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(Circulation Research. 1997;81:953.)
© 1997 American Heart Association, Inc.

Articles

Mechanisms of Action of Troglitazone in the Prevention of High Glucose–Induced Migration and Proliferation of Cultured Coronary Smooth Muscle Cells

Kenichi Yasunari, Masakazu Kohno, Hiroaki Kano, Koji Yokokawa, Mieko Minami, Junichi Yoshikawa

From The First Department of Internal Medicine, Osaka City (Japan) University Medical School.

Correspondence to Kenichi Yasunari, MD, The First Department of Internal Medicine, Osaka City University Medical School, 1–5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan.

Abstract

Abstract Recent findings suggest that high glucose levels may promote atherosclerosis in coronary vascular smooth muscle cells (VSMCs). To explore the intracellular mechanisms of action by which troglitazone affects this process, we examined the effect of troglitazone on the migration and growth characteristics of cultured rabbit coronary VSMCs. Treatment with chronic high glucose medium (22.2 mmol/L) for 5 days increased VSMC migration by 92%, [³H]thymidine incorporation by 135%, and cell number by 32% compared with VSMCs treated with normal glucose (5.5 mmol/L glucose+16.6 mmol/L mannose) medium. Troglitazone at 100 nmol/L and 1 µmol/L significantly suppressed high glucose–induced VSMC migration by 34% and 42%, respectively, the proliferative effect (as measured by cell number) by 17% and 27%, and [³H]thymidine incorporation by 45% and 60% (n=6, P<.05). The high glucose–induced impairment of insulin-mediated [³H]deoxyglucose uptake was blocked by a protein kinase C (PKC) inhibitor (calphostin C, 1 µmol/L) and was also improved by troglitazone without any change in insulin receptor number and affinity. The high glucose–induced insulin-mediated increase in cell number and in [³H]thymidine incorporation was suppressed by troglitazone. Troglitazone (1 µmol/L) also suppressed high glucose–induced phospholipase D activation, elevation of the cytosolic NADH/NAD⁺ ratio (as measured by the cytosolic ratio of lactate/pyruvate), and membrane-bound PKC activation. Flow cytometric DNA histogram analysis of cell cycle stage showed that high glucose–induced increase in the percentage of cells in the S phase was suppressed by 1 µmol/L troglitazone. These findings suggest that PKC may be a link between impairment of insulin-mediated glucose uptake and the increase in migration and proliferation induced by high glucose levels and that troglitazone may be clinically useful for the treatment of high glucose–induced coronary atherosclerosis.

Key Words: antioxidant • glucose • vascular smooth muscle • phospholipase D • protein kinase C

Hyperglycemia is probably an important causative factor in the development of macrovascular complications in patients with coronary artery disease both before¹ and after² percutaneous transmural coronary angioplasty. However, the mechanisms participating in accelerated atherosclerotic disease in patients with diabetes are unclear. VSMC migration and proliferation appear to play important roles in the development of atherosclerosis.³ Although hyperglycemia has been suggested to contribute to the development of complications in patients with diabetes, few studies have focused on the direct effect of elevated glucose levels on VSMCs.

It has been reported that elevated glucose-induced increases in the NADH/NAD⁺ ratio and glyco-oxidation products in diabetic humans and animals are evidence of diabetes-induced oxidative stress.⁴ However, it is unknown to what extent these parameters of increased oxidative stress reflect increased VSMC migration and proliferation.

Troglitazone, originally introduced for clinical use as an insulin sensitizer, has been shown to possess potent antioxidant properties.⁵ The finding that active oxygen species stimulate VSMC growth⁶ suggests that troglitazone might prevent active oxygen species–induced vascular growth. These observations led us to speculate that troglitazone may prevent high glucose–induced migration and hyperproliferation of VSMCs, possibly through its antioxidative effects.

Hyperglycemia⁷ and insulin resistance⁸ are also considered independent risk factors for coronary artery disease. However, it is unknown whether insulin resistance exists in coronary VSMCs and whether hyperinsulinemia directly promotes coronary VSMC proliferation.

The present study was, therefore, designed to investigate the effect of troglitazone on high glucose–induced coronary VSMC migration and proliferation and on high glucose and insulin-mediated glucose uptake and proliferation by coronary VSMCs. Possible intracellular mechanisms of action of troglitazone were also studied.

Materials and Methods

Materials
Type II collagenase, Nonidet P-40, and dithiothreitol were purchased from Sigma Chemical Co. DMEM, penicillin-streptomycin, trypsin EDTA (Versine), and FCS were purchased from GIBCO Laboratories. [³H]Thymidine, [³H]ethanolamine, ¹²⁵I-insulin, and the PKC assay system were purchased from Amersham Japan Co. Multiwell pipettes and flasks were purchased from Becton Dickinson & Co. Troglitazone and pioglitazone were donated by Sankyo Co. -Tocopherol was donated by Eisai Co.

