Abstract
Peripheral artery disease (PAD) is the most common cardiovascular disease associated with type 2 diabetes mellitus (T2DM). PAD is characterised by the narrowing or occlusion of systemic arteries impeding blood supply to the extremities. The prevalence of PAD is rapidly increasing because of the global T2DM pandemic and an ageing population. As PAD progresses to its most severe manifestation, termed critical limb ischaemia (CLI), surgical revascularisation becomes the preferred treatment option. Despite considerable advances in surgical revascularisation, up to 50% of patients with CLI are poor candidates for surgery or have failed revascularisation. Consequently, a large clinical cohort of patients is left with few viable treatment options
Angiogenesis is the formation of new capillaries from the pre-existing vasculature. In therapeutic angiogenesis, attempts are made to enhance blood vessel growth and augment perfusion. Therapeutic angiogenesis has been studied as a treatment option for PAD for over two decades. However, no therapeutic agents have successfully transitioned from bench to bedside, and the development of therapeutic angiogenesis treatments remains a major clinical challenge.
Ghrelin is a 28-amino acid peptide hormone most frequently studied in the context of energy homeostasis and metabolism. Emerging evidence suggests ghrelin may have a role in angiogenesis, making it a desirable agent for the treatment of PAD. However, what remains unknown is (1) the role of endogenous ghrelin in vascular homeostasis and its possible implications for PAD; (2) whether ghrelin induces therapeutic angiogenesis in an aged, T2DM murine model of PAD; and (3) what role endogenous ghrelin plays in humans with T2DM and non-diabetic PAD.
To investigate this, two preclinical studies and one clinical study were conducted. The first preclinical study utilised global ghrelin knockout mice and unilateral hindlimb ischaemia as an in vivo preclinical PAD model. Following 14 days of ischaemia, ghrelin knockout mice had a delayed perfusion recovery. Microcomputer tomography and histological analysis revealed a failure to induce reparative revascularisation. Microangiography demonstrated an impaired endothelial function in knockout mice with hindlimb ischaemia, likely further exacerbating the perfusion recovery. Molecular analysis revealed that impaired revascularisation following injury was associated with the dysregulation of several microRNAs (miRNAs) associated with angiogenesis (miRNA -126 & -132). This preclinical study highlights the importance of endogenous ghrelin in vascular homoeostasis, shown by ghrelin knockout mice having an endogenous defect in perfusion recovery following hindlimb ischaemia.
The second preclinical study investigated if exogenous ghrelin could induce therapeutic angiogenesis in an aged T2DM murine model of PAD. db/db mice were used as a murine model of T2DM and their non-diabetic db/+ littermates were used as controls. Unilateral hindlimb ischaemia was used as an in vivo preclinical PAD model. For 14 consecutive days post-surgery, either acylated-ghrelin, des-acylated ghrelin, or vehicle was administered. Exogenous acylated-ghrelin enhanced perfusion recovery during unilateral hindlimb ischaemia, with microcomputer tomography, microangiography, and histological analysis revealing an increase in neovascularisation and vasoreactivity in acylated-ghrelin treated db/db and db/+ mice. Des-acylated ghrelin demonstrated improvements in recovery following hindlimb ischaemia but to a lesser extent than acylated-ghrelin. Molecular analysis revealed a group of miRNAs (miRNA -126 & -132) which are associated with angiogenesis to be upregulated in both ghrelin-treated mice, shedding further light on the molecular mechanism by which ghrelin acts. This study demonstrated that acylated-ghrelin improves perfusion recovery in db/db and db/+ mice following ischaemia, through the induction of therapeutic angiogenesis, paving the way for acylated-ghrelin as a therapy for T2DM and non-diabetic PAD.
The final study in this thesis was a clinical study, in which a large cohort of PAD and T2DM-PAD patients is recruited, and the circulating plasma ghrelin concentrations measured. Analysis revealed a dysregulation of the ghrelin system in non-diabetic and T2DM-PAD, suggesting a role for ghrelin in human PAD.
In its entirety, this thesis demonstrates an important role of endogenous ghrelin in a preclinical murine model of PAD and human PAD, and T2DM-PAD. Furthermore, this thesis provides the first evidence to show exogenous ghrelin improves recovery from ischaemia in both a T2DM and non-diabetic murine model of PAD. These findings have significantly contributed to the understanding of ghrelin in PAD and provide the foundation for phase 1 clinical trials with exogenous ghrelin for the effective treatment of T2DM and non-diabetic PAD, thus potentially saving life and limb.