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Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFβ stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT.

Here, we review the highlights of cardiovascular basic science published in 2021 and early 2022 on behalf of the European Society of Cardiology Council for Basic Cardiovascular Science. We begin with non-coding RNAs which have emerged as central regulators cardiovascular biology, and then discuss how technological developments in single-cell ‘omics are providing new insights into cardiovascular development, inflammation, and disease. We also review recent discoveries on the biology of extracellular vesicles in driving either protective or pathogenic responses. The Nobel Prize in Physiology or Medicine 2021 recognized the importance of the molecular basis of mechanosensing and here we review breakthroughs in cardiovascular sensing of mechanical force. We also summarize discoveries in the field of atherosclerosis including the role of clonal haematopoiesis of indeterminate potential, and new mechanisms of crosstalk between hyperglycaemia, lipid mediators, and inflammation. The past 12 months also witnessed major advances in the field of cardiac arrhythmia including new mechanisms of fibrillation. We also focus on inducible pluripotent stem cell technology which has demonstrated disease causality for several genetic polymorphisms in long-QT syndrome and aortic valve disease, paving the way for personalized medicine approaches. Finally, the cardiovascular community has continued to better understand COVID-19 with significant advancement in our knowledge of cardiovascular tropism, molecular markers, the mechanism of vaccine-induced thrombotic complications and new anti-viral therapies that protect the cardiovascular system.

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture mechanics of the heterogeneous, highly collagenous plaque tissue, and how this is related to the tissue's fibrous structure, are not known yet. Existing pipelines to study plaque mechanics are limited to obtaining only gross mechanical characteristics of the plaque tissue, based on the assumption of structural homogeneity of the tissue. However, fibrous plaque tissue is structurally heterogeneous, arguably mainly due to local variation in the collagen fiber architecture. The mechano-imaging pipeline described here has been developed to study the heterogeneous structural and mechanical plaque properties. In this pipeline, the tissue's local collagen architecture is characterized using multiphoton microscopy (MPM) with second-harmonic generation (SHG), and the tissue's failure behavior is characterized under uniaxial tensile testing conditions using digital image correlation (DIC) analysis. This experimental pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue. The obtained knowledge is key to better understand, predict, and prevent atherosclerotic plaque rupture events.

Atherosclerosis is a lipid and inflammation-driven disease of the arteries that is characterized by gradual buildup of plaques in the vascular wall. A so-called vulnerable plaque, consisting of a lipid-rich necrotic core contained by a thin fibrous cap, may rupture and trigger thrombus formation, which can lead to ischemia in the heart (heart attack) or in the brain (stroke). In this study, we present a protocol to investigate the lipid composition of advanced human carotid plaques using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), providing a framework that should enable the discrimination of vulnerable from stable plaques based on lipid composition. We optimized the tissue preparation and imaging methods by systematically analyzing data from three specimens: two human carotid endarterectomy samples (advanced plaque) and one autopsy sample (early stage plaque). We show a robust data reduction method and evaluate the variability of the endarterectomy samples. We found diacylglycerols to be more abundant in a thrombotic area compared to other plaque areas and could distinguish advanced plaque from early stage plaque based on cholesteryl ester composition. We plan to use this systematic approach to analyze a larger dataset of carotid atherosclerotic plaques. Electronic supplementary material The online version of this article (10.1007/s13361-019-02254-y) contains supplementary material, which is available to authorized users.

The catastrophic mechanical rupture of an atherosclerotic plaque is the underlying cause of the majority of cardiovascular events. The infestation of vascular calcification in the plaques creates a mechanically complex tissue composite. Local stress concentrations and plaque tissue strength properties are the governing parameters required to predict plaque ruptures. Advanced imaging techniques have permitted insight into fundamental mechanisms driving the initiating inflammatory-driven vascular calcification of the diseased intima at the (sub-) micron scale and up to the macroscale. Clinical studies have potentiated the biomechanical relevance of calcification through the derivation of links between local plaque rupture and specific macrocalcification geometrical features. The clinical implications of the data presented in this review indicate that the combination of imaging, experimental testing, and computational modelling efforts are crucial to predict the rupture risk for atherosclerotic plaques. Specialised experimental tests and modelling efforts have further enhanced the knowledge base for calcified plaque tissue mechanical properties. However, capturing the temporal instability and rupture causality in the plaque fibrous caps remains elusive. Is it necessary to move our experimental efforts down in scale towards the fundamental (sub-) micron scales in order to interpret the true mechanical behaviour of calcified plaque tissue interactions that is presented on a macroscale in the clinic and to further optimally assess calcified plaques in the context of biomechanical modelling.