Cell Culture
VSMCs were grown from explants of 4-week-old male Japanese White rabbit coronary arteries by the explant method. Cells were identified as VSMCs on the basis of their morphological and growth characteristics as previously reported.^9,10 Briefly, VSMCs exhibited a typical "hill and valley" growth pattern and also exhibited positive fluorescence with antibodies against -smooth muscle actin but no fluorescence with antibodies against factor VIII antigen. VSMCs were grown in DMEM supplemented with 10% FCS. Cells from passages 3 to 5 were used and were subcultured after trypsinization on a weekly basis, since cells became confluent in 1 week. Each plate was replenished twice a week with fresh medium. For studies of cells under hyperglycemic conditions, cells were allowed to grow for 5 days in high glucose (22.2 mmol/L) medium before use in order to more closely simulate chronic hyperglycemia. As a control for osmolarity, cells were grown for 5 days in normal glucose (5.6 mmol/L glucose+16.6 mmol/L mannose) medium.

Experimental Protocol
In the migration experiment, VSMCs were allowed to grow for 5 days in high or normal glucose medium with 10% FCS in the presence or absence of troglitazone, pioglitazone, or -tocopherol. VSMCs were trypsinized and suspended at a concentration of 1.5x10⁵ cells/mL in DMEM without FCS supplemented with 0.4% BSA. Then VSMCs were used in the migration experiment described below. In growth experiments, VSMCs were allowed to grow for 5 days in high or normal glucose medium with 10% FCS in the presence or absence of troglitazone, pioglitazone, or -tocopherol. VSMCs were cultured in DMEM without FCS for 48 hours to induce quiescence. Then VSMCs were used in cell count experiments, in [³H]thymidine incorporation experiments, and in the measurement of PLD and PKC.

Migration Assay
The migration of VSMCs was assayed by a modified version of Boyden’s chamber method using microchemotaxis chambers (Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp) with pores 5.0 µm in diameter, as previously reported.¹¹ In all experiments, collagen-coated filters were used. Briefly, the membranes were treated with 0.5N acetic acid and then incubated for 48 to 72 hours at 25°C in a collagen solution (100 µg/mL type I collagen in 0.5N acetic acid). They were then air-dried. Cultured VSMCs were trypsinized and suspended at a concentration of 1.5x10⁵ cells/mL in DMEM supplemented with 0.4% BSA. Cell number was counted with an electronic cell counter (model ZB1, Coulter Electronics). A 200-µL volume of VSMC suspension (3.0x10⁴ cells) was placed in the upper chamber, and 40 µL of DMEM/0.4% BSA containing a migration factor was placed in the lower chamber. The chamber was incubated at 37°C under 5% CO₂ in air for 4 hours. After incubation, the filter was removed, and the VSMCs on the upper side of the filter were scraped off. The VSMCs that had migrated to the lower side of the filter were fixed in methanol, stained with Diff-Quick staining solution, and counted under a microscope for quantification of VSMC migration. Migration activity was expressed as the number of cells that had migrated per high-power field (x400). In experiments to determine the effects of troglitazone on VSMC migration, these agents were added to the lower chamber before the incubation.

Growth Curves
For determination of cell numbers, VSMCs were placed in six-well culture dishes at 2x10⁴/mL and grown in DMEM containing 10% FCS, which was changed every 72 hours. After the medium was aspirated, the same medium without FCS was applied for 48 hours. Cultures were washed with a calcium- and magnesium-free PBS and harvested with trypsin EDTA solution. Counts were performed with a Coulter counter.¹²

Determination of DNA Synthesis
Relative rates of DNA synthesis were assessed by determination of [³H]thymidine incorporation into trichloroacetic acid–precipitable material.¹³ Quiescent VSMCs grown in 24-well culture dishes were pulsed 4 hours with [³H]thymidine (10 µCi/mL), washed with cold calcium- and magnesium-free PBS, and incubated with 5% trichloroacetic acid at 4°C for 10 minutes. Cells were dissolved in 1N NaOH at 37°C for 30 minutes and then neutralized. Radioactivity was determined by liquid scintillation counting.

Flow Cytometric Analysis of Cell Cycle Stage
Quiescent VSMCs grown in flasks were detached with 0.25% trypsin at 37°C for 5 minutes and then pelleted by centrifugation (1000 rpm for 5 minutes). The cells were resuspended in 200 µL of solution A (trypsin, 30 mg/L; citric acid, 3.4 mmol/L; spermine, 1.5 mmol/L; Tris-HCl, 0.5 mmol/L; and Nonidet P-40, 2 mL/L). Ten minutes later, 150 µL of solution B (trypsin inhibitor, 500 mg/L; RNase, 100 mg/L; citric acid, 3.4 mmol/L; spermine, 1.5 mmol/L; Tris-HCl, 0.5 mmol/L, and Nonidet P-40, 2 mL/L) was added, and the mixture was left to stand for >10 minutes. All cell cycle samples¹⁴ were analyzed within 3 hours using a flow cytometer (Epics Profile). Red blood cells were used as an internal standard for DNA analysis.

