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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2207-2212.)
© 1995 American Heart Association, Inc.

Articles

Aldose Reductase Inhibitor Prevents Hyperproliferation and Hypertrophy of Cultured Rat Vascular Smooth Muscle Cells Induced by High Glucose

Kenichi Yasunari; Masakazu Kohno; Hiroaki Kano; Koji Yokokawa; Takeshi Horio; Junichi Yoshikawa

From the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan.

	Abstract

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Abstract Vascular remodeling is a key process in the pathophysiology of atherosclerosis. Recent evidence suggests that high glucose levels may function as a vascular smooth muscle growth and proliferation–promoting substance. To explore the role of the polyol pathway in this process, we examined the effect of an aldose reductase inhibitor (ARI), epalrestat, on the growth characteristics of cultured rat vascular smooth muscle cells (VSMCs). Epalrestat (10 nmol/L, 1 µmol/L) significantly suppressed the high glucose–induced proliferative effect as measured by [³H]thymidine incorporation by 67% and 82% in cell number, suggesting ARI as an antimitogenic factor. In VSMCs, epalrestat (10 nmol/L, 1 µmol/L) significantly suppressed the high glucose–induced incorporation of [³H]leucine by 45% and 58% with the concomitant reduction of the cell size estimated by flowcytometry. Epalrestat (1 µmol/L) also suppressed high glucose–induced intracellular NADH/NAD⁺ increase and membrane-bound protein kinase C activation. These results indicate that this ARI possesses an antiproliferative and antihypertrophic action on VSMCs induced by high glucose possibly through protein kinase C suppression.

Key Words: atherosclerosis • vascular smooth muscle cells • vascular remodeling • diabetes mellitus • protein kinase C

	Introduction

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Macrovascular complications related to atherosclerosis are the principal cause of mortality in a diabetic population.¹ Late complications of diabetes mellitus are mainly related to the involvement of arterial wall of both small and large vessels. The presence of such complications in almost all types of diabetes mellitus suggests that they have a common pathogenic mechanism, which is manifested by high blood glucose and related alterations.²

VSMC growth and proliferation seem to be important factors for the development of atherosclerosis.³ Recently it has been reported that high glucose increases membrane-bound PKC activity, which results in vascular hypertrophy and hyperplasia.⁴

Hyperglycemia itself leads to some metabolic abnormalities, such as increased polyol pathway activity.⁵ Previous studies have suggested that abnormal glucose metabolism, ie, accumulation of intracellular sorbitol, may also contribute to late complications of diabetes.⁶ This abnormal metabolism, which has been confirmed in experimental models,⁷ plays a key role in the development of diabetic complications. The existence of a polyol pathway in VSMCs⁸ as well as the arterial wall⁹ suggests that hyperglycemia may lead to the accumulation of sorbitol within VSMCs, contributing to their dysfunction and remodeling.

Therefore, the present study is designed to investigate whether activation of the polyol pathway is involved in high glucose–induced vascular hypertrophy and hyperproliferation and whether an ARI, epalrestat, prevents this glucose-induced process.

	Methods

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Materials
All tissue culture supplies were obtained from GIBCO Laboratories. Epalrestat [E-3-carboxymethyl-5-(2E-methyl-3-phenylpropenylidene) rhodanine]¹⁰ was a gift from Ono Pharmaceutical, Osaka, Japan. All other chemicals were analytic grade and were obtained from Sigma Chemical Co. Radioimmunoassay kits for PKC, [³H]thymidine, and [³H]leucine were purchased from Amersham Japan. Multiwell plates, pipettes, and flasks were purchased from Becton-Dickinson.

Cell Culture
VSMCs were grown from explants of 14-week-old Wistar rat aorta, with animals handled as described previously.¹¹ Cells were identified as VSMCs according to their morphological and growth characteristics as previously reported.^{12 13} VSMCs were grown in DMEM supplemented with 10% FCS. Passages three to five were used and were subcultured after trypsinization on a weekly basis since cells became confluent in 1 week.¹⁴ Media were changed every 3 days.

For studies of cells under hyperglycemic conditions, the cells were allowed to grow at least two passages in high glucose (22.5 mmol/L) DMEM before use. To more closely simulate chronic hyperglycemia, cells were passaged in high glucose rather than by adding glucose acutely. To control for osmolarity, cells were grown for at least two passages in 5.6 mmol/L glucose plus 16.6 mmol/L mannose. In preparation for experiments, the cells were made quiescent to look at proliferation, hypertrophy, and cell cycle by placing them for 48 hours in serum-free medium.