Highlights • We built a micro-spectroscopic photoacoustic imaging setup, allowing spectral imaging throughout the section. • Spectral differences were primarily due to differences in hydrocarbon chain length and number of unsaturated bonds. • PA signal intensity was most strongly correlated with sphingomyelin and cholesteryl ester area in MALDI-MSI. • The spectral feature of lipids abundant in necrotic core can be used for detection of atherosclerosis with features of instability., Atherosclerosis is a lipid-driven and an inflammatory disease of the artery walls. The composition of atherosclerotic plaque stratifies the risk of a specific plaque to cause a cardiovascular event. In an optical resolution photoacoustic microscopy setup, of 45 μm resolution, we extracted plaque lipid photoacoustic (PA) spectral signatures of human endarterectomy samples in the range of 1150–1240 nm, using matrix assisted laser desorption ionization mass spectrometry imaging as a reference. We found plaque PA signals to correlate best with sphingomyelins and cholesteryl esters. PA signal spectral variations within the plaque area were compared to reference molecular patterns and absorption spectra of lipid laboratory standards. Variability in the lipid spectroscopic features extracted by principal component analysis of all samples revealed three distinct components with peaks at: 1164, 1188, 1196 and 1210 nm. This result will guide the development of PA-based atherosclerosis disease staging capitalizing on lipidomics of atherosclerotic tissue.

Atherosclerosis is a disease characterized by the narrowing of the arteries as a result of plaque buildup. Lipid-rich plaques are hypothesized to be ‘vulnerable’ plaques with higher risks of rupture. In order to identify lipid biomarkers in-vivo, we use an in-house built photoacoustic imaging microscope system in tandem with mass spectrometer imaging for lipid full characterization. We show photoacoustic lipid spectra of sectioned human carotid endarterectomy samples on histological glass slides to unravel a photoacoustic lipid spectral histology ranging from 1140 nm to 1250 nm. We relate the spectral features identified by PAM to lipids found by mass spectrometry imaging.

Purpose Many radioligands have been developed for the visualization of atherosclerosis by targeting inflammation. However, interpretation of in vivo signals is often limited to plaque identification. We evaluated binding of some promising radioligands in an in vitro approach in atherosclerotic plaques with different phenotypes. Methods Tissue sections of carotid endarterectomy tissue were characterized as early plaque, fibro-calcific plaque, or phenotypically vulnerable plaque. In vitro binding assays for the radioligands [111In]In-DOTATATE; [111In]In-DOTA-JR11; [67Ga]Ga-Pentixafor; [111In]In-DANBIRT; and [111In]In-EC0800 were conducted, the expression of the radioligand targets was assessed via immunohistochemistry. Radioligand binding and expression of radioligand targets was investigated and compared. Results In sections characterized as vulnerable plaque, binding was highest for [111In]In-EC0800; followed by [111In]In-DANBIRT; [67Ga]Ga-Pentixafor; [111In]In-DOTA-JR11; and [111In]In-DOTATATE (0.064 ± 0.036; 0.052 ± 0.029; 0.011 ± 0.003; 0.0066 ± 0.0021; 0.00064 ± 0.00014 %Added activity/mm2, respectively). Binding of [111In]In-DANBIRT and [111In]In-EC0800 was highest across plaque phenotypes, binding of [111In]In-DOTA-JR11 and [67Ga]Ga-Pentixafor differed most between plaque phenotypes. Binding of [111In]In-DOTATATE was the lowest across plaque phenotypes. The areas positive for cells expressing the radioligand’s target differed between plaque phenotypes for all targets, with lowest percentage area of expression in early plaque sections and highest in phenotypically vulnerable plaque sections. Conclusions Radioligands targeting inflammatory cell markers showed different levels of binding in atherosclerotic plaques and among plaque phenotypes. Different radioligands might be used for plaque detection and discerning early from vulnerable plaque. [111In]In-EC0800 and [111In]In-DANBIRT appear most suitable for plaque detection, while [67Ga]Ga-Pentixafor and [111In]In-DOTA-JR11 might be best suited for differentiation between plaque phenotypes.

Background Imaging Somatostatin Subtype Receptor 2 (SST2) expressing macrophages by [DOTA,Tyr3]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST2 targeting ligand with a five times higher tumor uptake than DOTATATE, and holds promise to improve plaque imaging. The aim of this study was to evaluate the potential of DOTA-JR11 for plaque detection. Methods and Results Atherosclerotic ApoE−/− mice (n = 22) fed an atherogenic diet were imaged by SPECT/CT two hours post injection of [111In]In-DOTA-JR11 (~ 200 pmol, ~ 50 MBq). In vivo plaque uptake of [111In]In-DOTA-JR11 was visible in all mice, with a target-to-background-ratio (TBR) of 2.23 ± 0.35. Post-mortem scans after thymectomy and ex vivo scans of the arteries after excision of the arteries confirmed plaque uptake of the radioligand with TBRs of 2.46 ± 0.52 and 3.43 ± 1.45 respectively. Oil red O lipid-staining and ex vivo autoradiography of excised arteries showed [111In]In-DOTA-JR11 uptake at plaque locations. Histological processing showed CD68 (macrophages) and SST2 expressing cells in plaques. SPECT/CT, in vitro autoradiography and immunohistochemistry performed on slices of a human carotid endarterectomy sample showed [111In]In-DOTA-JR11 uptake at plaque locations containing CD68 and SST2 expressing cells. Conclusions The results of this study indicate DOTA-JR11 as a promising ligand for visualization of atherosclerotic plaque inflammation.

