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Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer ( p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly ( p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling.

Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 ( Jag1 ECKO ) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1 ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.

Atherosclerosis is a lipid driven chronic inflammatory disease that is characterized by the formation of plaques at predilection sites. These predilection sites (side branches, curved segments, and bifurcations) have often been associated with disturbed shear stress profiles. However, in addition to shear stress, endothelial cells also experience artery wall strain that could contribute to atherosclerosis progression. Herein, we describe a method to accurately obtain these shear stress and strain profiles. We developed a fluid-structure interaction (FSI) framework for modelling arteries within a commercially available package (Abaqus, version 6.14) that included known prestresses (circumferential, axial and pressure associated). In addition, we co-registered 3D histology to a micro-CT-derived 3D reconstruction of an atherosclerotic carotid artery from a cholesterol-fed ApoE -/- mouse to include the spatial distribution of lipids within a subject-specific model. The FSI model also incorporated a nonlinear hyperelastic material model with regionally-varying properties that distinguished between healthy vessel wall and plaque. FSI predicted a lower shear stress than CFD (~-12%), but further decreases in plaque regions with softer properties (~-24%) were dependent on the approach used to implement the prestresses in the artery wall. When implemented with our new hybrid approach (zero prestresses in regions of lipid deposition), there was significant heterogeneity in endothelial shear stress in the atherosclerotic artery due to variations in stiffness and, in turn, wall strain. In conclusion, when obtaining endothelial shear stress and strain in diseased arteries, a careful consideration of prestresses is necessary. This paper offers a way to implement them., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Background: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions., Methods: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE -/- ) mice and ApoE -/- mice with a genetic deletion of IRF5 (ApoE -/- Irf5 -/- ) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation., Results: Both lesion and necrotic core size were significantly reduced in ApoE -/- Irf5 -/- mice compared with IRF5-competent ApoE -/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c + macrophages was evident in ApoE -/- Irf5 -/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c + macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c - macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-β3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ., Conclusions: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c + macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis., (© 2017 The Authors.)

Exposure of endothelial cells to low and multidirectional blood flow is known to promote a pro-atherogenic phenotype. The mechanics of the vessel wall is another important mechano-stimulus within the endothelial cell environment, but no study has examined whether changes in the magnitude and direction of cell stretch can be pro-atherogenic. Herein, we developed a custom cell stretching device to replicate the in vivo stretch environment of the endothelial cell and examined whether low and multidirectional stretch promote nuclear translocation of NF-κB. A fluid-structure interaction model of the device demonstrated a nearly uniform strain within the region of cell attachment and a negligible magnitude of shear stress due to cyclical stretching of the cells in media. Compared to normal cyclical stretch, a low magnitude of cyclical stretch or no stretch caused increased expression of nuclear NF-κB (p = 0.09 and p < 0.001, respectively). Multidirectional stretch also promoted significant nuclear NF-κB expression, comparable to the no stretch condition, which was statistically higher than the low (p < 0.001) and normal (p < 0.001) stretch conditions. This is the first study to show that stretch conditions analogous to atherogenic blood flow profiles can similarly promote a pro-atherogenic endothelial cell phenotype, which supports a role for disturbed vessel wall mechanics as a pathological cell stimulus in the development of advanced atherosclerotic plaques.

Objective: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries., Approach and Results: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow., Conclusions: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites., (© 2016 The Authors.)

Background: Although disturbed flow is thought to play a central role in the development of advanced coronary atherosclerotic plaques, no causal relationship has been established. We evaluated whether inducing disturbed flow would cause the development of advanced coronary plaques, including thin cap fibroatheroma., Methods and Results: D374Y-PCSK9 hypercholesterolemic minipigs (n=5) were instrumented with an intracoronary shear-modifying stent (SMS). Frequency-domain optical coherence tomography was obtained at baseline, immediately poststent, 19 weeks, and 34 weeks, and used to compute shear stress metrics of disturbed flow. At 34 weeks, plaque type was assessed within serially collected histological sections and coregistered to the distribution of each shear metric. The SMS caused a flow-limiting stenosis, and blood flow exiting the SMS caused regions of increased shear stress on the outer curvature and large regions of low and multidirectional shear stress on the inner curvature of the vessel. As a result, plaque burden was ≈3-fold higher downstream of the SMS than both upstream of the SMS and in the control artery (P<0.001). Advanced plaques were also primarily observed downstream of the SMS, in locations initially exposed to both low (P<0.002) and multidirectional (P<0.002) shear stress. Thin cap fibroatheroma regions demonstrated significantly lower shear stress that persisted over the duration of the study in comparison with other plaque types (P<0.005)., Conclusions: These data support a causal role for lowered and multidirectional shear stress in the initiation of advanced coronary atherosclerotic plaques. Persistently lowered shear stress appears to be the principal flow disturbance needed for the formation of thin cap fibroatheroma., (© 2015 American Heart Association, Inc.)

Atherosclerosis may be triggered by an elevated net transport of lipid-carrying macromolecules from plasma into the arterial wall. We hypothesised that whether lesions are of the thin-cap fibroatheroma (TCFA) type or are less fatty and more fibrous depends on the degree of elevation of transport, with greater uptake leading to the former. We further hypothesised that the degree of elevation can depend on haemodynamic wall shear stress characteristics and nitric oxide synthesis. Placing a tapered cuff around the carotid artery of apolipoprotein E -/- mice modifies patterns of shear stress and eNOS expression, and triggers lesion development at the upstream and downstream cuff margins; upstream but not downstream lesions resemble the TCFA. We measured wall uptake of a macromolecular tracer in the carotid artery of C57bl/6 mice after cuff placement. Uptake was elevated in the regions that develop lesions in hyperlipidaemic mice and was significantly more elevated where plaques of the TCFA type develop. Computational simulations and effects of reversing the cuff orientation indicated a role for solid as well as fluid mechanical stresses. Inhibiting NO synthesis abolished the difference in uptake between the upstream and downstream sites. The data support the hypothesis that excessively elevated wall uptake of plasma macromolecules initiates the development of the TCFA, suggest that such uptake can result from solid and fluid mechanical stresses, and are consistent with a role for NO synthesis. Modification of wall transport properties might form the basis of novel methods for reducing plaque rupture.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors-mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current 'state of the art' on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

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., (© 2014 American Heart Association, Inc.)

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.

Objective: The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques., Methods and Results: Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development., Conclusions: The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in (1) and (2)). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells and in vivo in larger animals and humans. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation. In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics as confirmed by in vivo Magnetic Resonance Imaging (MRI): Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed. In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

Plaque rupture and subsequent thrombotic occlusion of the coronary artery account for as many as three quarters of myocardial infarctions. The concept of plaque stabilisation emerged about 20 years ago to explain the discrepancy between the reduction of cardiovascular events in patients receiving lipid lowering therapy and the small decrease seen in angiographic evaluation of atherosclerosis. Since then, the concept of a vulnerable plaque has received a lot of attention in basic and clinical research leading to a better understanding of the pathophysiology of the vulnerable plaque and acute coronary syndromes. From pathological and clinical observations, plaques that have recently ruptured have thin fibrous caps, large lipid cores, exhibit outward remodelling and invasion by vasa vasorum. Ruptured plaques are also focally inflamed and this may be a common denominator of the other pathological features. Plaques with similar characteristics, but which have not yet ruptured, are believed to be vulnerable to rupture. Experimental studies strongly support the validity of anti-inflammatory approaches to promote plaque stability. Unfortunately, reliable non-invasive methods for imaging and detection of such plaques are not yet readily available. There is a strong biological basis and supportive clinical evidence that low-density lipoprotein lowering with statins is useful for the stabilisation of vulnerable plaques. There is also some clinical evidence for the usefulness of antiplatelet agents, beta blockers and renin-angiotensin-aldosterone system inhibitors for plaque stabilisation. Determining the causes of plaque rupture and designing diagnostics and interventions to prevent them are urgent priorities for current basic and clinical research in cardiovascular area.

