Biological targeting of inflammation in atherosclerosis using iron oxide particles and MRI

Abstract

Atherosclerosis is now widely viewed as an inflammatory disease. Intraplaque inflammation drives the progression and destabilisation of atherosclerotic lesions, converting chronic stable asymptomatic lesions into acute lesions with ensuing clinical sequelae, including acute coronary syndrome, transient ischaemic attack or stroke. There is currently no clinical imaging technique available to assess the degree of inflammation associated with plaques. The purpose of this work is to develop and utilise novel in vivo magnetic resonance imaging (MRI) methodologies to assess inflammation in atherosclerotic plaques. This thesis describes the development of antibody-conjugated iron oxide particles targeted against endothelial adhesion molecules in order to act as a contrast agent for MRI of inflammation in atherosclerosis. This study aims at visualising and characterising atherosclerosis using targeted iron oxide particles as an MRI probe for detecting inflamed plaque disease in both human atherosclerotic tissue and an apolipoprotein E-deficient (ApoE-/-) mouse model.

This study is comprised of four main experimental stages. The initial in vitro feasibility study confirmed MRI detection of activated endothelial cells using anti-E-selectin antibody and anti-VCAM-1 antibody conjugated superparamagnetic iron oxide particles (SPIO). Subsequent ex vivo studies demonstrated MRI detection and characterisation of inflammatory markers on human atherosclerotic plaques using anti-VCAM-1 antibody and anti-E-selectin antibody conjugated SPIO with confirmatory immunohistochemistry. Further, the ex vivo in situ stage consisted of MRI detection of atherosclerotic lesions in the aortic root of ApoE-/- mice using a new improvised version of iron oxide particles – the dual antibody-conjugated microparticles of iron oxide (MPIO) against VCAM-1 and P-selectin. The final in vivo stage involved detection and characterisation of atherosclerotic lesions in the aortic root and aortic arch of ApoE-/- mice by in vivo MRI using the dual-targeted MPIO. Following this, an animal model (ApoE-/- mouse) with focal atherosclerotic lesions in carotid arteries was developed by means of peri-arterial cuff placement to allow in vivo molecular MRI using these probes.

The in vitro cellular model of endothelial inflammation demonstrated stimulated bovine aortic endothelial cells were detectable on MRI using targeted SPIO as a contrast agent, confirmed by immunocytochemistry. Inflammation of human atherosclerotic plaques was similarly detectable by ex vivo MRI. Further, we have demonstrated that the MR contrast effect induced by endothelial-bound dual-ligand MPIO quantitatively tracked with macrophage content within the aortic root lesions of ApoE-/- mice by in vivo MRI. In the final in vivo mouse carotid stage, we have utilised the shear stress modifying cuff to generate both stable and rupture-prone lesions in the murine carotid artery. Using dual-targeted MPIO, we have subsequently identified these high-risk inflamed carotid plaque lesions by in vivo MRA. The in vivo MRI combined with dual-targeted MPIO approach will potentially allow real time in vivo characterisation of plaque vulnerability, leading to accurate risk stratification in individual patients, thereby contributing a personalised approach to the management of carotid atherosclerotic disease in the future.