Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

EPA Pesticide Factsheets

Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

Cryopreservation and Recovery of Human Endometrial Epithelial Cells with High Viability, Purity, and Functional Fidelity

PubMed Central

Chen, Joseph C.; Hoffman, Jacquelyn R.; Arora, Ripla; Perrone, Lila A.; Gonzalez-Gomez, Christian J; Vo, Kim Chi; Laird, Diana J.; Irwin, Juan C.; Giudice, Linda C.

2015-01-01

Objective To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eEC) retaining molecular and functional characteristics of endometrial epithelium in vivo. Design This is an in vitro study using human endometrial cells. Setting University research laboratory. Patients Endometrial biopsies were obtained from premenopausal women undergoing benign gynecological procedures. Interventions Primary eEC were cryopreserved in 1% fetal bovine serum (FBS)/10% dimethyl sulfoxide (DMSO) in Defined Keratinocyte Serum Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium. Main Outcome Measures Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity. Results eEC recovered after cryopreservation (n=5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared to eSF, recovered eEC displayed increased (P<0.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<0.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eEC secrete levels of cytokines and growth factors comparable to freshly cultured eEC. Recovered eEC can formed a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN. Conclusion We have developed a protocol for cryopreservation of eEC in which recovered cells after thawing demonstrate morphological, transcriptomic, and functional characteristics of human endometrial epithelium in vivo. PMID:26515378

Specific gene delivery to liver sinusoidal and artery endothelial cells.

PubMed

Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

2013-09-19

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Molecular Signatures of Tissue-Specific Microvascular Endothelial Cell Heterogeneity in Organ Maintenance and Regeneration

PubMed Central

Nolan, Daniel J.; Ginsberg, Michael; Israely, Edo; Palikuqi, Brisa; Poulos, Michael G.; James, Daylon; Ding, Bi-Sen; Schachterle, William; Liu, Ying; Rosenwaks, Zev; Butler, Jason M.; Xiang, Jenny; Rafii, Arash; Shido, Koji; Rabbany, Sina Y.; Elemento, Olivier; Rafii, Shahin

2013-01-01

SUMMARY Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration. PMID:23871589

Functional roles of cell surface peptidases in reproductive organs

PubMed Central

2004-01-01

A number of biologically active peptides have been proposed to regulate function and differentiation of reproductive organs in an autocrine and/or paracrine fashion. Regulation of the local concentrations of these peptides is one of the important factors influencing their physiological effects on target cells. Membrane‐bound cell surface peptidases can activate or inactivate biologically active peptides before peptide factors access their receptors on the cell surface. Aminopeptidase A (EC 3.4.11.7), placental leucine aminopeptidase (EC 3.4.11.3), aminopeptidase‐N/CD13 (EC 3.4.11.2), dipeptidyl peptidases IV/CD26 (EC.3.4.14.5), carboxypeptidase‐M (EC 3.4.17.12), neutral endopeptidase/CD10 (EC 3.4.24.11) and endothelin converting enzyme‐1 (EC 3.4.23) are differentially expressed on the ovary, endometrium and placenta. The inhibition of enzyme activity affects steroid hormone production by granulosa and thecal cells, decidualization of endometrium and migration of extravillous trophoblasts. These findings suggest that membrane‐bound cell surface peptidases are local regulators for cellular growth and differentiation in reproductive organs by controlling extracellular concentration of peptide factors. (Reprod Med Biol 2004; 3: 165 –176) PMID:29662383

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila.

PubMed

Hara, Yusuke; Hirai, Keiichiro; Togane, Yu; Akagawa, Hiromi; Iwabuchi, Kikuo; Tsujimura, Hidenobu

2013-02-01

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. βFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression. Copyright © 2012 Elsevier Inc. All rights reserved.

Adhesion Molecule Expression and Function of Primary Endothelial Cells in Benign and Malignant Tissues Correlates with Proliferation

PubMed Central

Sievert, Wolfgang; Tapio, Soile; Breuninger, Stephanie; Gaipl, Udo; Andratschke, Nicolaus; Trott, Klaus-Rüdiger; Multhoff, Gabriele

2014-01-01

Background Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs). This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. Methodology/Principal Findings Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. Conclusions/Significance This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature. PMID:24632811

Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

PubMed Central

Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

2013-01-01

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

Microfabricated poly(ethylene glycol) templates enable rapid screening of triculture conditions for cardiac tissue engineering.