Evidence is accumulating that calcium-rich microdeposits in the vascular wall might play a crucial role in the onset and progression of atherosclerosis. Here we investigated an atherosclerotic lesion of the carotid artery in an established murine model, i.e. the apolipoprotein E-deficient (APOE(-/-) ) mouse to identify (i) the presence of microcalcifications, if any, (ii) the elemental composition of microcalcifications with special reference to calcium/phosphorus mass ratio and (iii) co-localization of increased concentrations of iron and zinc with microcalcifications. Atherosclerosis was induced by a flow-divider placed around the carotid artery resulting in low and high shear-stress regions. Element composition was assessed with a proton microprobe. Microcalcifications, predominantly present in the thickened intima of the low shear-stress region, were surrounded by areas with normal calcium levels, indicating that calcium-precipitation is a local event. The diameter of intimal microcalcifications varied from 6 to 70 μm. Calcium/phosphorus ratios of microcalcifications varied from 0.3 to 4.8, mainly corresponding to the ratio of amorphous calcium-phosphate. Increased iron and zinc concentrations commonly co-localized with microcalcifications. Our findings indicate that the atherosclerotic process in the murine carotid artery is associated with locally accumulated calcium, iron and zinc. The calcium-rich deposits resemble amorphous calcium phosphate rather than pure hydroxyapatite. We propose that the APOE(-/-) mouse, in which atherosclerosis was evoked by a flow-divider, offers a useful model to investigate the pathophysiological significance of accumulation of elements such as calcium, iron and zinc., (© 2010 The Authors. International Journal of Experimental Pathology © 2010 International Journal of Experimental Pathology.)

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., Conclusions: Spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries.

Interactions between flow and biological cells and tissues are intrinsic to the circulatory, respiratory, digestive and genitourinary systems. In the circulatory system, an understanding of the complex interaction between the arterial wall (a living multi-component organ with anisotropic, nonlinear material properties) and blood (a shear-thinning fluid with 45% by volume consisting of red blood cells, platelets, and white blood cells) is vital to our understanding of the physiology of the human circulation and the etiology and development of arterial diseases, and to the design and development of prosthetic implants and tissue-engineered substitutes. Similarly, an understanding of the complex dynamics of flow past native human heart valves and the effect of that flow on the valvular tissue is necessary to elucidate the etiology of valvular diseases and in the design and development of valve replacements. In this paper we address the influence of biomechanical factors on the arterial circulation. The first part presents our current understanding of the impact of blood flow on the arterial wall at the cellular level and the relationship between flow-induced stresses and the etiology of atherosclerosis. The second part describes recent advances in the application of fluid-structure interaction analysis to arterial flows and the dynamics of heart valves.

Atherosclerosis is currently appreciated as a disease with a large inflammatory component. The underlying mechanisms, which are uncovered in a rapid pace, are greatly interconnected and as such very complex. Nevertheless, for clinicians it is important have some degree of insight in these immunologic mechanisms in order to interpret the current research advances. The aim of this review is to supply clinicians with this knowledge, avoiding too much detail. All the relevant immunologic basics will be discussed at first, followed by the immunity related theories of atherosclerosis. Finally, current and new immune-modulatory therapies will be discussed.

Atherosclerosis is a chronic lipid-driven inflammatory disease of the arteries. Early lesions (fatty streaks) contain monocytes and T lymphocytes which are recruited from the circulation by adhesion to activated vascular endothelial cells (EC). This process is described as the leukocyte adhesion cascade. Atherogenesis occurs predominantly at branches and bends of the arterial tree that are exposed to relatively low or re-circulating blood flow. Here we briefly review the effects of blood flow and shear stress on the leukocyte adhesion cascade and endothelial cell function.

Atherosclerosis is a chronic inflammatory disease of arteries. It is triggered by proinflammatory mediators which induce adhesion molecules (eg, vascular cell adhesion molecule [VCAM]-1) in endothelial cells (ECs) by activating p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases by phosphorylation. Blood flow influences atherosclerosis by exerting shear stress (mechanical drag) on the inner surface of arteries, a force that alters endothelial physiology. Regions of the arterial tree exposed to high shear are protected from endothelial activation, inflammation, and atherosclerosis, whereas regions exposed to low or oscillatory shear are susceptible. We examined whether MAP kinase phosphatase (MKP)-1, a negative regulator of p38 and JNK, mediates the antiinflammatory effects of shear stress. We observed that expression of MKP-1 in cultured ECs was elevated by shear stress, whereas the expression of VCAM-1 was reduced. MKP-1 induction was shown to be necessary for the antiinflammatory effects of shear stress because gene silencing of MKP-1 restored VCAM-1 expression in sheared ECs. Immunostaining revealed that MKP-1 is preferentially expressed by ECs in a high-shear, protected region of the mouse aorta and is necessary for suppression of EC activation at this site, because p38 activation and VCAM-1 expression was enhanced by genetic deletion of MKP-1. We conclude that MKP-1 induction is required for the antiinflammatory effects of shear stress. Thus, our findings reveal a novel molecular mechanism contributing to the spatial distribution of vascular inflammation and atherosclerosis.

