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(Circulation Research. 1997;81:363-371.)
© 1997 American Heart Association, Inc.

Articles

High Glucose Concentrations Increase Endothelial Cell Permeability via Activation of Protein Kinase C

Albrecht Hempel, Christian Maasch, Ute Heintze, Carsten Lindschau, Rainer Dietz, Friedrich C. Luft, , Hermann Haller

From the Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Virchow Klinikum, Humboldt University of Berlin (Germany).

Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany. E-mail haller{at}mdc-berlin.de

	Abstract

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Abstract Endothelial cell permeability is impaired in diabetes mellitus and may be increased by high extracellular glucose concentrations. High glucose activates protein kinase C (PKC), a family of kinases vital to intracellular signaling. We tested the hypothesis that high glucose concentration activates PKC in endothelial cells and leads to an increase in endothelial cell permeability via distinct PKC isoforms. Porcine aortic endothelial cells were used, and the PKC isoforms , , , , and were identified in these cells. Glucose caused a rapid dose-dependent increase in endothelial cell permeability, with an EC₅₀ of 17.5 mmol/L. Phorbol 12-myristate 13-acetate (TPA) induced an increase in permeability very similar to that elicited by glucose. The effect of glucose and TPA was totally reversed by preincubating the cells with the PKC inhibitors staurosporine (10^-8 mol/L) and Goe 6976 (10^-8 mol/L). Downregulation of PKC by preincubation with TPA for 24 hours also abolished the effect of glucose and TPA on endothelial cell permeability. High glucose (20 mmol/L) caused an increase in PKC activity at 2, 10, and 30 minutes. Cell fractionation and Western blot analysis showed a glucose-induced translocation of PKC and PKC. Confocal microscopy confirmed the translocation and showed an association of PKC and PKC with nuclear structures and the cell membrane. Specific antisense oligodesoxynucleotides (ODNs) against PKC reduced the expression of the isoform, abolished the effects of glucose on endothelial cell permeability completely, and reduced the TPA effect significantly. In contrast, specific antisense ODNs against PKC had no effect on glucose-induced permeability and only a minor effect on the TPA-induced increase in permeability. We conclude that an increase in extracellular glucose leads to a rapid dose-dependent increase in endothelial cell permeability via the activiation of PKC and that this effect is mediated by the PKC isoform .

Key Words: permeability • glucose • hyperglycemia • protein kinase C • antisense oligodesoxynucleotide

	Introduction

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The leading cause of morbidity and mortality in patients with diabetes mellitus is accelerated small- and large-vessel disease.¹ Recent trials have underscored the importance of elevated glucose levels as an independent risk factor.² The mechanisms by which high glucose concentrations may induce vascular changes in diabetic patients are not well understood; however, several hypotheses, including direct effects on cellular signaling pathways and diminished Na⁺,K⁺- ATPase activity, have been suggested.³ One specific mechanism may involve the activation of PKC in vascular cells. PKC is associated with many vascular cell functions that are abnormal in diabetes, including cell contraction, basement membrane production, signal transduction for hormones and growth factors, and cell proliferation.^{4 5 6 7 8 9 10 11} Lee et al¹² found increased PKC activity in cultured capillary endothelial cells exposed to high glucose concentrations. PKC activation was also observed in kidneys, hearts, and retinas from diabetic rats, as well as in isolated glomeruli.¹¹ Williams et al¹³ showed that exposure of mesangial cells and vascular smooth muscle cells to elevated glucose concentrations caused a long-term increase in PKC activity. They and others observed that glucose-induced PKC activation interferes with agonist-induced intracellular calcium signaling.^{7 14 15 16} High glucose concentrations also result in an increased synthesis of DAG.¹⁷ Shiba et al¹⁸ studied retinal cells and subsequently proposed that the increased DAG synthesis may be responsible for the PKC activation. These observations suggest that activation of the PKC system by hyperglycemia may represent an important pathway by which adverse effects are initiated in diabetes.¹¹

PKC does not exist as a single enzyme but instead consists of several distinct isoforms that have different enzymatic properties and functions.^{19 20} The isoforms identified thus far have been grouped into three larger families, depending on their regulatory properties. Group I isoforms (, ßI, ßII, and ) depend on calcium and DAG for activation. Group II isoforms (, , and ) are solely activated by DAG, whereas group III isoforms ( and ) function independent of calcium ions or DAG for activation.^{21 22} It remains to be determined which of the PKC isoforms are activated by high glucose concentrations and which are responsible for mediating the cellular effects of hyperglycemia. We found that PKC, PKCß, PKC, and PKC are activated by high glucose concentrations in vascular smooth muscle cells.¹⁴ Inoguchi et al¹⁷ have suggested that high glucose concentration leads preferentially to an increased expression of PKC isoform ßII in cardiovascular tissue. A role for PKCß in diabetes has recently been suggested by the findings of Ishii et al,²³ who have demonstrated that an oral inhibitor of PKCß ameliorates vascular dysfunction in diabetic rats. However, this isoform is not uniformly expressed in all tissues. We were unable to detect PKCß expression in our endothelial cell preparation in an earlier study.²⁴ Therefore, it is possible that high glucose concentration exerts its intracellular effects in endothelial cells via other PKC isoforms.