PubMed

Iyer, Rohin K; Chiu, Loraine L Y; Radisic, Milica

2009-06-01

The purpose of this study was to design a simple system for cultivation of micro-scale cardiac organoids and investigate the effects of cellular composition on the organoid function. We hypothesized that cultivation of cardiomyocytes (CM) on preformed networks of fibroblasts (FB) and endothelial cells (EC) would enhance the structural and functional properties of the organoids, compared to simultaneously seeding the three cell types or cultivating enriched CM alone. Microchannels for cell seeding were created by photopolymerization of poly(ethylene glycol) diacrylate. In the preculture group the channels were seeded with a mixture of NIH 3T3 FB and D4T EC, following by addition of neonatal rat CM after 2 days of FB/EC preculture. The control microchannels were seeded simultaneously with FB/EC/CM (simultaneous triculture) or with enriched CM alone (enriched CM). Preculture resulted in cylindrical, contractile, and compact cardiac organoids that contained elongated CM expressing connexin-43 and cardiac troponin I. In contrast, simultaneous triculture resulted in noncontractile organoids with clusters of CM growing separately from elongated FBs and ECs. The staining for Connexin-43 was absent in the simultaneous triculture group. When fixed or frozen FB/EC were utilized as a preculture substrate for CM, noncontractile organoids were obtained; while preculture on a single cell type (either FB or EC) resulted in contractile organoids but with inferior properties compared to preculture with both FB/EC. These results emphasize the importance of living cells, presence of both nonmyocyte cell types as well as sequential seeding approach for cultivation of functional multicell type cardiac organoids. 2008 Wiley Periodicals, Inc.

Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells

PubMed Central

Belair, David G.; Whisler, Jordan A.; Valdez, Jorge; Velazquez, Jeremy; Molenda, James A.; Vickerman, Vernella; Lewis, Rachel; Daigh, Christine; Hansen, Tyler D.; Mann, David A.; Thomson, James A.; Griffith, Linda G.; Kamm, Roger D.; Schwartz, Michael P.; Murphy, William L.

2015-01-01

Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. PMID:25190668

AKT inhibition mitigates GRP78 (glucose-regulated protein) expression and contribution to chemoresistance in endometrial cancers.

PubMed

Gray, Michael J; Mhawech-Fauceglia, Paulette; Yoo, Eunjeong; Yang, Wangrong; Wu, Eijean; Lee, Amy S; Lin, Yvonne G

2013-07-01

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients. Copyright © 2012 UICC.

Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor.

PubMed

Aamelfot, Maria; Weli, Simon C; Dale, Ole B; Koppang, Erling O; Falk, Knut

2013-05-01

Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon. © 2013 Anatomical Society.

MIP-2 causes differential activation of RhoA in mouse aortic versus pulmonary artery endothelial cells

PubMed Central

Moldobaeva, Aigul; Baek, Amy; Wagner, Elizabeth M.

2008-01-01

Previously, we have shown that endothelial cell chemotaxis to the proangiogenic chemokine MIP-2 (macrophage inflammatory protein-2), is much greater in mouse aortic endothelial cells (EC) than pulmonary arterial endothelial cells (PA EC). This was true despite the observation that both cell types display comparable levels of the ligand receptor, CXCR2 (8). Since the systemic arterial circulation is proangiogenic in the adult lung and the pulmonary circulation is relatively resistant to neovascularization, we questioned whether the observed functional heterogeneity is related to inherent differences in cell signaling cascades of the two EC subtypes. Specifically, we measured activation of Rac1 and RhoA, both thought to be involved in EC cell migration. Rac1 showed inconsistent and minimal changes in both cell types after MIP-2 treatment (p>0.05). However, activated RhoA was increased upon exposure to MIP-2 only in aortic EC (61% increase; p<0.05). Decreased RhoA activation after treatment of aortic EC with specific siRNA for RhoA resulted in a functional decrease in EC chemotaxis to MIP-2 (17% increase; p<0.05). Additionally, increased RhoA activation in PA EC with adenoviral infection of RhoA caused an increase in PA EC chemotaxis to MIP-2 (46% increase; p<0.05). Inhibition of RhoA activity with the Rho kinase inhibitor, Y27632 blocked aortic EC chemotaxis and stress fiber formation. Thus, RhoA activation is increased after MIP-2 treatment in mouse aortic endothelial cells but not in pulmonary artery endothelial cells. We conclude that RhoA is part of a signaling pathway essential for aortic cell migration after CXCR2 ligation. This result provides one explanation for the difference in chemotaxis observed in these two endothelial subtypes that express similar levels of CXCR2. PMID:17662312

Lipid Droplet Biogenesis and Function in the Endothelium.

