Max L. Senders

Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis

Significance This study shows that an endothelialized microfluidic chip with controllable permeability can serve as a model for nanoparticle translocation across the permeable endothelium. Integration of this in vitro model and an in vivo rabbit model revealed that the extravasation of nanoparticles across the endothelium in atherosclerotic plaques depends on microvascular permeability. This approach represents a unique method for the assessment of nanoparticle behavior across the atherosclerotic endothelium, and may also serve as a valuable tool to study nanomedicine accumulation in a variety of other diseases. Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid–polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.

Atherosclerotic plaque targeting mechanism of long-circulating nanoparticles established by multimodal imaging.

Atherosclerosis is a major cause of global morbidity and mortality that could benefit from novel targeted therapeutics. Recent studies have shown efficient and local drug delivery with nanoparticles, although the nanoparticle targeting mechanism for atherosclerosis has not yet been fully elucidated. Here we used in vivo and ex vivo multimodal imaging to examine permeability of the vessel wall and atherosclerotic plaque accumulation of fluorescently labeled liposomal nanoparticles in a rabbit model. We found a strong correlation between permeability as established by in vivo dynamic contrast enhanced magnetic resonance imaging and nanoparticle plaque accumulation with subsequent nanoparticle distribution throughout the vessel wall. These key observations will enable the development of nanotherapeutic strategies for atherosclerosis.

Polyglucose nanoparticles with renal elimination and macrophage avidity facilitate PET imaging in ischaemic heart disease

Tissue macrophage numbers vary during health versus disease. Abundant inflammatory macrophages destruct tissues, leading to atherosclerosis, myocardial infarction and heart failure. Emerging therapeutic options create interest in monitoring macrophages in patients. Here we describe positron emission tomography (PET) imaging with 18F-Macroflor, a modified polyglucose nanoparticle with high avidity for macrophages. Due to its small size, Macroflor is excreted renally, a prerequisite for imaging with the isotope flourine-18. The particles short blood half-life, measured in three species, including a primate, enables macrophage imaging in inflamed cardiovascular tissues. Macroflor enriches in cardiac and plaque macrophages, thereby increasing PET signal in murine infarcts and both mouse and rabbit atherosclerotic plaques. In PET/magnetic resonance imaging (MRI) experiments, Macroflor PET imaging detects changes in macrophage population size while molecular MRI reports on increasing or resolving inflammation. These data suggest that Macroflor PET/MRI could be a clinical tool to non-invasively monitor macrophage biology.

Three-dimensional dynamic contrast-enhanced MRI for the accurate, extensive quantification of microvascular permeability in atherosclerotic plaques.

Atherosclerotic plaques that cause stroke and myocardial infarction are characterized by increased microvascular permeability and inflammation. Dynamic contrast‐enhanced MRI (DCE‐MRI) has been proposed as a method to quantify vessel wall microvascular permeability in vivo. Until now, most DCE‐MRI studies of atherosclerosis have been limited to two‐dimensional (2D) multi‐slice imaging. Although providing the high spatial resolution required to image the arterial vessel wall, these approaches do not allow the quantification of plaque permeability with extensive anatomical coverage, an essential feature when imaging heterogeneous diseases, such as atherosclerosis. To our knowledge, we present the first systematic evaluation of three‐dimensional (3D), high‐resolution, DCE‐MRI for the extensive quantification of plaque permeability along an entire vascular bed, with validation in atherosclerotic rabbits. We compare two acquisitions: 3D turbo field echo (TFE) with motion‐sensitized‐driven equilibrium (MSDE) preparation and 3D turbo spin echo (TSE). We find 3D TFE DCE‐MRI to be superior to 3D TSE DCE‐MRI in terms of temporal stability metrics. Both sequences show good intra‐ and inter‐observer reliability, and significant correlation with ex vivo permeability measurements by Evans Blue near‐infrared fluorescence (NIRF). In addition, we explore the feasibility of using compressed sensing to accelerate 3D DCE‐MRI of atherosclerosis, to improve its temporal resolution and therefore the accuracy of permeability quantification. Using retrospective under‐sampling and reconstructions, we show that compressed sensing alone may allow the acceleration of 3D DCE‐MRI by up to four‐fold. We anticipate that the development of high‐spatial‐resolution 3D DCE‐MRI with prospective compressed sensing acceleration may allow for the more accurate and extensive quantification of atherosclerotic plaque permeability along an entire vascular bed. We foresee that this approach may allow for the comprehensive and accurate evaluation of plaque permeability in patients, and may be a useful tool to assess the therapeutic response to approved and novel drugs for cardiovascular disease. Copyright

