Kohlhaas, Christine Frederike
Metabolic regulation of human vascular endothelial cell function in vitro.
PhD thesis, University of Glasgow.
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The vascular endothelium contributes to the maintenance of vascular health by regulating vascular tone and leukocyte adhesion, amongst others. The vasoregulatory actions of the endothelium are mediated through coordinated release of vasodilators such as nitric oxide (NO) and prostacyclin, and vasoconstrictors such as endothelin-1 and thromboxane A2. Endothelial NO is the principal vasodilator in the vasculature and is produced by endothelial nitric oxide synthase (eNOS). Insulin is a vasoactive hormone that exerts its vasodilatory effects through eNOS-mediated NO production. Endothelial function is impaired in a number of disorders, including insulin resistance, diabetes and atherosclerosis, leading to dysregulated vasodilation as well as increased monocyte adhesion and plaque formation (atherosclerosis). The underlying molecular mechanisms leading to endothelial dysfunction are still in question.
The work presented in this thesis addressed this question by investigating how insulin signalling and eNOS-mediated NO and superoxide production in human vascular endothelial cells are affected under experimental hyperinsulinaemia (chapter 3) and experimental hyperglycaemia (chapter 4). Atherogenic processes in human aortic endothelial cells (HAEC) were investigated by assessing monocyte adhesion under experimental hyperinsulinaemia (chapter 3), and by determining the contribution of NO and AMP-dependent kinase (AMPK) activity to the regulation of endothelial chemokine production (chapter 6). The potential of insulin to modify the subcellular distribution of eNOS was investigated in chapter 5.
Clinical hyperinsulinaemia correlates with attenuated NO-mediated vasodilation, but it is not clear how hyperinsulinaemia impairs eNOS-mediated NO production. The present study modelled hyperinsulinaemia in HAEC and demonstrated a blunted response of hyperinsulinaemic cells to Ca2+-stimulated, but not insulin-stimulated eNOS-mediated NO synthesis. To address the underlying mechanisms responsible, the protein expression levels of components of the metabolic and mitogenic insulin signalling pathways, and of the metabolic energy sensor, AMPK, were quantified. Experimental hyperinsulinaemia slightly and non-significantly increased basal and insulin-stimulated eNOSS1177 phosphorylation in a time-dependent manner, and the levels of eNOST495 increased following acute insulin stimulation under these conditions. No marked dysregulation of individual insulin signalling pathway components was identified as a potential cause, but increased activating AMPKT172 phosphorylation was found to be a potential cause of increased unstimulated eNOSS1177 phosphorylation under experimental hyperinsulinaemia. Monocyte adhesion to hyperinsulinaemic HAEC did not differ from control HAEC, indicating that experimental hyperinsulinaemia did not act as a proatherogenic factor in the present study.
Overt diabetes was simulated by experimental hyperglycaemia in human umbilical vein endothelial cells (HUVEC) and its effect on insulin-stimulated eNOS phosphorylation and endothelial superoxide production assessed. Insulin tended to stimulate phosphorylation of eNOSS615 and eNOSS1177, and decrease phosphorylation of eNOSS114, eNOST495 and eNOSS633 under control conditions. Experimental hyperglycaemia slightly reduced basal phosphorylation of Ser633 and significantly reduced insulin-stimulated phosphorylation of Ser114, but mildly increased basal Ser615 phosphorylation, indicating some dysregulation of eNOS phosphorylation. The upstream components of the metabolic insulin signalling pathway were not impaired in hyperglycaemic conditions.
The subcellular localisation of eNOS is thought to have implications for its function. This study showed that eNOS localises to the plasma membrane, the nucleus, the cytoplasm and, primarily, the perinuclear area of HAEC. Insulin stimulation did not affect this distribution. Phospho-eNOS species were found primarily at the plasma membrane, and insulin may modulate the abundance of an intracellular eNOST495 species.
Previous work in our laboratory on AMPK-mediated reduction of adhesion molecule expression has lead to the investigation of AMPK- and NO-mediated regulation of chemokine production in the present study. Inhibition of NO synthesis increased the production of monocyte chemoattractant protein (MCP)-1 in HAEC. AMPK activity downregulated TNFα-stimulated MCP-1 expression, and this was NO-dependent in the short-term, but may be NO-independent during prolonged AMPK activation. These data implicate NO and AMPK as antiatherogenic mediators in vascular endothelial cells in vitro.
Taken together, the data in this thesis provide further insight into some of the molecular mechanisms involved in endothelial function and their response to hyperinsulinaemia, hyperglycaemia and proatherogenic stimulation.
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