Growth Curves
For the determination of cell numbers, VSMCs were placed into six-well culture dishes at 2x10⁴/mL and grown in DMEM containing 10% FCS changed every 72 hours and then switched to the same medium without FCS for 2 days. Cultures were washed with calcium- and magnesium-free PBS [PBS(-)] and harvested with trypsin EDTA solution. Counts were performed by a Coulter counter.¹⁵

Determination of DNA and Protein Synthesis
Relative rates of DNA and protein syntheses were assessed by determination of [³H]thymidine and leucine incorporations, respectively, into TCA-precipitable material. Quiescent VSMCs grown in 24-well culture dishes were pulsed for 4 hours with [³H]thymidine (10 µCi/mL) or leucine (10 µCi/mL), washed with cold PBS(-), and incubated with 5% TCA at 4°C for 10 minutes. Cells were dissolved in 1N NaOH at 37°C for 30 minutes and neutralized. The radioactivity was determined by liquid scintillation counting.

Flowcytometric Analysis of Cell Size and Cell Cycle
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, spermin 1.5 mmol/L, Tris-HCl 0.5 mmol/L, Nonidet P-40 2 ml/L). Ten minutes after, 150 µL of solution B (trypsin inhibitor 500 mg/L, RNase 100 mg/L, citric acid 3.4 mmol/L, spermin 1.5 mmol/L, Tris HCl 0.5 mmol/L, Nonidet P-40 2 ml/L) was added and left for 10 minutes at room temperature. Solution C 150 µL (propidium iodide 622 µmol/L, spermin 3.0 mmol/L, citric acid 3.4 mmol/L, Tris-HCl 0.5 mmol/L, Nonidet P-40 2 ml/L) was then added and left for more than 10 minutes. All samples for cell size and cell cycle¹⁶ were analyzed within 3 hours on flowcytometer (EPICS PROFILE). Red blood cells were used as an internal standard of DNA analysis.

Cell Fractionation and Assay of PKC
VSMCs detached from culture dishes by incubation with DMEM were washed twice with an ice-cold assay buffer [50 mmol/L tris(hydroxymethyl)nitromethane (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] and were sonicated with three 10-second bursts. The homogenates were centrifuged at 100 000g for 60 minutes at 4°C, separating the cytosolic and particulate fractions. The cytosolic fraction was kept on ice with Nonidet P-40 added to a final concentration of 1%. The pellet resuspended in the assay buffer containing 1% Nonidet P-40 was stirred on ice for 1 hour and then was centrifuged at 100 000g for 30 minutes. PKC activity was measured by a modification of the method previously reported using the Amersham PKC assay system.¹⁷ In brief, a sample of the reaction mixture (50 mmol/L Tris/HCl [pH 7.5], 3 mmol/L calcium acetate, 2 mol% 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. 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 detected by scintillation counting. PKC assay was 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.

Metabolic and Biochemical Assays
VSMCs were incubated in 5.6 mmol/L or 22.2 mmol/L with or without epalrestat 1 µmol/L for 48 hours. Incubations were terminated by rapidly adding 6N perchloric acid to the culture medium with shaking. The tubes were then centrifuged and the supernatant was removed and was assayed for fructose by standard enzymatic methods.¹⁸ Prior to fructose analysis, extracts were treated with glucose oxidase¹⁹ to prevent interference with measurement of fructose by high glucose levels. The effect of elevated glucose levels on the cytosolic ratio of NADH/NAD⁺ was not measured directly but was inferred from changes in the ratio of lactate/pyruvate; the cytosolic ratio of these metabolites is a more reliable parameter of cytosolic ratio of NADH/NAD⁺ than measurement of pyridine nucleotides themselves in tissue extract.²⁰

Statistical Analysis
All values are expressed as mean±SD (n>6) in three to six separate experiments. ANOVA with subsequent Scheffé's modified t test was used to determine significant differences in multiple comparisons.²¹ A value of P<.05 was considered to be significant.