PKC activation in endothelial cells leads to an increase in endothelial cell permeability.^{25 26 27 28 29 30} Leakage of serum proteins, particularly albumin, through the endothelium is observed in retinal vessels early in diabetes mellitus.^{29 30} Similarly, an increase in endothelial permeability has been implicated in early diabetic nephropathy.^{31 32 33} Increased endothelial cell permeability in larger vessels leads to interstitial edema and may enhance cell proliferation and matrix production.³¹ The mechanisms of increased endothelial permeability in diabetes mellitus are unclear; however, high glucose concentrations have been implicated.^{34 35} The effect of high glucose concentration on PKC-regulated endothelial cell signaling has not been examined to our knowledge. We tested the hypothesis that high glucose concentration increases endothelial cell permeability for albumin via activation of PKC. We investigated the isoforms , , , , and , which are all expressed in endothelial cells.²⁴ We found that a high glucose concentration led to a rapid dose-dependent increase in endothelial cell permeability. The effect was mediated by activation of PKC. The isoforms and underwent translocation with high glucose concentrations; however, our results specifically implicate PKC.

	Materials and Methods

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Materials
Phorbol ester TPA, histone type III-S, DEAE-cellulose, and all other materials, if not stated otherwise, were purchased from Sigma. [-³²P]ATP was obtained from Amersham. 1,2-Diolein and phosphatidylserine were purchased from Avanti Polar Lipids. Cy3-conjugated anti-mouse IgG and anti-rabbit antibodies were from Dianova. ODNs were made by TIB Biomol. The PKC inhibitor Goe 6976 was obtained from Calbiochem. Addition of mannose (20 mmol/L) to the medium was used as an osmotic control in all experiments.

Measurement of Endothelial Cell Permeability
Porcine aortic endothelial cells were isolated, as previously described,^{35 36} by mechanically scraping the intima of the descending porcine aortas. Harvests from the cells were plated at a density of 10⁵ cells on 100-cm² plastic Petri dishes. The cells were cultured at 37°C in medium 199 with Earle's salt, supplemented with 100 IU/mL penicillin G, 20 µg/mL streptomycin, and 20% (vol/vol) NCS (GIBCO). The medium was renewed every second day. After 5 days, when the cells had grown to confluence, they were trypsinized in PBS (mmol/L: NaCl 137, KCl 2.7, KH₂PO₄ 1.5, and Na₂HPO₄ 8.0, at pH 7.4, with 0.05% [wt/vol] trypsin and 0.02% [wt/vol] EDTA) and seeded at a density of 7x10⁴ cells/cm² on either 24-mm round polycarbonate filters or 60-mm plastic Petri dishes for determination of albumin flux and determination of PKC activity, respectively. Experiments were performed with confluent monolayers, 4 days after seeding on filters. The purity of these cultures was >98% endothelial cells.³⁵

Macromolecule Permeability
The permeability across the endothelial cell monolayer was studied in a two-compartment system separated by a filter membrane as described previously.³⁶ Both compartments contained modified Tyrode's solution (mmol/L: NaCl 150, KCl 2.7, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.0, and HEPES 30, pH 7.4 at 37°C) supplemented with 10% (vol/vol) NCS. There was no hydrostatic pressure gradient between both compartments. The "luminal" compartment containing the monolayer had a volume of 2.5 mL, and the "abluminal" compartment had a volume of 10.5 mL. The fluid in the "abluminal" compartment was constantly stirred. Trypan blue–labeled albumin (60 µmol/L) was added to the luminal compartment. The appearance of Trypan blue–labeled albumin in the abluminal compartment was continuously monitored by pumping the liquid through a two-wavelength photometer (model Specord S-10, Carl Zeiss; wavelength, 580 nm for Trypan blue and 720 nm for control wavelength). Changes in the concentration of Trypan blue–labeled albumin were detected with a time delay of <12 seconds. The concentration of Trypan blue–labeled albumin in the luminal compartment was determined every 10 minutes of incubation. The concentration did not change significantly during the time course of the experiments. The albumin flux (F) across the monolayer was determined from the rise of albumin concentration ([A2]) in the abluminal compartment (volume V): F=d[A2]/dtxV, where t is time. Data were expressed as percentage of a defined control situation.

