Regeneration of Cryoinjury Induced Necrotic Heart Lesions in Zebrafish Is Associated with Epicardial Activation and Cardiomyocyte Proliferation

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death

Tissue Microenvironments Define and Get Reinforced by Macrophage Phenotypes in Homeostasis or during Inflammation, Repair and Fibrosis

Current macrophage phenotype classifications are based on distinct in vitro culture conditions that do not adequately mirror complex tissue environments. In vivo monocyte progenitors populate all tissues for immune surveillance which supports the maintenance of homeostasis as well as regaining homeostasis after injury. Here we propose to classify macrophage phenotypes according to prototypical tissue environments, e.g. as they occur during homeostasis as well as during the different phases of (dermal) wound healing. In tissue necrosis and/or infection, damage- and/or pathogen-associated molecular patterns induce proinflammatory macrophages by Toll-like receptors or inflammasomes. Such classically activated macrophages contribute to further tissue inflammation and damage. Apoptotic cells and antiinflammatory cytokines dominate in postinflammatory tissues which induce macrophages to produce more antiinflammatory mediators. Similarly, tumor-associated macrophages also confer immunosuppression in tumor stroma. Insufficient parenchymal healing despite abundant growth factors pushes macrophages to gain a profibrotic phenotype and promote fibrocyte recruitment which both enforce tissue scarring. Ischemic scars are largely devoid of cytokines and growth factors so that fibrolytic macrophages that predominantly secrete proteases digest the excess extracellular matrix. Together, macrophages stabilize their surrounding tissue microenvironments by adapting different phenotypes as feed-forward mechanisms to maintain tissue homeostasis or regain it following injury. Furthermore, macrophage heterogeneity in healthy or injured tissues mirrors spatial and temporal differences in microenvironments during the various stages of tissue injury and repair. Copyright (C) 2012 S. Karger AG, Base

Anabolic Therapies

The striking clinical benefits of intermittent parathyroid hormone in osteoporosis have begun a new era of skeletal anabolic agents. Recombinant human parathyroid hormone (rhPTH) (1–34) is the first US Food and Drug Administration–approved anabolic therapy. Its use has been limited by the need for subcutaneous injection. Newer delivery systems include transdermal and oral preparations. Newer anabolic therapies include monoclonal antibody to sclerostin, a potent inhibitor of osteoblastogenesis; and use of bone morphogenetic proteins and parathyroid hormone–related protein PTHrP, a calcium-regulating hormone similar to PTH

Wnt4 Enhances Murine Hematopoietic Progenitor Cell Expansion Through a Planar Cell Polarity-Like Pathway

Background: While the role of canonical (b-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. Methodology/Principal Findings: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while b-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. Conclusions/Significance: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis throug

Three-year survival of single- and two-surface ART restorations in a high-caries child population

The aim of this study was to evaluate the survival of single- and two-surface atraumatic restorative treatment (ART) restorations in the primary and permanent dentitions of children from a high-caries population, in a field setting. The study was conducted in the rainforest of Suriname, South America. ART restorations, made by four Dutch dentists, were evaluated after 6 months, 1, 2, and 3 years. Four hundred seventy-five ART restorations were placed in the primary dentition and 54 in first permanent molars of 194 children (mean age 6.09 ± 0.48 years). Three-year cumulative survivals of single- and two-surface ART restorations in the primary dentition were 43.4 and 12.2%, respectively. Main failure characteristics were gross marginal defects and total or partial losses. Three-year cumulative survival for single-surface ART restorations in the permanent dentition was 29.6%. Main failure characteristics were secondary caries and gross marginal defects. An operator effect was found only for two-surface restorations. The results show extremely low survival rates for single- and two-surface ART restorations in the primary and permanent dentitions. The variable success for ART may initiate further discussion about alternative treatment strategies, especially in those situations where choices have to be made with respect to a well-balanced, cost-effective package of basic oral health care

Quantifying the Evolution of Vascular Barrier Disruption in Advanced Atherosclerosis with Semipermeant Nanoparticle Contrast Agents

Acute atherothrombotic occlusion in heart attack and stroke implies disruption of the vascular endothelial barrier that exposes a highly procoagulant intimal milieu. However, the evolution, severity, and pathophysiological consequences of vascular barrier damage in atherosclerotic plaque remain unknown, in part because quantifiable methods and experimental models are lacking for its in vivo assessment.To develop quantitative nondestructive methodologies and models for detecting vascular barrier disruption in advanced plaques.Sustained hypercholesterolemia in New Zealand White (NZW) rabbits for >7-14 months engendered endothelial barrier disruption that was evident from massive and rapid passive penetration and intimal trapping of perfluorocarbon-core nanoparticles (PFC-NP: ∼250 nm diameter) after in vivo circulation for as little as 1 hour. Only older plaques (>7 mo), but not younger plaques (<3 mo) demonstrated the marked enhancement of endothelial permeability to these particles. Electron microscopy revealed a complex of subintimal spongiform channels associated with endothelial apoptosis, superficial erosions, and surface-penetrating cholesterol crystals. Fluorine ((19)F) magnetic resonance imaging and spectroscopy (MRI/MRS) enabled absolute quantification (in nanoMolar) of the passive permeation of PFC-NP into the disrupted vascular lesions by sensing the unique spectral signatures from the fluorine core of plaque-bound PFC-NP.The application of semipermeant nanoparticles reveals the presence of profound barrier disruption in later stage plaques and focuses attention on the disrupted endothelium as a potential contributor to plaque vulnerability. The response to sustained high cholesterol levels yields a progressive deterioration of the vascular barrier that can be quantified with fluorine MRI/MRS of passively permeable nanostructures. The possibility of plaque classification based on the metric of endothelial permeability to nanoparticles is suggested