PubMed

Kuo, Andrew; Lee, Monica Y; Sessa, William C

2017-04-14

Fatty acids (FA) are transported across the capillary endothelium to parenchymal tissues. However, it is not known how endothelial cells (EC) from large vessels process a postprandial surge of FA. This study was designed to characterize lipid droplet (LD) formation in EC by manipulating pathways leading to the formation and degradation of LD. In addition, several functions of LD-derived FA were assessed. LD were present in EC lining the aorta after the peak in plasma triglycerides initiated by a gavage of olive oil in mice, in vivo. Similarly, in isolated aorta, oleic acid treatment generates LD in EC ex vivo. Cultured EC readily form LD largely via the enzyme DGAT (diacylglycerol O-acyltransferase 1) and degrade LD via ATGL (adipocyte triglyceride lipase) after FA loading. Functionally, LD-derived FA are dynamically regulated and function to protect EC from lipotoxic stress and provide FA for metabolic needs. Our results delineate endothelial LD dynamics for the first time in vivo and in vitro. Moreover, LD formation protects EC from lipotoxic stress, regulates EC glycolysis, and provides a source of FA for adjacent cells in the vessel wall or tissues. © 2017 American Heart Association, Inc.

Redox Regulation of Endothelial Cell Fate

PubMed Central

Song, Ping; Zou, Ming-Hui

2014-01-01

Endothelial cells (ECs) are present throughout blood vessels and have variable roles in both physiological and pathological settings. EC fate is altered and regulated by several key factors in physiological or pathological conditions. Reactive nitrogen species and reactive oxygen species derived from NAD(P)H oxidases, mitochondria, or nitric oxide-producing enzymes are not only cytotoxic but also compose a signaling network in the redox system. The formation, actions, key molecular interactions, and physiological and pathological relevance of redox signals in ECs remain unclear. We review the identities, sources, and biological actions of oxidants and reductants produced during EC function or dysfunction. Further, we discuss how ECs shape key redox sensors and examine the biological functions, transcriptional responses, and post-translational modifications evoked by the redox system in ECs. We summarize recent findings regarding the mechanisms by which redox signals regulate the fate of ECs and address the outcome of altered EC fate in health and disease. Future studies will examine if the redox biology of ECs can be targeted in pathophysiological conditions. PMID:24633153

Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets.

PubMed

Vosen, Sarah; Rieck, Sarah; Heidsieck, Alexandra; Mykhaylyk, Olga; Zimmermann, Katrin; Bloch, Wilhelm; Eberbeck, Dietmar; Plank, Christian; Gleich, Bernhard; Pfeifer, Alexander; Fleischmann, Bernd K; Wenzel, Daniela

2016-01-26

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction.

PubMed

Shao, Yue; Mann, Jennifer M; Chen, Weiqiang; Fu, Jianping

2014-03-01

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system.

PubMed

Walz, A; Keck, C; Weber, H; Kissel, C; Pietrowski, D

2005-09-01

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

PubMed

Jonsson, Ing-Marie; Juuti, Jarmo T; François, Patrice; AlMajidi, Rana; Pietiäinen, Milla; Girard, Myriam; Lindholm, Catharina; Saller, Manfred J; Driessen, Arnold J M; Kuusela, Pentti; Bokarewa, Maria; Schrenzel, Jacques; Kontinen, Vesa P

2010-12-02

Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown. In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine. Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization.

PubMed

Walker, Christopher J; Rush, Craig M; Dama, Paola; O'Hern, Matthew J; Cosgrove, Casey M; Gillespie, Jessica L; Zingarelli, Roman A; Smith, Blair; Stein, Maggie E; Mutch, David G; Shakya, Reena; Chang, Chia-Wen; Selvendiran, Karuppaiyah; Song, Jonathan W; Cohn, David E; Goodfellow, Paul J

2018-05-01

Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.

MicroRNAs as Regulators of Endothelial Cell Functions in Cardiometabolic Diseases

PubMed Central

Araldi, Elisa; Suárez, Yajaira

2016-01-01

Endothelial cells (ECs) provide nutrients and oxygen essential for tissue homeostasis. Metabolic imbalances and other environmental stimuli, like cytokines or low shear stress, trigger endothelial inflammation, increase permeability, compromise vascular tone, promote cell proliferation and ultimately cause cell death. These factors contribute to EC dysfunction, which is crucial in the development of cardiometabolic diseases. microRNAs (miRNAs) are small non-coding RNAs that have important functions in the regulation of ECs. In the present review, we discuss the role of miRNAs in various aspects of EC pathology in cardiometabolic diseases like atherosclerosis, type 2 diabetes, obesity, and the metabolic syndrome, and in complication of those pathologies, like ischemia. We also discuss the potential therapeutic applications of miRNAs in promoting angiogenesis and neovascularization in tissues where the endothelium is damaged in different cardiometabolic diseases. PMID:26825686

The Anti-Inflammatory Cytokine Interleukin-19 Is Expressed in and Angiogenic for Human Endothelial Cells

PubMed Central

Jain, Surbhi; Gabunia, Khatuna; Kelemen, Sheri E.; Panetti, Tracee S.; Autieri, Michael V.

