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Abstract
Background
Oxidative stress contributes to vascular diseases in patients with diabetes. As the mechanism of development and progression of diabetic vascular complications is poorly understood, this study was aimed to assess the potential role of hyperglycemia-induced oxidative stress and to determine whether the oxidative stress is a major factor in hyperglycemia-induced migration of vascular smooth muscle cells (VSMCs).
Methods
We treated primary cultured rat aortic smooth muscle cells for 72 hours with medium containing 5.5 mM D-glucose (normal glucose), 30 mM D-glucose (high glucose) or 5.5 mM D-glucose plus 24.5 mM mannitol (osmotic control). We measured the migration of VSMCs and superoxide production. Immunoblotting of PKC isozymes using phoshospecific antibodies was performed, and PKC activity was also measured.
Results
Migration of VSMCs incubated under high glucose condition were markedly increased compared to normal glucose condition. Treatment with diphenyleneiodonium (DPI, 10 µmol/L) and superoxide dismutase (SOD, 500 U/mL) significantly suppressed high glucose-induced migration of VSMCs. Superoxide production was significantly increased in high glucose condition and was markedly decreased after treatment with DPI and SOD. High glucose also markedly increased activity of PKC-δ isozyme. When VSMCs were treated with rottlerin or transfected with PKC-δ siRNA, nitro blue tetrazolium (NBT) staining and NAD(P)H oxidase activity were significantly attenuated in the high glucose-treated VSMCs. Furthermore, inhibition of PKC-δ markedly decreased VSMC migration by high glucose.
Figures and Tables
 | Fig. 2Effect of high glucose on the migration of VSMCs. Data are shown as the mean ± SE from 4 experiments in each group. *P < 0.01 vs. normal glucose. NG, normal glucose; HG, high glucose.
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 | Fig. 3Effect of high glucose on the ROS production in vascular smooth muscle cell. Dihydroethidium (DHE) staining for superoxide anion (×100).
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 | Fig. 4Effect of various inhibitors such as DPI, NAC, SOD, L-NAME, and allopurinol on the superoxide generation in normal and high glucose-treated vascular smooth muscle cells (VSMCs). Data are shown as mean ± SE from 4 experiments in each group. *P < 0.05 vs. normal glucose. †P < 0.01 compared with corresponding values in each vehicle.
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 | Fig. 5Effects of various inhibitors, such as such as DPI (10 µmol/L), SOD (500 U/mL), allopurinol (Allo, 100 µmol/L), rotenone (Rot, 100 µmol/L), L-NAME (NMA, 10 µmol/L), and indomethacin (IND, 10 µmol/L), NADH-stimulated superoxide production in normal and high glucose-treated VSMCs. Data are shown as mean ± SE from 4 experiments in each group. *P < 0.01 vs. normal glucose. †P < 0.01 vs. vehicle (veh).
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 | Fig. 6Effects of high glucose on in vitro PKC activity in VSMCs. Results were expressed as a mean percentage of 5.5 mmol/L D-glucose ± SE from 4 independent experiments. *P < 0.01 vs. normal glucose (5.5 mM).
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 | Fig. 7Effects of high glucose on in vitro PKC activity and isozyme selective inhibition of PKC-δ in VSMCs. A. Confluent cultured VSMCs were treated with high glucose condition for 72 h. To verify the efficacy and selectivity of isozyme specific PKC inhibitor, VSMCs were treated with rottlerin (3 µmol/L) for 1 h before incubation with high glucose for 72 h, lysed and immunoblotted by using PKC isozyme-specific antibodies recognizing phosphorylated forms of PKC-δ. B. Quiescent VSMCs were transfected with PKC-δ siRNA prior to stimulation with high glucose for 72 h. Total PKC isozymes in the immunoprecipitates were verified by immunoblotting with selective antibodies. The bar graph represents the mean ± SE of three separate experiments. *P < 0.01 compared with normal glucose. †P < 0.01 compared with high glucose without pretreatment with rottlerin.
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 | Fig. 8Effect of transient transfections of PKC-δ siRNA on cellular superoxide production in normal and high glucose-treated VSMCs. Superoxide production was measured by NBT reduction. Results are presented as means ± SE for three independent experiments. *P < 0.05 vs. normal glucose. †P < 0.01 vs. vehicle.
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 | Fig. 9Effect of DPI, selective PKC inhibitor or PKC-δ siRNA on VSMC migration.Quiescent VSMCs were pretreated with DPI (10 µmol/L), rottlerin (3 µmol/L) for 1 h or transfected with PKC-δ siRNA prior to stimulation with high glucose. Results are obtained from three separate experiments and are expressed in the mean ± SE. *P < 0.05 compared with high glucose without treatment.
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