PKC Activity
PKC activity was measured in cultured confluent cells after cell fractionation into a cytosolic and a particulate fraction as previously described.^{37 38} Cell cultures were incubated in control medium (5 mmol/L glucose) or with high glucose concentration (20 mmol/L) for the described time periods. After lysis by sonification, the homogenate was spun in a TLA 100-2 rotor (Beckman) at 627 000g for 10 minutes, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in buffer containing 1.0% Triton X-100 and shaken at 4°C for 30 minutes. The dissolved pellet was then diluted with buffer to a final concentration of 0.5% Triton X-100 and centrifuged at 100 000 rpm for another 10 minutes. The supernatant was used as the particulate fraction. The assays were carried out according to standard procedures using the peptide KRTLRR (Bachem) as substrate.⁴⁰ Enzyme activity was measured in the presence or absence of phosphatidylserine, diolein, and calcium. Data presented in Fig 3 were calculated by subtracting nonspecific activity. With the peptide and ATP concentration used, the rates of kinase activity were linear over 20 minutes.

View larger version (58K): [in this window] [in a new window]   Figure 3. Effect of high glucose concentration (20 mmol/L) on PKC activity in the cytosolic and particulate fraction. Extracellular 20 mmol/L glucose led to a rapid increase of PKC activity in the particulate fraction, with a concomitant decrease in the cytosol. At 30 minutes, this effect was reversed (n=4).

Western Blotting
Western blot analysis was carried out as described previously.³⁹ After the experiments, the cultured endothelial cells were treated with ice-cold homogenization buffer (20 mmol/L Tris-HCl, pH 7.5, 250 mmol/L sucrose, 7.5 mmol/L EGTA, 10 mmol/L mercaptoethanol, 1 mmol/L phenylmethylsulfonyl fluoride, and 50 µmol/L leupeptin) and homogenized. The homogenate was then spun in a TLA 100-2 rotor (Beckman) at 100 000 rpm for 10 minutes, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in buffer containing 1.0% Triton X-100 and shaken at 4°C for 30 minutes. The homogenate was centrifuged at 100 000 rpm for another 10 minutes. The supernatant was used as the particulate fraction. Both PKC-containing fractions then underwent chromatography using 10% SDS-polyacrylamide gels. Protein from the cytosolic fraction (40 µg) and protein from the particulate fraction (10 µg) were loaded into each lane. The fractions were then electroblotted by the semidry technique onto polyvinylidene fluoride membranes (Immobilon-P, Millipore). The membranes were successively incubated, first with blocking buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, 10% nonfat dry milk powder (Merck), 0.2% (vol/vol) Tween 20, and 0.02% NaN₃ for 120 minutes at room temperature. The next incubation was conducted in affinity-purified isoenzyme-specific antibody diluted in incubation buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 1% BSA at room temperature. We used highly specific polyclonal antibodies directed against peptide sequences of PKC that reacted specifically with the , , , and subspecies of PKC (antibodies were from GIBCO, 1:80 to 1:100); the antibody against PKC was monoclonal and from UBI (1:200). A final incubation was carried out in Tris-buffered saline with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Pierce Chemicals). The membranes were thoroughly washed after each incubation with a buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 0.2% (vol/vol) Tween 20. Visualization was achieved by chemiluminescence (Renaissance, DuPont). The specificity of the antibodies used was established in previously published studies.^{14 24}

Immunocytochemistry
The techniques for confocal microscopy were as described previously.^{14 37} The cells were fixed with 4% paraformaldehyde and permeabilized with 80% methanol at -20°C. After incubation with 3% skimmed milk in PBS for 60 minutes, the preparation was incubated for 1 hour at room temperature with the PKC antibodies (see above) diluted in PBS with 0.1% BSA (1:80), washed twice with PBS, and then exposed to the secondary antibody (Cy3-conjugated anti-rabbit or anti-mouse IgG at 1:100, 1% BSA/PBS, Dianova) for 60 minutes. The preparation was mounted with 50% glycerol under a glass coverslip on a Nikon-Diaphot microscope. An MRC 600 confocal imaging system (Bio-Rad Laboratories) with an argon/krypton laser was used. At least 10 to 18 cells from each of at least six experiments were examined under each experimental condition. The results were reproduced by two separate investigators, and multiple experiments were performed. The observers were unaware of the experimental design and antibodies used.

Oligonucleotides
Phosphorothioate ODNs were purchased (TIB Molbiol). We selected an antisense ODN (ISIS 3521) against the human 3' untranslated region derived from the human PKC sequence (database, European Molecular Biology Laboratories).^{40 41} The antisense sequence used for PKC was 5' GTT.CTC.GCT.G GT.GAG.TTT CA 3'. The sense ODN sequence (5' TG.AAA.CTC.ACC.AGC.GAG.AAC 3'), a reverse ODN sequence (5' AC.TTT. GAG.TGG.TCG.CTC.TTG 3'), and a scrambled version (5' GAG.TTG. CTT.GCT. TAT. CGG. TC 3') were used as controls. The antisense sequence used for PKC against the human AUG start codon was 5' GCC.ATT.GAA.CAC.TAC CAT 3'. The sense ODN sequence (5' ATG.GTA.GTG.TTC.AAT.GGC 3') was used as a control. We used a cationic lipid solution (Lipofectin, GIBCO BAL, Life Technologies) to enhance the ODN uptake. For transfection, the cells were incubated with lipofectin (10 µg/mL) and ODN (1 µmol/L) in the absence of NCS at 37°C for 4 hours, washed two times with medium, and then incubated with medium plus 10% NCS and ODN (1 µmol/L) for another 4 hours. Afterwards, the medium was changed back to 10% NCS for 24 hours before the start of the experiments.