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe–/– mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe–/– mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP–immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates

Macrophage accumulation in atherosclerosis is directly linked to the destabilization and rupture of plaque, causing acute atherothrombotic events. Circulating monocytes enter the plaque and differentiate into macrophages, where they are activated by CD4+ T lymphocytes through CD40–CD40 ligand signalling. Here, we report the development and multiparametric evaluation of a nanoimmunotherapy that moderates CD40–CD40 ligand signalling in monocytes and macrophages by blocking the interaction between CD40 and tumour necrosis factor receptor-associated factor 6 (TRAF6). We evaluated the biodistribution characteristics of the nanoimmunotherapy in apolipoprotein E-deficient (Apoe–/–) mice and in non-human primates by in vivo positron-emission tomography imaging. In Apoe–/– mice, a 1-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, which was due to the impaired migration capacity of monocytes, as established by a transcriptome analysis. The rapid reduction of plaque inflammation by the TRAF6-targeted nanoimmunotherapy and its favourable toxicity profiles in both mice and non-human primates highlights the translational potential of this strategy for the treatment of atherosclerosis.A nanoparticle-based immunotherapy targeted to a protein responsible for signalling between monocytes and macrophages reduces plaque inflammation in atherosclerotic mice and appears to be safe in non-human primates.

Targeting myeloperoxidase in inflammatory atherosclerosis

Atherosclerosis is a chronic immune disorder driven by the accumulation of lipids in the vessel wall, resulting in the formation of plaques. Progression of the disease may ultimately lead to peripheral artery disease or the occurrence of clinical events, including stroke and myocardial infarction. Our view on the atherosclerosis pathophysiology is shifting from an emphasis on lipids to vascular immunology, particularly macrophage dynamics. The recently completed CANTOS trial unveiled a decrease in recurrent cardiovascular events through treatment with an interleukin-1b-blocking antibody as compared with placebo. Cumulative evidence supports the notion that macrophage inflammation is an important driver of disease progression with major involvement of the haematopoietic organs. Myeloid cells produced by these tissues are recruited by endothelial cells and enabled entry into the plaque. Monocyte recruitment is an important driver of atherosclerosis in early stages, while in progressed plaques macrophage proliferation seems to dominate vessel wall inflammation. Neutrophils are the most abundant myeloid cells present in the circulation. Apart from the release of neutrophil extracellular traps (NETs), neutrophils secrete granulocytes to aid in degradation of microbiomes and to regulate immune responses. Their cargo is discharged upon activation in the plasma or when interaction with microbiomes is initiated. One of the most important enzymes released by neutrophils through degranulation is myeloperoxidase (MPO). In patients with elevated levels of circulating MPO, increased cardiovascular events are observed while, in atherosclerotic lesions, MPO interacts with the extracellular matrix to form hypochlorous acid by converting chloride anion and hydrogen peroxide. The presence of MPO in plaques has been associated with endothelium apoptosis, superficial erosion, and lesion rupture (Take home figure). Altogether, it is becoming increasingly evident that MPO is a compelling imaging and therapeutic target in atherosclerosis. In this issue of the European Heart Journal, using a mouse model of atherosclerosis, Rashid et al. describe the evaluation of MPO magnetic resonance imaging (MRI) to detect unstable plaques. Additionally, the study explores MPO (Take home figure) as a target for directed therapy aimed at stabilizing the plaque phenotype. Mice with an Apoe genetic background, an established atherosclerosis model, underwent tandem stenosis after being fed a 6-week-long enriched Western diet. The surgical procedure involves partially ligating the right common carotid artery at two places distally from the innominate artery. If successful, this results in the formation of plaques that display several characteristics observed in unstable human plaques, including intraplaque haemorrhage, disruptive fibrous caps, intraluminal stenosis, and neovessels. First, the authors compare a MPO-targeted gadolinium (MPOGd) contrast agent with the generic Gd-DTPA (Magnevist). MPOGd was observed to accumulate and be retained in plaques up to 60 min after injection, while Gd-DTPA washed out more rapidly. Importantly, MPO-Gd administration allowed distinguishing stable from unstable plaques by using a T1-weighted MR sequence resulting in a hyperintense vessel wall. Validation studies confirmed the in vivo findings by establishing surrogate markers of MPO activity using liquid chromatography tandem mass spectrometry (LC-MS/ MS). To corroborate the agent’s specificity, MPO was knockedout in Apoe mice. The Apoe MPO double knockouts underwent tandem stenosis and were subjected to in vivo MRI after injection with MPO-Gd. Significantly less contrast was found compared with regular tandem stenosis Apoe mice. Immunohistochemistry with picrosirius red staining revealed a significantly increased fibrous cap thickness and cap to lesion height ratio in the Apoe MPO double knockouts. Rashid et al. subsequently studied whether blocking MPO in tandem stenosis Apoe mice through pharmacological inhibition with AZM198 showed similar findings compared with genetic deletion. Indeed, they found the MPO-Gd contrast and LC-MS/MS measurements to be very similar to the values observed in the Apoe MPO double knockout mice. However, MPO staining showed no difference in MPO expression compared with non-treated tandem