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.

Purpose of Review: This review describes evidence that shear stress acts through modulation of inflammation and by that process affects atherogenesis and plaque composition., Recent Findings: In low shear stress regions antiatherogenic transcription factors are downregulated and pro-atherogenic transcription factors are upregulated. Consequently, inflammatory cells may home low shear stress regions more easily to the plaques because of increased expression of adhesion factors, a decreased rolling speed and an increased expression of chemokines, thereby changing the composition of the plaques into a more vulnerable phenotype. In contrast, in advanced plaque development vascular lumen decreases and shear stress increases, especially upstream of the plaques. The predominant upstream location of lipids induces a prevalent upstream location of inflammatory cells leading to localized plaque rupture., Summary: Shear stress has been shown to play a role in plaque induction, plaque progression and plaque rupture. The mechanism for plaque induction seems to differ from the role of shear stress for plaque rupture, whereby the former mechanism is induced by low shear stress and the latter by high shear stress.

There is an urgent need for representative animal models where prospective examination of the events leading up to plaque rupture and the rupture process itself can be performed. Recently, reports have begun to emerge that apolipoprotein E and low density lipoprotein receptor knockout mice may spontaneously develop unstable atherosclerosis, with plaques in certain parts of the arterial tree showing features suggestive of plaque rupture. Here we discuss the problems inherent in applying definitions of plaque rupture as seen in human arteries to mice; the anatomic locations in mice where unstable plaques do and do not occur; methods of inducing plaque instability in mice; and how to assess plaque stability in mice. These considerations lead us to a number of general recommendations.

We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.

Background: Atherosclerosis is considered an inflammatory disease. Recent studies provided evidence for a predominant upstream location of plaque inflammation. The present study introduces a novel technique that evaluates the underlying mechanism of this spatial organization., Methods and Results: In hypercholesterolemic rabbits, atherosclerosis of the infrarenal aorta was induced by a combination of endothelial denudation and a high-cholesterol diet (2% cholesterol for 2 months). At the time of death, aortic vessel segments were dissected and reconstructed with a new technique that preserved the original intravascular ultrasound-derived lumen geometry. This enabled us to study the spatial relation of histological markers like macrophages, smooth muscle cells, lipids, gelatinolytic activity, and oxidized low-density lipoprotein. Results showed a predominant upstream localization of macrophages and gelatinase activity. Colocalization studies indicated that gelatinase activity was associated with macrophages and smooth muscle cells. Further analysis revealed that this was caused by subsets of smooth muscle cells and macrophages, which were associated with oxidized low-density lipoprotein accumulation., Conclusions: Upstream localization of a vulnerable plaque phenotype is probably due to an accumulation of oxidized low-density lipoprotein, which activates/induces subsets of smooth muscle cells and macrophages to gelatinase production.

Tissue harmonic imaging (THI) has been shown to increase image quality of medical ultrasound in the frequency range from 2 to 10 MHz and might, therefore, also be used to improve image quality in intravascular ultrasound (IVUS). In this study we constructed a prototype IVUS system that could operate in both fundamental frequency and second harmonic imaging modes. This system uses a conventional, continuously rotating, single-element IVUS catheter and was operated in fundamental 20 MHz, fundamental 40 MHz, and harmonic 40 MHz modes (transmit 20 MHz, receive 40 MHz). Hydrophone beam characterization measurements demonstrated the build-up of a second harmonic signal as a function of increasing pressure. Imaging experiments were conducted in both a tissue-mimicking phantom and in an atherosclerotic animal model in vivo. Acquisitions of fundamental 20 and 40 MHz and second harmonic acquisitions resulted in cross sections of the phantom and a rabbit aorta. The harmonic results of the imaging experiments showed the feasibility of intravascular THI with a conventional IVUS catheter both in a phantom and in vivo. The harmonic acquisitions also showed the potential of THI to reduce image artifacts compared to fundamental imaging.

Background: Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies., Methods and Results: We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38+/-0.68 versus 0.22+/-0.04); contained fewer smooth muscle cells (1.9+/-1.6% versus 26.3+/-9.7%), less collagen (15.3+/-1.0% versus 22.2+/-1.0%), and more lipids (15.8+/-0.9% versus 10.2+/-0.5%); and showed more outward vascular remodeling (214+/-19% versus 117+/-9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II-induced intraplaque hemorrhages occurred in the lowered shear stress regions only., Conclusions: Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.