Statistics
Statistical analysis was carried out on a Macintosh computer (Apple Inc) using a commercially available statistical program (Statview, Cricket Software Inc). Since the data feature substantial variability and are not uniformly distributed, we used nonparametric statistical tests, such as the sign test and Mann-Whitney test to analyze the data from the 6 to 10 separate experiments. A value of P<.05 was accepted as significant. References to increases or decreases in the following section are only so stated if statistically significant.

	Results

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We first investigated the effects of glucose on the albumin permeability of endothelial cells and compared the results with the effect of the phorbol ester TPA as shown in Fig 1 (n=6). High glucose concentration (20 mmol/L) led to a rapid increase in endothelial cell permeability. This effect began within minutes after incubation and reached its maximum at 30 minutes. Afterwards, despite the presence of glucose, the endothelial cell permeability declined and returned to basal levels within 100 minutes (Fig 1A). Lower concentrations of glucose (10 mmol/L) did not alter the rate of the permeability increase but induced lower maximum levels. Fig 1B shows the dose dependency of the glucose effect after 30 minutes. A significant increase was observed at 10 mmol/L glucose concentration. The EC₅₀ of the glucose effect was 17.5 mmol/L. Concentrations above 40 mmol/L did not increase the glucose-induced permeability further. Mannose (20 mmol/L), which was used as an osmotic control, had no significant effect on endothelial cell permeability. Further investigations of the glucose effect on endothelial cells showed that repetitive exposure had an additive effect. When glucose at a concentration of 10 mmol/L was replaced by control medium for 10 minutes and then readded, the endothelial cell permeability increased from 135±5.4% at the first exposure to 146±7.4% at the second exposure and to 153±7.6% after the third exposure (P<.05 compared with the first exposure). When we compared the effects of high glucose concentrations (20 mmol/L) with those of TPA (Fig 1C and 1D), we observed a similar time course and maximum response. Incubation of endothelial cells with TPA led to a rapid increase in endothelial cell permeability. A statistically significant effect was observed at 10 nmol/L TPA. The EC₅₀ of the TPA effect was 5x10^-8 mol/L. Concentrations above 1 µmol/L did not increase the TPA-induced permeability. We then investigated whether glucose and TPA had additive effects on albumin flux. However, concomitant exposure of endothelial cells to glucose (20 mmol/L) and TPA (10 nmol/L) had no additive effect on endothelial cell permeability (143±7.3%) (n=3).

View larger version (63K): [in this window] [in a new window]   Figure 1. Effect of glucose (A and B) and the phorbol ester TPA (C and D) on albumin flux across aortic endothelial monolayers. Data are percentage of control; 100% corresponds to a 4.8±0.5x10-6-cm/s permeability barrier formed by endothelial monolayer and filter support. Data are mean±SD after 30 minutes of incubation (n=6 separate experiments, *P<.05 vs control). A and B, Incubation of endothelial cells in high glucose concentration led to a rapid increase in endothelial cell permeability. A significant effect was observed at 10 mmol/L glucose concentration. The EC50 of the glucose effect was 17.5 mmol/L. Concentrations above 40 mmol/L did not increase the glucose-induced permeability. Furthermore, mannose (20 mmol/L) had no significant effect on endothelial cell permeability. C and D, Incubation of endothelial cells with TPA led to a rapid increase in endothelial cell permeability. A statistically significant effect was observed at 10 nmol/L TPA. The EC50 of the phorbol ester effect was 50 nmol/L. Concentrations above 1 µmol/L did not increase the TPA-induced permeability.

We next investigated the effects of PKC inhibition on glucose (20 mmol/L)–and TPA (100 nmol/L)–induced endothelial cell permeability to albumin (n=6). Fig 2 (left panel) shows the effect of different PKC inhibitors on permeability after 30 minutes of glucose exposure. Incubation of endothelial cells with staurosporine (10^-8 mol/L) 30 minutes before glucose exposure reduced the glucose-induced permeability to almost basal values. The PKC inhibitor Goe 6976 (10^-8 mol/L) also reduced the glucose-induced endothelial permeability (Fig 2, left panel). The inhibitors alone had no significant effect on endothelial cell permeability. These findings indicate that the effects of glucose are mediated by PKC. This assumption was supported by downregulation of PKC. Preincubation with TPA (100 nmol/L) for 24 hours abolished the effects of glucose (20 mmol/L). In the right panel of Fig 2 are shown the effects of PKC inhibition on TPA-induced endothelial permeability (n=6). As expected, staurosporine (10^-8 mol/L) and 24 hours of preincubation with TPA abolished the effect of TPA on endothelial cell permeability.

