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

Cysteamine inhibits lysosomal oxidation of low density lipoprotein in human macrophages and reduces atherosclerosis in mice

Background and aims: We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol) which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. Methods: LDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19-22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. Results: Confocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. Conclusions: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease

Elevated Uptake of Plasma Macromolecules by Regions of Arterial Wall Predisposed to Plaque Instability in a Mouse Model

Atherosclerosis may be triggered by an elevated net transport of lipid-carrying macromolecules from plasma into the arterial wall. We hypothesised that whether lesions are of the thin-cap fibroatheroma (TCFA) type or are less fatty and more fibrous depends on the degree of elevation of transport, with greater uptake leading to the former. We further hypothesised that the degree of elevation can depend on haemodynamic wall shear stress characteristics and nitric oxide synthesis. Placing a tapered cuff around the carotid artery of apolipoprotein E -/- mice modifies patterns of shear stress and eNOS expression, and triggers lesion development at the upstream and downstream cuff margins; upstream but not downstream lesions resemble the TCFA. We measured wall uptake of a macromolecular tracer in the carotid artery of C57bl/6 mice after cuff placement. Uptake was elevated in the regions that develop lesions in hyperlipidaemic mice and was significantly more elevated where plaques of the TCFA type develop. Computational simulations and effects of reversing the cuff orientation indicated a role for solid as well as fluid mechanical stresses. Inhibiting NO synthesis abolished the difference in uptake between the upstream and downstream sites. The data support the hypothesis that excessively elevated wall uptake of plasma macromolecules initiates the development of the TCFA, suggest that such uptake can result from solid and fluid mechanical stresses, and are consistent with a role for NO synthesis. Modification of wall transport properties might form the basis of novel methods for reducing plaque rupture

Recombinant cells and chimeraplasty as tools to study atheroprotective effects of apolipoprotein E

Apolipoprotein (apo) E is secreted by liver and macrophages and is atheroprotective, in part by contributing to plasma cholesterol homeostasis. Three common isoforms arise from single nucleotide polymorphisms (SNPs): the rarest variant, apoE2, differs from wild-type apoE3 by an R158C substitution, and causes recessive Type III hyperlipidaemia, while apoE4 (C112R) produces a dominant hyperlipidaemia. One aim of this thesis was to determine whether lipidated apoE3 particles, newly-secreted by recombinant Chinese hamster ovary (CHO), were biologically active, using CHO cells engineered to secrete antiatherogenic apoAI as positive controls. An ELISA was established to quantify levels of secreted apoE, while apoAI was measured by immunoblotting and scanning densitometry. Although secreted apoAI was about 55 % better at activating the cholesterol esterifying enzyme, LCAT (lecithin-cholesterol acyltransferase), than apoE after 0.5 (P<0.05) or 1 h (P<0.001) of incubation, both particles were equally effective at promoting cholesterol efflux. These data suggest that these atheroprotective actions may be normal physiological roles for apoE. The second aim was to test the hypothesis that: 'synthetic RNA-DNA oligonucleotides (chimeraplasts) can convert the APOE3 gene to mutant APOE2 and APOE4 in human hepatoblastoma (HepG2) and human monocyte-macrophage (THP-1) cells'. Such new cell lines, secreting dysfunctional apoE2 or apoE4, would allow the atheroprotective properties of the three main human apoE isoforms to be compared in future studies. Initially, CHOE3 and human embryonic kidney (HEK-293; ԑ3/ԑ3) cells, which are both readily transfected, were targeted with apoE3-to-apoE2 chimeraplast complexed with polyethylenimine (PEI); the expected conversions were detected. Dose-dependent conversions using apoE3-to-apoE2 or apoE3-to-apoE4 chimeraplast were also seen in HepG2 cells, as judged by direct sequencing and PCR-RFLP, whereas THP-1 cells proved refractory and the conversion was limited. Trials with end-protected all-DNA molecules proved unsuccessful. Increasing efficiency was subsequently investigated: by using different PEIs (linear vs. branched, and tagging with melittin, galactose or mannose); by centrifugation to enhance cell-complex contact; and by nuclear microinjection. Linear PEI (L-PEI) alone worked best with the apoE3-to-apoE2 chimeraplast, while galactose-4-PEI mixed with L-PEI (1:1) gave good conversion with the apoE3-to-apoE4 reagent. Unexpectedly, the conversion appeared unstable in both THP-1 and HepG2 cells after freeze-thawing and/ or repeated passaging. Attempts to understand this problem were then hampered by difficulties in synthesizing active chimeraplasts. In conclusion, although an emerging technology with enormous potential, chimeraplast-directed gene mutation/repair remains problematical and factors such as chimeraplast quality and design, gene target and cell type all require detailed study in future investigations

The thermodynamics of and strengthening due to co-clusters: general theory and application to the case of Al-Cu-Mg alloys

Co-clusters in ternary or higher order metallic alloys are metastable structures involving two or more distinct alloying atoms that retain the structure of the host lattice. A thermodynamic model based on a single interaction energy of dissimilar nearest neighbour alloying elements is presented, and a model for the strengthening due to these co-cluster dimers is derived. The model includes a new treatment of (short-) order strengthening relevant to these co-clusters and further encompasses modulus hardening and chemical hardening. The models are tested against data on a wide range of Al-Cu-Mg alloys treated at temperatures between 20 and 220ºC. Both quantitative calorimetry data on the enthalpy change due to co-cluster formation and strengthening due to co-clusters is predicted well. It is shown that in general (short-range) order strengthening will be the main strengthening mechanism

Targeted molecular iron oxide contrast agents for imaging atherosclerotic plaque

Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

Tracer uptake along vessels in uncuffed mice.

<p>Fluorescence intensity along the length of carotid arteries of mice without cuffs; data from left and right carotids have been combined. Rhodamine-labelled tracer was administered to the mice (“Rho”) or was omitted to assess autofluorescence (“Auto”). Mean (dark line) +1 SEM (light line), n = 4.</p

Blood flow velocities measured conventional and reversed cuff.

<p>Mean, peak and diastolic blood flow velocities in cm/s, measured by Doppler ultrasound, with the cuff in the conventional and reversed orientation. Velocity was measured at the upstream (“LC-U”) and downstream (“LC-D”) ends of the cuff in the left carotid, and in the uncuffed right carotid (“RC”). Values are averages for ten cardiac cycles.</p><p>Blood flow velocities measured conventional and reversed cuff.</p