Uncovering the Interaction between Undercarboxylated Osteocalcin and Vascular Function in Normoglycaemic and Hyperglycaemic Environments

Tacey, Alexander ORCID: 0000-0002-0718-9665 (2020) Uncovering the Interaction between Undercarboxylated Osteocalcin and Vascular Function in Normoglycaemic and Hyperglycaemic Environments. PhD thesis, Victoria University.


Endothelial dysfunction is the initiating process in the development of atherosclerosis and cardiovascular disease (CVD) and is a significant predictor of future adverse cardiovascular events. Increasing evidence suggests a link between vascular function and the skeleton, an association that may be mediated by bone-derived proteins such as osteocalcin (OC). OC is an osteoblast-derived, vitamin K-dependent protein that primarily exists in two biological forms. Carboxylated osteocalcin (cOC) is involved in bone formation and undercarboxylated osteocalcin (ucOC) is suggested to be the bioactive form of the protein responsible for regulating energy metabolism and glucose homeostasis. As such, ucOC may be targeted as a therapeutic treatment for metabolic diseases such as diabetes. In humans, the association of OC with endothelial dysfunction and CVD is conflicting, with some suggesting that OC is associated with beneficial effects in the vasculature and others reporting adverse effects. Research in animals suggest that in vivo OC treatment improves vascular function. However, corresponding improvements in metabolic outcomes suggest that the improvements in vascular function may occur indirectly, due to improvements in energy metabolism. As such, the primary aim of this thesis is to investigate if ucOC has a direct biological effect on vascular function in normoglycaemic and hyperglycaemic environments in preclinical models and humans. This was examined in four studies. Study 1: Hyperglycaemia is a pathological condition that has a toxic effect on blood vessels and is a major risk factor for atherosclersosis and CVD. However, it is unclear whether the dysfunction caused by hyperglycaemia is blood vessel specific and whether the dysfunction is exacerbated by an atherogenic diet. It was important to identify which blood vessels developed dysfunction for subsequent studies to assess the vasoactive role of ucOC. Abdominal aorta, iliac and mesenteric arteries were dissected from male New Zealand White Rabbits following either a four week normal or atherogenic diet (n = 6 – 12 per group). The arteries were incubated ex vivo in normal or high glucose solutions (20 mM or 40 mM) for 2 h and isometric tension myography was used to determine endothelial-dependent vasodilation. The atherogenic diet reduced blood vessel relaxation, as measured by area under the curve (AUC), by 25% (p < 0.05) in the aorta, 17% (p = 0.06) in the iliac artery and 40% (p = 0.07) mesenteric artery. In the aorta of the atherogenic diet-fed rabbits the 20 mM glucose incubation altered EC50, thereby reducing the potency of acetylcholine (p < 0.05), and tended to reduce Emax and AUC in the normal diet-fed rabbits. Incubation of the iliac artery from atherogenic diet-fed rabbits in 40 mM glucose also altered EC50, reducing the potency of acetylcholine (p < 0.05). No dysfunction occurred in the mesentery with high glucose incubation following either the normal or atherogenic diet. High glucose-induced endothelial dysfunction appears to be blood vessel specific; the aorta may be the optimal artery to study potential therapeutic treatments of hyperglycaemia-induced endothelial dysfunction. Study 2: In Study 1, we established that acute high glucose incubations and an atherogenic diet cause endothelial dysfunction in rabbit aorta. As such, this study examined the biological effect of ucOC on blood vessel function in rabbit aorta ex vivo, as well as determining the effect of ucOC on markers of endothelial function in human cells in vitro. Isometric tension and immunohistochemistry techniques were used on the aorta of male New Zealand White Rabbits and human aortic endothelial cells (HAEC) were cultured to assess the effect of ucOC in normal and high glucose environments. Overall, ucOC, both 10 ng/ml and 30 ng/ml, did not significantly alter acetylcholine- induced blood vessel relaxation in rabbits (p > 0.05). The ucOC treatment did not cause any significant changes in the immunoreactivity of cellular signalling markers (endothelial nitric oxide synthase, protein kinase B, mammalian target of rapamycin and nitrotyrosine) in rabbit aorta (p > 0.05). In