View larger version (39K): [in this window] [in a new window]   Figure 2. Effect of PKC inhibitors on glucose-induced (left panel) and TPA-induced (right panel) endothelial cell permeability at 30 minutes. PKC inhibitors were added 30 minutes before glucose (20 mmol/L) or TPA (100 nmol/L) exposure (n=6). Left, Staurosporine (Stauro, 10-8 mol/L) reduced the glucose-induced permeability almost completely. Similar inhibitory effects were observed with the PKC inhibitor Goe 6976 (Gö, 10-8 mol/L). Preincubation with TPA (100 nmol/L) for 24 hours also abolished the effects of glucose (20 mmol/L). Right, Stauro (10-8 mol/L) and 24-hour preincubation with TPA abolished the TPA effect on endothelial cell permeability. The inhibition of the calcium-sensitive PKC isoforms with Gö (10-8 mol/L) had a smaller inhibitory effect on TPA-induced permeability compared with glucose. Data are percentage of control; 100% corresponds to a 4.8±0.5x10-6-cm/s permeability barrier formed by endothelial monolayer and filter support. Data are mean±SD after 30 or 60 minutes of incubation (n=6 separate experiments, *P<.05 vs control).

Fig 3 shows the effect of high glucose concentration on PKC activity after cell fractionation into a cytosolic and a particulate fraction (n=3). Under resting conditions (5 mmol/L glucose), PKC activity was mostly located in the cytosol. Glucose (20 mmol/L) induced a rapid increase in membrane-bound (particulate) PKC activity, with a concomitant decrease in the cytosolic fraction. Increased PKC activity was observed at 2 minutes and stayed elevated at 10 minutes. However, the effect of glucose on PKC translocation was less after 30 minutes, and the PKC activity in the particulate fraction decreased. Mannose (20 mmol/L) had no significant effect on PKC distribution.

We then used confocal microscopy to examine the effect of high glucose concentration (20 mmol/L) on the intracellular distribution of PKC isoform immunoreactivity. Fig 4 shows the immunoreactivity of PKC, PKC, PKC, PKC, and PKC under control conditions (left panels) and after 30 minutes of 20 mmol/L glucose (middle panels) (representative of three experiments). High glucose induced changes in intracellular distribution of PKC isoforms and . Barely detectable expression of PKC was visible under resting conditions. High glucose induced an increase of PKC immunoreactivity in the cytosol and in the perinuclear region. PKC under resting conditions showed a punctuated pattern in the cytosol. Exposure to high glucose concentrations increased immunoreactivity of PKC mostly into distinct areas of the nucleus and in the cytosol. The intracellular distribution of the PKC isoforms , , and was not influenced after 30 minutes of 20 mmol/L glucose. PKC showed a fibrillar pattern in the cytosol under both conditions. PKC immunoreactivity showed a punctuated pattern, both in the cytosol and in the nuclear region, which was not influenced by high glucose concentration. PKC immunoreactivity appeared as a peri-nuclear ring and was not affected by high glucose concentration. The lack of effect of 20 mmol/L glucose on PKC isoforms , , and was also confirmed after 2, 5, and 10 minutes (data not shown). When 20 mmol/L mannose was used for osmotic control, the intracellular distributions of PKC, PKC, and PKC were not altered. However, we observed a slight increase in the perinuclear immunoreactivity of PKC (data not shown).

View larger version (163K): [in this window] [in a new window]   Figure 4. Effect of high glucose concentration (20 mmol/L) and TPA (100 nmol/L) on the intracellular distribution of PKC isoforms , , , , and under control conditions (left panels, control), after 30 minutes of 20 mmol/L glucose (middle panels), and after 30 minutes of 100 nmol/L TPA (right panels). High glucose concentration induced changes in intracellular distribution of PKC isoforms and . In contrast, PKC isoforms , , and were not influenced by high glucose concentration. TPA induced changes in intracellular distribution of PKC isoforms , , and and, to a lesser extent, of PKC isoform (representative of three experiments). The graded color bar indicates different PKC concentrations, where blue, green, yellow, and red represent increasing PKC concentrations.

