardial infarction. We analyzed the phosphorylation of protein kinase Cß₂ (PKCß₂) at serine/threonine residues, which may contribute to the endothelial dysfunction during acute hyperglycemia. Furthermore, this study was designed to investigate whether selective blockade of this regulatory mechanism may prevent the development of endothelial hyperadhesiveness.

Methods and Results— Incubation of the human aortic endothelial cells with high glucose (22.2 mmol/L) resulted in significant increase of vascular cell adhesion molecule (VCAM)-1 protein expression (172±15% versus control; P<0.01). Phorbol 12-myristate 13-acetate, a potent activator of PKC, mimicked the effect of high glucose on VCAM-1 expression. High glucose led to a rapid increase (181±22% versus control; P<0.01) of membrane-bound PKCß, reflecting activation of this enzyme. The nonselective inhibitor of PKCß₁ and PKCß₂ isoforms LY379196, as well as CGP53353, a highly selective inhibitor of PKCß₂, prevented in a dose-dependent manner upregulation of VCAM-1. Incubation with high glucose was associated with increased PKCß₂ phosphorylation at the Ser-660 residue, and both LY379196 and CGP53353 prevented this event. Exposure of the cells to high glucose also reduced the protein level of the inhibitory subunit of nuclear factor-B, IB, leading to its enhanced binding activity. Selective inhibition of PKCß abolished IB degradation.

Conclusions— Our findings demonstrate for the first time that phosphorylation of Ser-660 represents a selective regulatory mechanism for glucose-induced upregulation of VCAM-1. Therefore, PKCß₂-selective inhibitors may be promising drugs for treatment of endothelial dysfunction during acute hyperglycemia and possibly in diabetes.

Key Words: diabetes mellitus • cell adhesion molecules • endothelium • inhibitors

	Introduction

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Stress hyperglycemia during myocardial infarction is associated with an increased risk of in-hospital mortality in patients with and without diabetes.^1,2 Furthermore, after stroke, the severity of hyperglycemia correlates strongly with mortality and morbidity in diabetic and nondiabetic patients.³ One of the possible mechanisms that could explain the worse outcomes in acute hyperglycemia is glucose-induced upregulation of adhesion molecules in endothelial cells.^4,5 Expression of adhesion molecules leads to rolling and migration of activated white blood cells into the vessel wall. Under such conditions, endothelial cells become a target for cytokines secreted by adherent leukocytes that in turn result in inflammation and activation of the coagulation cascade. Subsequent induction of tissue factor expression is one of the crucial steps in thrombus formation. However, the precise molecular mechanism by which hyperglycemia regulates adhesion molecule transcription is still unclear.

See p 7

Glucose upregulates protein kinase C (PKC).^6,7 PKC comprises several structurally related serine/threonine kinases classified in 3 groups. The "conventional" or "classical" PKCs include PKC , ß₁, ß₂ and . These isoforms can be activated by Ca²⁺ and/or by diacylglycerol (DAG) as well as phorbol esters. The "novel" PKC , , can also be activated by DAG and phorbol esters but are Ca²⁺ independent. The "atypical" PKCs, which include PKC and PKC, are unresponsive to Ca²⁺/DAG and phorbol esters.⁸ Three mechanisms of activation of PKC exist: (1) phosphorylation, (2) ligand binding (DAG, phospholipids and Ca²⁺), and (3) pairing with PKC-binding proteins, which leads to subcellular localization of this enzyme.⁹ Particularly interesting is the modulation of PKC activity by phosphorylation of serine and threonine amino acid residues within its catalytic and regulatory domains.¹⁰ For some proteins, it is known that phosphorylation of distinct amino acid residues can not only dramatically increase the catalytic activity of the enzyme but also even change substrate specificity.¹¹

The main problem in the development of drugs targeting PKC is a ubiquitous participation of PKC in different signaling cascades from a wide array of receptors (convergence), resulting in multiple cellular effects (ie, proliferation, apoptosis, gene expression).¹² Thus, nonspecific blockade of PKC would be associated with severe side effects for whole organism.¹³ Several studies have strongly implicated activation of PKCß in the pathogenesis of the vascular complications of diabetes.^14,15 However, a precise characterization of the regulatory mechanism PKCß isoform involved is still missing. This prompted us to investigate the regulation of PKCß₂ isoform in human aortic endothelial cells (HAECs) exposed to high glucose. Use of selective inhibitors specifically interfering with this subtype is challenging. In this study, we addressed serine/threonine phosphorylation of PKCß₂ to define selective pharmacological tools against endothelial hyperadhesiveness under high-glucose conditions.

