Molecular MRI of mouse atherosclerosis

Abstract

Atherosclerosis is a disease that mainly affects the large arteries and is characterized by accumulation of lipids and fibrous tissue in the vessel wall intima. The major risk of atherosclerosis is thrombus formation, which may lead to for example myocardial infarction or stroke. Molecular imaging is the non-invasive visualization and measurement of biological processes at the molecular and cellular level within a living organism. The detection and classification of atherosclerotic plaques with molecular imaging is currently being explored. This thesis addresses the potential role of magnetic resonance imaging for molecular imaging of atherosclerosis. Specifically, the aim of this thesis was to develop several MRI contrast agents targeted towards atherosclerotic plaque and to study the utility of these agents in vivo in MRI studies of a refined mouse model of atherosclerosis. Chapter 1 gives an introduction to this thesis and shortly explains the main subjects involved. A short overview of the pathology of atherosclerosis is given, followed by introducing molecular imaging of atherosclerosis. MRI contrast agents used in this thesis are briefly introduced as well. The mouse model involved placement of a tapered cast around the carotid artery of apoE-/- mice. This results in the formation of vascular lesions on both sides of the cast. Upstream a lesion with more vulnerable characteristics and downstream a lesion with more stable characteristics was formed. This mouse model was used throughout the thesis. The MR characterization of the above mouse model is described in chapter 2. Angiography, high-resolution T1- and T2- weighted black blood, and phase-contrast flow velocity imaging was performed on the carotid arteries of these mice. MR angiography showed that blood flow through the right carotid artery was preserved and confirmed the tapered nature of the constriction. Laminar flow with low wall shear stress was measured upstream of the cast, which is related to the development of a vulnerable lesion. Apparent flow velocities were low downstream to the cast, which is consistent with the occurrence of vortices or an oscillatory nature of the flow. This explains the formation of a stabilized lesion at this position. Detailed characterization of the vascular properties of the mouse model was performed in chapter 3. Vascular endothelium in atherosclerotic plaques is known to be more permeable than healthy vessel wall endothelium. In this study three differently sized paramagnetic contrast agents were employed to study their ability to produce T1-weighted MRI contrast enhancement by their passive accumulation, in stable and vulnerable mouse carotid atherosclerotic plaques at two stages of development. The two smallest of the tested contrast agents, i.e. Gd-HP-DO3A and micelles, resulted in contrast permeate the vessel wall in all plaque phenotypes on T1-weighted MR images. Liposomes, on the other hand, were too large to accumulate and caused no significant contrast enhancement in the atherosclerotic plaques. These findings were also confirmed using fluorescence microscopy. Histology showed that phenotype differences as well as sizes of the plaques were larger at 9 weeks than at 6 weeks after cast placement. These results imply that when suitably modified with targeting ligands, the fast permeation of Gd-HP-DO3A into plaques make low molecular weight contrast agents suitable for imaging of abundant targets inside atherosclerotic plaques. The lack of intraplaque accumulation of liposomes makes these particles a good candidate for imaging of plaque-associated vascular markers, because these contrast materials cause little background enhancement and the endothelial markers are directly accessible from the lumen. Micelles accumulate into atherosclerotic plaques on a longer timescale, which makes them suitable for imaging of less abundant markers inside atherosclerotic plaques. Collagen plays an important role in the stabilization of atherosclerotic plaques, and limited collagen content may represent a risk for plaque rupture. Imaging of collagen as a key plaque component could help in risk assessment, monitoring the progression of the disease, and evaluating the efficacy of therapy. In chapter 4 the collagen-binding protein CNA35 was conjugated to paramagnetic and fluorescent micelles as a tool for MR and fluorescent imaging of collagen in atherosclerotic lesions. The above described mouse model of experimental atherosclerosis, developing lesions with different collagen contents, was used to assess the ability of the micelles to produce differential MR contrast enhancement. Molecular MRI of collagen with CNA35-micelles was shown to discriminate collagen-rich from relatively collagen-poor lesions. Ex vivo fluorescence microscopy supported the in vivo findings and showed pronounced accumulation of CNA35 micelles in the higher collagen content plaques, whereas control mutant-CNA35-micelles showed much less accumulation. A significantly higher signal enhancement in the collagen-richer plaque was observed for collagen-specific micelles as compared to control micelles. Migration of leukocytes into tissues is a common feature of inflammatory diseases such as atherosclerosis. Vascular adhesion molecules, like vascular cell adhesion molecule-1 (VCAM-1), are important in mediating leukocyte adhesion to endothelial cells and diapedesis to underlying tissue. Target-specific imaging of VCAM-1 expression is valuable, because it serve as a surrogate marker of inflammatory cell recruitment into atherosclerotic plaques. The efficacy of positive (T1-weighted) and negative (T2-weighted) MRI contrast approaches for imaging VCAM-1 expression in atherosclerotic plaques was explored in chapter 5. For this purpose VCAM 1-targeted paramagnetic liposomes as well as superparamagnetic micellar iron oxides were developed. In vitro specificity was shown on VCAM-1 expressing mouse heart endothelioma (H5V) cells. Vessel wall contrast changes were evaluated by comparing MRI scans acquired before and at 24 hours after injection of VCAM-1-targeted or control particles. The measured MRI contrast changes agreed with the spatial distribution of VCAM-1 expression in the plaque for both contrast agents. Highest contrast changes were observed at the shoulder of the plaques in regions of highest VCAM-1levels. These in vivo data were in line with ex vivo microscopy. Chapter 6 concludes this thesis with a summarizing discussion of the work presented and future perspectives of MRI of atherosclerosis.