The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells

INTRODUCTION: The membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated. METHODS: We examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model. RESULTS: Parental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system. CONCLUSION: Our findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis

Impaired flow-induced arterial remodeling in DOCA-salt hypertensive rats

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10 +/- 1 x 10(3) to 17 +/- 2 x 10(3) mu m(2); 6 weeks: 13 +/- 2 x 10(3) to 24 +/- 3 x 10(3) mu m(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (empty set: 385 +/- 13 to 463 +/- 14 mu m). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 x 10(3) +/- 1 x 10(3) mu m(2)). The diameter of the HF arteries of normotensive rats increased (empty set: 463 +/- 22 mu m) but no expansion occurred in the HF arteries of hypertensive rats (empty set: 471 +/- 16 mu m). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (empty set: 419 +/- 13 to 475 +/- 16 mu m). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean empty set between 235 and 290 mu m), and infiltration of monocyte/ macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/ macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition. Hypertension Research (2012) 35, 1093-1101; doi:10.1038/hr.2012.94; published online 12 July 201

Cx47 fine-tunes the handling of serum lipids but is dispensable for lymphatic vascular function.

Mutations in the gap junction protein connexin47 (Cx47) are associated with lymphedema. However, the role of Cx47 in lymphatic pathophysiology is unknown. We demonstrate that Cx47 is expressed in lymphatic endothelial cells by whole-mount immunostaining and qPCR. To determine if Cx47 plays a role in lymphatic vessel function we analysed Cx47-/- mice. Cx47-deficiency did not affect lymphatic contractility (contractile amplitude or frequency) or lymphatic morphology (vessel diameter or number of valves). Interstitial fluid drainage or dendritic cell migration through lymphatic vessels was also not affected by Cx47-deficiency. Cx47 is dispensable for long-chain fatty acid absorption from the gut but rather promotes serum lipid handling as prolonged elevated triglyceride levels were observed in Cx47-deficient mice after oral lipid tolerance tests. When crossed with Apolipoprotein E-deficient (Apoe-/-) mice, LDL-cholesterol was decreased in young Cx47-/-Apoe-/- adults as compared to Apoe-/- mice, which was inverted later in life. Finally, advanced atherosclerotic plaques in thoracic-abdominal aortas of 15 months-old mice tended to be larger in Cx47-/-Apoe-/- mice. These plaques contained fewer macrophages but similar amounts of T lymphocytes, collagen and lipids than plaques of Apoe-/- mice. In conclusion, Cx47 is expressed in lymphatic endothelium and seems modestly implicated in multiple aspects of lymphatic pathophysiology

Calcitonin Gene-Related Peptide Selectively Relaxes Contractile Responses to Endothelin-1 in Rat Mesenteric Resistance Arteries □ S

ABSTRACT We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wiremyography, and pharmacological tools. ET A -and ET B -receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET B -receptors, NOsynthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET Aantagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 M) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET A -mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K ϩ channels

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism.-receptor complexes.-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1

Communication in production animal medicine: modelling a complex interaction with the example of dairy herd health medicine

<p>Abstract</p> <p>Background</p> <p>The importance of communication skills in veterinary medicine is increasingly recognised. Appropriate communication skills towards the client are of utmost importance in both companion animal practice and production animal field and consultancy work. The need for building a relationship with the client, alongside developing a structure for the consultation is widely recognised and applies to both types of veterinary practice.</p> <p>Results</p> <p>Veterinary advisory practice in production animal medicine is, however, characterised by a more complex communication on different levels. While the person-orientated communication is a permanent process between veterinarian and client with a rather personal perspective and defines the roles of interaction, the problem-orientated communication deals with emerging difficulties; the objective is to solve an acute health problem. The solution - orientated communication is a form of communication in which both veterinarian and client address longstanding situations or problems with the objective to improve herd health and subsequently productivity performance. All three forms of communication overlap.</p> <p>Conclusions</p> <p>Based on this model, it appears useful for a veterinary practice to offer both a curative and an advisory service, but to keep these two separated when deemed appropriate. In veterinary education, the strategies and techniques necessary for solution orientated communication should be included in the teaching of communication skills.</p