Metabolic and Biochemical Assays
VSMCs were incubated in normal or high glucose medium with or without troglitazone (1 µmol/L) for 48 hours. Incubations were terminated by rapidly adding 3N perchloric acid to the culture medium with shaking. The tubes were then centrifuged, and the supernatant was removed. The effect of elevated glucose levels on the cytosolic lactate/pyruvate ratio is a more reliable indicator of the cytosolic NADH/NAD⁺ ratio than are measurements of pyridine nucleotides themselves in tissue extracts.¹⁵

Insulin Binding
VSMCs cultured in 24-well dishes were rinsed with assay buffer (pH 7.8) (HEPES, 100 mmol/L; NaCl, 120 mmol/L; KCl, 5 mmol/L; MgSO₄, 1.2 mmol/L; and glucose, 8 mmol/L; along with 0.1% bovine serum albumin) and incubated for 30 minutes at 37°C in 250 µL buffer containing 25 µL¹²⁵ I-insulin (25 000 cpm) and 25 µL buffer (for total binding) or 25 µL unlabeled insulin or IGF-1 at specified concentrations. The assay was terminated by cooling plates on ice. Aliquots of supernatants were counted for free radioactivity. Medium was removed, and cells were then washed with three times with fresh ice-cold assay buffer and solubilized in 0.2 mol/L acetic acid (pH 2.5) containing 0.5 mmol/L NaCl for 6 minutes to extract only surface-bound ligands.⁷ Radioactivity was measured in a gamma counter. Nonspecific binding of ¹²⁵I-insulin was defined as binding in the presence of 0.1 µmol/L unlabeled insulin. Binding data were graphically analyzed, and dissociation constants were determined from Scatchard plots.

Glucose Transport Analysis
For glucose transport studies, VSMCs were grown to confluence and, on the day of the experiment, were incubated with PSS containing 145 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES, 1 mmol/L Mg₂SO₄, 0.5 mmol/L Na₂HPO₄, and 1.5 mmol/L CaCl₂. Cells were acclimatized in PSS for 1 hour, after which the buffer was replaced with PSS containing vehicle (0.01 mmol/L HCl) or 100 µU/L porcine insulin. After 20 minutes of this pretreatment, solutions were replaced with identical solutions containing tracer amounts (700 pmol/L) of [³H]DOG. Transport of [³H]DOG was allowed to proceed for 5 minutes. Wells were then aspirated and washed three times with ice-cold PSS. Cells were solubilized with 0.5 mol/L NaOH and neutralized with HCl, and the mixture was quantitatively transferred (using three washes with PSS) to scintillation vials. All experiments were repeated a minimum of four times using four replicates per group per experiment.

PLD Activity Measured by Ethanolamine Release
VSMCs in 35-mm dishes were cultured in medium containing [³H]ethanolamine (5 µCi/mL per dish) for 24 hours to label cellular phosphatidylethanolamine. After removal of the labeling medium, the cells were washed twice with buffer A (20 mmol/L HEPES [pH 7.4], 120 mmol/L NaCl, 5 mmol/L glucose+16.6 mmol/L mannose, or 22.2 mmol/L glucose). After a 0.5- to 1-hour incubation with buffer A, the reaction was terminated by removing buffer A and adding 0.75 mL methanol. The cells were harvested by gentle scraping. The dishes were then washed again with 0.75 mL methanol. Distilled water (0.6 mL) and 0.75 mL chloroform were added to the cells. The aqueous and lipid phases were obtained from the cell extracts by adding 0.75 mL chloroform and 0.75 mL water, followed by centrifugation (3000g for 10 minutes). Fractionation of ethanolamine metabolites from the aqueous phases was performed on Dowex 50w H⁺-packed columns (1-mL bed volume, Bio-Rad Econo columns) as previously described.¹⁶ The initial flow-through (1 mL) along with the following 3 mL water contained glycerophosphoethanolamine. Ethanolamine phosphate was eluted with 15 mL water. Finally, ethanolamine was eluted with 8 mL of 1 mol/L HCl.

