Kim Van der Heiden

Activation of Nrf2 in Endothelial Cells Protects Arteries From Exhibiting a Proinflammatory State

Objective—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. Methods and Results—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. Conclusions—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.

Primary Cilia Sensitize Endothelial Cells for Fluid Shear Stress

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. Developmental Dynamics 237:725–735, 2008.

Expression patterns of Tgfβ1–3 associate with myocardialisation of the outflow tract and the development of the epicardium and the fibrous heart skeleton

Transforming growth factor‐beta (Tgfβ) is essential for normal embryogenesis. The cardiac phenotypes obtained after knockout of each of the three mammalian isoforms suggest different roles during morphogenesis. We studied cardiovascular expression of Tgfβ1–3 in parallel tissue sections of normal mouse embryos from 9.5 to 15.5 days post coitum (dpc) by using radioactive in situ hybridisation. The Tgfβ isoforms are differentially expressed in unique and in overlapping patterns during cardiovascular development. In the vessels, Tgfβ1 is found in the intima, whereas Tgfβ2 and ‐β3 are mainly present in the media and adventitia of the great arteries. Tgfβ1 is present in the endocardium at all stages examined. The Tgfβ2 signal in the endocardium of the atrioventricular canal and outflow tract (9.5 dpc) shifts during epithelial–mesenchymal transformation toward the mesenchymal cushions (10.5–11.5 dpc) after which it exhibits a marked spatiotemporal expression pattern as the cushion differentiation progresses (11.5–15.5 dpc). The myocardium underlying the endocardial cushions and the atrial muscular septum are intensely positive for Tgfβ2 at early stages (9.5–11.5 dpc) and expression decreases at 12.5 days. In contrast to earlier reports, we find marked overlap of Tgfβ2 and ‐β3 expression. Tgfβ3 expression shows a characteristic distribution in the mesenchymal cushions, suggesting a role in cushion differentiation, possibly additional to Tgfβ2. From 14.5 dpc onward, a strong Tgfβ3 signal is found in the fibrous septum primum of the atrium and in the fibrous skeleton of the heart. Special attention was paid to the proepicardial organ and its derivatives. The proepicardial organ strongly expresses Tgfβ2 as early as 9.5 days, and all isoforms are present in the epicardium from 12.5 dpc onward. The spatiotemporal cardiovascular expression of Tgfβ1–3 supports both specific and complementary functions during cardiovascular development that might explain functional redundancy between the Tgfβ‐isoforms. The information provided favors novel roles of Tgfβ1–3 in epicardial development, of Tgfβ2 in myocardialisation, and of Tgfβ3 in differentiation of the fibrous structures of the heart. Developmental Dynamics 227:431–444, 2003.

The effects of stenting on shear stress: relevance to endothelial injury and repair

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.

Disturbed blood flow induces RelA expression via c-Jun N-terminal kinase 1: a novel mode of NF-κB regulation that promotes arterial inflammation.

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. # Novelty and Significance {#article-title-52}Rationale: The nuclear factor (NF)-&kgr;B pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-&kgr;B are defined, the mechanisms that regulate the expression levels of NF-&kgr;B transcription factors are uncertain. Objective: We studied the signaling mechanisms that regulate RelA NF-&kgr;B 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-&kgr;B expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-&kgr;B 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-&kgr;B expression and inflammation in murine carotid arteries via JNK1. Conclusions: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-&kgr;B expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK–NF-&kgr;B crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

Fluid Shear Stress and Inner Curvature Remodeling of the Embryonic Heart. Choosing the Right Lane

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.

c-Jun N-Terminal Kinase Primes Endothelial Cells at Atheroprone Sites for Apoptosis

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.

Deciphering the Endothelial Shear Stress Sensor

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 …

Tgfβ/Alk5 signaling is required for shear stress induced klf2 expression in embryonic endothelial cells

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. Developmental Dynamics 240:1670–1680, 2011.

Primary cilia as biomechanical sensors in regulating endothelial function

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.