| dc.description.abstract | Type 2 diabetes (T2DM) is associated with microvascular complications i.e. diabetic retinopathy, nephropathy and neuropathy, each having life altering effects. Dysfunction of the endothelial cells (ECs) that line all blood vessels plays a critical role in the pathogenesis of these conditions. Current diabetic therapies aim to normalise glycaemia to reduce the widespread pathogenic effects of raised glucose, including hyperglycaemia mediated damage to the vasculature and ECs. However, therapies that directly improve endothelial function could be hugely beneficial to individuals with diabetes. In particular, if an existing treatment has vascular benefits then it could be administered to targeted patients rapidly without the need for lengthy clinical trials. Increasing evidence suggests that incretin-based anti-diabetic therapies (GLP1 analogues: exenatide and liraglutide; and dipeptidyl peptidase IV enzyme (DPPIV) inhibitors: sitagliptin and vildagliptin) possess vascular effects beyond promoting insulin secretion. Several large clinical studies have reported that the GLP-1 analogues and DPPIV inhibitors have ameliorative effects on cardiovascular events in individuals with T2DM, supporting previous in-vitro and animal studies which show potential benefits of the therapies on EC function. However, very little of the available data examines the effects of the therapies on the microvasculature in humans and since the microvascular complications are so impactful to patients, a greater understanding of this area is critical. Thus the aim of this thesis was to examine the effect of the naturally occurring incretin GLP-1 7-36 and anti-diabetic therapies (GLP-1 analogues: exenatide and liraglutide; and dipeptidyl peptidase IV enzyme (DPPIV) inhibitors: sitagliptin and vildagliptin) on microvascular EC responses, specifically proliferation, NO production and inflammatory responses, as assessed by alteration of surface levels of the key adhesion molecules ICAM-1 and E-selectin. All studies were carried out using a human cerebral microvascular EC cell line – HCMEC/D3. Proliferation was investigated using commercially available WST-8, BrdU and CyQUANTR DNA staining assays in addition to manual cell counts. Activation of pro-proliferative signalling pathways in response to the incretin-based treatments was examined using a commercially available phosphokinase array and phospho-specific ELISAs. Activation of the NO pathway was examined by quantifying nitrate as a surrogate for NO and also by studying eNOS phosphorylation using a specific ELISA. Changes in EC inflammatory responses were examined using cell based ELISAs to quantify changes in surface levels of the adhesion molecules E-selectin and ICAM-1. These studies showed, for the first time, that GLP-1 7-36 and incretin-based treatments increased microvascular EC proliferation and that the intracellular proliferative pathways activated included upregulation (as assessed by changes in phosphorylation) of AKT, ERK1/2 and PRAS40. Further investigation using specific inhibitors indicated PRAS40 phosphorylation was significantly reduced by the inclusion of the AKT inhibitor MK2206 for all treatments, indicating that AKT activation was upstream of PRAS40. Since PRAS40 is known to be involved in mTORC1 activation these data suggest that the incretin-based therapies induce proliferation, at least in part, by activation of the AKT / PRAS40 / mTORC proliferative pathway. This was supported by the observation that proliferation, as assessed by BrdU inclusion, induced by all treatments was significantly inhibited by rapamycin, a specific inhibitor of the mTORC pathway. Interestingly, studies using the GLP-1 receptor (GLP-1R) inhibitor, exendin 9-39 indicated that only GLP-1 7-36 induced BrdU incorporation was GLP-1R dependent. NO production was increased by GLP-1 7-36 and all incretin-based therapies studied, as was eNOS phosphorylation, suggesting that incretin-based treatments promote NO production in microvascular ECs. Unexpectedly, all incretin-based treatments studied induced a significant increase in surface levels of ICAM-1 on the surface of HCMEC/D3 cells and nonsignificant increases in levels of E-selectin. Conversely, none of the treatments altered TNF-α induced increases in ICAM-1 or E-selectin. These data, suggest that the incretin-based treatments do not have an anti-inflammatory effect in HCMEC/D3 cells, and instead are actually pro-inflammatory In conclusion, the naturally occurring incretin GLP-1 7-36 and incretin-based therapies have profound effects on HCMEC/D3 microvascular ECs. This thesis showed, for the first time that the therapies not only induce proliferation by a mechanism that appears to involve the AKT / PRAS40 / mTORC signalling cascade, but that they also induce NO production in these cells. Modulation of both of these cellular responses could be highly beneficial therapeutically, beyond the known antihyperglycaemic effects of the therapies. In contrast, the observation that these therapies may have a pro-inflammatory effect on microvascular ECs is clearly undesirable. Further studies to elucidate the effects of the therapies on microvascular ECs are required to fully understand whether these incretin-based therapies could actually ameliorate the microvascular compilations of T2DM. | en_GB |