Carotid atherosclerosis is a risk factor for ischemic stroke, one of the main causes of mortality and disability worldwide. The disease is characterized by plaques, heterogeneous deposits of lipids, and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis, but the nature of their involvement is not fully understood. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 different lipid signals, originating of >90 uniquely assigned species, in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-directed multivariate analysis. These data allowed us to extract the spatial lipid patterns associated with morphological plaque features in advanced plaques from a symptomatic population, revealing spatial lipid patterns in atherosclerosis and their relation to histological tissue type. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, whereas diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. These results demonstrate a clear colocalization between plaque features and specific lipid classes, as well as individual lipid species in high-risk atherosclerotic plaques.

Pulmonary hypertension (PH) is a devastating disease with a poor outcome. Progressive remodeling of the pulmonary microvasculature leads to an increase in pulmonary vascular stiffness as well as to an increase in pulmonary vascular resistance. These alterations in mechanical characteristics of the pulmonary vasculature further contribute to the progression of pulmonary vascular disease. This chapter will focus on how changes in vascular stiffness affect mechanical forces acting on the endothelium and smooth muscle cells and how these changes in mechanical forces in turn contribute to the development and progression of pulmonary vascular disease with a focus on the role of mechanosensing and mechanotransduction in these processes.

Aims Atherosclerotic plaque development has been associated with wall shear stress (WSS). However, the multidirectionality of blood flow, and thus of WSS, is rarely taken into account. The purpose of this study was to comprehensively compare five metrics that describe (multidirectional) WSS behaviour and assess how WSS multidirectionality affects coronary plaque initiation and progression. Methods and results Adult familial hypercholesterolaemic pigs (n = 10) that were fed a high-fat diet, underwent imaging of the three main coronary arteries at three-time points [3 (T1), 9 (T2), and 10–12 (T3) months]. Three-dimensional geometry of the arterial lumen, in combination with local flow velocity measurements, was used to calculate WSS at T1 and T2. For analysis, arteries were divided into 3 mm/45° sectors (n = 3648). Changes in wall thickness and final plaque composition were assessed with near-infrared spectroscopy–intravascular ultrasound, optical coherence tomography imaging, and histology. Both in pigs with advanced and mild disease, the highest plaque progression rate was exclusively found at low time-averaged WSS (TAWSS) or high multidirectional WSS regions at both T1 and T2. However, the eventually largest plaque growth was located in regions with initial low TAWSS or high multidirectional WSS that, over time, became exposed to high TAWSS or low multidirectional WSS at T2. Besides plaque size, also the presence of vulnerable plaque components at the last time point was related to low and multidirectional WSS. Almost all WSS metrics had good predictive values for the development of plaque (47–50%) and advanced fibrous cap atheroma (FCA) development (59–61%). Conclusion This study demonstrates that low and multidirectional WSS promote both initiation and progression of coronary atherosclerotic plaques. The high-predictive values of the multidirectional WSS metrics for FCA development indicate their potential as an additional clinical marker for the vulnerable disease., Graphical Abstract Graphical Abstract

Acute myocardial infarction is one of the leading causes of death in the western world. Timely implementation of reperfusion therapy has resulted in increased survival and is currently the optimal treatment for acute MI. Death of cardiomyocytes following ischemia results in “danger signals” that elicit an inflammatory reaction that is crucial for removal of cell debris, wound healing, and generation of scar tissue. However, when the inflammatory response is excessive in duration and/or magnitude, it can result in exacerbated tissue damage and adverse remodeling contributing to the pathogenesis of heart failure. In order to successfully develop and implement inflammation modulating treatments that result in an optimal balance between healing, scar formation, and remodeling, visualization and characterization of the inflammatory response are crucial. Based on the imaging date, patient selection and precise targeting and timing of anti-inflammatory treatment can be realized. Various imaging techniques are under development and are being evaluated for this purpose. In this chapter we discuss the biology of post-MI inflammation and remodeling and relevant animal models and provide an overview of potentially promising imaging strategies.

Atherosclerotic plaques prone to rupture may cause acute myocardial infarction (MI) but can also heal without causing an event. Certain common histopathological features, including inflammation, a thin fibrous cap, positive remodelling, a large necrotic core, microcalcification, and plaque haemorrhage are commonly found in plaques causing an acute event. Recent advances in imaging techniques have made it possible to detect not only luminal stenosis and overall coronary atherosclerosis burden but also to identify such adverse plaque characteristics. However, the predictive value of identifying individual adverse atherosclerotic plaques for future events has remained poor. In this Position Paper, the relationship between vulnerable plaque imaging and MI is addressed, mainly for non-invasive assessments but also for invasive imaging of adverse plaques in patients undergoing invasive coronary angiography. Dynamic changes in atherosclerotic plaque development and composition may indicate that an adverse plaque phenotype should be considered at the patient level rather than for individual plaques. Imaging of adverse plaque burden throughout the coronary vascular tree, in combination with biomarkers and biomechanical parameters, therefore holds promise for identifying subjects at increased risk of MI and for guiding medical and invasive treatment.

