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Investigating the actions of ghrelin on cerebral artery function and the brain post-stroke.

thesis
posted on 23.02.2017, 23:31 by Ku, Jacqueline Mei Qi
Cerebral endothelial dysfunction is associated with several stroke risk factors, resulting in dysregulated brain perfusion and an increased susceptibility to stroke. Evidence suggests that deficits in vasoprotective nitric oxide (NO) caused primarily by Nox2-NADPH oxidase-driven oxidative stress (excess levels of reactive oxygen species) is a common underlying cause of cerebral endothelial dysfunction associated with these conditions. Thus, this highlights the potential for identifying therapeutic agents to treat endothelial dysfunction associated with risk factors for stroke prevention. Despite advances in our understanding on mechanisms of neuronal cell death after stroke, translation of this knowledge into effective stroke therapies has been, thus far, unsuccessful. Importantly, endothelial dysfunction occurs at all levels of the cerebral vascular tree following ischaemic stroke including at the level of the blood-brain barrier (BBB), which can accelerate neuronal injury and potentially increase the risk of a recurrent stroke. Thus, the identification of novel drugs that target both neuronal and vascular injury mechanisms may be a more effective therapeutic approach. Ghrelin-related peptides, namely acylated ghrelin, des-acylated ghrelin and obestatin, are produced primarily by the stomach and are best known for their neuroendocrine actions. Importantly, emerging evidence suggests that these peptides may have neuroprotective actions, as well as vasoprotective actions in the systemic circulation. Taken together, these findings raise the possibility that ghrelin-related peptides may have important protective roles in the cerebral circulation, and may target neuro- and vaso-protective mechanisms after ischaemic stroke. As such, the main aims of this thesis were to firstly investigate the actions of ghrelin-related peptides on mouse cerebral blood vessels by examining their effects on NO and superoxide levels, and secondly test whether post-stroke treatment of ghrelin-related peptides limits brain injury and BBB disruption in a mouse model of cerebral ischaemia and reperfusion.In Chapter 2, we demonstrated that exogenous des-acylated ghrelin or obestatin are potent stimulators of vasodilator NO, and des-acylated ghrelin suppresses Nox2 oxidase-derived superoxide levels in mouse cerebral arteries. In contrast, acylated ghrelin had no effect on cerebral vascular tone or superoxide levels, likely due to the fact that its receptor, GHSR1a, is not expressed in cerebral arteries. Vasodilator responses to des-acylated ghrelin or obestatin were sustained following antagonism of GHSR1a, and responses to des-acylated ghrelin were unaffected in GHSR-deficient mice. Thus, des-acylated ghrelin and obestatin elicit cerebral vascular responses through the activation of unidentified ghrelin receptor(s). Next, using ghrelin-deficient mice, we found that a deficiency in ghrelin-related peptides was associated with impaired cerebral artery vasodilator NO capacity and augmented superoxide levels, indicating that des-acylated ghrelin and/or obestatin presumably regulate cerebral artery NO and superoxide levels in vivo. Collectively, these findings reveal for the first time that both exogenous and endogenous des-acylated ghrelin and obestatin regulate two important and inter-related regulatory mechanisms of cerebral vascular function. As such, we propose that these peptides may be novel regulators of cerebral vascular function and potentially brain perfusion. To date, the majority of studies infer that ghrelin-related peptides may also have protective actions in the systemic vasculature of humans and rats. Unexpectedly, in Chapter 3 we found that, in contrast to these studies, exogenous acylated ghrelin, des-acylated ghrelin or obestatin had no effect on tone or superoxide levels in mouse thoracic aorta or mesenteric arteries. Furthermore, the GHSR1a receptor was not expressed in mouse thoracic aorta. Moreover, NO vasodilator capacity and superoxide levels were unaffected in the thoracic aorta of ghrelin-deficient mice, suggesting that endogenous ghrelin-related peptides do not modulate NO production and superoxide levels in mouse systemic arteries. Thus, taken together with previously published work, this study reveals potential species (human and rat vs. mouse) and regional (cerebral vs. systemic) differences in vascular responses to ghrelin-related peptides, and suggests that the mouse may not be a suitable species to study their potential roles in regulating systemic vascular function. In Chapter 4, we characterized two in vitro BBB models of ischaemia and reperfusion using an immortalised mouse cerebral endothelial cell line (bEnd.3 cells) in combination with oxygen glucose deprivation with or without reoxygenation (RO). After exposure of bEnd.3 cells to OGD (to simulate cerebral ischaemia), we observed endothelial hyperpermeability, and associated loss of tight junction proteins (occludin and claudin-5), and cell death after prolonged exposure periods (16-24 hours). Following exposure of bEnd.3 cells to 1 h OGD followed by 23 h RO (to simulate cerebral ischaemia and reperfusion), we found that RO exacerbated hyperpermeability, which was associated with a loss of tight junction proteins (ZO-1 and occludin), but not cell death. Although both models have limitations, the findings of this study will facilitate their use in future studies of post-stroke BBB disruption and for the testing of novel therapeutics. In Chapter 5, we examined whether post-stroke treatment with acylated ghrelin or des-acylated ghrelin improves stroke outcome in a mouse model of focal cerebral ischaemia and reperfusion, and examined whether des-acylated ghrelin can limit BBB disruption by measuring its effect on BBB permeability. Post-stroke treatment of mice with des-acylated ghrelin treatment reduced brain injury and swelling, as well as potentially reducing the number of apoptotic cells in peri-infarct brain regions. Previous studies have reported that pre-stroke treatment with acylated ghrelin is protective in rodents models of cerebral ischaemia and reperfusion. However, here we found that post-stroke treatment with acylated ghrelin failed to improve stroke outcome at the dose we tested. Next, we found that des-acylated markedly reduced BBB permeability in mice after stroke, indicative of reduced BBB disruption. Also, using the in vitro BBB model of OGD + RO established in Chapter 4, we found that des-acylated ghrelin attenuated the increase in permeability of bEnd.3 cells caused by OGD + RO by potentially preventing the disruption of tight junctions. Although more research is needed, the findings of this Chapter raise the possibility that des-acylated ghrelin or longer acting analogues may represent novel stroke therapies. Collectively, this thesis demonstrates previously unrecognised protective roles for des-acylated ghrelin and obestatin in the cerebral vasculature and shows the existence of novel ghrelin receptor(s) in cerebral vessels. Furthermore, this thesis demonstrates that post-stroke treatment with des-acylated ghrelin can improve stroke outcome by limiting brain injury and BBB disruption following cerebral ischaemia and reperfusion. Although further studies are needed, these findings raise the possibility that these peptides could be employed to treat cerebral endothelial dysfunction associated with cardiovascular risk factors, as well as limit brain injury associated with stroke by targeting neuronal and vascular mechanisms.

History

Principal supervisor

Christopher Sobey

Additional supervisor 1

Alyson Miller

Year of Award

2016

Department, School or Centre

Pharmacology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences