Tissue-engineered collagenous fibrous cap models to systematically elucidate atherosclerotic plaque rupture.

A significant amount of vascular thrombotic events are associated with rupture of the fibrous cap that overlie atherosclerotic plaques. Cap rupture is however difficult to predict due to the heterogenous composition of the plaque, unknown material properties, and the stochastic nature of the event. Here, we aim to create tissue engineered human fibrous cap models with a variable but controllable collagen composition, suitable for mechanical testing, to scrutinize the reciprocal relationships between composition and mechanical properties. Myofibroblasts were cultured in 1 × 1.5 cm-sized fibrin-based constrained gels for 21 days according to established (dynamic) culture protocols (i.e. static, intermittent or continuous loading) to vary collagen composition (e.g. amount, type and organization). At day 7, a soft 2 mm ∅ fibrin inclusion was introduced in the centre of each tissue to mimic the soft lipid core, simulating the heterogeneity of a plaque. Results demonstrate reproducible collagenous tissues, that mimic the bulk mechanical properties of human caps and vary in collagen composition due to the presence of a successfully integrated soft inclusion and the culture protocol applied. The models can be deployed to assess tissue mechanics, evolution and failure of fibrous caps or complex heterogeneous tissues in general.

SPECT/CT imaging of inflammation and calcification in human carotid atherosclerosis to identify the plaque at risk of rupture.

BACKGROUND: Calcification and inflammation are atherosclerotic plaque compositional biomarkers that have both been linked to stroke risk. The aim of this study was to evaluate their co-existing prevalence in human carotid plaques with respect to plaque phenotype to determine the value of hybrid imaging for the detection of these biomarkers. METHODS: Human carotid plaque segments, obtained from endarterectomy, were incubated in [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt), targeting Leukocyte Function-associated Antigen-1 (LFA-1) on leukocytes. By performing SPECT/CT, both inflammation from DANBIRT uptake and calcification from CT imaging were assessed. Plaque phenotype was classified using histology. RESULTS: On a total plaque level, comparable levels of calcification volume existed with different degrees of inflammation and vice versa. On a segment level, an inverse relationship between calcification volume and inflammation was evident in highly calcified segments, which classify as fibrocalcific, stable plaque segments. In contrast, segments with little or no calcification presented with a moderate to high degree of inflammation, often coinciding with the more dangerous fibrous cap atheroma phenotype. CONCLUSION: Calcification imaging alone can only accurately identify highly calcified, stable, fibrocalcific plaques. To identify high-risk plaques, with little or no calcification, hybrid imaging of calcification and inflammation could provide diagnostic benefit.

The Correlation Between Wall Shear Stress and Plaque Composition in Advanced Human Carotid Atherosclerosis.

The role of wall shear stress (WSS) in atherosclerotic plaque development is evident, but the relation between WSS and plaque composition in advanced atherosclerosis, potentially resulting in plaque destabilization, is a topic of discussion. Using our previously developed image registration pipeline, we investigated the relation between two WSS metrics, time-averaged WSS (TAWSS) and the oscillatory shear index (OSI), and the local histologically determined plaque composition in a set of advanced human carotid plaques. Our dataset of 11 carotid endarterectomy samples yielded 87 histological cross-sections, which yielded 511 radial bins for analysis. Both TAWSS and OSI values were subdivided into patient-specific low, mid, and high tertiles. This cross-sectional study shows that necrotic core (NC) size and macrophage area are significantly larger in areas exposed to high TAWSS or low OSI. Local TAWSS and OSI tertile values were generally inversely related, as described in the literature, but other combinations were also found. Investigating the relation between plaque vulnerability features and different combinations of TAWSS and OSI tertile values revealed a significantly larger cap thickness in areas exposed to both low TAWSS and low OSI. In conclusion, our study confirmed previous findings, correlating high TAWSS to larger macrophage areas and necrotic core sizes. In addition, our study demonstrated new relations, correlating low OSI to larger macrophage areas, and a combination of low TAWSS and low OSI to larger cap thickness.

An MRI-based method to register patient-specific wall shear stress data to histology.