	Methods

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Cell Culture
HAECs were obtained from Clonetics and grown in gelatin-coated flasks in optimized endothelial growth medium (Clonetics) supplemented with 10% FCS and without hydrocortisone. The cells were detached by exposure to trypsin/EDTA for 120 seconds in HEPES-buffered saline and reseeded in collagen-coated 6-cm cell-culture dishes or 24-multiwell plates. Cells were first grown to confluence in humidified air, 5% CO₂ at 37°C. Confluent cells were maintained in endothelial growth medium containing 2% FCS. They were incubated with control (5.5 mmol/L) and at an elevated glucose concentration (22.2 mmol/L). Cells between the third and sixth subpassages were used.

Drugs
The selective inhibitor of PKCß₂ CGP53353¹⁶ was kindly provided by Dr Doriano Fabbro (Novartis Pharma AG, Basel, Switzerland); the PKC inhibitor LY379196 (specific for ß₁ and ß₂ isoforms) was provided by Eli Lilly.¹⁷ Calphostin C and phorbol 12-myristate 13-acetate (PMA) were purchased from Calbiochem.

Western Blotting
HAECs were washed twice with PBS and harvested in the extraction buffer (120 mmol/L sodium chloride, 50 mmol/L Tris, 20 mmol/L sodium fluoride, 1 mmol/L benzamidine, 1 mmol/L DTT, 1 mmol/L EDTA, 6 mmol/L EGTA, 15 mmol/L sodium pyrophosphate, 0.8 µg/mL leupeptin, 30 mmol/L p-nitrophenyl phosphate, 0.1 mmol/L PMSF, and 1% NP-40) for immunoblotting. All cell debris was removed by centrifugation at 12 000g for 10 minutes at 4°C. The samples (20 µg) were treated with 5x Laemmli’s SDS-PAGE sample buffer (0.35 mol/L Tris-Cl, pH 6.8, 15% SDS, 56.5% glycerol, 0.0075% bromophenol blue), followed by heating at 95°C for 3 minutes, and then subjected to 8% SDS-PAGE gel for electrophoresis. The proteins were then transferred onto Immobilon-P filter papers (Millipore AG) with a semidry transfer unit (Hoefer Scientific). The membranes were then blocked by use of 5% skim milk in PBS-Tween buffer (0.1% Tween 20; pH 7.5) for 1 hour and incubated with the antibody anti-human vascular cell adhesion molecule (VCAM)-1 (R&D), anti-PKCß (Gibco), anti-IB, anti-phospho-PKC/ß₂ (Thr641) (all from Cell Signaling Technology), and anti-phospho-PKCß₂ (Santa Cruz). The immunoreactive bands were detected by an enhanced chemiluminescence system (Amersham).

Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay
HAECs cultured in 60-mm plastic dishes were washed twice with PBS and harvested into 1.5-mL tubes by scraping in 1 mL of ice-cold hypotonic buffer (mmol/L: 10 KCl, 10 HEPES, 0.1 EDTA, 0.1 EGTA, 1.0 DTT, 1.0 PMSF) + 6 mL of Nonidet P40 (a final concentration of 0.6%) and incubated on ice for 20 minutes. Cells were homogenized by vigorous vortexing for 10 seconds. The homogenate was centrifuged (3000 rpm) at 4°C for 10 minutes to obtain a pellet of nuclei. The isolated nuclei in the pellet were resuspended in 40 mL of ice-cold hypertonic buffer (mmol/L: 400 NaCl, 20 HEPES, 1.0 EDTA, 1.0 EGTA, 1.0 DTT, 1.0 PMSF). Nuclear proteins were extracted by incubation of the homogenate on ice for 20 minutes with intermittent vortexing. The supernatant containing nuclear proteins was collected after centrifugation at 8000 rpm, 4°C for 10 minutes. The nuclear protein was then transferred into a new precooled tube and stored at –70°C until use. Protein concentration was determined by Bio-Rad Protein Assay.