These results indicated that high glucose concentration exerts an effect on PKC isoforms and in endothelial cells. This assumption was confirmed by cell fractionation into a cytosolic and a particulate fraction, followed by Western blotting after 30 minutes of exposure to high glucose concentration. The cytosolic fraction contained the soluble proteins; the particulate fraction consisted of cell membranes and nuclear proteins. In Fig 5 are shown the results of these translocation experiments for PKC and PKC (representative of three experiments). PKC showed a single band at 82 kD and was mostly located in the cytosolic fraction under resting conditions. Glucose (20 mmol/L) induced a translocation to the particulate fraction. PKC, at 90 kD, was also located primarily in the cytosolic fraction and was translocated to the particulate fraction upon exposure to high glucose.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effect of high glucose concentration (20 mmol/L) on the translocation of PKC isoforms (left panel) and (right panel) from the cytosolic to the particulate fraction. Glucose (20 mmol/L) induced a translocation of PKC and PKC from the cytosolic to the particulate fraction (representative of three experiments).

In the following experiments, we concentrated on PKC and PKC and investigated the hypothesis that these isoforms are responsible for the glucose-induced changes in endothelial cell permeability. Fig 6 shows confocal photomicrographs on the time course of the effects of 20 mmol/L glucose on the intracellular distribution of PKC (Fig 6, top panel) and PKC (Fig 6, bottom panel) (representative of three experiments). Glucose (20 mmol/L) had a rapid effect on the intracellular distribution of these isoforms. For PKC at 5 minutes, an intense staining of the cell nuclei was present, whereas in the cytosol a more diffuse pattern was observed. The nuclear immunoreactivity had decreased after 10 minutes. After 30 minutes of high glucose exposure, the cell nuclei were almost free of PKC immunoreactivity, which by this time was mostly located in the perinuclear area. PKC (Fig 6, bottom panel) in resting cells showed a weak punctuated pattern of distribution. Glucose induced a more fibrillar pattern in the cytosol and toward the cell membrane. After 10 minutes of glucose exposure, the cytosol showed a coarse focal pattern of PKC immunoreactivity and an enhanced immunoreactivity at the cell membrane. This focal distribution was further enhanced at 30 minutes. We observed a slight increase in nuclear immunoreactivity after 5 minutes, which increased after 10 and 30 minutes of high glucose concentration.

View larger version (108K): [in this window] [in a new window]   Figure 6. Confocal photomicrographs on the time course of the effects of 20 mmol/L glucose on the intracellular distribution of PKC (top panel) and PKC (bottom panel). Top, Glucose induced a translocation of PKC toward the perinuclear area and into the nucleus. In the cytosol, a more diffuse pattern was present, and the PKC immunoreactivity showed an association with the cell membrane. Bottom, PKC in resting cells showed a perinuclear pattern of distribution. Glucose induced a focal pattern in the cytosol and at the cell membrane. Nuclear immunoreactivity of PKC increased slowly and appeared to be concentrated in the nucleus after 30 minutes (representative of three experiments).

For the inhibition of PKC and PKC, we applied antisense ODN to specifically suppress expression of the respective PKC isoform. Using an antisense ODN against the 3' untranslated region of PKC, we investigated its effect on the expression of this PKC isoform in endothelial cells. Fig 7 (top panel) shows a Western blot analysis of PKC after transfection with antisense ODN compared with a sense ODN control. The endothelial cells were incubated with 1 µmol/L ODN together with lipofectin (10 µg/mL), as described above. Antisense ODN led to a downregulation of PKC to 27.5% compared with control (n=3). In contrast, protein levels of PKC were not affected by exposure of the endothelial cells to antisense ODN against PKC. Lipofectin alone had no effect on PKC expression levels. We then used an antisense ODN against the 3' untranslated region of PKC and investigated its effect on the expression of this PKC isoform in endothelial cells. Fig 7 (bottom panel) shows a Western blot analysis of PKC after exposure to antisense ODN compared with a sense ODN control. The endothelial cells were incubated with 1 µmol/L ODN together with lipofectin (10 µg/mL). Antisense ODN led to a downregulation of PKC to 43% compared with control (n=3). In contrast, protein levels of PKC were not affected by exposure of the endothelial cells to antisense ODN against PKC.

View larger version (33K): [in this window] [in a new window]   Figure 7. Western blot analysis of antisense ODN against PKC (top panel) and PKC (bottom panel) as described in "Materials and Methods" (n=3; the bars show mean densitometric data±SD of these experiments). Western blots were stained with PKC-specific antibodies as indicated. Antisense ODN against PKC led to a significant downregulation of PKC compared with control or sense-treated cells. PKC protein levels were not influenced by antisense ODN against PKC. Antisense ODN against PKC led to a significant downregulation of PKC compared with control or sense-treated cells. Protein levels of PKC were not influenced by antisense ODN against PKC.