Purpose Atherosclerotic plaque development and progression signifies a complex inflammatory disease mediated by a multitude of proinflammatory leukocyte subsets. Using single photon emission computed tomography (SPECT) coupled with computed tomography (CT), this study tested a new dual-isotope acquisition protocol to assess each radiotracer’s capability to identify plaque phenotype and inflammation levels pertaining to leukocytes expressing leukocyte function-associated antigen-1 (LFA-1) and the leukocyte subset of proinflammatory macrophages expressing somatostatin receptor subtype-2 (SST2). Individual radiotracer uptake was quantified and the presence of corresponding immunohistological cell markers was assessed. Methods Human symptomatic carotid plaque segments were obtained from endarterectomy. Segments were incubated in dual-isotope radiotracers [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt) and [99mTc]Tc-[N0–14,Asp0,Tyr3]-octreotate ([99mTc]Tc-Demotate 2) before scanning with SPECT/CT. Plaque phenotype was classified as pathological intimal thickening, fibrous cap atheroma or fibrocalcific using histology sections based on distinct morphological characteristics. Plaque segments were subsequently immuno-stained with LFA-1 and SST2 and quantified in terms of positive area fraction and compared against the corresponding SPECT images. Results Focal uptake of co-localising dual-radiotracers identified the heterogeneous distribution of inflamed regions in the plaques which co-localised with positive immuno-stained regions of LFA-1 and SST2. [111In]In-Danbirt and [99mTc]Tc-Demotate 2 uptake demonstrated a significant positive correlation (r = 0.651; p = 0.001). Fibrous cap atheroma plaque phenotype correlated with the highest [111In]In-Danbirt and [99mTc]Tc-Demotate 2 uptake compared with fibrocalcific plaques and pathological intimal thickening phenotypes, in line with the immunohistological analyses. Conclusion A dual-isotope acquisition protocol permits the imaging of multiple leukocyte subsets and the pro-inflammatory macrophages simultaneously in atherosclerotic plaque tissue. [111In]In-Danbirt may have added value for assessing the total inflammation levels in atherosclerotic plaques in addition to classifying plaque phenotype.

Carotid atherosclerosis is one of the main causes of stroke, mortality and disability worldwide. The disease is characterized by plaques, heterogeneous depositions of lipids and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 lipids in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-driven multivariate analysis. These data allowed us to compare the spatial lipid patterns with morphological plaque features that have been associated with enhanced risk of symptoms. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, while diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. This is the first study to systematically investigate spatial lipid patterns in atherosclerosis, analyze their relation to histological tissue type, and to demonstrate a clear co-localization between plaque features and specific lipid classes and individual lipid molecules in high-risk atherosclerotic plaques.

Mouse models are a crucial and often used tool to provide insight into the underlying mechanisms of human atherosclerosis. However, mice profoundly differ from humans in lipoprotein synthesis and metabolism, key factors in atherosclerotic plaque formation. Mouse models often require genetic and dietary modifications to mimic human pathophysiology, shifting from a high-density lipoprotein to an low-density lipoprotein dominant lipoprotein profile. We examined the suitability of mice with a humanized liver as a model for lipoprotein studies and studies on plaque formation, given the central role of hepatocytes in lipoprotein synthesis and metabolism. Our results show a progressive humanization of the mouse liver and a humanized lipoprotein profile. However, no atherosclerotic plaque formation was observed in the studied time frame, despite presence of functional macrophages and application of a high cholesterol western-type diet. The humanized-liver mouse model therefore might require further modifications to induce atherosclerosis, yet seems a valuable model for in vivo studies on lipoprotein metabolism. (C) 2020 The Authors. Published by Elsevier Inc.

The aim of this manuscript is to provide guidelines for appropriate use of CFD to obtain reproducible and reliable wall shear stress maps in native and instrumented human coronary arteries. The outcome of CFD heavily depends on the quality of the input data, which include vessel geometrical data, proper boundary conditions, and material models. Available methodologies to reconstruct coronary artery anatomy are discussed in ‘Imaging coronary arteries: a brief review’ section. Computational procedures implemented to simulate blood flow in native coronary arteries are presented in ‘Wall shear stress in native arteries’ section. The effect of including different geometrical scales due to the presence of stent struts in instrumented arteries is highlighted in ‘Wall shear stress in stents’ section. The clinical implications are discussed in ‘Clinical applications’ section, and concluding remarks are presented in ‘Concluding remarks’ section.

Background Imaging Somatostatin Subtype Receptor 2 (SST2) expressing macrophages by [DOTA,Tyr3]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST2 targeting ligand with a five times higher tumor uptake than DOTATATE, and holds promise to improve plaque imaging. The aim of this study was to evaluate the potential of DOTA-JR11 for plaque detection. Methods and Results Atherosclerotic ApoE−/− mice (n = 22) fed an atherogenic diet were imaged by SPECT/CT two hours post injection of [111In]In-DOTA-JR11 (~ 200 pmol, ~ 50 MBq). In vivo plaque uptake of [111In]In-DOTA-JR11 was visible in all mice, with a target-to-background-ratio (TBR) of 2.23 ± 0.35. Post-mortem scans after thymectomy and ex vivo scans of the arteries after excision of the arteries confirmed plaque uptake of the radioligand with TBRs of 2.46 ± 0.52 and 3.43 ± 1.45 respectively. Oil red O lipid-staining and ex vivo autoradiography of excised arteries showed [111In]In-DOTA-JR11 uptake at plaque locations. Histological processing showed CD68 (macrophages) and SST2 expressing cells in plaques. SPECT/CT, in vitro autoradiography and immunohistochemistry performed on slices of a human carotid endarterectomy sample showed [111In]In-DOTA-JR11 uptake at plaque locations containing CD68 and SST2 expressing cells. Conclusions The results of this study indicate DOTA-JR11 as a promising ligand for visualization of atherosclerotic plaque inflammation. Electronic supplementary material The online version of this article (doi:10.1007/s12350-020-02046-y) contains supplementary material, which is available to authorized users.