Double-stranded synthetic oligonucleotide to consensus sequence for NF-B (5-AGTTGAGGGGACTTTCCCAGG-3) (Promega) was 5' end-labeled with [g-³²P]ATP and T4 polynucleotide kinase (Promega). The mixture of 2 mL [g-³²P]ATP, 4 mL oligonucleotide probe, 2 mL 10x kinase buffer (Promega), 2 mL T4 polynucleotide kinase (Promega), and 10 mL H₂O was incubated at 37°C for 20 minutes, and then 2 mL of 0.5 mol/L EDTA was added to inactivate kinase. In addition, 178 mL of TE buffer (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA) was added. Protein/DNA binding reaction was carried out in 20 mL of mixture containing a binding buffer (Promega) (50 mmol/L NaCl, 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 10 mmol/L Tris-HCl, pH 7.5, and 0.05 g/L poly[dIdC] in 4% glycerol), nuclear protein (5 to 10 mg), and ³²P-labeled oligonucleotide (2 mL). The mixture was incubated for 30 minutes at room temperature. After the incubation, the sample was loaded onto 7.5% denaturing polyacrylamide gel (5 mL 30% acrylamide/bis acrylamide, 2.5 mL 5x TBE, 25 mL TEMED, 12.3 mL H₂O, 200 mL 10% ammonium persulfate) at 200 V for 1 hour in an electrophoresis buffer (0.5x TBE) containing 45 mmol/L Tris, 45 mmol/L boric acid, and 1.0 mmol/L EDTA. The gel was subjected to autoradiography at –70°C for 24 hours.

Cell Fractionation
PKCß translocation was measured by Western blotting after cell fractionation into a cytosolic and a particulate fraction. Cells were harvested and sonicated, and samples were centrifuged at 100 000g for 1 hour at 4°C. The supernatant was used as the cytosolic fraction. The pellet was resuspended in 40 mL of buffer containing 1% Triton X-100 and 0.1% SDS. Cell debris was separated by centrifugation, and the supernatant was used as the particulate detergent-soluble fraction.

Statistical Analysis
Results are expressed as mean±SEM, and n indicates number of experiments. Statistical evaluation of the data was performed with Student’s t test for simple comparison between 2 values when appropriate. For multiple comparisons, results were analyzed by ANOVA followed by Fisher’s test. A value of P<0.05 was considered statistically significant.

	Results

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Effect of Glucose and Phorbol Ester on VCAM-1 Expression
Incubation of HAECs with medium containing high glucose (22.2 mmol/L) resulted in a significant increase of VCAM-1 protein expression (172±15% versus control; n=6; P<0.01). The nonselective PKC inhibitor calphostin C (300 nmol/L) prevented such VCAM-1 upregulation, suggesting an involvement of PKC (Figure 1A). PMA (1 µmol/L), a potent activator of "classic" and "novel" isoforms of PKC, mimicked the effect of high glucose on VCAM-1 expression (352±52% versus control; n=5; P<0.01, Figure 1B).

View larger version (19K): [in this window] [in a new window]   Figure 1. Expression of vascular cell adhesion molecule-1 (VCAM-1) in HAECs exposed to control (5.5 mmol/L) as well as high concentration of glucose (22.2 mmol/L; A) and PMA (1 µmol/L; B). Effect of high glucose concentration on PKCß accumulation in particulate fraction at 60 minutes (C). Representative Western blots and densitometric quantification are shown. C, Data are mean±SEM (n=5 to 6). *P<0.05 vs control; **P<0.05 vs glucose.

Role of PKC ß₂ Inhibition on Glucose- and PMA-Induced VCAM-1 Expression
Replacement of the medium from 5.5 to 22.2 mmol/L glucose led to a fast and significant increase (181±22% versus control; n=5; P<0.01) of detergent-soluble membrane-bound PKCß, reflecting activation of this enzyme (Figure 1C). Treatment of the cells with the inhibitor of ß₁ and ß₂ isoforms LY379196 as well as with the selective PKCß₂ inhibitor CGP53353 prevented glucose-induced VCAM-1 upregulation in a dose-dependent manner (Figure 2A). Hence, activation of PKCß₂ regulates VCAM-1 expression in human endothelial cells acutely exposed to elevated glucose. Furthermore, LY379196 and CGP53353 were able to blunt the PMA-stimulated VCAM-1 expression (Figure 2B). Both inhibitors were also able to diminish VCAM-1 expression in control cells (data not shown), indicating that PKCß₂ maintains the basal level of VCAM-1 in HAECs.

View larger version (31K): [in this window] [in a new window]   Figure 2. Representative Western blots showing dose-dependent effect of PKCß inhibitors LY379196 and selective PKCß2 inhibitor CGP53353 on glucose (A) and PMA-induced upregulation of VCAM-1 protein expression (B). Similar findings were observed in 3 independent experiments.