	Results

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Inhibition of High Glucose–Induced Cell Proliferation by Epalrestat
As shown in Fig 1, cell numbers cultured in high (22.2 mmol/L) glucose were higher than those cultured in normal (5.6 mmol/L) glucose. An ARI, epalrestat, exhibited dose-dependent inhibitory effect of this high (22.2 mmol/L) glucose–induced cell proliferation of VSMCs, although epalrestat itself did not change the number cultured in 5.6 mmol/L glucose. In these experiments, VSMCs upon confluence were cultured in FCS-free media for 2 days to induce quiescence. Epalrestat 1 µmol/L and 10 nmol/L reduced the number of VSMCs cultured in 22.2 mmol/L glucose 12% and 4%, respectively. As shown in Table 1, L-glucose did not increase the cell number, and D-glucose increased the cell number in a dose-dependent manner (5.6 to 22.2 mmol/L).

View larger version (28K): [in this window] [in a new window]   Figure 1. Bar graph shows cell number of VSMCs cultured for two passages in 5.6 mmol/L or 22.2 mmol/L glucose with or without indicated doses of an aldose reductase inhibitor, epalrestat. Cells were plated in the medium containing 10% FCS and after reaching confluence were grown in media without FCS for 48 hours. Cell number was measured by Coulter counter. *P<.05.

View this table: [in this window] [in a new window]   Table 1. Effect of Glucose Dose Dependency on Cell Number, [3H]Thymidine, and [3H]Leucine Incorporation

Antiproliferative and Antihypertrophic Action of Epalrestat on Postconfluent VSMCs
Fig 2 shows the effect of epalrestat on [³H]thymidine and leucine incorporations of postconfluent VSMCs in the normal glucose (5.6 mmol/L) of FCS-free media or stimulated by high glucose (22.2 mmol/L). Epalrestat inhibited DNA and protein syntheses of VSMCs. The maximal inhibition was seen at 100 nmol/L for both [³H]thymidine and [³H]leucine incorporations. As shown in Table 1, L-glucose did not increase [³H]thymidine and leucine incorporations compared with mannose-treated cells. Glucose increased [³H]thymidine and leucine incorporation in a dose-dependent manner (5.6 to 22.2 mmol/L). Epalrestat did not cause the loss of cells at the confluent state. Two passages after the addition of epalrestat, fewer than 1% of cells were found to be present in the supernatant media. Cell viability also was checked by trypan blue staining, confirming that more than 99% of the cells were alive.

View larger version (28K): [in this window] [in a new window]   Figure 2. Bar graphs show DNA and protein synthesis. Incorporation of [3H]thymidine into DNA and [3H]leucine into protein synthesis after chronic high glucose stimulation of quiescent VSMCs with the indicated dose of an ARI, epalrestat. Experimental details are given in "Methods." Increases in [3H]thymidine or [3H]leucine incorporation are each expressed relative to the mean of [3H] content (100%) in their respective controls. Values represent mean±SD of six determinations in three to four different cell preparations. *P<.05.

Flowcytometric Analysis
Fig 3 shows the histogram of cell size of postconfluent VSMCs defined by flow cytometric analysis. Chronic epalrestat treatment tended to reduce the cell size and caused a significant left-hand shift in cell size in high (22.2 mmol/L) glucose–treated cell groups. This result further confirmed the inhibitory effect of epalrestat on cellular hypertrophy of VSMCs. However, epalrestat 1 µmol/L did not change the cell size of VSMCs cultured in 5.6 mmol/L glucose.

View larger version (28K): [in this window] [in a new window]   Figure 3. A, Histogram shows relative cell size of postconfluent cells as measured by flowcytometric analysis. VSMCs were plated on T-25 flasks and cultured in DMEM with 5.6 mmol/L glucose (top), 22.2 mmol/L glucose (middle), and 22.2 mmol/L glucose plus epalrestat 1 µmol/L(M) (bottom) until confluent. Cultures were maintained in serum-free media for 2 days to induce quiescence. The data presented are typical of four such experiments. y axis shows cell numbers and x axis represents cell size. B, Graph shows inhibitory effect of epalrestat on chronic high glucose–induced hypertrophy of VSMCs. Relative cell size was measured by flowcytometry as described in "Methods." *P<.05.

VSMCs cultured without FCS for 48 hours show 100% G₀-G₁ stage. Chronic high glucose (22.2 mmol/L) treatment itself did not change the cell cycle (data not shown). As shown in Table 1, PDGF 1 ng/mL treatment for 24 hours induced the changes of cell cycle from G₀-G₁ to S (17.7%) and G₂-M (14.0%) stage. High glucose treatment with PDGF 1 ng/mL facilitated the change of the cell cycle from G₀-G₁ to S (20.7%) and G₂-M (15.3%). Epalrestat 1 µmol/L suppressed this high glucose–induced change.

