Sandra M. Bovens

Molecular MRI of murine atherosclerotic plaque targeting NGAL: a protein associated with unstable human plaque characteristics

AIMS Neutrophil gelatinase-associated lipocalin (NGAL) is an effector molecule of the innate immune system. One of its actions is the prolongation of matrix metalloproteinase-9 (MMP-9) activity by the formation of a degradation-resistant NGAL/MMP-9 complex. We studied NGAL in human atherosclerotic lesions and we examined whether NGAL could act as a target for molecular imaging of atherosclerotic plaques. METHODS AND RESULTS Increased levels of NGAL and the NGAL/MMP-9 complex were associated with high lipid content, high number of macrophages, high interleukin-6 (IL-6) and IL-8 levels, and low smooth muscle cell content in human atherosclerotic lesions obtained during carotid endarterectomy (n= 122). Moreover, plaque levels of NGAL tended to be higher when intra-plaque haemorrhage (IPH) or luminal thrombus was present (n= 77) than without the presence of IPH or thrombus (n= 30). MMP-9 and -8 activities were strongly related to NGAL levels. The enhancement on magnetic resonance (MR) images of the abdominal aorta of ApoE(-/-)/eNOS(-/-) mice was observed at 72 h after injection of NGAL/24p3-targeted micelles. The specificity of these results was validated by histology, and co-localization of micelles, macrophages, and NGAL/24p3 was observed. CONCLUSION NGAL is highly expressed in atheromatous human plaques and associated with increased MMP-9 activity. NGAL can be detected in murine atherosclerotic arteries using targeted high-resolution MR imaging. Therefore, we conclude that NGAL might serve as a novel imaging target for the detection of high-risk plaques.

Current Status of Clinical Magnetic Resonance Imaging for Plaque Characterisation in Patients with Carotid Artery Stenosis

OBJECTIVE The article aims to provide an overview of the literature that assessed the agreement between magnetic resonance imaging (MRI) and histology for specific carotid plaque characteristics associated with vulnerability in terms of sensitivity and specificity. METHODS A systematic search strategy was conducted in MEDLINE and EMBASE databases resulting in 1084 articles. Finally, we included 17 papers. Due to variation in presentation, especially in MRI and histology methods, a pooled analysis could not be performed. RESULTS Two studies were performed on a 3.0-T MRI scanner; all other studies were performed on a 1.5-T scanner. Most performed sequences were two-dimensional (2D) and three-dimensional (3D) T1-weighted and all histology protocols varied slightly. Our results indicate that calcification, fibrous cap, intraplaque haemorrhage and lipid-rich necrotic cores can be identified with moderate-to-good sensitivity and specificity. CONCLUSIONS Based on current literature, it appears premature for routine application of MRI as an imaging modality to assess carotid plaque characteristics associated with plaque vulnerability. Although MRI still holds promise, clinical application for plaque characterisation would require consensus regarding MRI settings and confirmation by histology. Predefined protocols for histology and MR imaging need to be established.

Evaluation of infarcted murine heart function: comparison of prospectively triggered with self-gated MRI

Measurement of cardiac function is often performed in mice after, for example, a myocardial infarction. Cardiac MRI is often used because it is noninvasive and provides high temporal and spatial resolution for the left and right ventricle. In animal cardiac MRI, the quality of the required electrocardiogram signal is variable and sometimes deteriorates over time, especially with infarcted hearts or cardiac hypertrophy. Therefore, we compared the self‐gated IntraGateFLASH method with a prospectively triggered FLASH (fast low‐angle shot) method in mice with myocardial infarcts (n = 16) and in control mice (n = 21). Mice with a myocardial infarct and control mice were imaged in a vertical 9.4‐T MR system. Images of contiguous 1‐mm slices were acquired from apex to base with prospective and self‐gated methods. Data were processed to calculate cardiac function parameters for the left and right ventricle. The signal‐to‐noise and contrast‐to‐noise ratios were calculated in mid‐ventricular slices. The signal‐to‐noise and contrast‐to‐noise ratios of the self‐gated data were higher than those of the prospectively gated data. Differences between the two gating methods in the cardiac function parameters for both left and right ventricle (e.g. end‐diastolic volumes) did not exceed the inter‐observer variability in control or myocardial infarcted mice. Both methods gave comparable results with regard to the cardiac function parameters in both healthy control mice and mice with myocardial infarcts. Moreover, the self‐gated method provided better signal‐to‐noise and contrast‐to‐noise ratios when the acquisition time was equal. In conclusion, the self‐gated method is suitable for routine use in cardiac MRI in mice with myocardial infarcts as well as in control mice, and obviates the need for electrocardiogram triggering and respiratory gating. In both gating methods, more than 10 frames per cardiac cycle are recommended. Copyright