Finally, we examined whether the specific downregulation of PKC and PKC with antisense ODN influenced the glucose- and TPA-induced increase in endothelial cell permeability (Fig 8, n=6). The results for glucose are shown in Fig 8, left panel. Endothelial cells were incubated with lipofectin (10 µg/mL), antisense ODN, sense ODN, or scrambled ODN against PKC or PKC before exposure to high glucose concentration (20 mmol/L). Antisense ODN for PKC almost completely inhibited the increase in glucose-induced endothelial cell permeability (Fig 8, left panel). Both the initial increase and the maximum response to glucose were significantly reduced. Sense and scrambled ODN for PKC had no effect on the glucose-induced permeability. In contrast to the effects of antisense against PKC, the antisense ODN against PKC did not reduce the glucose-induced permeability significantly.

View larger version (39K): [in this window] [in a new window]   Figure 8. Effect of antisense ODN against PKC, sense, scrambled ODN, or antisense against PKC and PKC on glucose (20 mmol/L)–induced (left panel) and TPA-induced (right panel) endothelial cell permeability. Endothelial cells were exposed to ODN with lipofectin (10 µg/mL) 24 hours before exposure to 20 mmol/L glucose or 100 nmol/L TPA (n=6). Left, Antisense against PKC significantly reduced the glucose-induced permeability (P<.01), whereas the control sense and scrambled ODN, the ODN against PKC and PKC, or lipofectin alone had no significant effect. Right, Antisense ODN against PKC reduced the TPA-induced increase in endothelial cell permeability, whereas sense and scrambled ODN had no significant effect on the TPA-induced permeability. In contrast to the glucose-induced permeability, ODN against PKC reduced the TPA-induced permeability by 30% (*P<.05).

The right panel of Fig 8 shows the effect of the ODNs on phorbol ester (100 nmol/L TPA)–induced increase in permeability (n=6). Antisense ODN and sense ODN against the respective PKC isoforms were added 24 hours before exposure to TPA (100 nmol/L). Antisense ODN for PKC reduced the increase in endothelial cell permeability significantly. As in the case of high glucose, the initial increase and the maximum response to glucose were significantly reduced (Fig 8, right panel). Sense and scrambled ODN for PKC had only a slight nonsignificant effect on the glucose-induced permeability. In contrast with the experiments using glucose, the antisense ODN against PKC had a significant effect on TPA-induced permeability.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used the albumin flux across cultured endothelial cell monolayers to assess the effects of glucose on endothelial cell permeability and to investigate the hypothesis that the glucose-induced increase in permeability is mediated via PKC. We relied on an antisense ODN approach to delineate which PKC isoform mediated the effects of glucose. We found that glucose led to a rapid dose-dependent increase in endothelial cell permeability. The effect was associated with activation of PKC and was similar to the effects of a phorbol ester on endothelial cell permeability. High glucose concentration induced an intracellular translocation of PKC isoforms and . Finally, we showed that PKC, rather than PKC, was responsible for the glucose-induced increase in endothelial cell permeability.

Lynch et al²⁶ were the first to describe that PKC activation is an important signal transduction pathway that increases the transport of macromolecules across endothelial monolayers. Several reports subsequently supported this finding in vitro.^{27 28} PKC activation plays a role in the increased endothelial cell permeability induced by other agonists, such as bradykinin,⁴² thrombin,²⁷ and endothelins.⁴³ Thus, in addition to an increase in cytosolic calcium, the activation of PKC appears to be a major determinant of an increase in endothelial cell permeability.⁴⁴ However, the exact mechanism of PKC action within endothelial cells is not clear. Exposure to phorbol ester leads to phosphorylation and redistribution of the cytoskeletal proteins caldesmon and vimentin, in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.²⁸ This observation indicates that PKC activation increases endothelial permeability by an interaction with cytoskeletal proteins.⁴⁴ This hypothesis is supported by recent observations in epithelial cells, where PKC-dependent actin reorganization leads to modulation of intercellular permeability.⁴⁵ The confocal photomicrographs in the present study indirectly support an interaction of PKC with cytosolic proteins. Glucose induced a rapid redistribution of PKC and PKC from the cytosol to the nucleus to the cell membrane. Possible binding partners of PKC within the cytosol and the nucleus should be the topic of future studies.

The present study is the first direct evidence that PKC plays a distinct role in mediating endothelial cell permeability by glucose. We used confocal microscopy and found a glucose-induced altered PKC distribution and an increased immunoreactivity of this isoform in endothelial cells. However, we were not able to confirm an increase in protein content by Western blotting. Therefore, we carried out additional experiments with different polyclonal and monoclonal antibodies for PKC. We used polyclonal antibodies (Biomol, Calbiochem, and GIBCO) and an additional monoclonal antibody (Transduction Laboratories). In general, the effects of glucose analyzed with the different antibodies were similar. We observed faint staining with a cytosolic distribution under resting conditions, followed by an appearance of PKC immunoreactivity in the perinuclear region and in the nucleus. PKC immunoreactivity was also observed in cell membrane structures; however, this staining was less pronounced. With the above antibodies, we observed the increase in immunoreactivity after exposure to glucose. Similar alterations have been observed by us previously.^{14 24 39} We believe that the increase in immunoreactivity is due to local accumulation of the protein after stimulation. In addition, unfolding of the protein after activation may make it more accessible to the antibodies. Thus, the total amount of PKC may be no different, as reflected by Western blotting. An alternative explanation may be that inactive PKC is stored in specific cellular vesicles.