Supplemental Digital Content is available in the text., Objective: In an adult porcine model of familial hypercholesterolemia (FH), coronary plaque development was characterized. To elucidate the underlying mechanisms of the observed inter-individual variation in disease severity, detailed lipoprotein profiles were determined. Approach and Results: FH pigs (3 years old, homozygous LDLR R84C mutation) received an atherogenic diet for 12 months. Coronary atherosclerosis development was monitored using serial invasive imaging and histology. A pronounced difference was observed between mildly diseased pigs which exclusively developed early lesions (maximal plaque burden, 25% [23%–34%]; n=5) and advanced-diseased pigs (n=5) which developed human-like, lumen intruding plaques (maximal plaque burden, 69% [57%–77%]) with large necrotic cores, intraplaque hemorrhage, and calcifications. Advanced-diseased pigs and mildly diseased pigs displayed no differences in conventional risk factors. Additional plasma lipoprotein profiling by size-exclusion chromatography revealed 2 different LDL (low-density lipoprotein) subtypes: regular and larger LDL. Cholesterol, sphingosine-1-phosphate, ceramide, and sphingomyelin levels were determined in these LDL-subfractions using standard laboratory techniques and high-pressure liquid chromatography mass-spectrometry analyses, respectively. At 3 months of diet, regular LDL of advanced-diseased pigs contained relatively more cholesterol (LDL-C; regular/larger LDL-C ratio 1.7 [1.3–1.9] versus 0.8 [0.6–0.9]; P=0.008) than mildly diseased pigs, while larger LDL contained more sphingosine-1-phosphate, ceramides, and sphingomyelins. Larger and regular LDL was also found in plasma of 3 patients with homozygous FH with varying LDL-C ratios. Conclusions: In our adult FH pig model, inter-individual differences in atherosclerotic disease severity were directly related to the distribution of cholesterol and sphingolipids over a distinct LDL profile with regular and larger LDL shortly after the diet start. A similar LDL profile was detected in patients with homozygous FH.

SummaryRupture of atherosclerotic plaques is the main cause of acute cardiovascular events. Animal models of plaque rupture are rare but essential for testing new imaging modalities to enable diagnosis of the patient at risk. Moreover, they enable the design of new treatment strategies to prevent plaque rupture. Several animal models for the study of atherosclerosis are available. Plaque rupture in these models only occurs following severe surgical or pharmaceutical intervention. In the process of plaque rupture, composition, biology and mechanics each play a role, but the latter has been disregarded in many animal studies. The biomechanical environment for atherosclerotic plaques is comprised of two parts, the pressure-induced stress distribution, mainly - but not exclusively – influenced by plaque composition, and the strength distribution throughout the plaque, largely determined by the inflammatory state. This environment differs considerably between humans and most animals, resulting in suboptimal conditions for plaque rupture. In this review we describe the role of the biomechanical environment in plaque rupture and assess this environment in animal models that present with plaque rupture.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stent deployment following balloon angioplasty is used routinely to treat coronary artery disease. These interventions cause damage and loss of endothelial cells (EC), and thus promote in-stent thrombosis and restenosis. Injured arteries are repaired (intrinsically) by locally derived EC and by circulating endothelial progenitor cells which migrate and proliferate to re-populate denuded regions. However, re-endothelialization is not always complete and often dysfunctional. Moreover, the molecular and biomechanical mechanisms that control EC repair and function in stented segments are poorly understood. Here, we propose that stents modify endothelial repair processes, in part, by altering fluid shear stress, a mechanical force that influences EC migration and proliferation. A more detailed understanding of the biomechanical processes that control endothelial healing would provide a platform for the development of novel therapeutic approaches to minimize damage and promote vascular repair in stented arteries.

Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothelial behaviour in altered gravity might contribute to new insights towards angiogenesis modu- lation. This study aimed at characterizing EC behaviour after hypergravity exposure (more than 1g), with special focus on cytoskeleton architecture and capillary-like structure formation. Herein, human umbilical vein ECs (HUVECs) were cultured under two-dimensional and three-dimensional conditions at 3g and 10g for 4 and 16 h inside the large diameter centrifuge at the European Space Research and Technology Centre (ESTEC) of the European Space Agency. Although no significant tendency regarding cyto- skeleton organization was observed for cells exposed to high gâ s, a slight loss of the perinuclear localization of b-tubulin was observed for cells exposed to 3g with less pronounced peripheral bodies of actin when compared with 1g control cells. Additionally, hypergravity exposure decreased the assembly of HUVECs into capillary-like structures, with a 10g level significantly reducing their organization capacity. In conclusion, short-term hypergravity seems to affect EC phenotype and their angiogenic potential in a time and g-level-dependent manner.Â, This work was supported by the Spin Your Thesis! 2014 programme, organized by ESA Education Office. Additional funding include the European Regional Development Fund (ERDF) through the Programa Operacional Factores de Competitividade – COMPETE, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, the research grant PEst-C/SAU/LA0002/2011, and co-financed by the North Portugal Regional Operational Programme (ON.2-O Novo Norte) in the framework of the project NORTE-01-0145-FEDER-000012, under the National Strategic Reference Framework (NSRF). Acknowledgements. The experiments reported here were performed in the framework of the Spin Your Thesis! 2014 programme, organized by ESA Education Office. The authors are grateful to staff of ESA Education Office, Natacha Callens and Lily Ha, for support, and to Alan Dowson for the technical support before and during the campaign. R.C.-A. acknowledges the PhD grant SFRH/BD/96593/2013 from FCT-Fundação para a Ciência e a Tecnologia