2010-01-01

OBJECTIVE The expression and effects of anti-inflammatory interleukins on endothelial cell (EC) activation and development of angiogenesis is uncharacterized. The purpose of this study is to characterize the expression and function of Interleukin-19 (IL-19), a recently described Th2 anti-inflammatory interleukin on EC pathophysiology. METHODS and RESULTS We demonstrate by immunohistochemistry and immunoblot that IL-19 is expressed in inflamed, but not normal human coronary endothelium, and can be induced in cultured human EC by serum and bFGF. IL-19 is mitogenic, chemotactic, and promotes cell EC spreading. IL-19 activates the signaling proteins STAT3, p44/42, and Rac1. In functional ex vivo studies, IL-19 promotes cord-like structure formation of cultured EC and also enhances microvessel sprouting in the mouse aortic ring assay. IL-19 induces tube formation in matrigel plugs in vivo. CONCLUSIONS These data are the first to report expression of the anti-inflammatory interleukin IL-19 in EC, and the first to indicate that IL-19 is mitogenic and chemotactic for EC, and can induce the angiogenic potential of EC. PMID:20966397

Thymic stromal lymphopoietin-induced HOTAIR activation promotes endothelial cell proliferation and migration in atherosclerosis

PubMed Central

Peng, Yudong; Meng, Kai; Jiang, Lili; Zhong, Yucheng; Yang, Yong; Lan, Yin

2017-01-01

Endothelial cells’ (EC) injury is a major step for the pathological progression of atherosclerosis. Recent study demonstrated that thymic stromal lymphopoietin (TSLP) exerts a protective role in atherosclerosis. However, the effect of TSLP and the exact molecular mechanism involved in EC remains unknown. In the present study, we found that long noncoding RNA (lncRNA) HOTAIR was much lower in EC from atherosclerotic plaque. Functional assays showed that HOTAIR facilitated cell proliferation and migration, and suppressed apoptosis in EC. Moreover, we demonstrated that TSLP functions upstream of HOTAIR. We found that serum level of TSLP was decreased in atherosclerosis patients and serum TSLP level positively correlated with HOTAIR expression in EC. Further investigation demonstrated that TSLP activated HOTAIR transcription through PI3K/AKT-IRF1 pathway and then regulates the EC proliferation and migration. TSLP-HOTAIR axis also plays a protective role in low-density lipoprotein (ox-LDL)-induced EC injury. Taken together, TSLP-HOTAIR may be a potential therapy for EC dysfunction in atherosclerosis. PMID:28615347

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

PubMed

Clarkin, Claire E; King, Aileen J; Dhadda, Paramjeet; Chagastelles, Pedro; Nardi, Nance; Wheeler-Jones, Caroline P; Jones, Peter M

2013-03-01

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation. Copyright © 2013 AlphaMed Press.

Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

PubMed Central

Ginsberg, Michael; James, Daylon; Ding, Bi-Sen; Nolan, Daniel; Geng, Fuqiang; Butler, Jason M; Schachterle, William; Pulijaal, Venkat R; Mathew, Susan; Chasen, Stephen T; Xiang, Jenny; Rosenwaks, Zev; Shido, Koji; Elemento, Olivier; Rabbany, Sina Y; Rafii, Shahin

2012-01-01

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders. PMID:23084400

Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development.

PubMed

Zhao, Xiaofeng; Peng, Xu; Sun, Shaogang; Park, Ann Y J; Guan, Jun-Lin

2010-06-14

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)-specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.

Sex Differences Influencing Micro- and Macrovascular Endothelial Phenotype In Vitro.

PubMed

Huxley, Virginia H; Kemp, Scott S; Schramm, Christine; Sieveking, Steve; Bingaman, Susan; Yu, Yang; Zaniletti, Isabella; Stockard, Kevin; Wang, Jianjie