Cell Fractionation and Assay of PKC
VSMCs were washed twice with an ice-cold assay buffer (50 mmol/L Tris-HCl [pH 7.5] buffer containing 2 mmol/L EDTA, 2 mmol/L EGTA, 0.25 mol/L sucrose, 10 mmol/L 2-mercaptoethanol, 0.21 mmol/L leupeptin, and 0.23 mmol/L phenylmethylsulfonyl fluoride). Cells were then scraped and were sonicated with three 10-second bursts. The homogenates were centrifuged at 100 000g for 60 minutes at 4°C to separate the cytosolic and particulate fractions. The cytosolic fraction was kept on ice for 1 hour and then centrifuged at 100 000g for 30 minutes. PKC activity was measured by a modified version of a method previously reported¹⁷ using the Amersham PKC assay system. In brief, a sample of the reaction mixture (50 mmol/L Tris-HCl [pH 7.5], 3 mmol/L calcium acetate, 100 µmol/L -phosphatidyl-L-serine, 1 µmol/L PMA, 225 µmol/L substrate peptide, 7.5 mmol/L dithiothreitol, and 0.05% [wt/vol] sodium azide) was mixed with magnesium [³²P]ATP and incubated at 25°C for 15 minutes. The substrate peptide interacts with 8% of PKC activity with cAMP-dependent protein kinase, with 13% of PKC activity with phosphorylase kinase, and with 38% of PKC activity with proteolytic PKC fragment but does not interact with hexokinase or myosin light chain kinase. An acidic reaction-quenching reagent was added to stop the reaction. Phosphorylated peptide was separated on binding paper. After the paper was washed, the extent of phosphorylation was measured by scintillation counting. Results of the PKC assay were linear for 15 minutes. PKC activity was determined by subtracting the initial rate of protein kinase activity (in the absence of activators) from the initial rate of protein kinase activity in the presence of phosphatidylserine, calcium acetate, and PMA.

Statistical Methods
Statistical analysis was performed by ANOVA and Scheffé’s modified t test.¹⁸ Values of P<.05 were considered significant.

Results

Inhibition of High Glucose–Induced Cell Migration by Troglitazone
PDGF-BB (1 ng/mL) stimulated the migration of VSMCs in a time-dependent manner. VSMC migration induced by PDGF-BB was increased during the initial 4 hours of incubation, after which VSMC migration showed a significant decline.¹¹ Therefore, subsequent studies of VSMC migration were performed with cells incubated for 4 hours.

PDGF-BB stimulated the migration of VSMCs in a dose-dependent (0.1 to 1 ng/mL) manner (Fig 1A). Therefore, 1 ng/mL PDGF-BB stimulation was used in subsequent studies. PDGF-BB–stimulated migration of VSMCs was enhanced in high glucose–treated cells, but this enhancement was completely suppressed by coincubation with 1 µmol/L troglitazone (Fig 1A).

View larger version (30K): [in this window] [in a new window]   Figure 1. A, Effects of 4 hours of incubation in normal glucose (NG, 5.6 mmol/L glucose+16.6 nmol/L mannose) or high glucose (HG, 22.2 mmol/L glucose) with or without 1 µmol/L troglitazone (TRO) on PDGF-BB–stimulated migration of coronary VSMCs. NS indicates not significantly different from NG. *P<.05 compared with NG. B, Effect of troglitazone, pioglitazone, and -tocopherol on glucose-potentiated migration of cultured coronary VSMCs stimulated with 1 ng/mL PDGF-BB for 4 hours. Migration activities are expressed as the number of cells per high-power field (HPF), and values are given as mean±SD of four measurements. NS indicates not significantly different. *P<.05.

The effect of troglitazone on the migration of VSMCs treated with PDGF-BB (1 ng/mL) is shown in Fig 1B. Troglitazone, pioglitazone, and -tocopherol inhibited glucose-potentiated PDGF-directed VSMC migration. This inhibition was concentration dependent, and the suppression of migration was greater for troglitazone-treated cells than for pioglitazone-treated cells. - Tocopherol (1 µmol/L) completely inhibited high glucose–potentiated PDGF-directed migration. Although nonstimulated VSMCs exhibited low migration activity, troglitazone did not inhibit their basal migration activity.

Inhibition of High Glucose–Induced Cell Proliferation by Troglitazone
As shown in Fig 2, cell proliferation in high glucose medium was greater than that in normal glucose medium. Troglitazone inhibited the high glucose–induced cell proliferation of VSMCs. There was no difference in the numbers of 5.6 mmol/L glucose–treated cells and 5.6 mmol/L glucose+16.6 mol/L mannose–treated cells. In these experiments, VSMCs on confluence were cultured in FCS-free medium for 48 hours to induce quiescence. Troglitazone (100 nmol/L and 1 µmol/L) reduced the number of VSMCs cultured in high glucose medium by 17% and 27%, respectively (Fig 2). Pioglitazone (100 nmol/L and 1 µmol/L) also reduced the number of VSMCs cultured in high glucose medium, although the reduction was smaller than that for cells treated with troglitazone. -Tocopherol (1 µmol/L) completely suppressed the high glucose–induced increase in cell number. Cell viability was checked by trypan blue staining, which confirmed that >99% of the cells were alive.