Development and Multiparametric Evaluation of Experimental Atherosclerosis in Rabbits

Several animal models have been developed to study atherosclerosis. Here we present a rabbit atherosclerosis model generated by surgical denudation of the aortic endothelium in combination with a high-fat and cholesterol-enriched diet. This model is characterized by the formation of vascular lesions that exhibit several hallmarks of human atherosclerosis. Due to the rabbits relative large size, as compared to rodents, this model is suited for the imaging-guided evaluation of novel therapeutic strategies using clinical scanners. In this chapter, we present an extensive outline of the procedures to induce aortic atherosclerotic lesions in rabbits as well as methods to evaluate the disease, including noninvasive in vivo multiparametric imaging and histopathology.

Author Correction: Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates

In the version of this Article originally published, the surname of the author Edward A. Fisher was spelt incorrectly as ‘Fischer’. This has now been corrected.

Nanobody-Facilitated Multiparametric PET/MRI Phenotyping of Atherosclerosis

OBJECTIVES This study sought to develop an integrative positron emission tomography (PET) with magnetic resonance imaging (MRI) procedure for accurate atherosclerotic plaque phenotyping, facilitated by clinically approved and nanobody radiotracers. BACKGROUND Noninvasive characterization of atherosclerosis remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated. METHODS We screened 3 nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule (VCAM)-1, lectin-like oxidized low-density lipoprotein receptor (LOX)-1, and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with copper-64 (64Cu), were extensively evaluated in Apoe–/– mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography, and histological analyses. RESULTS The 3 nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with gallium-68 (68Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68Ga-MMR–PET, plaque burden by T2-weighted MRI, and neovascularization by dynamic contrast-enhanced (DCE) MRI. Additionally, inflammation and microcalcifications were evaluated by fluorine-18 (18F)-labeled fluorodeoxyglucose (18F-FDG) and 18F-sodium fluoride (18F-NaF) PET, respectively. We observed an increase in all the aforementioned measures as disease progressed, and the imaging signatures correlated with histopathological features. CONCLUSIONS We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MRI protocol that allows noninvasive assessment of different processes relevant to atherosclerosis progression. This approach allows the multiparametric study of atherosclerosis and can aid in early stage anti-atherosclerosis drug trials.