Thin-Cap Fibroatheroma Rupture Is Associated With a Fine Interplay of Shear and Wall Stress

In this review, we summarized the effect of mechanical factors (shear and wall stress) on thin-cap fibroatheroma formation and rupture. To make this review understandable for a biology-oriented audience, we start with detailed definitions of relevant mechanical metrics. We then describe how biomechanics has supported histopathologic efforts to understand the basis of plaque rupture. In addition to plaque rupture, biomechanics also contributes toward the progression of thin-cap fibroatheroma through a multitude of reported mechanobiological mechanisms. We thus propose a new mechanism whereby both shear stress and wall stress interact to create thin-cap fibroatheromas. Specifically, when regions of certain blood flow and wall mechanical stimuli coincide, they synergistically create inflammation within the cellular environment that can lead to thin-cap fibroatheroma rupture. A consequence of this postulate is that local shear stress is not sufficient to cause rupture, but it must coincide with regions of local tissue stiffening and stress concentrations that can occur during plaque progression. Because such changes to the wall mechanics occur over a micrometer scale, high spatial resolution imaging techniques will be necessary to evaluate this hypothesis and ultimately predict plaque rupture in a clinical environment.

Systems biology of the functional and dysfunctional endothelium

This review provides an overview of the effect of blood flow on endothelial cell (EC) signalling pathways, applying microarray technologies to cultured cells, and in vivo studies of normal and atherosclerotic animals. It is found that in cultured ECs, 5-10% of genes are up- or down-regulated in response to fluid flow, whereas only 3-6% of genes are regulated by varying levels of fluid flow. Of all genes, 90% are regulated by the steady part of fluid flow and 10% by pulsatile components. The associated gene profiles show high variability from experiment to experiment depending on experimental conditions, and importantly, the bioinformatical methods used to analyse the data. Despite this high variability, the current data sets can be summarized with the concept of endothelial priming. In this concept, fluid flows confer protection by an up-regulation of anti-atherogenic, anti-thrombotic, and anti-inflammatory gene signatures. Consequently, predilection sites of atherosclerosis, which are associated with low-shear stress, confer low protection for atherosclerosis and are, therefore, more sensitive to high cholesterol levels. Recent studies in intact non-atherosclerotic animals confirmed these in vitro studies, and suggest that a spatial component might be present. Despite the large variability, a few signalling pathways were consistently present in the majority of studies. These were the MAPK, the nuclear factor-κB, and the endothelial nitric oxide synthase-NO pathways.

Seven-Tesla Magnetic Resonance Imaging of Atherosclerotic Plaque in the Significantly Stenosed Carotid Artery A Feasibility Study