2018-06-09

Endothelial dysfunction is an early hallmark of multiple disease states that also display sex differences with respect to age of onset, frequency, and severity. Results of in vivo studies of basal and stimulated microvascular barrier function revealed sex differences difficult to ascribe to specific cells or environmental factors. The present study evaluated endothelial cells (EC) isolated from macro- and/or microvessels of reproductively mature rats under the controlled conditions of low-passage culture to test the assumption that EC phenotype would be sex-independent. The primary finding was that EC, regardless of where they are derived, retain a sex-bias in low-passage culture, independent of varying levels of reproductive hormones. Implications of the work include the fallacy of expecting a universal set of mechanisms derived from study of EC from one sex and/or one vascular origin to apply uniformly to all EC under unstimulated conditions no less in the disease state. Vascular endothelial cells (EC) are heterogeneous with respect to phenotype reflecting at least organ of origin, location within the vascular network, and physical forces. Sex, as an independent influence on EC functions in health or etiology, susceptibility, and progression of dysfunction in numerous disease states, has been largely ignored. The current study focussed on EC isolated from aorta (macrovascular) and skeletal muscle vessels (microvascular) of age-matched male and female rats under identical conditions of short term (passage 4) culture. We tested the hypothesis that genomic sex would not influence endothelial growth, wound healing, morphology, lactate production, or messenger RNA and protein expression of key proteins (sex hormone receptors for androgen (AR) and oestrogen (ERα and ERβ); PECAM-1 and VE-CAD mediating barrier function; α v β 3 and N-Cadherin influencing matrix interactions; ICAM-1 and VCAM-1 mediating EC/white cell adhesion). The hypothesis was rejected as EC origin (macro- versus microvessel) and sex influenced multiple phenotypic characteristics. Statistical model analysis of EC growth demonstrated an hierarchy of variable importance, recapitulated for other phenotypic characteristics, wherein predictions assuming EC homogeneity < Sex < Vessel Origin < Sex and Vessel Origin. Further, patterns of EC mRNA expression by vessel origin and by sex did not predict protein expression. Overall the study demonstrated that accurate assessment of sex-linked EC dysfunction first requires understanding of EC function by position in the vascular tree and by sex. Results from a single EC tissue source/species/sex cannot provide universal insight into the mechanisms regulating in vivo endothelial function in health, no less disease. (250) This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

In vitro characteristics of endothelial cells prepared from human cerebral arteriovenous malformation lesions using a novel method.

PubMed

Hao, Q; Chen, X L; Ma, L; Ye, X; Wang, H; Wang, T T; Hu, Y; Zhao, Y L

2018-03-01

The cerebral arteriovenous malformation (cAVM) is a usual and continually unaware reason of heamorrhage and seizure. It contains of feeder arteries, drain veins and abnormal vessel nets. However, pathologic mechanisms of the development of cAVM are unknown. The purpose of this study was to explore a novel protocol to isolate, culture and passage endothelial cells (ECs) from human cAVM lesions. We developed a protocol for isolating and growing ECs from eight patients with cAVM. The tissues were microsurgically removed from cAVM lesion and were digested by 0.25% Trypsin-EDTA, and cultured in ECM medium. ECs were selected by FACS and confirmed their EC origin by immunocytochemistry of the basic expression patterns of CD31 and CD34. LDL-uptake and capillary tube formation were used to determine their functional features. The isolated cAVM-ECs exhibited contact inhibition of growth and appearance of rounded cobblestone. cAVM-ECs were CD31- and CD34-positive. In functional assays, cAVM-ECs were able to uptake LDL and form capillary tubes. cAVM-ECs from younger patients proliferated faster than that from elders, and cAVM-ECs were less stable than normal artery ECs. In addition, cAVM-ECs appeared to more easily transform into mesenchymal cells than normal artery ECs. Using the protocol, isolated cAVM-ECs are stably established, and retain their endothelial phenotypes. These cAVM-ECs may provide a biological tool to examine molecular phenotypes and mechanisms responsible for human cAVM. Copyright © 2017 Elsevier Inc. All rights reserved.

Entorhinal Cortical Ocean Cells Encode Specific Contexts and Drive Context-Specific Fear Memory

PubMed Central

Kitamura, Takashi; Sun, Chen; Martin, Jared; Kitch, Lacey J; Schnitzer, Mark J; Tonegawa, Susumu

2016-01-01

Summary Forming distinct representations and memories of multiple contexts and episodes is thought to be a crucial function of the hippocampal-entorhinal cortical network. The hippocampal dentate gyrus (DG) and CA3 are known to contribute to these functions but the role of the entorhinal cortex (EC) is poorly understood. Here, we show that Ocean cells, excitatory stellate neurons in the medial EC layer II projecting into DG and CA3, rapidly form a distinct representation of a novel context and drive context-specific activation of downstream CA3 cells as well as context-specific fear memory. In contrast, Island cells, excitatory pyramidal neurons in the medial EC layer II projecting into CA1, are indifferent to context-specific encoding or memory. On the other hand, Ocean cells are dispensable for temporal association learning, for which Island cells are crucial. Together, the two excitatory medial EC layer II inputs to the hippocampus have complementary roles in episodic memory. PMID:26402611

Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel.

