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Abstract
Background
Oxidative stress is thought to be one of the causative factors contributing to macrovascular complications in diabetes. However, the mechanisms of development and progression of diabetic vascular complications are poorly understood. We hypothesized that PKC-δ isozyme contributes to ROS generation and determined their roles in the critical intermediary signaling events in high glucose-induced proliferation of vascular smooth muscle (VSM) cells.
Methods
We treated primary cultured rat aortic smooth muscle cells for 72 hours with medium containing 5.5 mmol/L D-glucose (normal glucose), 30 mmol/L D-glucose (high glucose) or 5.5 mmol/L D-glucose plus 24.5 mmol/L mannitol (osmotic control). We then measured cell number, BrdU incorporation, cell cycle and superoxide production in VSM cells. Immunoblotting of PKC isozymes using phoshospecific antibodies was performed, and PKC activity was also measured.
Results
High glucose increased VSM cell number and BrdU incorporation and displayed significantly greater percentages of S and G2/M phases than compared to 5.5 mmol/L glucose and osmotic control. The nitroblue tetrazolium (NBT) staining in high glucose-treated VSM cell was more prominent compared with normal glucose-treated VSM cell, which was significantly inhibited by DPI (10 µM), but not by inhibitors for other oxidases. High glucose also markedly increased activity of PKC-δ isozyme. When VSM cells were treated with rottlerin, a specific inhibitor of PKC-δ or transfected with PKC-δ siRNA, NBT staining and NAD(P)H oxidase activity were significantly attenuated in the high glucose-treated VSM cells. Furthermore, inhibition of PKC-δ markedly decreased VSM cell number by high glucose.
Figures and Tables
Fig. 1
Effect of high glucose on vascular smooth muscle cell proliferation (cell count) (A) and DNA synthesis (BrdU incorporation) of rat VSM cells (B).
Each VSM cells were incubated with media containing normal glucose (5.5 mmol/L D-glucose) or high glucose (30 mmol/L D-glucose) up to 72 h. At the end of incubation, cell number was measured by counting trypsinized cells with a hematocytometer and the incorporation of BrdU into VSM cells was examined using a microplate reader.
Data are obtained from three separate experiments and are expressed in the mean ± SE.
*p < 0.05 compared with normal glucose for 24 h.
†p < 0.01 compared with normal glucose for 72 h.
‡p < 0.01 compared with normal glucose for 72 h.
Fig. 2
Effect of various inhibitors such as DPI (10 µmol/L), NAC (10 µmol/L), SOD (500 U/mL), L-NAME (10 µmol/L), and allopurinol (10 µmol/L) on superoxide generation in normal and high glucose-treated VSM cells.
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.
Fig. 3
Effects of various inhibitors, 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 VSM cells.
Data are shown as mean ± SE from 4 experiments in each group.
*p < 0.01 vs. normal glucose.
†p < 0.01 vs. vehicle.
Fig. 4
Effects of high glucose on in vitro PKC activity in VSM cells.
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.
Fig. 5
Effects of high glucose on in vitro PKC activity and isozyme selective inhibition of PKC in VSM cells.
Confluent cultured VSM cells were treated with high glucose condition for 72 h. To verify the efficacy and selectivity of isozyme specific PKC inhibitors, VSM cells were treated with G6976 (1 µmol/L) and GF109203X (10 µmol/L) (A), rottlerin (3 µmol/L) and GF109203X (10 µmol/L) (B) 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-α and PKC-βII (A), PKC-δ and PKC-ζ (B). 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 < 0.05 compared with high glucose without pretreatment with G6976.
†p < 0.01 compared with normal glucose.
‡p < 0.01 compared with high glucose without pretreatment with rottlerin.
Fig. 6
Effect of selective PKC-δ inhibitor or PKC-δ siRNA on p38 MAP kinase. Quiescent VSM cells were pretreated with rottlerin (3 µmol/L) for 1 h or transfected with PKC-δ siRNA prior to stimulation with high glucose for 72 h. p38 MAP kinase activiy was assessed by immunoblotting with antibodies specific for phosphorylated p38 MAP kinase.
The bar graph represents the mean fold increases in three separate experiments.
Results are expressed as the mean ± SE.
*p < 0.01 compared with normal glucose.
†p < 0.01 compared with high glucose without treatment.
Fig. 7
Effect of transient transfections of PKC-δ siRNA on cellular superoxide production in normal and high glucose-treated VSM cells. Superoxide production was measured by NBT reduction.
Results are presented as means ± SEM for three independent experiments.
*p < 0.05 vs. normal glucose.
†p < 0.01 vs. vehicle.
Fig. 8
Effect of selective PKC inhibitor or PKC-δ siRNA on VSM cell proliferation (cell count). Quiescent VSM cellswere pretreated with rottlerin (3 µmol/L) for 1 h or transfected with PKC-δ siRNA prior to stimulation with high glucose for 72 h. At the end of incubation, cell number was measured by counting trypsinized cells with a hematocytometer.
Results are obtained from three separate experiments and are expressed in the mean ± SE.
*p < 0.01 compared with normal glucose.
†p < 0.05 compared with high glucose without treatment.
Table 1
Effect of High Glucose on the Cell Cycle Analysis Using Flow Cytometry
Cells were grown in 25-mm2 flasks and synchronized, and then the cell cycle was initiated by incubation with DMEM containing normal or high glucose for 72 h. The distribution of the cell cycle was examined as described in METHODS. Data are expressed as the percent distribution of cell cycle phases G0/G1, S, and G2/M in normal and high glucose. Similar results were obtained in three separate experiments.
*p < 0.01 compared with normal glucose.
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