Wall shear stress (WSS), the frictional force exerted on endothelial cells by blood flow, is hypothesised to influence atherosclerotic plaque growth and composition. We developed a methodology for image registration of MR and histology images of advanced human carotid plaques and corresponding WSS data, obtained by MRI and computational fluid dynamics. The image registration method requires four types of input images, in vivo MRI, ex vivo MRI, photographs of transversally sectioned plaque tissue and histology images. These images are transformed to a shared 3D image domain by applying a combination of rigid and non-rigid registration algorithms. Transformation matrices obtained from registration of these images are used to transform subject-specific WSS data to the shared 3D image domain as well. WSS values originating from the 3D WSS map are visualised in 2D on the corresponding lumen locations in the histological sections and divided into eight radial segments. In each radial segment, the correlation between WSS values and plaque composition based on histological parameters can be assessed. The registration method was successfully applied to two carotid endarterectomy specimens. The resulting matched contours from the imaging modalities had Hausdorff distances between 0.57 and 0.70 mm, which is in the order of magnitude of the in vivo MRI resolution. We simulated the effect of a mismatch in the rigid registration of imaging modalities on WSS results by relocating the WSS data with respect to the stack of histology images. A 0.6 mm relocation altered the mean WSS values projected on radial bins on average by 0.59 Pa, compared to the output of original registration. This mismatch of one image slice did not change the correlation between WSS and plaque thickness. In conclusion, we created a method to investigate correlations between WSS and plaque composition.

Perspectives on Small Animal Radionuclide Imaging; Considerations and Advances in Atherosclerosis.

This review addresses nuclear SPECT and PET imaging in small animals in relation to the atherosclerotic disease process, one of our research topics of interest. Imaging of atherosclerosis in small animal models is challenging, as it operates at the limits of current imaging possibilities regarding sensitivity, and spatial resolution. Several topics are discussed, including technical considerations that apply to image acquisition, reconstruction, and analysis. Moreover, molecules developed for or applied in these small animal nuclear imaging studies are listed, including target-directed molecules, useful for imaging organs or tissues that have elevated expression of the target compared to other tissues, and molecules that serve as substrates for metabolic processes. Differences between animal models and human pathophysiology that should be taken into account during translation from animal to patient as well as differences in tracer behavior in animal vs. man are also described. Finally, we give a future outlook on small animal radionuclide imaging in atherosclerosis, followed by recommendations. The challenges and solutions described might be applicable to other research fields of health and disease as well.

Imaging of atherosclerosis, targeting LFA-1 on inflammatory cells with 111In-DANBIRT.

BACKGROUND: 111In-DOTA-butylamino-NorBIRT (DANBIRT) is a novel radioligand which binds to Leukocyte Function-associated Antigen-1 (LFA-1), expressed on inflammatory cells. This study evaluated 111In-DANBIRT for the visualization of atherosclerotic plaque inflammation in mice. METHODS AND RESULTS: ApoE-/- mice, fed an atherogenic diet up to 20 weeks (n = 10), were imaged by SPECT/CT 3 hours post injection of 111In-DANBIRT (~ 200 pmol, ~ 40 MBq). Focal spots of 111In-DANBIRT were visible in the aortic arch of all animals, with an average Target-to-Background Ratio (TBR) of 1.7 ± 0.5. In vivo imaging results were validated by ex vivo SPECT/CT imaging, with a TBR up to 11.5 (range 2.6 to 11.5). Plaques, identified by Oil Red O lipid-staining on excised arteries, co-localized with 111In-DANBIRT uptake as determined by ex vivo autoradiography. Subsequent histological processing and in vitro autoradiography confirmed 111In-DANBIRT uptake at plaque areas containing CD68 expressing macrophages and LFA-1 expressing inflammatory cells. Ex vivo incubation of a human carotid endarterectomy specimen with 111In-DANBIRT (~ 950 nmol, ~ 190 MBq) for 2 hours showed heterogeneous plaque uptake on SPECT/CT, after which immunohistochemical analysis demonstrated co-localization of 111In-DANBIRT uptake and CD68 and LFA-1 expressing cells. CONCLUSIONS: Our results indicate the potential of radiolabeled DANBIRT as a relevant imaging radioligand for non-invasive evaluation of atherosclerotic inflammation.

The effect of the heart rate lowering drug Ivabradine on hemodynamics in atherosclerotic mice.