HAEC, ucOC did not attenuate endothelin 1, interleukin 6, vascular adhesion molecule 1, monocyte chemoattractant protein 1 or lactate dehydrogenase, all of which were increased in response to high glucose treatment (p > 0.05). In conclusion, the results of this study suggest that ucOC has no direct influence on endothelial function in rabbit aorta ex vivo or in human endothelial cells in vitro. Study 3: In this study we examined whether ucOC is related to blood pressure and vascular function in older adults and whether ucOC has a direct effect on endothelial function in the carotid artery of rabbits. To undertake the study, we used perfusion myography, which allows for the examination of whole vessel segments with pulsatile flow and pressure that mimics an endogenous environment. In older adults, ucOC, blood pressure, pulse wave velocity (PWV) and brachial artery flow mediated dilation (BAFMD) were measured (n = 38, 26 post-menopausal women and 12 men, mean age 73 ± 1 years). In male New Zealand White Rabbits, the vasoactivity of the carotid artery was assessed following a four week normal or atherogenic diet. An ucOC dose response curve (0.3 – 45 ng/ml) was administered following incubation of the arteries for 2 h in either normal or high glucose conditions. The concentration of ucOC was higher in normotensive older adults compared to those with stage 2 hypertension (34%, p < 0.05), particularly in women (43%, p < 0.01), but not men (p > 0.05). In all participants, higher ucOC was also associated with lower PWV (p < 0.05), but not BAFMD (p > 0.05). In rabbits, ucOC at any dose did not cause an alteration in the vasoactivity of the carotid artery, following either a normal or atherogenic diet (p > 0.05). In conclusion, ucOC is associated with vascular function in older adults, exclusively in post-menopausal women, but it has no direct effect on endothelial function in rabbit carotid arteries. Study 4: Vitamin K is a regulator of OC carboxylation, with higher vitamin K intake known to reduce circulating levels of ucOC. As ucOC was associated with vascular function in adults in Study 3, we tested the hypothesis that a suppression of ucOC following an increase in dietary vitamin K1 would exhibit a relative worsening of cardiometabolic risk factors. Men (n = 20) and women (n = 10) aged 62 ± 10 years participated in a randomised, controlled, crossover study. Participants were split into high and low responder subgroups following a four week high vitamin K1 diet (HK) of increased leafy green vegetables. High and low responders were defined based on the median percent reduction (30%) in ucOC following the HK diet. Blood pressure (resting and 24 h), arterial stiffness, plasma glucose and lipid concentrations, and serum OC forms were assessed. Following the HK diet, ucOC and ucOC/tOC were suppressed more (p < 0.01) in high responders (41% and 29% respectively) than in low responders (12% and 10% respectively). The reductions in ucOC and ucOC/tOC were not associated with changes in blood pressure, PWV, plasma glucose or lipid concentrations in the high responders (p > 0.05). The results from this study suggest that the suppression of ucOC via consumption of leafy green vegetables has no negative effects on cardiometabolic health, perhaps, in part, due to compensatory mechanisms, such as increased nitric oxide. General conclusions: Overall, the results of this thesis suggest that ucOC does not have a direct biological role in the regulation of endothelial function in rabbit arteries and human endothelial cells. In humans there is some association between ucOC and vascular function but the suppression of circulating ucOC does not influence vascular function or cardiovascular risk factors. As ucOC was not found to have a detrimental effect on vascular function, it may be targeted as a therapeutic treatment for metabolic diseases, such as T2DM, without a risk of adverse effects on the vasculature.

Item type Thesis (PhD thesis)
URI https://vuir.vu.edu.au/id/eprint/42182
Subjects Current > FOR (2020) Classification > 3201 Cardiovascular medicine and haematology
Current > FOR (2020) Classification > 3208 Medical physiology
Current > Division/Research > Institute for Health and Sport
Keywords thesis by publication; endothelial dysfunction; cardiovascular disease; vascular function; skeleton; osteocalcin; OC; undercarboxylated osteocalcin; ucOC; hyperglycaemia; glucose; atherogenic diet; rabbits; endothelial function; vitamin K
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