It has recently been suggested that the intracellular effects of high glucose concentration are mostly mediated by the PKC isoform ßII.¹⁷ In a previous study, we observed a translocation of PKC isoform ß in response to an increase in glucose concentration in vascular smooth muscle cells.¹⁴ Ishii et al²³ have recently demonstrated that an oral inhibitor of PKCß ameliorates vascular dysfunction in diabetic rats. However, we could not detect PKCß-specific immunoreactivity in our endothelial cell preparation. The lack of PKCß expression in human umbilical vein endothelial cells has recently been confirmed using reverse-transcriptase polymerase chain reaction.²⁴ Therefore, our results indicate that the effects of high glucose concentration in various cell types are mediated by different PKC isoforms. Furthermore, we cannot rule out the possibility that endothelial cells from other origins express PKCß or that this PKC isoform is induced during cell differentiation.²⁴ Whether activation of PKC plays a role in glucose-induced endothelial permeability in vivo is presently under investigation.

The results with TPA suggest that PKC also may play a role in the phorbol ester–mediated increase in endothelial cell permeability. We have recently shown that TPA alters the intracellular distribution of PKC both in the cytosol and in the nuclear area,²⁴ indicating that this isoform associates with cytoskeletal structures. In addition, because TPA influences other PKC isoforms besides and , we cannot rule out the possibility that more PKC isoforms are involved in the phorbol ester–induced increase in endothelial cell permeability. Our results suggest that the glucose-mediated effects on PKC isoforms are more specific and directly mediated solely by PKC. Using a PKC inhibitor specific for calcium-dependent PKC isoforms, Northover and Northover²⁷ recently suggested that PKC plays a role in microvascular permeability. A PKC translocation in endothelial cells has been described by us and others.^{33 46} However, cytoskeletal substrates or binding partners for PKC have not yet been identified. Jaken et al⁴⁷ showed earlier that PKC can be associated with focal contacts in fibroblasts. PKC has also been found in junctional complexes.⁴⁸ Analysis of specific functions for PKC isoforms have been hampered by the lack of specific inhibitors. Thus, several authors have relied on an antisense ODN approach. Dean et al⁴⁰ showed that the specific inhibition of PKC reduced the increase in intercellular adhesion molecule-1 expression on human A549 cells in response to phorbol ester. Antisense complementary to the mRNA initiation codon regions for PKC and PKCß downregulated these isoforms and inhibited insulin-induced glucose uptake, whereas DAG generation was not impaired.⁵ The antisense ODN inhibition in these studies was, as in our experiments, specific for PKC; no significant effect on other PKC isoforms was observed. This experience demonstrates that an antisense ODN approach can be used for delineation of the specific PKC effects in signal transduction and cell physiology. Early experiments have shown that this approach can also be used in vivo. Intraperitoneal injection of ODN in mice caused a dose-dependent ODN sequence–dependent reduction in PKC mRNA.⁴³ Thus, this approach may possibly be applied to prevent glucose-induced vascular changes in vivo.

We believe that our observations might be clinically relevant; however, we cannot be certain. Ours is clearly not a model of diabetes mellitus. Our endothelial cells were not harvested from the microvasculature, where much of diabetic vasculopathy occurs. Our cell culture system is likely to be much more permeable than the permeability occurring in living blood vessels. The endothelial cells in our system are by no means under physiological conditions. Our 20 mmol/L glucose challenges were high and transient; the hyperglycemic insults of diabetes mellitus are lower and longer lasting. Nevertheless, we feel that our observations are important because they suggest a role for PKC in mediating a glucose-related effect in endothelial cells.

In summary, we observed a rapid dose-dependent increase in endothelial cell permeability for albumin with high glucose concentrations. The effect of high glucose concentration was similar to the increase in permeability induced by the phorbol ester TPA and inhibited by various PKC inhibitors, suggesting that the glucose effect was mediated by PKC. We demonstrated that high glucose concentration increased PKC activity rapidly and induced a redistribution of PKC and PKC. A specific antisense ODN against PKC essentially abolished the effect of high glucose concentration on endothelial cell permeability. Our results suggest that the effect of high glucose concentration on PKC-mediated cellular processes in endothelial cells is mainly due to the activation of PKC.

	Selected Abbreviations and Acronyms

 

DAG = 1,2-diacyl-sn-glycerol NCS = newborn calf serum ODN = oligodesoxynucleotide PKC = protein kinase C TPA = phorbol 12-myristate 13-acetate

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG He-2119/5-1).

Received January 15, 1997; accepted June 18, 1997.

	References

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