PubMed

Sarmiento, Daniela; Montorfano, Ignacio; Cerda, Oscar; Cáceres, Mónica; Becerra, Alvaro; Cabello-Verrugio, Claudio; Elorza, Alvaro A; Riedel, Claudia; Tapia, Pablo; Velásquez, Luis A; Varela, Diego; Simon, Felipe

2015-03-01

A hallmark of severe inflammation is reactive oxygen species (ROS) overproduction induced by increased inflammatory mediators secretion. During systemic inflammation, inflammation mediators circulating in the bloodstream interact with endothelial cells (ECs) raising intracellular oxidative stress at the endothelial monolayer. Oxidative stress mediates several pathological functions, including an exacerbated EC migration. Because cell migration critically depends on calcium channel-mediated Ca(2+) influx, the molecular identification of the calcium channel involved in oxidative stress-modulated EC migration has been the subject of intense investigation. The transient receptor potential melastatin 4 (TRPM4) protein is a ROS-modulated non-selective cationic channel that performs several cell functions, including regulating intracellular Ca(2+) overload and Ca(2+) oscillation. This channel is expressed in multiple tissues, including ECs, and contributes to the migration of certain immune cells. However, whether the TRPM4 ion channel participates in oxidative stress-mediated EC migration is not known. Herein, we investigate whether oxidative stress initiates or enhances EC migration and study the role played by the ROS-modulated TRPM4 ion channel in oxidative stress-mediated EC migration. We demonstrate that oxidative stress enhances, but does not initiate, EC migration in a dose-dependent manner. Notably, we demonstrate that the TRPM4 ion channel is critical in promoting H2O2-enhanced EC migration. These results show that TRPM4 is a novel pharmacological target for the possible treatment of severe inflammation and other oxidative stress-mediated inflammatory diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Lipid Droplet Biogenesis and Function in the Endothelium

PubMed Central

Kuo, Andrew; Lee, Monica Y.; Sessa, William C.

2017-01-01

Rationale Fatty acids (FA) are transported across the capillary endothelium to parenchymal tissues. However, it is not known how endothelial cells (EC) process a post-prandial surge of FA. Objective This study was designed to characterize lipid droplet (LD) formation and function in EC. Methods and Results LD form and degrade in EC in vivo after FA loading. In cultured EC, LD synthesis and turnover is dynamic and function to protect EC from lipotoxic stress and provide FA for metabolic needs. Conclusions Our results delineate endothelial LD dynamics for the first time, demonstrating their protective role in lipotoxicity, FA utilization and mobilization. PMID:28119423

Hypomethylation associated enhanced transcription of trefoil factor-3 mediates tamoxifen-stimulated oncogenicity of ER+ endometrial carcinoma cells.

PubMed

Pandey, Vijay; Zhang, Min; Chong, Qing-Yun; You, Mingliang; Raquib, Ainiah Rushdiana; Pandey, Amit K; Liu, Dong-Xu; Liu, Liang; Ma, Lan; Jha, Sudhakar; Wu, Zheng-Sheng; Zhu, Tao; Lobie, Peter E

2017-09-29

Tamoxifen (TAM) is widely used as an adjuvant therapy for women with breast cancer (BC). However, TAM possesses partial oestrogenic activity in the uterus and its use has been associated with an increased incidence of endometrial carcinoma (EC). The molecular mechanism for these observations is not well understood. Herein, we demonstrated that forced expression of Trefoil factor 3 ( TFF3) , in oestrogen receptor-positive (ER+) EC cells significantly increased cell cycle progression, cell survival, anchorage-independent growth, invasiveness and tumour growth in xenograft models. Clinically, elevated TFF3 protein expression was observed in EC compared with normal endometrial tissue, and its increased expression in EC was significantly associated with myometrial invasion. TAM exposure increased expression of TFF3 in ER+ EC cells and its elevated expression resulted in increased oncogenicity and invasiveness. TAM-stimulated expression of TFF3 in EC cells was associated with hypomethylation of the TFF3 promoter sequence and c-JUN/SP1-dependent transcriptional activation. In addition, small interfering ( si) RNA -mediated depletion or polyclonal antibody inhibition of TFF3 significantly abrogated oncogenicity and invasiveness in EC cells consequent to TAM induction or forced expression of TFF3. Hence, TAM-stimulated upregulation of TFF3 in EC cells was critical in promoting EC progression associated with TAM treatment. Importantly, inhibition of TFF3 function might be an attractive molecular modality to abrogate the stimulatory effects of TAM on endometrial tissue and to limit the progression of EC.

