Control of tissue morphology by Fasciclin III-mediated intercellular adhesion.

Morphogenesis is dependent on the orchestration of multiple developmental processes to generate mature functional organs. However, the signalling pathways that coordinate morphogenesis and the mechanisms that translate these signals into tissue shape changes are not well understood. Here, we demonstrate that changes in intercellular adhesion mediated by the transmembrane protein Fasciclin III (FasIII) represent a key mediator of morphogenesis. Using the embryonic Drosophila hindgut as an in vivo model for organogenesis, we show that the tightening of hindgut curvature that normally occurs between embryonic stage 12 and 15 to generate the characteristic shepherd's crook shape is dependent on localised JAK/STAT pathway activation. This localised pathway activity drives the expression of FasIII leading to its subcellular lateralisation at a stage before formation of septate junctions. Additionally, we show that JAK/STAT- and FasIII-dependent morphogenesis also regulates folds within the third instar wing imaginal disc. We show that FasIII forms homophilic intercellular interactions that promote intercellular adhesion in vivo and in cultured cells. To explore these findings, we have developed a mathematical model of the developing hindgut, based on the differential interfacial tension hypothesis (DITH) linking intercellular adhesion and localised surface tension. Our model suggests that increased intercellular adhesion provided by FasIII can be sufficient to drive the tightening of tube curvature observed. Taken together, these results identify a conserved molecular mechanism that directly links JAK/STAT pathway signalling to intercellular adhesion and that sculpts both tubular and planar epithelial shape.

In vivo mapping of vascular inflammation using the translocator protein tracer 18F-FEDAA1106.

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.

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.

Loss of vps54 function leads to vesicle traffic impairment, protein mis-sorting and embryonic lethality.

The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect.

Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells.

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.

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

Role of nuclear factor kappaB in cardiovascular health and disease.

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.

Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinflammatory activation at sites that are resistant to atherosclerosis.

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.

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.

Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo.

Intracranial aneurysms are associated with disturbed velocity patterns, and chronic inflammation, but the relevance for these findings are currently unknown. Here, we show that (disturbed) shear stress induced by vortices is a sufficient condition to activate the endothelial NF-kB pathway, possibly through a mechanism of mechanosensor de-activation. We provide evidence for this statement through in-vitro live cell imaging of NF-kB in HUVECs exposed to different flow conditions, stochastic modelling of flow induced NF-kB activation and induction of disturbed flow in mouse carotid arteries. Finally, CFD and immunofluorescence on human intracranial aneurysms showed a correlation similar to the mouse vessels, suggesting that disturbed shear stress may lead to sustained NF-kB activation thereby offering an explanation for the close association between disturbed flow and intracranial aneurysms.

Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.

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.