The heart rate lowering drug Ivabradine was shown to improve cardiac outcome in patients with previous heart failure. However, in patients without heart failure, no beneficial effect of Ivabradine was observed. Animal studies suggested a preventive effect of Ivabradine on atherosclerosis which was due to an increase in wall shear stress (WSS), the blood flow-induced frictional force exerted on the endothelium, triggering anti-inflammatory responses. However, data on the effect of Ivabradine on WSS is sparse. We aim to study the effect of Ivabradine on (i) the 3D WSS distribution over a growing plaque and (ii) plaque composition. We induced atherosclerosis in ApoE-/- mice by placing a tapered cast around the right common carotid artery (RCCA). Five weeks after cast placement, Ivabradine was administered via drinking water (15 mg/kg/day) for 2 weeks, after which the RCCA was excised for histology analyses. Before and after Ivabradine treatment, animals were imaged with Doppler Ultrasound to measure blood velocity. Vessel geometry was obtained using contrast-enhanced micro-CT. Time-averaged WSS during systole, diastole and peak WSS was subsequently computed. Ivabradine significantly decreased heart rate (459 ± 28 bpm vs. 567 ± 32 bpm, p < 0.001). Normalized peak flow significantly increased in the Ivabradine group (124.2% ± 40.5% vs. 87.3% ± 25.4%, p < 0.05), reflected by an increased normalized WSS level during systole (110.7% ± 18.4% vs. 75.4% ± 24.6%, p < 0.05). However, plaque size or composition including plaque area, relative necrotic core area and macrophage content were not altered in mice treated with Ivabradine compared to controls. We conclude that increased WSS in response to Ivabradine treatment did not affect plaque progression in a murine model.

Temporal and spatial changes in wall shear stress during atherosclerotic plaque progression in mice.

Wall shear stress (WSS) is involved in atherosclerotic plaque initiation, yet its role in plaque progression remains unclear. We aimed to study (i) the temporal and spatial changes in WSS over a growing plaque and (ii) the correlation between WSS and plaque composition, using animal-specific data in an atherosclerotic mouse model. Tapered casts were placed around the right common carotid arteries (RCCA) of ApoE-/- mice. At 5, 7 and 9 weeks after cast placement, RCCA geometry was reconstructed using contrast-enhanced micro-CT. Lumen narrowing was observed in all mice, indicating the progression of a lumen intruding plaque. Next, we determined the flow rate in the RCCA of each mouse using Doppler Ultrasound and computed WSS at all time points. Over time, as the plaque developed and further intruded into the lumen, absolute WSS significantly decreased. Finally at week 9, plaque composition was histologically characterized. The proximal part of the plaque was small and eccentric, exposed to relatively lower WSS. Close to the cast a larger and concentric plaque was present, exposed to relatively higher WSS. Lower WSS was significantly correlated to the accumulation of macrophages in the eccentric plaque. When pooling data of all animals, correlation between WSS and plaque composition was weak and no longer statistically significant. In conclusion, our data showed that in our mouse model absolute WSS strikingly decreased during disease progression, which was significantly correlated to plaque area and macrophage content. Besides, our study demonstrates the necessity to analyse individual animals and plaques when studying correlations between WSS and plaque composition.

Live Observation of Atherosclerotic Plaque Disruption in Apolipoprotein E-Deficient Mouse.

AIM: The actual occurrence of spontaneous plaque rupture in mice has been a matter of debate. We report on an in vivo observation of the actual event of possible plaque disruption in a living ApoE(-/-) mouse. METHODS AND RESULTS: During live contrast-enhanced ultrasonography of a 50-week-old ApoE(-/-) male mouse, symptoms suggesting plaque disruption in the brachiocephalic artery were observed. Histological analysis confirmed the presence of advanced atherosclerotic lesions with dissections and intraplaque hemorrhage in the affected brachiocephalic trunk, pointing towards plaque rupture as the cause of the observed event. However, we did not detect a luminal thrombus or cap rupture, which is a key criterion for plaque rupture in human atherosclerosis. CONCLUSION: This study reports the real-time occurrence of a possible plaque rupture in a living ApoE(-/-) mouse.

Measurements of the wall shear stress distribution in the outflow tract of an embryonic chicken heart.

In order to study the role of blood-tissue interaction in the developing chicken embryo heart, detailed information about the haemodynamic forces is needed. In this study, we present the first in vivo measurements of the three-dimensional distribution of wall shear stress (WSS) in the outflow tract (OFT) of an embryonic chicken heart. The data are obtained in a two-step process: first, the three-dimensional flow fields are measured during the cardiac cycle using scanning microscopic particle image velocimetry; second, the location of the wall and the WSS are determined by post-processing flow velocity data (finding velocity gradients at locations where the flow approaches zero). The results are a three-dimensional reconstruction of the geometry, with a spatial resolution of 15-20 microm, and provides detailed information about the WSS in the OFT. The most significant error is the location of the wall, which results in an estimate of the uncertainty in the WSS values of 20 per cent.