[Show abstract][Hide abstract] ABSTRACT: Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-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 18F-FEDAA1106 and 2-deoxy-2-[18F]fluoro-d-glucose (18F-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 18F-FEDAA1106 or 18F-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 18F-FEDAA1106 (p < .01) or FDG (p < .05). However, the 18F-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 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.
[Show abstract][Hide abstract] ABSTRACT: Stent deployment following balloon angioplasty is used routinely to treat coronary artery disease (CAD). 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 (EPC) which migrate and proliferate to re-populate denuded regions. However, re-endothelialisation 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 minimise damage and promote vascular repair in stented arteries.
Cardiovascular Research 04/2013; · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The need for noninvasive imaging to distinguish stable from vulnerable atherosclerotic plaques is evident. Activated macrophages play a role in atherosclerosis and express folate receptor folate receptor β (FR-β). The feasibility of folate targeting to detect atherosclerosis was demonstrated in human and mouse plaques, and it was suggested that molecular imaging of FR-β through folate conjugates might be a specific marker for plaque vulnerability. However, these studies did not allow differentiation between stable and vulnerable atherosclerotic plaques. We investigated the feasibility of a folate-based radiopharmaceutical (111In-EC0800) with high-resolution animal single-photon emission computed tomography/computed tomography (SPECT/CT) to differentiate between stable and vulnerable atherosclerotic plaques in apolipoprotein E-/- mice in which we can induce plaques with the characteristics of stable and vulnerable plaques by placing a flow-modifying cast around the common carotid artery. Both plaques showed 111In-EC0800 uptake, with higher uptake in the vulnerable plaque. However, the vulnerable plaque was larger than the stable plaque. Therefore, we determined tracer uptake per plaque volume and demonstrated higher accumulation of 111In-EC0800 in the stable plaque normalized to plaque volume. Our data show that 111In-EC0800 is not a clear-cut marker for the detection of vulnerable plaques but detects both stable and vulnerable atherosclerotic plaques in a mouse model of atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krüppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfβ)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfβ-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfβ ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfβ/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process.
[Show abstract][Hide abstract] ABSTRACT: 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.
We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation.
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.
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.
Circulation Research 02/2011; 108(8):950-9. · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International review of cell and molecular biology 01/2011; 290:87-119. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atherosclerosis develops predominantly at branches and bends in arteries that are exposed to disturbed flow which exerts low, oscillatory shear stress on endothelial cells (ECs). We demonstrated that c-Jun N-terminal kinase (JNK) is activated in ECs at atherosusceptible but not atheroprotected sites. Transcriptome profiling of cultured ECs treated with a pharmacological inhibitor revealed that JNK functions as a positive regulator of NF-kappaB transcription factors, which promote inflammation by inducing inflammatory molecules (eg, VCAM-1). This observation was confirmed by silencing of JNK1 and ATF2 (a downstream transcription factor), which led to reduced NF-kappaB expression in cultured ECs. We validated our findings by demonstrating that EC expression of NF-kappaB and VCAM-1 and the accumulation of CD68-positive macrophages was elevated at atherosusceptible sites compared with atheroprotected sites in aortas of wild-type mice. Genetic deletion of JNK1 suppressed NF-kappaB and VCAM-1 expression, and reduced macrophage accumulation at the atherosusceptible site, indicating that JNK1 positively regulates NF-kappaB expression and inflammation. To establish a causal relationship between shear stress and JNK activity, we altered blood flow in the murine carotid artery by placing a constrictive cuff. We observed that low, oscillatory shear stress can enhance JNK activity, NF-kappaB and VCAM-1 expression in ECs and promote macrophage accumulation in arteries. We conclude that JNK1-ATF2 signalling promotes EC activation and inflammation at atheroprone sites exposed to low, oscillatory shear stress by enhancing NF-kappaB expression. Our findings illuminate a novel level of cross-talk between the NF-kappaB and JNK signalling pathways that may influence the spatial distribution of atherosclerotic lesions.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
Spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries.