A comparison of the pro-angiogenic potential of human induced pluripotent stem cell derived endothelial cells and induced endothelial cells in a murine model of peripheral arterial disease.

PubMed

Clayton, Zoe E; Yuen, Gloria S C; Sadeghipour, Sara; Hywood, Jack D; Wong, Jack W T; Huang, Ngan F; Ng, Martin K C; Cooke, John P; Patel, Sanjay

2017-05-01

Endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) promote angiogenesis, and more recently induced endothelial cells (iECs) have been generated via fibroblast trans-differentiation. These cell types have potential as treatments for peripheral arterial disease (PAD). However, it is unknown whether different reprogramming methods produce cells that are equivalent in terms of their pro-angiogenic capabilities. We aimed to directly compare iPSC-ECs and iECs in an animal model of PAD, in order to identify which cell type, if any, displays superior therapeutic potential. IPSC-ECs and iECs were generated from human fibroblasts, and transduced with a reporter construct encoding GFP and firefly luciferase for bioluminescence imaging (BLI). Endothelial phenotype was confirmed using in vitro assays. NOD-SCID mice underwent hindlimb ischaemia surgery and received an intramuscular injection of either 1×10 6 iPSC-ECs, 1×10 6 iECs or control vehicle only. Perfusion recovery was measured by laser Doppler. Hindlimb muscle samples were taken for histological analyses. Perfusion recovery was enhanced in iPSC-EC treated mice on day 14 (Control vs. iPSC-EC; 0.35±0.04 vs. 0.54±0.08, p<0.05) and in iEC treated mice on days 7 (Control vs. iEC; 0.23±0.02 vs. 0.44±0.06, p<0.05), 10 (0.31±0.04 vs. 0.64±0.07, p<0.001) and 14 (0.35±0.04 vs. 0.68±0.07, p<0.001) post-treatment. IEC-treated mice also had greater capillary density in the ischaemic gastrocnemius muscle (Control vs. iEC; 125±10 vs. 179±11 capillaries/image; p<0.05). BLI detected iPSC-EC and iEC presence in vivo for two weeks post-treatment. IPSC-ECs and iECs exhibit similar, but not identical, endothelial functionality and both cell types enhance perfusion recovery after hindlimb ischaemia. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

PubMed

Kono, Ken; Hiruma, Hitomi; Kobayashi, Shingo; Sato, Yoji; Tanaka, Masaru; Sawada, Rumi; Niimi, Shingo

2016-01-01

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

miR-145 targets the SOX11 3’UTR to suppress endometrial cancer growth

PubMed Central

Chang, Lei; Yuan, Zhongfu; Shi, Huirong; Bian, Yangyang; Guo, Ruixia

2017-01-01

To explore the functions of SOX (Sex determining Region Y-related HMG-box) family genes in endometrial cancer (EC) and determine the influence of miR-145/SOX11 on EC cell functions. The relationship between miR-145 and SOX11 was confirmed using TargetScan, miRNA databases and dual-luciferase reporter gene assays. The expression of SOX11 mRNA in tissue specimens was examined using RT-qPCR, while SOX11 protein expression in tissues and cell lines were detected through immunohistochemistry (IHC) and western blotting. After transfection using Lipofectamine 2000, the proliferation, migration, invasion and apoptosis of ECC-1 and HEC-1-A cells were assessed through colony formation, transwell and flow cytometry assays. The correlation of SOX11 expression with the prognosis outcomes of patients was analyzed using Kaplan-Meier analysis and the log-rank test. SOX11 showed high expression in EC, which is negatively correlated with a poor prognostic outcome of EC patients. The expression of miR-145 was lower in EC tissues than in adjacent tissues. MiR-145 significantly reduced the expression of SOX11. In ECC-1 cells, miR-145 suppressed the propagation, migration, and invasion of cells and promoted cell apoptosis. MiR-145 also inhibited the proliferation, migration, and invasion of HEC-1-A cells and facilitated cell apoptosis by inhibiting SOX11. MiR-145 targeted site 3 (3615) of the SOX11 3’UTR to affect the expression of SOX11. MiR-145 and its target gene SOX11 could serve as diagnostic markers for EC. MiR-145 targets the SOX11 3’UTR to inhibit its expression and suppress the propagation and metastasis of EC cells. PMID:29218252

Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.