Possible Involvement of PKC in the Antiproliferative and Antihypertrophic Action of Epalrestat on VSMCs Induced by High Glucose
Recently, a role of PKC in mediating protein synthesis in VSMCs has been suggested. Accordingly, we examined the possible involvement of PKC in the antiproliferative and antihypertrophic actions of epalrestat. As shown in Fig 4, membrane-bound PKC was increased by high glucose treatment. But this increase was significantly reduced by 1 µmol/L epalrestat.

View larger version (33K): [in this window] [in a new window]   Figure 4. Bar graph shows distribution of PKC activities between membranous and cytosolic fractions in VSMCs. Results are mean±SD. VSMCs were grown to confluence and then kept in DMEM containing the indicated concentration of glucose with or without epalrestat for at least two passages before PKC activity measurement as described in "Methods." *P<.05.

Effect of Aldose Reductase Inhibitor Added In Vitro on Glucose-Induced Metabolic Changes
Fructose levels were significantly increased from 13±2 µmol/L (5.6 mmol/L glucose) to 25±2 µmol/L (22.2 mmol/L glucose) (n=6, P<.05). Epalrestat 1 µmol/L significantly decreased glucose-induced increase in fructose levels from 28±2 µmol/L (22.2 mmol/L glucose) to 16±3 µmol/L (22.2 mmol/L glucose plus epalrestat) (n=6, P<.05).

Effect of Elevated Glucose and Aldose Reductase Inhibitors on Cytosolic Redox State of VSMCs
To obtain a more reliable assessment of VSMC cytosolic NADH/NAD⁺, lactate/pyruvate ratios were measured in VSMCs separated from culture medium. As shown in Fig 6, lactate/pyruvate ratios in 5 mmol/L glucose-induced VSMCs were almost the same as those in extracts in VSMC plus medium. Nevertheless, the ratios increased 30% after 48 hours of incubation of 22.2 mmol/L versus 5.6 mmol/L glucose, closely corresponding to the increases (35%) observed in VSMC plus medium after the same duration of incubation. Epalrestat 1 µmol/L prevented this increase (Fig 6).

View larger version (17K): [in this window] [in a new window]   Figure 6. Plots show effect of 5.6 mmol/L or 22.2 mmol/L glucose±epalrestat 1 µmol/L on the VSMCs plus medium lactate/pyruvate ratio (A) or on the VSMC lactate/pyruvate ratio (B). VSMCs in 5.6 or 22.2 mmol/L glucose±epalrestat, an ARI, were incubated for 48 hours. Data are expressed as mean±SD of six determinations. *P<.05.

	Discussion

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In the present study, we have shown that high glucose induces hyperplasia and hypertrophy of VSMCs in culture. This study confirms the previous report of the high glucose–induced mitogenic and hypertrophic actions.⁴ Furthermore, in the present study, we have demonstrated for the first time that epalrestat, an ARI, prevents high glucose–induced hyperplasia and hypertrophy of VSMCs in a concentration-dependent manner. Aldose reductase was expressed in rat cultured VSMCs.⁸ It also has been reported that plasma glucose levels were 29.8±2.6 mmol/L for streptozotocin-induced diabetic rats.²² Glucose-induced increase in cell number was observed in 11.1 to 22.2 mmol/L. The concentration of epalrestat used in this study is within the circulating level after the administration.²³ Thus, it seems plausible to speculate that an ARI, epalrestat, may prevent vascular hypertrophy and hyperplasia in vivo. However, we should be careful about translating our results into findings in humans. It has been reported that early diabetes mellitus in rats is associated with increased blood flow,²⁴ which can be contrary to high glucose–induced vascular hyperproliferation and hypertrophy.

Cultured VSMCs of bovine,²⁵ porcine,⁴ and rat²⁶ origin grown in the presence of high glucose media have been shown to exhibit increased activity of membrane-bound PKC. This has been attributed to an increased formation of diacylglycerol from glycolytic intermediates such as dihydroxy-acetone phosphate and glyceraldehyde-3-phosphate.²⁴ Involvement of PKC in cell growth and proliferation has been postulated. Indeed, it has been reported that PMA increases the rate of DNA, RNA, and protein synthesis.²⁷ In the present study, epalrestat also inhibited the high glucose–induced PKC activation. To our knowledge, this is the first report that an ARI decreases membrane-bound PKC activity. Thus, it has been postulated that high glucose increases PKC activity through the polyol pathway in VSMCs, which results in the hyperplasia and hypertrophy of VSMCs.

