Molecular Mechanisms Involved In Hyperglycemia-induced Vascular Endothelial Dysfunction And The Protective Role Of Resveratrol

BackgroundEpidemiologic data indicate that the risk of developing atherosclerosis and cardiovascular complications is 3-fold to 4-fold higher for patients with type 2 diabetes compared to the general population, and that 80 percents of diabetic patients dies of cardiovascular diseases. Endothelial dysfunction (ED) plays a key role in the initial stage of atherosclerosis, and is involved in the whole process of atherosclerotic lesions. Hyperglycemic can increase the production of reactive oxygen species (ROS) and decrease the nitric oxide (NO) bioavailability, and subsequently induce the vascular ED. The strategies for the protection of vascular endothelial function may reduce the risk of atherosclerosis and its disability rate and mortality in patients with diabetes.Resveratrol, trans-3, 4', 5-trihydroxyestilbene, is a kind of natural polyphenols produced by several plants, such as grape, mulberry, peanut and giant knotweed rhizome. The unique anti-oxidative effect of resveratrol provides a comprehensive protective role in cardiovascular system. Recently, increasing evidences suggest that resveratrol plays a beneficial effect against diabetes and cardiovascular diseases. Some cellular and animal studies suggest that the beneficial effects of resveratrol on plasma glucose control and vascular protection require AMP-activated protein kinase (AMPK) activity.AMPK, a recently indentified key molecule regulating energy metabolism, plays a pivotal role in cellular energy homeostasis and adaptation reaction, as a sensor of cellular energy status. Accumulative studies demonstrate that AMPK is not only an energy sensor but also a powerful effecter, which is involved in several metabolic processes via its phosphorylating action. Although the regulatory mechanism of AMPK activation is not completely elucidated, activation of AMPK can enhance the insulin sensitivity and reduce the complications of type 2 diabetes mellitus through regulating cellular signal transduction, metabolism and gene expression. Increasing evidences suggest that AMPK may be a linker between energy metabolism and circulatory diseases. Recent studies have demonstrated that AMPK can activate endothelial nitric oxide synthesis (eNOS), and consequently inhibit oxidative stress. Although it is reported that resveratrol can protect from cardiovascular complications of diabetes, the underlying precise molecular mechanisms have not been clarified.ObjectivesThe present study was to test the hypotheses: 1) resveratrol can promote the phosphorylation and activation of eNOS, inhibit hyperglycemia-induced overproduction of superoxide anion in cultured human umbilical vein endothelial cells (HUVECs), and finally improve hyperglycemia-induced impairment of endothelium-dependent vasodilatation; 2) the protection effect of resveratrol on the improvement of endothelial dysfunction is mediated by the activation of AMPK.Methods1. After the cultured HUVECs were treated with varying concentrations of resveratrol for different duration time, the expression and phosphorylation of eNOS were measured by Western blotting and the production of NO was detected by Griess assay. Additionally, L-NAME was used to test the involvement of eNOS enzyme in the effect of resveratrol on the production of NO.2. After the cultured HUVECs were incubated with inhibitors of several kinases, the expression and phosphorylation of eNOS were measured by Western blotting and the production of NO was detected by Griess assay.3. Test the effect of resveratrol on the expression and phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) using Western blotting assay. In addition, test the effect of resveratrol on hyperglycemia-induced changes in the expression and phosphorylation of AMPK and ACC by Western blotting.4. Test the effect of high glucose on the production of superoxide anion by Griess assay and the inhibitory effect of resveratrol on hyperglycemia-induced overproduction of superoxide, and investigate the involvement of AMPK in the protective action of resveratrol using AMPK inhibitor compound C (CC). 5. Test the vasodilatation action of resveratrol in phenylephrine constricted mouse aortic rings treated with or without endothelium denudation, L-NAME, indomethacin and CC. Test the effect of resveratrol on acetylcholine (Ach)-induced vasodilatation in mouse aortic rings with or with out incubation of high glucose.Results1. Treatment with resveratrol for 60 min dose-dependently increased the phosphorylation of eNOS in cultured HUVECs. Additionally, resveratrol (30μM) time-dependently increased the phosphorylation of eNOS in cultured HUVECs. Moreover, resveratrol significantly increased the production of NO, which can be attenuated by the incubation of L-NAME, indicating the resveratrol can increase the NO bioavailability through time- and dose-dependently activating eNOS.2. Resveratrol (100μM) significantly increased the production of NO in cultured HUVECs, which can be attenuated by CC (a specific inhibitor of AMPK) but not the inhibitors of PKA and PI3K, indicating that resveratrol-induced eNOS activation and NO production were mediated by AMPK activation.3. Resveratrol significantly enhanced the phosphorylation of AMPK and ACC but not the total expression of AMPK and ACC, while treatment of high glucose remarkably reduced the phosphorylation of AMPK and ACC, which can by reversed by the treatment of resveratrol (30μM), indicating that resveratrol can protect from hyperglycemia-induced reduction in the activation of AMPK through activating ACC.4. Resveratrol significantly inhibited hyperglycemia-induced overproduction of superoxide anion, which can be attenuated by the incubation of CC, indicating that AMPK activation is required in resveratrol-induced reduction in superoxide production.5. Resveratrol dose-dependently dilated the phenylephrine-constricted mouse aortic rings with intact endothelium, which can be partially attenuated by endothelium denudation, L-NAME and CC, but unaffected by indomethacin, indicating that resveratrol-induced vasodilatation is required AMPK/eNOS pathway.6. High glucose but not isotonic mannitol significantly impaired Ach-induced vasodilatation, which can be patially reversed by resveratrol. However, treatment with CC can significantly block the beneficial effect of resveratrol, indicating that resveratrol can protect the aortae from hyperglycemia-induced endothelial dysfunction through activating AMPK.Conclusions1. Resveratrol can promote the phosphorylation and activation of eNOS, increase the NO production, inhibit hyperglycemia-induced overproduction of superoxide anion in cultured human umbilical vein endothelial cells (HUVECs), and finally improve hyperglycemia-induced impairment of endothelium-dependent vasodilatation.2. The protection effect of resveratrol on the improvement of endothelial dysfunction is mediated by the activation of AMPK.