View larger version (34K): [in this window] [in a new window]   Figure 2. Number of coronary VSMCs after stimulation with 22.2 mmol/L glucose or 5.6 mmol/L glucose+16.6 nmol/L mannose with or without the indicated dose of troglitazone, pioglitazone, or -tocopherol. Cells were plated in medium containing 10% FCS. Cells were grown in media with 0.1% FCS for 48 hours. Cell number was determined with a Coulter counter. NS indicates not significantly different. *P<.05.

Fig 3 shows the effect of troglitazone on [³H]thymidine incorporation by postconfluent VSMCs in FCS-free normal or high glucose medium. Troglitazone (100 nmol/L and 1 µmol/L) inhibited DNA synthesis by VSMCs by 45% and 60%, respectively. Pioglitazone (100 µmol/L and 1 µmol/L) also reduced [³H]thymidine incorporation by VSMCs cultured in high glucose, although the reduction was smaller than that for cells treated with troglitazone. -Tocopherol (1 µmol/L) completely suppressed the high glucose–induced increase in [³H]thymidine incorporation. Troglitazone did not cause a loss of cells in the confluent state. Five days after the addition of troglitazone, <1% of the cells were found to be present in the supernatant medium. Cell viability was also checked by trypan blue staining, which confirmed that >99% of the cells were alive.

View larger version (32K): [in this window] [in a new window]   Figure 3. Incorporation of [3H]thymidine into DNA of coronary VSMCs after incubation with 22.2 mmol/L glucose or 5.6 mmol/L glucose+16.6 mmol/L mannose for 5 days with the indicated doses (µmol/L) of troglitazone, pioglitazone, or -tocopherol. Experimental details are given in "Materials and Methods." Values are mean±SD of six determinations for three or four different cell preparations. NS indicates not significantly different. *P<.05.

Flow Cytometric Analysis
VSMCs cultured without FCS for 48 hours were in the G₀-G₁ stage (100%). Chronic high glucose treatment itself without growth factors did not change the stage of the cell in the cell cycle (data not shown). Mean±SD data are shown in the Table. PDGF-BB (1 ng/mL) treatment for 24 hours changed the stage of the cell in the cell cycle from G₀-G₁ to S (18.0%) and G₂-M (2.8%). High glucose treatment changed the stage of the cell in the cell cycle from G₀-G₁ to S (28.1%) and G₂-M (4.6%). Troglitazone (1 µmol/L) eliminated these high glucose–induced changes.

View this table: [in this window] [in a new window]   Table 1. Flow Cytometric DNA Histogram Analysis of Stage in Cell Cycle

Effect of Troglitazone on Glucose-Induced Increase in VSMC Cytosolic NADH/NAD⁺ Ratio
For more reliable assessment of the VSMC cytosolic NADH/NAD⁺ ratio, lactate/pyruvate ratios were measured in VSMCs plus medium. As shown in Fig 4, the ratio was 91.7% higher with high glucose than with normal glucose medium after 24 hours of incubation. Troglitazone (1 µmol/L) significantly reduced the extent of this increase.

View larger version (15K): [in this window] [in a new window]   Figure 4. Plots show the effect of 5.5 mmol/L glucose+ 16.6 mmol/L mannose or 22.2 mmol/L glucose with or without troglitazone (1 µmol/L) on the coronary VSMC+medium lactate/pyruvate ratio. VSMCs were incubated for 24 hours. Values are mean±SD of six determinations. NS indicates not significantly different. *P<.05.

Insulin Binding
The specific binding of ¹²⁵I-insulin was 0.8±0.1% of total ¹²⁵I-insulin added. The concentration needed to obtain half-maximal displacement (IC₅₀) was estimated to be 160 pmol/L. IGF-1 was >10 000 times less potent than insulin in displacing ¹²⁵I-insulin (Fig 5). Scatchard plots obtained using a model that assumes two independent binding sites revealed a high-affinity insulin receptor with a dissociation constant (K_d) of 9.5±1.5x10^-9 mol/L and a binding capacity (B_max) of 8.9±0.7 x10³ sites/cell and a low-affinity receptor with a K_d of 4.3x10^-8 mol/L. Incubation of cells with 22.2 mmol/L glucose for 48 hours before binding changed neither the K_d (8.1±1.6x10^-9 mol/L) nor the B_max (9.7±0.6x10³ sites/cell) for specific binding at 37°C for 30 minutes.

View larger version (14K): [in this window] [in a new window]   Figure 5. A, Displacement of 125I-insulin (43.9 pmol/L) by unlabeled insulin (n=4). Cells were incubated with insulin or IGF-1 for 30 minutes at 37°C in binding buffer. Values are mean±SD. SD was 2.6% to 15.0% of the mean value. B, Saturation binding and Scatchard plot for specific binding of 125I-insulin to high glucose–treated () and untreated () cells. Values are means of four measurements. SD was 5.0% to 16.0% of the mean value.