The mechanisms of this antiproliferative and antihypertrophic action of an ARI, epalrestat, remain to be elucidated. However, as shown in Fig 5, high glucose may act as a pseudohypoxic agent. In hypoxic tissues, vascular changes are closely related to an increase in NADH/NAD⁺ (because of impaired oxidation of NADH to NAD⁺) and associated metabolic imbalances. An increase in cytosolic-free NADH/NAD⁺ also is observed in tissues exposed to elevated glucose levels at normal tissue PO₂. In contrast to hypoxia, the redox imbalance induced by elevated glucose levels is largely the result of increased oxidation of sorbitol to fructose coupled to reduction of NAD⁺ to NADH in the second step of the sorbitol pathway (Fig 5). Thus, chronic glucose treatment increases the intracellular sorbitol level as already reported.²⁸ An increased sorbitol level increases D-fructose and NADH, which results in the increase in the ratio of NADH/NAD⁺ (Fig 6). Increased NADH/NAD⁺ accelerates the pathway from the dihydroxyacetone phosphate to phosphatidic acid, which increases the diacylglycerol levels and results in the PKC activation (Fig 5). An ARI, epalrestat, may prevent the accumulation of intracellular sorbitol, which decreases the ratio of NADH/NAD⁺ as shown in Fig 6 and results in the decrease in phosphatidic acid. As a result, PKC activity enhanced by high glucose was significantly decreased, which may play some role in antihypertrophic as well as antihyperproliferative effects on VSMCs induced by high glucose.

View larger version (37K): [in this window] [in a new window]   Figure 5. Schematic shows imbalances in glucose, sorbitol, and lipid metabolism linked to hyperglycemic pseudohypoxia and vascular remodeling. Pharmacological interventions of ARIs that prevent glucose-induced vascular remodeling are shown.

Glucose 22.2 mmol/L did not change the cell cycle (data not shown). However, PDGF 1 ng/mL significantly changed the cell cycle from G₀-G₁ stage to S and G₂-M stage, suggesting that PDGF is a competent growth factor.²⁹ Glucose 22.2 mmol/L with PDGF significantly increased VSMCs in the S and G₂-M stages, suggesting that glucose is not a competent growth factor but a progression growth factor. The ARI epalrestat 1 µmol/L inhibited this increase. Since epalrestat decreases membrane-bound PKC activity, it is plausible to speculate that high glucose-induced change in cell cycle is due to PKC activation and that this is blocked by an ARI possibly through PKC suppression. In fact, the published results obtained with synthetic inhibitors of PKC show a clearer picture. H-7 inhibited the restimulation of quiescent rat VSMCs by both PDGF and epidermal growth factor.³⁰ Staurosporine was effective in quiescent rabbit VSMCs restimulated with serum,³¹ and K252a had similar effects in bovine carotid VSMCs.³² We have also obtained the results that a PKC inhibitor, PKC(19-36) 1 µmol/L, prevents the hyperproliferation and hypertrophy induced by chronic high glucose (data not shown).

PKC-mediated inhibition of VSMC proliferation has been reported.³³ A previous study used phorbol esters to elucidate the regulatory roles of PKC in cell proliferation. However, prolonged incubation with phorbol esters leads to downregulation of PKC activity, making studies with these esters difficult to interpret. In contrast, elevations in extracellular glucose induce sustained increases in PKC activity and thus differ markedly from the responses observed with hormonal and pharmacological stimulation of PKC.

In summary, an ARI, epalrestat, prevents vascular hyperproliferation and hypertrophy induced by high glucose. This action may be, at least in part, mediated by the suppression of PKC activation by high glucose.

	Selected Abbreviations and Acronyms

 

ARI = aldose reductase inhibitor DMEM = Dulbecco's modified Eagle's medium FCS = fetal calf serum PBS = phosphate-buffered saline PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate VSMC = vascular smooth muscle cell

View this table: [in this window] [in a new window]   Table 2. DNA Histogram Analysis of Cell Cycle by Flowcytometry

	Acknowledgments

 
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, by Osaka City University Medical Research Foundation, and by Japan Research Foundation for Chronic Diseases and Rehabilitation (RFCDR-Japan). The authors would like to thank Atsumi Ohnishi for excellent technical assistance.

	Footnotes

 
Reprint requests to Kenichi Yasunari, MD, Division of Hypertension and Atherosclerosis, First Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan.

Received April 4, 1995; accepted June 9, 1995.

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