Rationale: The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. Objective: We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. Methods and Results: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. Conclusions: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK–NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

Supplemental Digital Content is available in the text., Rationale: Blood flow–induced shear stress controls endothelial cell (EC) physiology during atherosclerosis via transcriptional mechanisms that are incompletely understood. The mechanosensitive transcription factor TWIST is expressed during embryogenesis, but its role in EC responses to shear stress and focal atherosclerosis is unknown. Objective: To investigate whether TWIST regulates endothelial responses to shear stress during vascular dysfunction and atherosclerosis and compare TWIST function in vascular development and disease. Methods and Results: The expression and function of TWIST1 was studied in EC in both developing vasculature and during the initiation of atherosclerosis. In zebrafish, twist was expressed in early embryonic vasculature where it promoted angiogenesis by inducing EC proliferation and migration. In adult porcine and murine arteries, TWIST1 was expressed preferentially at low shear stress regions as evidenced by quantitative polymerase chain reaction and en face staining. Moreover, studies of experimental murine carotid arteries and cultured EC revealed that TWIST1 was induced by low shear stress via a GATA4-dependent transcriptional mechanism. Gene silencing in cultured EC and EC-specific genetic deletion in mice demonstrated that TWIST1 promoted atherosclerosis by inducing inflammation and enhancing EC proliferation associated with vascular leakiness. Conclusions: TWIST expression promotes developmental angiogenesis by inducing EC proliferation and migration. In addition to its role in development, TWIST is expressed preferentially at low shear stress regions of adult arteries where it promotes atherosclerosis by inducing EC proliferation and inflammation. Thus, pleiotropic functions of TWIST control vascular disease and development.

Objective—Atherosclerosis is a focal disease that occurs predominantly at branches and bends of the arterial tree. Endothelial cells (EC) at atherosusceptible sites are prone to injury, which can contribute to lesion formation, whereas EC at atheroprotected sites are resistant. The c-Jun N-terminal kinase (JNK) is activated constitutively in EC at atherosusceptible sites but is inactivated at atheroprotected sites by mitogen-activated protein kinase phosphatase-1 (MKP-1). Here, we examined the effects of JNK activation on EC physiology at atherosusceptible sites.Methods and Results—We identified transcriptional programs regulated by JNK by applying a specific pharmacological inhibitor to cultured EC and assessing the transcriptome using microarrays. This approach and subsequent validation by gene silencing revealed that JNK positively regulates the expression of numerous proapoptotic molecules. Analysis of aortae of wild-type, JNK1−/−, and MKP-1−/−mice revealed that EC at an atherosusceptible site express proapoptotic proteins and are primed for apoptosis and proliferation in response to lipopolysaccharide through a JNK1-dependent mechanism, whereas EC at a protected site expressed lower levels of proapoptotic molecules and were protected from injury by MKP-1.Conclusion—Spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries.

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Kruppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress. Developmental Dynamics 237:725–735, 2008. © 2008 Wiley-Liss, Inc.