ObjectivesThe objective of this study was to assess the feasibility of carotid vessel wall imaging at 7.0 for T magnetic resonance imaging (MRI) in a series of patients with a symptomatic greater than 70% stenosis of the internal carotid artery. Materials and MethodsFirst, a series of 6 healthy volunteers were scanned at 3.0 T and 7.0 T MRI to perform a signal-to-noise ratio comparison between these 2 field strengths. Second, in patients with a greater than 70% stenosed carotid artery, a 7.0 T MRI protocol, consisting of a dual-echo turbo spin echo sequence (echo times of 45 and 150 milliseconds) and a T1-weighted turbo spin echo sequence, was obtained. Lumen and vessel wall were delineated for interobserver and intraobserver reproducibility, and signal intensity distribution in the most severely stenosed part of the internal carotid artery was correlated with different plaque components on histopathologic findings. ResultsThe mean (SD) signal-to-noise ratio in the vessel wall was 42 (12) at 7.0 T and 24 (4) at 3.0 T. Nineteen patients were included, but technical issues yielded carotid MRI data of 14 patients available for the final analysis. Of these patients, 4 were diagnosed with stroke, 7 were diagnosed with a transient ischemic attack, and 3 were diagnosed with amaurosis fugax. Intraclass correlation coefficient of the agreements of lumen and vessel wall determination between 2 observers and between the repeated measures of 1 observer were above 0.80 in both 3.0 T and 7.0 T data sets of the healthy volunteers and also in the 7.0 T data set of the patients. Signal hyperintensity in the 7.0 T magnetic resonance images was inversely proportional to calcification. Other correlations between plaque components and signal intensity could not be confirmed. ConclusionsThis first series of patients with carotid atherosclerotic plaque who were scanned at 7.0 T MRI shows that 7.0 T MRI enables to adequately determine lumen and vessel wall areas. Signal hyperintensity in these 7.0 T magnetic resonance images was inversely proportional to calcification. However, at this stage, no other correlations between histologic findings and vessel wall contrast were found. Implementation of in vivo high-resolution 7.0 T MRI of plaque components for risk stratification remains challenging. Future development of hardware and software is still needed to attain a more robust setup and to enable complete plaque characterization, similar to what is currently possible with multiple MRI sequences at 1.5 T and 3.0 T MRI.

7.0 T MRI detection of cerebral microinfarcts in patients with a symptomatic high-grade carotid artery stenosis.

In the current study, the presence of cerebral cortical microinfarcts (CMIs) was evaluated in a series of 21 patients with a symptomatic high-grade > 50% stenosis of the carotid artery. A T2-weighted fluid-attenuated inversion recovery sequence and a T1-weighted turbo field echo sequence of the brain were obtained at 7.0 Tesla magnetic resonance imaging. Primary study endpoint was the number of CMIs and macroinfarcts. In total, 53 cerebral infarcts (35 macroinfarcts; 18 CMIs) were found ipsilateral to the symptomatic carotid artery, in 14 patients (67%). In four of these patients, both CMIs and macroinfarcts were visible. In the contralateral hemisphere, seven infarcts (five macroinfarcts and two CMIs) were found in five patients (24%). In the ipsilateral hemispheres, the number of CMIs and macroinfarcts were significantly correlated (P = 0.02). Unpaired comparison of medians showed that the number of CMIs in the ipsilateral hemisphere was significantly higher than the number of CMIs in the contralateral hemisphere (P = 0.04). No significant correlation was found between stenosis grade and the number of any infarct. The current study shows that in symptomatic patients with significant extracranial carotid artery stenosis, CMIs are part of the total cerebrovascular burden and these CMIs prevail with a similar pattern as observed macroinfarcts.

BLT1 antagonist LSN2792613 reduces infarct size in a mouse model of myocardial ischaemia–reperfusion injury

AIMS Restoration of coronary blood flow is crucial in the treatment of acute myocardial infarction. Reperfusion, however, induces ischaemia-reperfusion (IR) injury, which further deteriorates myocardial function. The innate immune system plays an important role in this process, mediating rapid influx of immune cells into the reperfused myocardium. Leukotriene B4 is an important leucocyte chemoattractant, performing its actions through binding to its specific receptor BLT1. We hypothesized that treatment with LSN2792613, a selective BLT1 antagonist, reduces infarct size (IS) in a mouse model of myocardial IR injury. METHODS AND RESULTS Male C57Bl/6J mice were subjected to myocardial ischaemia for 30 min by surgical coronary artery ligation, followed by reperfusion. Mice received either LSN2792613 or vehicle, three times daily (orally) for up to 72 h after reperfusion. BLT1 inhibition with LSN2792613 reduced IS compared with vehicle treatment (26.9 ± 2.7 vs. 34.9 ± 2.2%, P = 0.030) at 24 h after reperfusion. The levels of IL-6 and keratinocyte chemoattractant were reduced in the infarcted tissue of LSN2792613-treated mice. Reduced apoptosis in LSN2792613-treated mice was suggested by increased levels of phosphorylated JNK and GSK3α/β, and confirmed by flow cytometric analysis showing less apoptotic and necrotic cardiomyocytes in the infarcted myocardium. Echocardiography at 4 weeks after myocardial IR showed a slightly higher ejection fraction and stroke volume in mice treated with LSN2792613 compared with vehicle-treated mice, whereas left ventricular volumes were comparable. CONCLUSION Selective BLT1 inhibition with LSN2792613 reduces inflammation and apoptosis following IR, resulting in reduced IS, and therefore might be a promising strategy to prevent myocardial IR injury.