Role of Ser-660 and Thr-641 Phosphorylation in PKCß₂-Mediated VCAM-1 Expression
Western blotting with antibodies against phosphorylated PKCß₂ at specific amino residues revealed that incubation of the cells with high glucose increased Ser-660 phosphorylation within the catalytic domain of this molecule (242±60% versus control; n=5; P<0.01; Figure 3). Treatment of the cells with PMA also elicited similar phosphorylation of Ser-660 (355±38% versus control; n=5; P<0.01; Figure 3). The time course of glucose-induced Ser-660 phosphorylation was consistent with the rapid accumulation of membrane-bound PKCß. The inhibitor of both PKCß isoforms LY379196 as well as the selective PKCß₂ inhibitor CGP53353 blunted PMA-induced Ser-660 phosphorylation in dose-dependent manner (Figure 4), suggesting that phosphorylation of PKCß₂ at Ser-660 may thus represent a selective regulatory mechanism for glucose-induced upregulation of VCAM-1.

View larger version (21K): [in this window] [in a new window]   Figure 3. Time course of phosphorylation of PKCß2 at Ser-660 amino acid residue during exposure to high glucose and PMA. Phosphorylation was assessed by Western blotting in cell lysate. Representative blots and densitometric quantification are shown. Data are mean±SEM (n=5). *P<0.05 vs control.

View larger version (48K): [in this window] [in a new window]   Figure 4. Representative Western blots showing dose-dependent effect of PKCß inhibitor LY379196 and selective PKCß2 inhibitor CGP53353 on PMA-induced phosphorylation of PKCß2 at Ser-660 amino acid residue. Similar findings were observed in 3 independent experiments.

Interestingly enough, phosphorylation at the other amino acid residue, Thr-641, was not affected by high glucose (data not shown). Incubation of glucose-treated cells with the PKCß₂ inhibitors did not affect the phosphorylation of Thr-641 (data not shown).

Effect of High Glucose and PMA on NF-B Activation and IB Degradation
We also determined the effect of high glucose on NF-B and compared the results with the effect of PMA. Incubation with elevated glucose increased NF-B binding activity compared with control cells. The increase in NF-B activity was a relatively early event, occurring within 1 hour after addition of glucose. Maximal increase in NF-B activity appeared at 2 and 4 hours, and this effect was reversed at 24 hours (data not shown). Quantitatively, the increase in NF-B activity after exposure to elevated glucose for 2 hours was 297±71% versus control cells (n=3, P<0.05, Figure 5A). High glucose–induced activation of NF-B was inhibited by calphostin C.

View larger version (25K): [in this window] [in a new window]   Figure 5. Effect of high glucose on NF-B binding activity in nuclear fractions of HAECs exposed to 22.2 mmol/L glucose for 2 hours in presence and absence of calphostin C (3x10–7 mol/L). Representative gel mobility shift assay and densitometric quantification of NF-B activation. Data are mean±SEM (n=3). *P<0.05 vs control; **P<0.05 vs glucose; (A) representative Western blots showing time course of glucose- and PMA-induced degradation of inhibitory subunit of NF-B, IB. Similar findings were observed in 3 independent experiments (B).

Accordingly, incubation of the cells with high glucose or treatment with PMA led to decreased protein levels of the inhibitory subunit of NF-B, IB, because of increased degradation of this factor (Figure 5B). Both nonselective and selective inhibitors of PKCß₂ abolished PMA-induced IB degradation (Figure 6).

View larger version (52K): [in this window] [in a new window]   Figure 6. Representative Western blots showing dose-dependent effect of PKCß inhibitor LY379196 and selective PKCß2 inhibitor CGP53353 on PMA-induced degradation of inhibitory subunit of NF-B, IB. Similar findings were observed in 3 independent experiments.

	Discussion

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The cell culture model used in this study reflects endothelial dysfunction in clinical settings of acute hyperglycemia, which occurs in both nondiabetic and diabetic patients after myocardial infarction or stroke.¹⁸ Transient changes in the expression of adhesion molecules in response to acute hyperglycemia trigger local inflammation and in turn contribute to clinical events.

In this study, we demonstrated for the first time that incubation of HAECs with high glucose leads to PKCß₂-dependent upregulation of VCAM-1 expression. Several lines of evidence support this conclusion. First, stimulation of the cells with glucose induced "translocation" of PKCß from the cytosolic cellular fraction to the particulate detergent-soluble fraction. Such compartmentalization is typical for activation of classic PKC. Second, we observed a selective Ser-660 phosphorylation within the catalytic domain of PKCß₂. Third, selective inhibition of PKCß₂ was able to abolish phosphorylation of Ser-660 as well as upregulation of VCAM-1.