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular pathologies are still the primary cause of death worldwide. The molecular mechanisms behind these pathologies have not been fully elucidated. Unravelling them will bring us closer to therapeutic strategies to prevent or treat cardiovascular disease. One of the major transcription factors that has been linked to both cardiovascular health and disease is NF-kappaB (nuclear factor kappaB). The NF-kappaB family controls multiple processes, including immunity, inflammation, cell survival, differentiation and proliferation, and regulates cellular responses to stress, hypoxia, stretch and ischaemia. It is therefore not surprising that NF-kappaB has been shown to influence numerous cardiovascular diseases including atherosclerosis, myocardial ischaemia/reperfusion injury, ischaemic preconditioning, vein graft disease, cardiac hypertrophy and heart failure. The function of NF-kappaB is largely dictated by the genes that it targets for transcription and varies according to stimulus and cell type. Thus NF-kappaB has divergent functions and can protect cardiovascular tissues from injury or contribute to pathogenesis depending on the cellular and physiological context. The present review will focus on recent studies on the function of NF-kappaB in the cardiovascular system.
[Show abstract][Hide abstract] ABSTRACT: Proinflammatory mediators influence atherosclerosis by inducing adhesion molecules (eg, VCAM-1) on endothelial cells (ECs) via signaling intermediaries including p38 MAP kinase. Regions of arteries exposed to high shear stress are protected from inflammation and atherosclerosis, whereas low-shear regions are susceptible. Here we investigated whether the transcription factor Nrf2 regulates EC activation in arteries.
En face staining revealed that Nrf2 was activated in ECs at an atheroprotected region of the murine aorta where it negatively regulated p38-VCAM-1 signaling, but was expressed in an inactive form in ECs at an atherosusceptible site. Treatment with sulforaphane, a dietary antioxidant, activated Nrf2 and suppressed p38-VCAM-1 signaling at the susceptible site in wild-type but not Nrf2(-/-) animals, indicating that it suppresses EC activation via Nrf2. Studies of cultured ECs revealed that Nrf2 inactivates p38 by suppressing an upstream activator MKK3/6 and by enhancing the activity of the negative regulator MKP-1.
Nrf2 prevents ECs at the atheroprotected site from exhibiting a proinflammatory state via the suppression of p38-VCAM-1 signaling. Pharmacological activation of Nrf2 reduces EC activation at atherosusceptible sites and may provide a novel therapeutic strategy to prevent or reduce atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: In order to study the role of blood-tissue interaction in the developing chicken embryo heart, detailed information about the haemodynamic forces is needed. In this study, we present the first in vivo measurements of the three-dimensional distribution of wall shear stress (WSS) in the outflow tract (OFT) of an embryonic chicken heart. The data are obtained in a two-step process: first, the three-dimensional flow fields are measured during the cardiac cycle using scanning microscopic particle image velocimetry; second, the location of the wall and the WSS are determined by post-processing flow velocity data (finding velocity gradients at locations where the flow approaches zero). The results are a three-dimensional reconstruction of the geometry, with a spatial resolution of 15-20 microm, and provides detailed information about the WSS in the OFT. The most significant error is the location of the wall, which results in an estimate of the uncertainty in the WSS values of 20 per cent.
Journal of The Royal Society Interface 05/2009; 7(42):91-103. · 3.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 Krüppel-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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular development is directed or modulated by genetic and epigenetic factors. The latter include blood flow-related shear stress and blood pressure-related circumferential strain. This review focuses on shear stress and its effects on endothelial cells lining the inner surfaces of the heart and blood vessels. Flow characteristics of the embryonic blood, like velocity, viscosity and periodicity, are taken into account to describe the responses of endothelial cells to shear stress and the sensors for this friction force. The primary cilium, which is an integral part of the shear sensor, connects to the cytoskeletal microtubules and transmits information about the level and direction of blood flow into the endothelial cell. When the heart remodels from a more or less straight into a c-shaped tube the sharp curvature, in combination with the small vessel dimensions and high relative viscosity, directs the highest shear stress to the inner curvature of this pump. This proves to be an important epigenetic modulator of cardiac morphogenesis because when shear stress is experimentally altered inner curvature remodeling is affected which leads to the development of congenital cardiovascular anomalies. The best of both worlds, mechanics and biology, are used here to describe early cardiogenesis.
The Scientific World Journal 01/2008; 8:212-22. · 1.22 Impact Factor