Histological validation of iron-oxide and gadolinium based MRI contrast agents in experimental atherosclerosis: The do's and don't's

MRI using targeted contrast agents (CA) has emerged as a promising technique to study atherothrombotic disease in vivo. Particularly, the use of targeted Gd and lipid-based nanoparticles has enabled detailed in vivo imaging of various molecular markers of atherosclerotic plaque pathophysiology. For validation purposes, it is crucial that nanoparticle accumulation in the plaque, cellular association and localization can be assessed by ex vivo immuno-histology or fluorescence microscopy of tissue sections. In this review we discuss the various methods that are available for histological evaluation of targeted MRI contrast agents such as lipid-based nanoparticles and iron oxide particles. We discuss the detection of these contrast agents in paraffin-embedded and in cryopreserved tissue sections of atherosclerotic plaques. During the embedding procedure in paraffin, most components of targeted lipid-based nanoparticles are generally washed out, though the actual targeting moieties may be retained in the embedded sections. Therefore staining of the antibody-antigen complex provides a suitable way to visualize the presence of the nanoparticle in the plaque. In cryosections, the localization of nanoparticles can be assessed directly by measuring the fluorescence of an incorporated fluorophore or by secondary stainings of the Gd-containing DTPA lipids or the iron oxide particles. With certain secondary stainings, be it for the contrast agent or for co-localization with the target, the contrast agent itself may interfere with standard histological protocols, yielding false positive results. The here presented techniques enable proper visualization of MR contrast agent accumulation and localization in atherosclerotic plaque, which will provide the validation necessary to advance these lipid-based nanoparticles to the clinic.

PLACD-7T Study: Atherosclerotic Carotid Plaque Components Correlated with Cerebral Damage at 7 Tesla Magnetic Resonance Imaging

Introduction: In patients with carotid artery stenosis histological plaque composition is associated with plaque stability and with presenting symptomatology. Preferentially, plaque vulnerability should be taken into account in pre-operative work-up of patients with severe carotid artery stenosis. However, currently no appropriate and conclusive (non-) invasive technique to differentiate between the high and low risk carotid artery plaque in vivo is available. We propose that 7 Tesla human high resolution MRI scanning will visualize carotid plaque characteristics more precisely and will enable correlation of these specific components with cerebral damage. Study objective: The aim of the PlaCD-7T study is 1: to correlate 7T imaging with carotid plaque histology (gold standard); and 2: to correlate plaque characteristics with cerebral damage ((clinically silent) cerebral (micro) infarcts or bleeds) on 7 Tesla high resolution (HR) MRI. Design: We propose a single center prospective study for either symptomatic or asymptomatic patients with haemodynamic significant (70%) stenosis of at least one of the carotid arteries. The Athero-Express (AE) biobank histological analysis will be derived according to standard protocol. Patients included in the AE and our prospective study will undergo a pre-operative 7 Tesla HR-MRI scan of both the head and neck area. Discussion: We hypothesize that the 7 Tesla MRI scanner will allow early identification of high risk carotid plaques being associated with micro infarcted cerebral areas, and will thus be able to identify patients with a high risk of periprocedural stroke, by identification of surrogate measures of increased cardiovascular risk.