Monitoring Vascular Permeability and Remodeling After Endothelial Injury in a Murine Model Using a Magnetic Resonance Albumin-Binding Contrast Agent

Background-Despite the beneficial effects of vascular interventions, these procedures may damage the endothelium leading to increased vascular permeability and remodeling. Re-endothelialization of the vessel wall, with functionally and structurally intact cells, is controlled by endothelial nitric oxide synthase (NOS3) and is crucial for attenuating adverse effects after injury. We investigated the applicability of the albumin-binding MR contrast agent, gadofosveset, to noninvasively monitor focal changes in vascular permeability and remodeling, after injury, in NOS3-knockout (NOS3(-/-)) and wild-type (WT) mice in vivo.Methods and Results-WT and NOS3(-/-) mice were imaged at 7, 15, and 30 days after aortic denudation or sham-surgery. T-1 mapping (R-1=1/T-1, s(-1)) and delayed-enhanced MRI were used as measurements of vascular permeability (R-1) and remodeling (vessel wall enhancement, mm(2)) after gadofosveset injection, respectively. Denudation resulted in higher vascular permeability and vessel wall enhancement 7 days after injury in both strains compared with sham-operated animals. However, impaired re-endothelialization and increased neovascularization in NOS3(-/-) mice resulted in significantly higher R-1 at 15 and 30 days post injury compared with WT mice that showed re-endothelialization and lack of neovascularization (R-1 [s(-1)]=15 days: (-/-)(NOS3)4.02 [interquartile range, IQR, 3.77-4.41] versus (WT)2.39 [IQR, 2.35-2.92]; 30 days: (-/-)(NOS3)4.23 [IQR, 3.94-4.68] versus (WT)2.64 [IQR, 2.33-2.80]). Similarly, vessel wall enhancement was higher in NOS3(-/-) but recovered in WT mice (area [mm(2)]=15 days: (-/-)(NOS3)5.20 [IQR, 4.68-6.80] versus (WT)2.13 [IQR, 0.97-3.31]; 30 days: (-/-)(NOS3)7.35 [IQR, 5.66-8.61] versus (WT)1.60 [IQR, 1.40-3.18]). Ex vivo histological studies corroborated the MRI findings.Conclusions-We demonstrate that increased vascular permeability and remodeling, after injury, can be assessed noninvasively using an albumin-binding MR contrast agent and may be used as surrogate markers for evaluating the healing response of the vessel wall after injury.</p

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

BS43 A new collagen III-specific MRI imaging probe to assess cardiac fibrosis

Heart failure (HF) has reached epidemic proportions, affecting approximately 64 million people globally and is the main cause of death and disability.1 Myocardial fibrosis, characterised by changes in the amount and/or distribution of collagen I and III, impairs cardiac function and relates to adverse outcomes of HF.2 3 Clinically, we rely on indirect or surrogate measurements of collagen in the myocardium and current targeted molecular imaging probes are limited to collagen I. Here, we report the discovery of a peptide selective for collagen III and a strategy to develop an imaging probe with superior properties for in vivo molecular magnetic resonance imaging (MRI) applications. A small peptide was screened and selected from a library of peptides with potential to bind to collagen identified based on protein-protein interaction studies. The peptide was conjugated to a DOTA-chelator and labelled with Europium [Eu(III)] for in vitro binding assays; gallium (68Ga) for in vivo PET/CT biodistribution; and gadolinium [Gd(III)] for in vivo MRI studies. The probe was further modified to increase the number of Gd(III) per peptide (from one to four) to amplify and prolong the MRI signal. The probe was validated using a surgical mouse model of myocardial infarction (MI). In vivo MRI was performed at days 10 and 21 post-MI (n=7). Imaging findings were validated with tissue analysis. A negative control probe, carrying a scrambled peptide sequence was used. All MRI experiments were performed at a 3 Tesla clinical MRI scanner. In vitro binding assays showed that the probe has a good affinity towards collagen III (Kd= 5.2±1.3µM) that is in the ideal range for a molecular imaging probe.4 Lack of binding of the scrambled probe (negative control) proved the specificity our probe (figure 1A). In vivo PET/CT biodistribution showed favourable pharmacokinetics with fast blood clearance and no unspecific binding (figure 1B). In vivo cardiac MRI showed selective late gadolinium enhancement (LGE) of the fibrotic scar at day 10 which decreased by day 21. This observation is expected as collagen III naturally gets replaced by collagen I at the later stages of cardiac fibrosis. The imaging data are validated histologically showing co-localisation of the MRI signal with collagen III (green colour) at day 10 and reduction of collagen III at day 21 (figure 2). Importantly, no enhancement was observed using the negative control probe and a clinically approved non-collagen targeting probe (Gadovist). We have developed a new molecular imaging probe specific for collagen type III. Using this probe, we have successfully imaged - previously undetectable - collagen III in cardiac fibrosis. This approach may enable early detection and characterisation of cardiac fibrosis in vivo allowing staging of disease and monitoring of therapie