Insulin-Stimulated [³H]DOG Uptake
Uptake of [³H]DOG into cells was linear between 0 and 15 minutes of incubation regardless of glucose concentration. Insulin-stimulated [³H]DOG uptake was observed at a concentration of 1 pmol/L and was increased at a concentration of 1 nmol/L (Fig 6A). After 5 days of incubation with 22.2 mmol/L glucose, insulin-stimulated [³H]DOG uptake was significantly decreased; this decrease was prevented by coincubation with 1 µmol/L calphostin C, a PKC inhibitor, and by coincubation with 100 nmol/L troglitazone (Fig 6B). Glucose treatment did not change basal [³H]DOG transport activity. Mannose had no effect on basal and insulin-stimulated [³H]DOG uptake.

View larger version (23K): [in this window] [in a new window]   Figure 6. Insulin-stimulated [3H]-DOG uptake. A, Concentration-response curve for the effect of insulin on VSMC glucose transport in high glucose–treated () and untreated () cells is shown. All cells were treated with insulin for 20 minutes before the addition of [3H]DOG, as described in "Materials and Methods." B, VSMCs were grown to confluence and kept in DMEM containing the indicated concentration of glucose with or without troglitazone for 5 days before measurement of insulin (100 µU/mL)–stimulated [3H]DOG uptake, as described in "Materials and Methods." Values are mean±SD of three determinants in three different cell preparations. NS indicates not significantly different. *P<.05.

Effect of High Glucose on Insulin-Stimulated VSMC Proliferation
Cell number and [³H]thymidine incorporation were measured to study the effect of high glucose on insulin-mediated VSMC growth.

High glucose (22.2 mmol/L) treatment for 5 days enhanced insulin-stimulated increase in cell number and [³H]thymidine incorporation. These changes were completely eliminated by coincubation with troglitazone (1 µmol/L) (Fig 7).

View larger version (22K): [in this window] [in a new window]   Figure 7. Insulin-mediated growth of 5.6 mmol/L glucose+ 16.6 mmol/L mannose–or 22.2 mmol/L glucose–treated VSMCs with () or without () troglitazone (1 µmol/L). A, Insulin-induced increase in cell number. B, Insulin-induced [3H]thymidine incorporation. NS indicates not significantly different. *P<.05 compared with values obtained without troglitazone.

PLD Activities With Normal and High Glucose Media
In order to evaluate one possible mechanism of suppression of high glucose–induced VSMC migration and proliferation, PLD activities in VSMCs in normal glucose and high glucose media were measured. As shown in Fig 8, PLD activities in high glucose–treated cells were greater than those in normal glucose–treated cells. VSMCs were coincubated with troglitazone (1 µmol/L) in either normal or high glucose medium for 5 days before measurement of PLD activity. When high glucose medium was coincubated with troglitazone (1 µmol/L), high glucose–induced PLD activation was significantly reduced. -Tocopherol (1 µmol/L) completely eliminated glucose-induced PLD activation.

View larger version (27K): [in this window] [in a new window]   Figure 8. PLD activities in normal glucose–and high glucose–treated cells. VSMCs were grown to confluence and kept in DMEM containing the indicated concentration of glucose with or without troglitazone (1 µmol/L) for 5 days before PLD activity measurement, as described in "Materials and Methods." Values are mean±SD. NS indicates not significantly different. *P<.05.

PKC Activities in Normal Glucose and High Glucose Media
To evaluate one possible mechanism of suppression of high glucose–induced VSMC proliferation, PKC activities in VSMCs in normal and high glucose media were measured. As shown in Fig 9, membrane-bound (particulate) PKC activities in high glucose–treated cells were greater than those in normal glucose–treated cells, with a corresponding decrease in cytosolic PKC activities. VSMCs were coincubated with troglitazone (1 µmol/L) in either normal or high glucose medium for 5 days before the determination of PKC activity. With normal glucose medium, troglitazone did not reduce basal PKC activity. When high glucose medium was coincubated with troglitazone (1 µmol/L), glucose-induced PKC activation was significantly reduced. -Tocopherol (1 µmol/L) completely eliminated high glucose–induced PKC activation (Fig 9).

View larger version (35K): [in this window] [in a new window]   Figure 9. The bar graph shows the distribution of PKC activities in cytosolic and particulate fractions of VSMCs. Values are mean±SD. VSMCs were grown to confluence and then kept in DMEM containing the indicated concentration of glucose with or without troglitazone for 5 days before PKC activity was measured, as described in "Materials and Methods." NS indicates not significantly different. *P<.05.

Discussion

In the present study, we showed for the first time that troglitazone reduces high glucose–induced migration and hyperproliferation of coronary VSMCs in a concentration-dependent manner. This preventive effect was observed for concentrations of 100 nmol/L to 1 µmol/L troglitazone, which are less than those obtained with oral administration.¹⁹ This suggests that troglitazone might prevent VSMC migration and proliferation in vivo. In fact, Law et al²⁰ have reported that troglitazone suppresses intimal hyperplasia in a balloon injury model of rat aorta.