Several pioneering studies on the role of different PKC isoforms in development of diabetic microangiopathies and macroangiopathies have demonstrated an involvement of PKCß.^14,15 A putative mechanism for the preferential activation of PKCß is the accumulation of DAG.¹⁹ Indeed, PKCß isoforms are more sensitive to DAG than other isoforms, especially in the presence of lower concentrations of Ca²⁺.²⁰ The principal biochemical pathway leading to accumulation of DAG in the hyperglycemic condition is its de novo synthesis.¹⁹ De novo synthesis of DAG is not accompanied by elevation of intracellular Ca²⁺, in contrast to the hydrolysis of phosphatidylinositol, which simultaneously produces DAG and inositol 1,4,5-trisphosphate. Therefore, diacylglycerol generated from the de novo pathway in the absence of a parallel rise in Ca²⁺ may preferentially activate the PKCß rather than the PKC isoform.²¹

Of particular interest is the fact that in our study, both PKC inhibitors did not affect the phosphorylation of Thr-641. These findings not only rule out phosphorylation of Thr-641 as a regulatory mechanism for glucose-induced upregulation of VCAM-1 expression but also may indicate that the catalytic activity of PKCß₂ is not completely shut down even with very high concentrations of these drugs. Therefore, PKCß₂ may still participate in physiological life-supporting processes.²² Indeed, we did not observe either an increase of cell death rate or changes of the cell shape by use of LY379196 as well as CGP53353 (data not shown). The absence of apparent cytotoxic effect together with favorable inhibition of glucose-induced VCAM-1 upregulation in the endothelium makes the PKCß₂ inhibitor a promising drug for treatment of endothelial dysfunction during acute hyperglycemia.

The molecular link between activation of PKC and upregulation of VCAM-1 expression under high-glucose conditions might involve NF-B activation.²³ The human gene encoding VCAM-1 contains in its promoter region 2 binding sites for NF-B.²⁴ As already reported, cells exposed to high glucose as well as vascular tissue from diabetic patients show increased binding activity of NF-B to promoter regions of many inflammatory genes.^4,25,26 However, which PKC isoform was responsible for glucose-induced NF-B activation in arterial endothelial cells remained unclear.

Distinct PKCs stimulate NF-B in a different manner, because the activation of NF-B comprises several different crucial steps, including degradation of its cytoplasmic inhibitor, IB.²⁷ Currently, 5 distinct IB proteins have been shown to functionally retain NF-B in the cytoplasm and render it inactive. Of the different IB proteins, the best-described is IB.²⁸ IB is phosphorylated by serine kinases after stimulation such as oxidative stress²⁹ or cytokines.³⁰ Phosphorylation of IB targets the IB for ubiquitination and rapid degradation by 26S proteasomes.³¹ The degradation of IB then allows the unbound NF-B to translocate into the nucleus, where it can transactivate the enhancer elements of many proinflammatory genes.³²

According to our results, glucose-induced Ser-660 phosphorylation of PKCß₂ leads to an IB-dependent mechanism of NF-B activation and hence, upregulation of VCAM-1 expression (Figure 7). Indeed, treatments with both high glucose and PMA exerted a degradation of IB protein, which was completely prevented by selective inhibition of PKCß₂.

View larger version (18K): [in this window] [in a new window]   Figure 7. Schematic representation of intracellular signaling that modulates upregulation of VCAM-1 expression in HAECs exposed to high glucose. LY379196 and CGP53353 abolish glucose-induced Ser-660 phosphorylation of PKCß2 and hence IB degradation, preventing activation of NF-B and VCAM-1 expression. IB, inhibitory subunit of NF-B. After IB degradation, NF-B heterodimers (p50:p65) traslocate into nucleus, where they upregulate transcription of target genes.

Furthermore, treatment with PKCß inhibitor improves endothelium-dependent NO-mediated vasodilation.³³ Thus, an additional link between inhibition of PKCß₂ and prevention of IB degradation observed in our study may be provided by the finding that NO itself is able to inhibit NF-B activation and VCAM-1 expression by stabilization of IB protein.²⁸

In conclusion, our findings demonstrate for the first time that phosphorylation of Ser-660 represents a selective regulatory mechanism for glucose-induced upregulation of VCAM-1. These results are relevant in understanding the intracellular signaling associated with pathological hyperadhesiveness of arterial endothelium in acute hyperglycemia and provide a new pharmacological target to protect the vessels in periods of transient increase of blood sugar.

	Acknowledgments

 
This work was supported in part by the Swiss National Research Foundation (32-67202.01 to Dr Cosentino), the Swiss Heart Foundation, the Italian Ministry of Health (ICS 030.6/RF00-49), and a Research Training Grant of the European Society of Cardiology (to Dr Kouroedov).

	Footnotes

 
Presented in part at the 76th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 2003, and published in abstract form (Circulation. 2003;108[suppl IV]:IV-304).

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