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

Imaging of Dysfunctional Elastogenesis in Atherosclerosis Using an Improved Gadolinium-Based Tetrameric MRI Probe Targeted to Tropoelastin

Dysfunctional elastin turnover plays a major role in the progression of atherosclerotic plaques. Failure of tropoelastin cross-linking into mature elastin leads to the accumulation of tropoelastin within the growing plaque, increasing its instability. Here we present Gd4-TESMA, an MRI contrast agent specifically designed for molecular imaging of tropoelastin within plaques. Gd4-TESMA is a tetrameric probe composed of a tropoelastin-binding peptide (the VVGS-peptide) conjugated with four Gd(III)-DOTA-monoamide chelates. It shows a relaxivity per molecule of 34.0 ± 0.8 mM-1 s-1 (20 MHz, 298 K, pH 7.2), a good binding affinity to tropoelastin (KD = 41 ± 12 μM), and a serum half-life longer than 2 h. Gd4-TESMA accumulates specifically in atherosclerotic plaques in the ApoE-/- murine model of plaque progression, with 2 h persistence of contrast enhancement. As compared to the monomeric counterpart (Gd-TESMA), the tetrameric Gd4-TESMA probe shows a clear advantage regarding both sensitivity and imaging time window, allowing for a better characterization of atherosclerotic plaques

PET performance evaluation of a pre-clinical SiPM-Based MR-Compatible PET Scanner

We have carried out a PET performance evaluation a silicon photo-multiplier (SiPM) based PET scanner designed for fully simultaneous pre-clinical PET/MR studies. The PET scanner has an inner diameter of 20 cm with an LYSO crystal size of 1.3 by 1.3 by 10 mm. The axial PET field of view (FOV) is 30.2 mm. The PET detector modules, which incorporate SiPMs, have been designed to be MR-compatible allowing them to be located directly within a Philips Achieva 3T MR scanner. The spatial resolution of the system measured using a point source in a non-active background, is just under 2.3 mm full width at half maximum (FWHM) in the transaxial direction when single slice rebinning (SSRB) and 2D filtered back-projection (FBP) is used for reconstruction, and 1.3 mm FWHM when resolution modeling is employed. The system sensitivity is 0.6% for a point source at the center of the FOV. The true coincidence count rate shows no sign of saturating at 30 MBq, at which point the randoms fraction is 8.2%, and the scatter fraction for a rat sized object is approximately 23%. Artifact-free images of phantoms have been obtained using FBP and iterative reconstructions. The performance is currently limited because only one of three axial ring positions is populated with detectors, and due to limitations of the first-generation detector readout ASIC used in the system. The performance of the system as described is sufficient for simultaneous PET-MR imaging of rat-sized animals and large organs within the mouse. This is demonstrated with dynamic PET and MR data acquired simultaneously from a mouse injected with a dual-labeled PET/MR probe