Atherosclerotic lesions preferentially localize near side branches or curved vessels. During the last few decades, research has been shown that low or low and oscillating shear stress is associated with plaque location. Despite ample evidence, the precise mechanism is unknown. This is mainly because of a lack of appropriate animal models. We describe two novel methods to study the hypothesis that shear stress acts through endothelial gene expression or shear stress acts through localizing of inflammation. Both literature evidence and own findings support a role for both mechanisms in atherosclerosis.

Atherosclerosis is intimately coupled to blood flow by the presence of predilection sites. The coupling is through mechanotransduction of endothelial cells and approximately 2000 gene are associated with this process. This paper describes a new platform to study and identify new signalling pathways in endothelial cells covering an atherosclerotic plaque. The identified networks are synthesized in primary cells to study their reaction to flow. This synthetic approach might lead to new insights and drug targets. [ABSTRACT FROM AUTHOR]

Aims: Studies in animals and patients indicate that rapamycin affects vasodilatation differently in outer and inner curvatures of blood vessels. We evaluated in this study whether rapamycin affects endothelial nitric oxide synthase (eNOS) responsiveness to shear stress under normo- and hypercholesteraemic conditions to explain these findings. [ABSTRACT FROM PUBLISHER]

Focuses on the role of innate and adaptive immunity in atherosclerosis. Details of physiology of innate as well as adaptive immune system; Subdivisions of adaptive immunity system; Description of process of activation of innate and adaptive immunity in atherosclerosis.

Macrophages, a significant component of atherosclerotic plaques vulnerable to acute complications, can be pro-inflammatory (designated M1), regulatory (M2), lipid- (Mox) or Heme-induced (Mhem). We showed previously that low (LSS) and oscillatory (OSS) shear stress cause thin-cap fibroatheroma and stable smooth muscle cell-rich plaque formation respectively in ApoE-knockout (ApoE −/− ) mice. Here we investigated whether different shear stress conditions relate to specific changes in macrophage polarization and plaque morphology by applying a shear stress-altering cast to the carotid arteries of high fat-fed ApoE −/− mice. The M1 markers iNOS and IRF5 were highly expressed in macrophage-rich areas of LSS lesions compared to OSS lesions 6 weeks after cast placement, while the M2 marker Arginase-1, and Mox/Mhem markers HO-1 and CD163 were elevated in OSS lesions. Our data indicates shear stress could be an important determinant of macrophage polarization in atherosclerosis, with low shear promoting M1 programming. [ABSTRACT FROM AUTHOR]

Abstract: Aims: Wall shear stress differentially regulates the arginase pathway in carotid arteries perfused ex vivo. Specific patterns of wall shear stress can locally determine atherosclerotic plaque size and composition in vivo. The present work investigates the effects of arginase inhibition on shear stress induced plaque composition. Methods and results: Carotid arteries of apolipoprotein E deficient mice were exposed to high (HSS), low (LSS) and oscillatory (OSS) shear stress conditions by the placement of a local shear stress modifier device for 9 weeks with or without the administration of the arginase inhibitor N-ω-Hydroxy-nor-L-arginine (nor-Noha) (10 mg/kg, i.p., 5 days/week). Carotid arginase activity was measured by colorimetric determination of urea. Atherosclerotic plaque size and composition, arginase expression and cellular localization were assessed by immunohistochemistry. Arginase activity was significantly increased in both LSS and OSS regions as compared to HSS. In the lesions, arginase II isoform co-localized preferentially with EC. Inhibition of arginase by nor-Noha decreased arginase activity and reduced plaque size in both LSS and OSS regions. Arginase inhibition affected mainly the composition of plaques developed in LSS regions by decreasing the total vascular ROS, the number of macrophages, apoptosis rate, lipid and collagen contents. Conclusions: Arginase activity is modulated by patterns of wall shear stress in vivo. Chronic inhibition of vascular arginase decreased the size of atherosclerotic lesions in both OSS and LSS regions, whereas changes on plaque composition were more pronounced in plaques induced by LSS. We identified wall shear stress as a key biomechanical regulator of arginase during plaque formation and stability. [Copyright &y& Elsevier]