We have also demonstrated for the first time that high glucose medium increases migration of coronary VSMCs and that troglitazone dose-dependently inhibits this increase in migration. The precise mechanisms of this increase in migration by high glucose and inhibition in migration by troglitazone remain to be determined. However, it has been reported that high glucose medium increases the production of active oxygen species²¹ and that the suppression of increased active oxygen species by catalase and by the antioxidant N-acetylcysteine results in suppression of migration of VSMCs.²² These findings suggest that oxidative stress by high glucose may cause increased migration of VSMCs and that the antioxidants troglitazone and -tocopherol may prevent this increase. We have also shown in this study that PLD activity was increased in high glucose–treated cells and that this increase was prevented by troglitazone and -tocopherol. Since PLD increases the formation of diacylglycerol,²³ which may play a role in migration,²⁴ suppression of PLD by troglitazone or -tocopherol may suppress the increase in VSMC migration induced by high glucose.

There are at least three possible mechanisms of the inhibition of high glucose–induced hyperproliferation of coronary VSMCs by troglitazone. The first is the blockade of oxidative modification of LDLs, as already reported.⁴ The second is suppression of the sorbitol pathway; we have already reported the importance of this pathway in high glucose–induced hyperproliferation of VSMCs.¹⁴ In the present study, we showed that the increase in the NADH/NAD⁺ ratio, which is an indicator of sorbitol pathway activity,⁴ induced by high glucose medium is suppressed by troglitazone, suggesting that troglitazone may suppress the activity of this pathway. In fact, the antioxidant vitamin C is reported to suppress the activity in the sorbitol pathway induced by high glucose.²⁵ The third possible mechanism is suppression of the oxidative stress induced by high glucose. It has been reported that production of active oxygen species can occur as a result of glyco-oxidation (nonenzymatic glycation)²¹ and increased PKC activity induced by incubation in high glucose medium.²⁶ We also showed in the present study that -tocopherol suppressed high glucose–induced migration and proliferation of VSMCs. We have also observed that increased concentrations of the scavenging enzyme catalase suppressed high glucose–induced VSMC proliferation (data not shown). These findings suggest that oxidative stress may play a role in the high glucose–induced hyperproliferation of VSMCs.

We also showed in the present study that troglitazone and -tocopherol suppress the high glucose–induced increase in membrane-bound PKC activity. This finding is explained in part by the suppression of increase in the NADH/NAD⁺ ratio induced by high glucose.¹⁴ Since activated PKC produces active oxygen species²⁷ and active oxygen species stimulate vascular smooth muscle cell growth,⁶ it is reasonable to assume that this suppression of increase in PKC activity by troglitazone and -tocopherol plays a role in the suppression of high glucose–mediated VSMC hyperproliferation. We and others have in fact demonstrated that PKC inhibitors prevent high glucose–induced hyperproliferation in vitro^16,28 and vascular dysfunction in vivo²⁹ through suppression of PKC. Since PLD is reported to activate PKC through the formation of diacylglycerol in VSMCs,³⁰ troglitazone may inhibit PLD, resulting in the suppression of PKC activity.

Cell cycle analysis was also performed to determine the mechanism of high glucose–induced hyperplasia. Glucose (22.2 mmol/L) alone without growth factors did not change the stage of cells in the cell cycle (data not shown). Glucose (22.2 mmol/L) with PDGF-BB (1 ng/mL) significantly increased the percentage of VSMCs in the S and G₂-M stages, suggesting that glucose is not a competence growth factor but is a progression growth factor. Troglitazone (100 nmol/L) inhibited this increase. Since troglitazone decreases membrane-bound PKC activity and activation of PKC is required for cell cycle progression and S-phase entry of VSMCs,¹⁶ the high glucose–induced change in stage in the cell cycle may be due to PKC activation, and this change may be blocked by troglitazone.

Troglitazone, a thiazolidinedione compound, is presently being tested as a new oral antidiabetic agent. There is evidence from animal studies and clinical trials with non–insulin-dependent diabetes mellitus patients that troglitazone may reduce insulin resistance,^31,32 which is consistent with the findings of the present study. The molecular mechanism of reducing insulin resistance is not understood. The possible involvement of PPAR in insulin responsiveness of adipocytes has been reported.³³ Although little is known concerning PPAR function in VSMCs, the finding that pioglitazone suppresses mitogen-induced³⁴ and high glucose–induced VSMC migration and growth suggests that this receptor may play a role in the mechanism of action of troglitazone and that the effect of troglitazone is not solely as an antioxidant. Troglitazone may possess a more potent hybrid activity as an antioxidizing thiazolidinedione. It has also been reported that troglitazone interferes with hyperglycemia-induced PKC-mediated insulin receptor kinase.³⁵ Therefore, suppression of PKC by troglitazone, possibly as a result of its antioxidant effect, may play a role in reducing insulin resistance.

