Fenofibrate Improves Vascular Endothelial Function In Diabetic Mice

Background and purpose:Recently high morbidity and mortality of diabetes mellitus has heavily burdened people economically and mentally around the world. Microvascular and macrovascular complications were major causes of disability and death in diabetic patients. High blood glucose sabotaged the integrity of blood vessels and induced endothelial dysfunction. Therefore, it is of great importance to improve endothelial function in diabetic individuals.Fenofibrate (FF) is an agonist of peroxisome proliferator-activated receptor a (PPARa), clinically used for primary hypercholesterolemia or mixed dyslipidemia. It is reported fenofibrate plays an important role in apoptosis and inflammation. In 2010, the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial conducted by Institute of Cardiology demonstrated that fenofibrate reduced the incidence of cardiovascular events in diabetes patients.Nitric oxide (NO), which could be produced by endothelial nitric oxide synthase (eNOS), is involved in the regulation of vascular function. Physiologically, NO suppresses inflammation and vascular hyperplasia. Endothelial dysfunction could induce oxidative stress with large amount of super oxide anion (O2-), which accelerates NO inactivation and decreases NO bioavailability.Adenosine monophosphate activated protein kinase (AMPK) is a critical regulator in energy metabolism and could be activated by oxidative stress. AMPK keeps the balance of blood glucose and regulates the expression of downstream eNOS. It was demonstrated that activation of AMPK ameliorated vascular dysfunction by increasing the phosphorylation of eNOS. To understand the cardiovascular protective effect of fenofibrate, the current study was designed to investigate the effects of fenofibrate on endothelial dysfunction in streptozotocin (STZ)-induced diabetic C57 mice and in high glucose-induced mouse aortic endothelial cells (MAECs) injury, focusing on the role of fenofibrate on AMPK and eNOS.Methods:1. In-vivo experimentC57 mice aged 4 weeks old were randomly divided into three groups:control group (Control), STZ-induced diabetic group (STZ), fenofibrate treated diabetic group (STZ+ FF). STZ-induced diabetic model was established by intraperitoneal injection of STZ (dissolved in sodium citrate buffer) with a dose of 60 mg/kg for consecutive 5 days. After 4 weeks, mice in STZ+FF were administrated fenofibrate of 100 mg/kg/d in gavage for consecutive 14 days. Level of serum triglyceride (TG) was quantified using TG test kits and level of serum high-density lipoprotein cholesterol (HDL-C) was quantified using HDL-C test kits. Endothelial function of extracted mouse aorta was examined by evaluating acetylcholine (ACh)-induced endothelium-dependent relaxation combined with phenhylephrine (Phe)-induced vasoconstriction and sodium nitroprusside (SNP)-induced endothelium-independent relaxation. Superoxide onion (02’) was determined using dihydroethidium (DHE) staining of aorta. Expression of eNOS and phosphorylated eNOS were examined by Western blotting.In-vitro experimentMouse aortic endothelial cells were identified as vWF positive cells using immunofluorescence. MAECs cultured in vitro was divided into 3 groups and treated with normal glucose (5.5 mmol/L), high glucose (HG 33mmol/L), HG+FF (33 mmol/μM+5μM) for 24 h respectively. Adhesion of MAECs was assessed by vitronectin assay. Matrigel-based tube formation assay was adopted to evaluate the angiogenesis of MAECs. Intracellular NO level was detected by DAF-FM staining and intracellular O2- level was detected by DHE staining. Expression of eNOS and AMPK were examined by Western blotting.Results:Role of fenofibrate on endothelium-dependent vasodilation in STZ-induced diabetic miceFenofibrate decreased the level of serum TG in diabetic miceSerum TG level was significantly increased in STZ group compared to Control, and fenofibrate treatment significantly decreased the level of TG in STZ+FF group compared to STZ group. Fenofibrate increased the level of serum HDL-C in diabetic miceSerum HDL-C level did not change in STZ group compared to Control, and fenofibrate treatment significantly increased the level of HDL-C in STZ+FF group compared to STZ group.Fenofibrate ameliorated impaired endothelium-dependent relaxation in diabetic miceACh-induced endothelium-dependent relaxation in mouse aorta was markedly impaired in diabetic mice, and fenofibrate treatment significantly ameliorated diabetes-impaired ACh-induced endothelium-dependent relaxation in mouse aorta. Fenofibrate did not alter Phe-induced vasoconstriction and SNP-induced endothelium-independent relaxation. L-NAME cancelled the protective effect of fenofibrate on endothelium-dependent relaxation. It was demonstrated that fenofibrate ameliorated impaired endothelium-dependent relaxation in diabetic mice. Fenofibrate decreased the level of intracellular O2- in diabetic mouse aortaIntracellular O-" level was significantly increased in diabetic mice aorta compared with control, and fenofibrate treatment significantly decreased the level of intracellular O2- in STZ+FF compared to STZ. Fenofibrate did not alter the expression of eNOS in diabetic mouse aortaExpression of eNOS and phosphorylation of eNOS had no statistical significance among the three groups.Role of fenofibrate on endothelial dysfunction induced by high glucose Cell identificationMouse aortic endothelial cells cultured in vitro was stained using vWF and Hochest 33258. Double positive cells accounted for 82%. Fenofibrate improved adhesion ability in high glucose-treated MAECsAdhesion ability was significantly impaired by high glucose treatment compared with control, and fenofibrate treatment significantly improved the ability of adhesion in HG+ FF group compared to HG group. Fenofibrate improved tube formation ability in high glucose-treated MAECsTube formation ability was significantly impaired by high glucose treatment compared with control, and fenofibrate treatment significantly improved the ability of tube formation in HG+FF group compared to HG group. Fenofibrate increased the level of intracellular NO in high glucose-treated MAECsIntracellular NO level was significantly decreased in high glucose-treated MAECs compared with control, and fenofibrate treatment significantly increased the level of intracellular NO in HG+FF group compared to HG group.5) Fenofibrate increased the level of intracellular O2- in high glucose-treated MAECs Intracellular O2- level was significantly increased in high glucose-treated MAECs compared with the control, and fenofibrate treatment significantly decreased the level of intracellular O2- in HG+FF group compared to HG group.6) Fenofibrate increased phosphorylation of eNOS and AMPK in high glucose-treated MAECsExpression of p-eNOS and p-AMPK were significantly increased in high glucose-treated MAECs compared with control, and fenofibrate treatment significantly decreased phosphorylation of eNOS and AMPK in HG+FF group compared to HG group.Conclusion:Fenofibrate could ameliorate endothelium-dependent vasodilation in diabetic mice and relieve high glucose-induced endothelial dysfunction, which was possibly related to activation of eNOS and AMPK phosphorylation.