Atherosclerosis develops in the arterial system at sites of low as well as low and oscillating shear stress. Previously, we demonstrated a shear-related distribution of ciliated endothelial cells in the embryonic cardiovascular system and postulated that the primary cilium is a component of the shear stress sensor, functioning as a signal amplifier. This shear-related distribution is reminiscent of the atherosclerotic predilection sites. Thus, we determined whether a link exists between location and frequency of endothelial primary cilia and atherogenesis. We analyzed endothelial ciliation of the adult aortic arch and common carotid arteries of wild type C57BL/6 and apolipoprotein-E-deficient mice. Primary cilia are located at the atherosclerotic predilection sites, where flow is disturbed, in wild type mice and they occur on and around atherosclerotic lesions in apolipoprotein-E-deficient mice, which have significantly more primary cilia in the aortic arch than wild type mice. In addition, common carotid arteries were challenged for shear stress by application of a restrictive cast, resulting in the presence of primary cilia only at sites of induced low and disturbed shear. In conclusion, these data relate the presence of endothelial primary cilia to regions of atherogenesis, where they increase in number under hyperlipidemia-induced lesion formation. Experimentally induced flow disturbance leads to induction of primary cilia, and subsequently to atherogenesis, which suggests a role for primary cilia in endothelial activation and dysfunction. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Background: Wall shear stress (WSS) is involved in the pathophysiology of atherosclerosis. The correlation between WSS and atherosclerosis can be investigated over time using a WSS-manipulated atherosclerotic mouse model. To determine WSS in vivo, detailed 3D geometry of the vessel network is required. However, a protocol to reconstruct 3D murine vasculature using this animal model is lacking. In this project, we evaluated the adequacy of eXIA 160, a small animal contrast agent, for assessing murine vascular network on micro-CT. Also, a protocol was established for vessel geometry segmentation and WSS analysis. Methods: A tapering cast was placed around the right common carotid artery (RCCA) of ApoE(-/-) mice (n = 8). Contrast-enhanced micro-CT was performed using eXIA 160. An innovative local threshold-based segmentation procedure was implemented to reconstruct 3D geometry of the RCCA. The reconstructed RCCA was compared to the vessel geometry using a global threshold-based segmentation method. Computational fluid dynamics was applied to compute the velocity field and WSS distribution along the RCCA. Results: eXIA 160-enhanced micro-CT allowed clear visualization and assessment of the RCCA in all eight animals. No adverse biological effects were observed from the use of eXIA 160. Segmentation using local threshold values generated more accurate RCCA geometry than the global threshold-based approach. Mouse-specific velocity data and the RCCA geometry generated 3D WSS maps with high resolution, enabling quantitative analysis of WSS. In all animals, we observed low WSS upstream of the cast. Downstream of the cast, asymmetric WSS patterns were revealed with variation in size and location between animals. Conclusions: eXIA 160 provided good contrast to reconstruct 3D vessel geometry and determine WSS patterns in the RCCA of the atherosclerotic mouse model. We established a novel local threshold-based segmentation protocol for RCCA reconstruction and WSS computation. The observed differences between animals indicate the necessity to use mouse-specific data for WSS analysis. For our future work, our protocol makes it possible to study in vivo WSS longitudinally over a growing plaque.

In this review, the role of wall shear stress in the chicken embryonic heart is analyzed to determine its effect on cardiac development through regulating gene expression. Therefore, background information is provided for fluid dynamics, normal chicken and human heart development, cardiac malformations, cardiac and vitelline blood flow, and a chicken model to induce cardiovascular anomalies. A set of endothelial shear stress-responsive genes coding for endothelin-1 (ET-1), lung Krüppel-like factor (LKLF/KLF2), and endothelial nitric oxide synthase (eNOS/NOS-3) are active in development and are specifically addressed.

Atherosclerosis-associated diseases are the main cause of mortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions that may become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animal models mimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models.

Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18 F-FEDAA1106 and 2-deoxy-2-[ 18 F]fluoro-Dglucose ( 18 F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18 F-FEDAA1106 or 18 F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18 F-FEDAA1106 (p , .01) or FDG (p , .05). However, the 18 F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18 F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18 F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.

Increased levels of Reactive oxygen species (ROS) lead to vascular complications like atherosclerosis. Important producers of ROS are the family of NADPH oxidases (NOXes). NOX subtypes have recently been shown to be responsive to blood flow-induced shear stress. Shear stress is also responsible for the focal nature of atherosclerosis by regulating expression of the athero-protective transcription factors KLF2 and Nrf2. These transcription factors inhibit pro-inflammatory and promote antioxidant gene expression. High, pulsatile blood flow in straight vessel segments induces KLF2 and Nrf2. In contrast, disturbed flow at vessel bends and bifurcations inhibits them, thus rendering the vessel wall susceptible to systemic risk factors for vascular disease. Several studies indicate that ROS might influence KLF2/Nrf2 expression and/or activation. In this study we set out to determine the interrelationship between shear stress-induced NOX subtypes and KLF-2/Nrf2 activation. HUVEC were exposed to 1.5 Pascal pulsatile shear stress (athero-protective) for 6 days in IBIDI flow chambers and compared to static conditions (athero-prone). NOX 1 and NOX 5 were expressed at undetectable levels for PCR analyses in static HUVEC. NOX 2 and NOX 4 were expressed in static HUVEC. Upon exposure to shear stress, NOX 2 expression decreased to undetectable levels while NOX 4 was reduced by 50%. This shift in NOX 2/NOX 4 balance resulted in a ROS shift from O 2- to H 2 O 2 , as determined by DHE and CMDCF staining. Subsequently, we silenced NOX 2 and 4 by transducing HUVEC with lenti-viral shRNAs, using non-targeting shRNA and non-transduced cells as controls. Silencing did not affect static or shear stress-induced KLF2 and Nrf2 expression, activation, or target gene expression. However, we did observe an Nrf2-independent effect of NOX silencing on antioxidant expression, which is currently under further investigation. Our preliminary results show a shift in NOX/ROS balance under shear stress, without affecting KLF2 or Nrf2. Together with the anti-oxidant activities of KLF2 and Nrf2, this shift in oxidant balance might be causative in the quiescent, anti-atherogenic effect of high, pulsatile shear stress on ECs.