Diabetes is associated not only with high glucose levels but also, at least in patients with non–insulin-dependent diabetes mellitus, with increased plasma insulin levels.³⁶ Therefore, characterization of the interaction between high glucose and insulin would have important clinical implications. Since hyperglycemia^1,37 and insulin^2,38 have been implicated in the genesis of macroangiopathy and coronary artery disease in patients with diabetes, we also investigated the effect of high glucose on insulin-mediated [³H]DOG uptake. Chronic glucose treatment decreased insulin-mediated [³H]DOG uptake (possibly via insulin resistance) without any change in insulin receptor number or affinity (Fig 6B), consistent with the findings of a previous study.³⁹ This result indicates that chronic exposure of VSMCs to glucose induces an increase in insulin resistance in the glucose transport effector system. Glucose may impair the efficacy of recruitment of intracellular glucose carriers to the cell surface by insulin. This is the first demonstration that troglitazone improves impaired insulin response in vascular tissue. This impairment of response may be due to increased PKC activity, since activation of PKC increases insulin resistance⁴⁰ and PKC inhibitor prevented this impairment. Since troglitazone, which was synthesized with an -tocopherol substitution in an effort to obtain an antioxidative effect, was shown to prevent the glucose-induced inhibition of insulin receptor response in cultured fibroblasts,¹¹ it may be that troglitazone restores high glucose–induced insulin resistance, possibly as a result of its antioxidative effect in VSMCs. Normalization of PKC activity or translocation by troglitazone may play a role in this effect. In fact, prevention by PKC inhibitors of glucose-induced insulin resistance in rat fat cells has been reported.⁴⁰ PKC may thus be a link between insulin resistance and increased migration and proliferation of coronary VSMCs induced by high glucose medium.⁴¹ In addition, impaired insulin-mediated glucose uptake is an indicator of increased PKC activity.

Impairment of insulin-mediated glucose uptake and increase in growth of VSMCs by high glucose medium may provide evidence for a new hypothesis concerning the mechanism of insulin resistance at the cellular level. It has been reported that VSMCs possess receptors for insulin and IGF-1.^42,43 The IGF-1 receptors form a common pathway for the growth effects of both insulin and IGF-1 in VSMCs.⁴⁴ The effects on glucose uptake³⁹ and growth⁴⁴ of IGF-1 were 10 to 100 times more potent than those of insulin. These findings suggest that PKC activation in VSMCs may impair glucose uptake and increase growth of VSMCs through effects on IGF-1 receptors. However, as shown in the present and another study,⁴⁵ overlap in signaling due to IGF-1 receptor interaction may be minimal, since IGF-1 did not displace labeled insulin in VSMCs (Fig 7A). Therefore, impairment of glucose uptake and increase in growth of VSMCs through effects on insulin receptors resulting from PKC activation can be also postulated to occur at least at physiological concentrations of <100 µU/mL (0.7 nmol/L),⁴⁶ because insulin-mediated [³H]DOG uptake increased at concentrations above 1 nmol/L, suggesting the involvement of IGF-1 receptors at concentrations above this level.

It has recently been reported that troglitazone inhibited PDGF-directed migration and basic fibroblast growth factor–induced proliferation of VSMCs from rat aorta via inhibition of mitogen-activated protein kinase signaling.²⁰ The present study further demonstrated that troglitazone improved insulin resistance and inhibited high glucose–potentiated PDGF-directed migration and high glucose–induced proliferation of rabbit coronary VSMCs via inhibition of PLD and PKC activation, possibly as a result of an antioxidant effect. This finding may be clinically useful, since troglitazone is mainly used in diabetic patients.

In conclusion, our findings suggest that oxidative stress may play a role in the vascular migration and hyperproliferation induced by high glucose stimulation. Our findings suggest that troglitazone might be useful in the treatment of high glucose–induced coronary atherosclerosis. The intracellular mechanism of action of troglitazone may include, in part, the suppression of PLD activation, PKC activation, and an increased percentage of cells in the S phase induced by high glucose, and this suppression may be the result of intracellular antioxidative effects of troglitazone. PKC may be a link between impairment of insulin-mediated glucose uptake and accelerated migration and proliferation induced by high glucose.

Selected Abbreviations and Acronyms

DOG = deoxyglucose IGF = insulin-like growth factor PDGF = platelet-derived growth factor PKC = protein kinase C PLD = phospholipase D PMA = phorbol 12-myristate 13-acetate PPAR = peroxisome proliferator-activated receptor VSMC = vascular smooth muscle cell

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, by grants from the Osaka City University Medical Research Foundation, Uehara Memorial Foundation, and Kimura Memorial Foundation, and by a Japan Heart Foundation Grant for Research on Hypertension and Vascular Metabolism. We would like to thank Atsumi Ohnishi for excellent technical assistance.

Received December 30, 1996; accepted August 29, 1997.

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