Targeted Molecular Iron Oxide Contrast Agents for Imaging Atherosclerotic Plaque

Overview: Cardiovascular disease remains a leading cause of death worldwide, with vulnerable plaque rupture the underlying cause of many heart attacks and strokes. Much research is focused on identifying an imaging biomarker to differentiate stable and vulnerable plaque. Magnetic Resonance Imaging (MRI) is a non-ionising and non-invasive imaging modality with excellent soft tissue contrast. However, MRI has relatively low sensitivity (micromolar) for contrast agent detection compared to nuclear imaging techniques. There is also an increasing emphasis on developing MRI probes that are not based on gadolinium chelates because of increasing concerns over associated systemic toxicity and deposits1. To address the sensitivity and safety concerns of gadolinium this project focused on the development of a high relaxivity probe based on superparamagnetic iron oxide nanoparticles for the imaging of atherosclerotic plaque with MRI. With development, this may facilitate differentiating stable and vulnerable plaque in vivo. Aim: To develop a range of MRI contrast agents based on superparamagnetic iron oxide nanoparticles (SPIONs), and test them in a murine model of advanced atherosclerosis. Methods: Nanoparticles of four core sizes were synthesised by thermal decomposition and coated with poly(maleicanhydride-alt-1-octadecene) (PMAO), poly(ethyleneimine) (PEI) or alendronate, then characterised for core size, hydrodynamic size, surface potential and relaxivity. On the basis of these results, one candidate was selected for further studies. In vivo studies using 10 nm PMAO-coated SPIONs were performed in ApoE-/- mice fed a western diet and instrumented with a perivascular cuff on the left carotid artery. Control ApoE-/- mice were fed a normal chow diet and were not instrumented. Mice were scanned on a 3T MR scanner (Philips Achieva) with the novel SPION contrast agent, and an elastin-targeted gadolinium agent that was shown previously to enable visualisation of plaque burden. Histological analysis was undertaken to confirm imaging findings through staining for macrophages, CX3CL1, elastin, tropoelastin, and iron. Results: The lead SPION agent consisted of a 10 nm iron oxide core with poly(maleicanhydride-alt-1-octadecene), (-36.21 mV, r2 18.806 mmol-1/s-1). The irregular faceting of the iron oxide core resulted in high relaxivity and the PMAO provided a foundation for further functionalisation on surface -COOH groups. The properties of the contrast agent, including the negative surface charge and hydrodynamic size, were designed to maximise circulation time and evade rapid clearance through the renal system or phagocytosis. In vitro testing showed that the SPION agent was non-toxic. In vivo results show that the novel contrast agent accumulates in similar vascular regions to a gadolinium-based contrast agent (Gd-ESMA) targeted to elastin, which accumulates in plaque. There was a significant difference in SPION signal between the instrumented and the contralateral non-instrumented vessels in diseased mice (p = 0.0411, student’s t-test), and between the instrumented diseased vessel and control vessels (p = 0.0043, 0.0022, student’s t-test). There was no significant difference between the uptake of either contrast agent between stable and vulnerable plaques (p = 0.3225, student’s t-test). Histological verification was used to identify plaques, and Berlin Blue staining confirmed the presence of nanoparticle deposits within vulnerable plaques and co-localisation with macrophages. Conclusion: This work presents a new MRI contrast agent for atherosclerosis which uses an under-explored surface ligand, demonstrating promising properties for in vivo behaviour, is still in circulation 24 hours post-injection with limited liver uptake, and shows good accumulation in a murine plaque model

Increased Vascular Permeability Measured With an Albumin-Binding Magnetic Resonance Contrast Agent Is a Surrogate Marker of Rupture-Prone Atherosclerotic Plaque:MRI of Plaque Permeability and Instability

BACKGROUND: Compromised structural integrity of the endothelium and higher microvessel density increase vascular permeability. We investigated whether vascular permeability measured in vivo by magnetic resonance imaging using the albumin-binding contrast agent, gadofosveset, is a surrogate marker of rupture-prone atherosclerotic plaque in a rabbit model. METHODS AND RESULTS: New Zealand white rabbits (n=10) were rendered atherosclerotic by cholesterol-diet and endothelial denudation. Plaque rupture was triggered with Russell’s viper venom and histamine. Animals were imaged pre-triggering, at 3 and 12 weeks, to quantify plaque area, vascular permeability, vasodilation, and stiffness and post-triggering to identify thrombus. Plaques identified on the pretrigger scans were classified as stable or rupture-prone based on the absence or presence of thrombus on the corresponding post-trigger magnetic resonance imaging, respectively. All rabbits had developed atherosclerosis, and 60% had ruptured plaques. Rupture-prone plaques had higher vessel wall relaxation rate (R(1); 2.30±0.5 versus 1.86±0.3 s(−1); P<0.001), measured 30 minutes after gadofosveset administration, and higher R(1)/plaque area ratio (0.70±0.06 versus 0.47±0.02, P = 0.01) compared with stable plaque at 12 weeks. Rupture-prone plaques had higher percent change in R(1) between the 3 and 12 weeks compared with stable plaque (50.80±7.2% versus 14.22±2.2%; P<0.001). Immunohistochemistry revealed increased vessel wall albumin and microvessel density in diseased aortas and especially in ruptured plaque. Electron microscopy showed lack of structural integrity in both luminal and microvascular endothelium in diseased vessels. Functionally, the intrinsic vasodilation of the vessel wall decreased at 12 weeks compared with 3 weeks (18.60±1.0% versus 23.43±0.8%; P<0.001) and in rupture-prone compared with stable lesions (16.40±2.0% versus 21.63±1.2%; P<0.001). Arterial stiffness increased at 12 weeks compared with 3 weeks (5.00±0.1 versus 2.53±0.2 m/s; P<0.001) both in animals with stable and rupture-prone lesions. CONCLUSIONS: T1 mapping using an albumin-binding contrast agent (gadofosveset) could quantify the changes in vascular permeability associated with atherosclerosis progression and rupture-prone plaques

Molecular Imaging in Ischemic Heart Disease

Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

Tropoelastin a novel marker for plaque progression and instability:MRI of tropoelastin in atherosclerosis

International audienceElastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques. We developed gadolinium-labeled tropoelastin-specific magnetic resonance contrast agents (Gd-TESMAs) for tropoelastin imaging in animal models