Antihypertensive Treatment Differentially Affects Vascular Sphingolipid Biology in Spontaneously Hypertensive Rats

We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A2, cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A2. This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR. Methods and Findings For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20–25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A2. The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction. Conclusion In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions

Human cerebrovascular contractile receptors are upregulated via a B-Raf/MEK/ERK-sensitive signaling pathway

<p>Abstract</p> <p>Background</p> <p>Cerebral ischemia results in a rapid increase in contractile cerebrovascular receptors, such as the 5-hydroxytryptamine type 1B (5-HT_1B), angiotensin II type 1 (AT₁), and endothelin type B (ET_B) receptors, in the vessel walls within the ischemic region, which further impairs local blood flow and aggravates tissue damage. This receptor upregulation occurs via activation of the mitogen-activated protein kinase pathway. We therefore hypothesized an important role for B-Raf, the first signaling molecule in the pathway. To test our hypothesis, human cerebral arteries were incubated at 37°C for 48 h in the absence or presence of a B-Raf inhibitor: SB-386023 or SB-590885. Contractile properties were evaluated in a myograph and protein expression of the individual receptors and activated phosphorylated B-Raf (p-B-Raf) was evaluated immunohistochemically.</p> <p>Results</p> <p>5-HT_1B, AT₁, and ET_Breceptor-mediated contractions were significantly reduced by application of SB-590885, and to a smaller extent by SB-386023. A marked reduction in AT₁receptor immunoreactivity was observed after treatment with SB-590885. Treatment with SB-590885 and SB-386023 diminished the culture-induced increase of p-B-Raf immunoreactivity.</p> <p>Conclusions</p> <p>B-Raf signaling has a key function in the altered expression of vascular contractile receptors observed after organ culture. Therefore, specific targeting of B-Raf might be a novel approach to reduce tissue damage after cerebral ischemia by preventing the previously observed upregulation of contractile receptors in smooth muscle cells.</p

Morphological characterization and immunohistochemical detection of the proinflammatory cytokines IL-1β, IL-17A, and TNF-α in lung lesions associated with contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease, is characterized by massive inflammation of the lung especially during the acute clinical stage of infection. Tissue samples from cattle, experimentally infected with Mycoplasma mycoides subsp. mycoides Afadé, were subjected to histopathological and immunohistochemical examination in order to provide insight into innate immune pathways that shape inflammatory host responses. Lung lesions were characterized by vasculitis, necrosis, and increased presence of macrophages and neutrophils, relative to uninfected animals. The presence of three cytokines associated with innate inflammatory immune responses, namely, IL-1β, IL-17A, and TNF-α, were qualitatively investigated in situ. Higher cytokine levels were detected in lung tissue samples from CBPP-affected cattle compared to samples derived from an uninfected control group. We therefore conclude that the cytokines TNF-α and IL-1β, which are prevalent in the acute phase of infections, play a role in the inflammatory response seen in the lung tissue in CBPP. IL-17A gets released by activated macrophages and attracts granulocytes that modulate the acute phase of the CBPP lesions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11250-016-0994-9) contains supplementary material, which is available to authorized users

Democratic Education: A Theoretical Review (2006–2017)

This theoretical review examines how democratic education is conceptualized within educational scholarship. Three hundred and seventy-seven articles published in English language peer-reviewed journals between 2006 and 2017 are discursively analyzed. Democratic education functions as a privileged nodal point of different political discourses. Two discourses against (elitist and neoliberal) and six discourses pro democratic education (liberal, deliberative, multiculturalist, participatory, critical, and agonistic) construct its meaning. It is argued that the different versions of democratic education respond to various (a) ontological and epistemological assumptions, (b) normative approaches to democracy, and (c) conceptions of the relationship between education and politics. For educational policy, the review provides a critique of elitist and neoliberal policies and support for participatory decision making across discourses. Recommendations for educational practice are made by identifying pedagogies across democratic education scholarship as well as specific pedagogies for each discourse