Abstract: High laminar shear stress (HLSS) is vasculoprotective partly through induction of Kruppel-like factor 2 (KLF2). Connexin37 (Cx37) is highly expressed in endothelial cells (ECs) of healthy arteries, but not in ECs overlying atherosclerotic lesions. Moreover, Cx37 deletion in apolipoprotein E-deficient (ApoE−/−) mice increases susceptibility to atherosclerosis. We hypothesized that shear stress, through KLF2 modulation, may affect Cx37 expression in ECs. Cx37 expression and gap-junctional intercellular (GJIC) dye transfer are prominent in the straight portion of carotid arteries of ApoE−/− mice, but are reduced at the carotid bifurcation, a region subjected to oscillatory flow. Shear stress-modifying vascular casts were placed around the common carotid artery of ApoE−/− mice. Whereas Cx37 expression was conserved in HLSS regions, it was downregulated to ~50% in low laminar or oscillatory flow regions. To study the mechanisms involved, HUVECs or bEnd.3 cells were exposed to flow in vitro. Cx37 and KLF2 expression were increased after 24h of HLSS. Interestingly, shear-dependent Cx37 expression was significantly reduced after silencing of KLF2. Moreover after exposure to simvastatin, a well-known KLF2 inducer, KLF2 binds to the Cx37 promoter region as shown by ChIP. Finally, GJIC dye transfer was highly reduced after KLF2 silencing and was increased after exposure to simvastatin. HLSS upregulates the expression of Cx37 in ECs by inducing its transcription factor KLF2, which increases intercellular communication. Therefore, this effect of shear stress on Cx37 expression may contribute to the synchronization of ECs and participate in the protective effect of HLSS. [Copyright &y& Elsevier]

Abstract: Angiotensin II (ATII)-mediated hypertension increases the risk for acute coronary events, which may be caused by augmented collagen degradation. Interstitial fibers of collagen type I in the plaque can be degraded by MMP8 and MMP13 specifically. Indeed high MMP8 levels have been correlated with ruptured plaques in patients. To study the contribution of ATII in plaque rupture, we evaluated its effect on MMP8 and MMP13 activity on the vulnerable lesions using an extravascular device that induces regions of pro-atherogenic shear stress in the carotid arteries of ApoE KO mice. This triggers the growth of lesions with a “vulnerable” macrophage-rich phenotype (referred to as upstream lesions) and lesions with a “stable” fibrotic phenotype (referred to as downstream lesions). ATII administration increased mean blood pressure, and increased the incidence of intra-plaque hemorrhages (IPH) from 30% to 73% of the animals in the upstream segments. The area of IPH was also increased by 5-fold. No IPHs were observed in the downstream lesions of the control group or the ATII group. In addition, ATII treatment doubled the size of upstream and downstream lesions. Upstream lesions in the ATII group were decreased in collagen content by 3-fold, contained 2-fold higher MMP8 and MMP13 levels, with a 2- and 3-fold increase in collagen type I degradation by MMP8 and MMP13 respectively compared to the upstream lesions in the control group. Gene expression analysis showed general increase in procollagens and TIMPs expression in response to ATII. However, ATII also decreased procollagen 5α3 expression in downstream lesions and decreased TIMP4 expression in upstream lesions. These data show that ATII promotes a “stable” fibrotic phenotype by inducing severe intra-plaque hemorrhages, characterized by increased degradation of interstitial collagen I via an MMP-mediated (MMP8 and MMP13) mechanism. [Copyright &y& Elsevier]

Abstract: The average low shear stress (SS) is known to determine predilection sites of atherosclerotic plaques. However, as plaques encroach into the lumen and thereby increase SS, interpretation of patient-specific data obtained at one moment in time regarding the influence of SS in the generation of atherosclerosis is not straightforward. This study aims to compare two methods of data analysis for the aid of data interpretation: (a) point-wise analysis of the raw data, (b) global analysis: to assess the history related natural SS distribution in coronary arteries by averaging the data in the axial vessel direction. Normal to mildly diseased human coronary arteries were investigated applying a combination of 3-D reconstruction technique and computational fluid dynamics (CFD). Point-wise analysis relating local wall thickness to local SS showed in only 4% of the cases an inverse relationship. In contrast, averaging the data in the axial vessel direction, showed in 38% a significant inverse relation between wall thickness and SS, resulting in an average negative slope of −0.70±0.46mm/Pa. These data suggest that using a geometry guided way of data averaging may reveal history related effects of SS, which in part explains localization of atherosclerotic plaques. [Copyright &y& Elsevier]