| Popis: |
Endothelial dysfunction, characterised by decreased NO synthesis, is associated with an increased risk of cardiovascular complications in patients with type 1 diabetes mellitus, type 2 diabetes mellitus (T2DM) and insulin resistance. The dysfunctional endothelium exhibits impaired insulin-stimulated NO generation and vasodilatation, as well as increased pro-inflammatory and pro-atherogenic signalling, which promote the development of atherosclerosis and hypertension. Despite cardiovascular complications being the leading cause of mortality and morbidity in people with diabetes and insulin resistance, the mechanism of underlying macrovascular endothelial dysfunction is poorly understood. In this study, the effects of experimental hyperglycaemia (eHG) and hyperinsulinaemia (eHI) were examined on endothelial cell phenotype and NO production in cultured human arterial endothelial cells. eHG markedly impaired ionomycin-stimulated NO production and insulin-stimulated eNOS activation and NO production in human aortic endothelial cells (HAECs), having similar effects in human coronary artery endothelial cells (HCAECs). IR/Akt/eNOS pathway activation and calcium signalling were preserved in eHG, suggesting that eHG targets eNOS directly, decreasing enzymatic activity. In addition to diminished eNOS function, eHG decreased mitochondrial activity in HAECs while preserving total mitochondrial mass and morphology. Intriguingly, inhibition of insulin-stimulated NO production and mitochondrial function by eHG were rescued by an AMP-activated protein kinase (AMPK) activator, AICAR, yet, the AMPK-dependence of these effects remains to be investigated. Ionomycin-stimulated NO production was impaired and insulin-stimulated NO production tended to reduce in HAECs cultured in eHI media. eHI decreased insulin-stimulated endothelial NO synthase (eNOS) activation, suggesting that the decrease in NO bioavailability in eHI is due to impaired eNOS activation. Furthermore, eHI abolished acute insulin-stimulated NO synthesis and phosphorylation of protein kinase B (Akt) Ser473 and eNOS Ser1177 in HAECs, and also tended to increase basal Akt Ser473, eNOS Ser1177 and G-protein-coupled receptor kinase interacting protein 1 (GIT1) Tyr554 phosphorylation, implying an impairment of insulin signalling. The inhibition of insulin-stimulated NO production by eHI was restored completely by GIT1 downregulation with siRNA and partially restored by inhibiting c-Src-dependent GIT1 Tyr554 phosphorylation. Desensitisation of the insulin receptor (IR)/Akt/eNOS pathway and altered GIT1-eNOS binding dynamics may therefore contribute to the decreased eNOS activity in eHI. Neither eHI nor eHG had any marked effect on reactive oxygen species generation or proinflammatory signalling yet von Willebrand factor levels and secretion were augmented in eHI-treated HAECs. Therefore, chronic insulin treatment may increase the risk of thrombosis by promoting coagulation. The effects of eHI and eHG were observed to partially replicate phenotypical changes observed in HAECs from donors with T2DM. HAECs from people with T2DM exhibited impaired insulin- and ionomycin-stimulated NO generation, decreased insulin-stimulated GIT1-eNOS binding and reduced eNOS Ser1177 phosphorylation. Unlike eHI and eHG, HAECs from people with T2DM also exhibited increased H2O2 generation and mitochondrial network fragmentation and loss of endothelial cell identity, suggesting that T2DM HAECs represent a more profound state of diabetic endothelial dysfunction. Overall, the results presented in this thesis demonstrate that eHG and eHI impair eNOS function and NO production in cultured HAECs, mimicking in part the phenotype observed in T2DM HAECs. Moreover, altered GIT1-eNOS binding and diminished mitochondrial function may contribute to diabetic endothelial dysfunction, yet, further investigation of GIT1 and mitochondrial function in eNOS regulation is necessary to design an effective therapeutic strategy. |