The single nonmotile primary cilium, protruding several microns from the apical surface, contains cytoskeletal elements in a specific fashion. It consists of 9 circularly arranged microtubule doublets, as revealed by transmission electron microscopy,1 but lacks the central ones characteristic for motile cilia and flagellae. The primary cilium is anchored to the basal body and is thereby connected to the cytoskeletal apparatus. Sorokin1 concluded that primary cilia were probably vestigial remnants of motile cilia. We now know that ciliary functions are abundant. Examples include processes involving Hedgehog and Wnt signaling2 and determining left–right asymmetry.3 Cilia functioning as sensory antennas in insect ears and the human retina are well established.4 Because of the widespread presence of primary cilia, it does not come as a surprise that many diseases, often syndromic, are caused by ciliary dysfunction. Several diseases are related to disruption of intraflagellar transport in the case of mutation in, for example, Polaris, Kif3a or various Bbs proteins that can lead to such conditions as obesity and Bardet Biedl Syndrome.5 Other cilium-related proteins involve cell membrane–bound calcium channels such as the complex formed by polycystin-1 and -2, encoded from Pkd1 and Pkd2 , respectively. Mutations cause polycystic kidney diseases.6,7 Article p 1161 Primary cilia were first described in the cardiovascular system in human embryos and adults more than 20 years ago.8 The geometry of the heart and vascular tree strongly influences hemodynamics with repercussions on the pattern of ciliation. Endothelial ciliation is restricted to areas of low and oscillatory …

Biomechanical forces like shear stress, the frictional force generated by the blood flow, and wall strain, generated by the blood pressure, have long been known to play a role in vascular development and in maintaining vascular homeostasis. Since the 1960s a role for these forces in the pathobiology of various cardiovascular diseases, including atherosclerosis and aneurysm formation, have become evident. To understand the interplay between mechanical forces, vascular biology and vascular disease, interaction between engineers and biomedical scientists with different knowledge bases, languages and cultures, is required. To move forward in this interdisciplinary field, an annual symposium was set up that seeks to bridge the gap between biomechanics, vascular biology, and clinical research with the main objective to apply biomechanical research to cardiovascular diseases. [...]

Extracellular matrix degradation in the aortic wall plays an important role in the formation and progression of aneurysms. In mice, a deficiency of the extracellular matrix protein fibulin-4, induces matrix metalloproteinases (MMP) upregulation and transforming growth factor-β dysregulation. This results in the formation of aneurysms in the thoracic aorta of homozygous Fibulin-4 R/R mice. In humans, aneurysms usually develop in the abdominal aorta during aging and are most often associated with atherosclerosis. To investigate the molecular mechanisms underlying the interaction of aneurysmatic and atherosclerotic aortic disease we developed a new mouse model. Fibulin-4 knockdown (Fibulin-4 +/R ) mice, which display mild extracellular matrix changes, were crossbred with atherosclerotic ApoE -/- mice. The double mutant mice were fed a high fat diet for 10 or 20 weeks starting at 9 weeks after birth. Thoracic and abdominal aortic architecture were assessed in vivo using high-resolution ultrasound. MMP activity in the aorta was determined using fluorescent imaging with protease-activatable near-infrared fluorescence probes. Following sacrifice, plaque burden was evaluated through quantitative oil-red-o staining and histological analysis. Already after 10 weeks of high fat diet, ApoE -/- Fibulin-4 +/R mice displayed increased matrix metalloproteinase activity in the abdominal aorta and increased plaque formation at the level of the visceral arteries as compared with ApoE -/- Fibulin-4 +/+ mice, with further increases observed after 20 weeks of high fat diet. Surprisingly, after 20 weeks high fat diet the aortic diameter increased up to 60% in the aortic arch and 20% in the abdominal aorta in the ApoE -/- Fibulin-4 +/R mice as compared with ApoE -/- Fibulin-4 +/= mice. This novel mutant mouse model is the first viable murine model which mimics the clinical situation of coexistent aneurysm formation and atherogenesis. It provides the opportunity to unravel the biological processes underlying aortic wall degeneration and to identify markers that elucidate key events in the early stages of the pathogenic sequence that culminates in an aneurysm. We identified that partial degradation of the extracellular matrix under the influence of atherosclerosis leads to aortic dilatation. In addition, we show that protease-activatable fluorescent sensors are promising new tools to monitor non-invasively the enhanced matrix metalloproteinases activity in the aortic wall that precedes aortic wall degeneration.

Depending on the pattern of blood flow to which they are exposed and their proliferative status, vascular endothelial cells can present a primary cilium into the flow compartment of a blood vessel. The cilium modifies the response of endothelial cells to biomechanical forces. Shear stress, which is the drag force exerted by blood flow, is best studied in this respect. Here we review the structural composition of the endothelial cilia and the current status of knowledge about the relation between the presence of primary cilia on endothelial cells and the shear stress to which they are exposed.

The cardiovascular system is exposed to biochemical and biomechanical signals. Various sensors for these signals have been described and they contribute to cardiovascular development, maintenance of vessel integrity during adult life, and to pathogenesis. In the past 10years, primary cilia, membrane-covered, rod-like cellular protrusions, were discovered on multiple cell types of the cardiovascular system. Primary cilia are sensory organelles involved in several key (developmental) signaling pathways and in chemo- and mechanosensing on a myriad of cell types. In the embryonic and adult cardiovascular system, they have been demonstrated to function as shear stress sensors on endothelial cells and could act as strain sensors on smooth muscle cells and cardiomyocytes and as chemosensors on fibroblasts. This review will cover their occurrence and elaborate on established and possible functions of primary cilia in the cardiovascular system.