Opposing Effects of the Angiopoietins on the Thrombin-Induced Permeability of Human Pulmonary Microvascular Endothelial Cells

BACKGROUND: Angiopoietin-2 (Ang-2) is associated with lung injury in ALI/ARDS. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1. METHODOLOGY/PRINCIPAL FINDINGS: Permeability was assessed by measuring macromolecule passage and transendothelial electrical resistance (TEER). Angiopoietins did not affect basal permeability. Nevertheless, they had opposing effects on the thrombin-induced permeability, in particular in the initial phase. Ang-2 enhanced the initial permeability increase (passage, P = 0.010; TEER, P = 0.021) in parallel with impairment of VE-cadherin organization without affecting VE-cadherin Tyr685 phosphorylation or increasing RhoA activity. Ang-2 also increased intercellular gap formation. Ang-1 preincubation increased Rac1 activity, enforced the VE-cadherin organization, reduced the initial thrombin-induced permeability (TEER, P = 0.027), while Rac1 activity simultaneously normalized, and reduced RhoA activity at 15 min thrombin exposure (P = 0.039), but not at earlier time points. The simultaneous presence of Ang-2 largely prevented the effect of Ang-1 on TEER and macromolecule passage. CONCLUSIONS/SIGNIFICANCE: Ang-1 attenuated thrombin-induced permeability, which involved initial Rac1 activation-enforced cell-cell junctions, and later RhoA inhibition. In addition to antagonizing Ang-1, Ang-2 had also a direct effect itself. Ang-2 sensitized the initial thrombin-induced permeability accompanied by destabilization of VE-cadherin junctions and increased gap formation, in the absence of increased RhoA activity

Mammary stem cells have myoepithelial cell properties.

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.This work was funded by Cancer Research UK, Breast Cancer Campaign, the University of Cambridge, Hutchison Whampoa Limited, La Ligue Nationale Contre le Cancer (Equipe Labelisée 2013) and a grant from Agence Nationale de la Recherche ANR- 08-BLAN-0078-01 to M.A.G.This is the author accepted manuscript. The final version is available from Nature at http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb3025.html

Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms

Vascular endothelial growth factor C promotes cervical cancer metastasis via up-regulation and activation of RhoA/ROCK-2/moesin cascade

<p>Abstract</p> <p>Background</p> <p>The elevated expression of vascular endothelial growth factor C (VEGF-C) is correlated with clinical cervical cancer metastasis and patient survival, which is interpreted by VEGF-C functions to stimulate angiogenesis and lymphatic genesis. However, the direct impact of VEGF-C on cervical cancer cell motility remains largely unknown.</p> <p>Methods</p> <p>In this study, we investigated the effects of VEGF-C on actin cytoskeleton remodeling and on cervical cancer cell migration and invasion and how the actin-regulatory protein, moesin regulated these effects through RhoA/ROCK-2 signaling pathway.</p> <p>Results</p> <p>On cervical carcinoma cell line SiHa cells, exposure of VEGF-C triggered remodeling of the actin cytoskeleton and the formation of membrane ruffles, which was required for cell movement. VEGF-C significantly enhanced SiHa cells horizontal migration and three-dimensional invasion into matrices. These actions were dependent on increased expression and phosphorylation of the actin-regulatory protein moesin and specific moesin siRNA severely impaired VEGF-C stimulated-cell migration. The extracellular small GTPase RhoA/ROCK-2 cascade mediated the increased moesin expression and phosphorylation, which was discovered by the use of Y-27632, a specific inhibitor of Rho kinase and by transfected constitutively active, dominant-negative RhoA as well as ROCK-2 SiRNA. Furthermore, in the surgical cervical specimen from the patients with FIGO stage at cervical intra-epithelial neoplasia and I-II cervical squamous cell carcinoma, the expression levels of moesin were found to be significantly correlated with tumor malignancy and metastasis.</p> <p>Conclusions</p> <p>These results implied that VEGF-C promoted cervical cancer metastasis by upregulation and activation of moesin protein through RhoA/ROCK-2 pathway. Our findings offer new insight into the role of VEGF-C on cervical cancer progression and may provide potential targets for cervical cancer therapy.</p