PubMed

Abdi, Khadar; Lai, Chun-Hsiang; Paez-Gonzalez, Patricia; Lay, Mark; Pyun, Joon; Kuo, Chay T

2018-04-25

Specialized, differentiated cells often perform unique tasks that require them to maintain a stable phenotype. Multiciliated ependymal cells (ECs) are unique glial cells lining the brain ventricles, important for cerebral spinal fluid circulation. While functional ECs are needed to prevent hydrocephalus, they have also been reported to generate new neurons: whether ECs represent a stable cellular population remains unclear. Via a chemical screen we found that mature ECs are inherently plastic, with their multiciliated state needing constant maintenance by the Foxj1 transcription factor, which paradoxically is rapidly turned over by the ubiquitin-proteasome system leading to cellular de-differentiation. Mechanistic analyses revealed a novel NF-κB-independent IKK2 activity stabilizing Foxj1 in mature ECs, and we found that known IKK2 inhibitors including viruses and growth factors robustly induced Foxj1 degradation, EC de-differentiation, and hydrocephalus. Although mature ECs upon de-differentiation can divide and regenerate multiciliated ECs, we did not detect evidence supporting EC's neurogenic potential.

Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells.

PubMed

Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J; Tarle, Susan A; Kaigler, Darnell

2014-04-01

In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential, which could make them a very attractive cell population for utilization in regenerative cell therapies.

Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation.

PubMed

Choudhary, Mayur; Naczki, Christine; Chen, Wenhong; Barlow, Keith D; Case, L Douglas; Metheny-Barlow, Linda J

2015-05-23

Proper functional association between mural cells and endothelial cells (EC) causes EC of blood vessels to become quiescent. Mural cells on tumor vessels exhibit decreased attachment to EC, which allows vessels to be unstable and proliferative. The mechanisms by which tumors prevent proper association between mural cells and EC are not well understood. Since gap junctions (GJ) play an important role in cell-cell contact and communication, we investigated whether loss of GJ plays a role in tumor-induced mural cell dissociation. Mural cell regulation of endothelial proliferation was assessed by direct co-culture assays of fluorescently labeled cells quantified by flow cytometry or plate reader. Gap junction function was assessed by parachute assay. Connexin 43 (Cx43) protein in mural cells exposed to conditioned media from cancer cells was assessed by Western and confocal microscopy; mRNA levels were assessed by quantitative real-time PCR. Expression vectors or siRNA were utilized to overexpress or knock down Cx43. Tumor growth and angiogenesis was assessed in mouse hosts deficient for Cx43. Using parachute dye transfer assay, we demonstrate that media conditioned by MDA-MB-231 breast cancer cells diminishes GJ communication between mural cells (vascular smooth muscle cells, vSMC) and EC. Both protein and mRNA of the GJ component Connexin 43 (Cx43) are downregulated in mural cells by tumor-conditioned media; media from non-tumorigenic MCF10A cells had no effect. Loss of GJ communication by Cx43 siRNA knockdown, treatment with blocking peptide, or exposure to tumor-conditioned media diminishes the ability of mural cells to inhibit EC proliferation in co-culture assays, while overexpression of Cx43 in vSMC restores GJ and endothelial inhibition. Breast tumor cells implanted into mice heterozygous for Cx43 show no changes in tumor growth, but exhibit significantly increased tumor vascularization determined by CD31 staining, along with decreased mural cell support detected by NG2 staining. Our data indicate that i) functional Cx43 is required for mural cell-induced endothelial quiescence, and ii) downregulation of Cx43 GJ by tumors frees endothelium to respond to angiogenic cues. These data define a novel and important role for maintained Cx43 function in regulation of vessel quiescence, and suggest its loss may contribute to pathological tumor angiogenesis.

Combined effects of physiologically relevant disturbed wall shear stress and glycated albumin on endothelial cell functions associated with inflammation, thrombosis and cytoskeletal dynamics.

PubMed

Maria, Zahra; Yin, Wei; Rubenstein, David Alan

2014-07-01

Diabetes mellitus is a major risk factor in the development of cardiovascular diseases (CVDs). The presence of advanced glycation end-products (AGEs) promotes CVDs by upregulating endothelial cell (EC) inflammatory and thrombotic responses, in a similar manner as disturbed shear stress. However, the combined effect of disturbed shear stress and AGEs on EC function has yet to be determined. Our goal was to evaluate these effects on EC responses. ECs were incubated with AGEs for 5 days. ECs were then subjected to physiological or pathological shear stress. Cell metabolic activity, surface expression of intercellular adhesion molecule-1, thrombomodulin, connexin-43 and caveolin-1, and cytoskeleton organization were quantified. The results show that irreversibly glycated albumin and pathological shear stress increased EC metabolic activity, and upregulated and downregulated the EC surface expression of intercellular adhesion molecule-1 and thrombomodulin, respectively. Expression of connexin-43, caveolin-1 and cytoskeletal organization was independent of shear stress; however, the presence of irreversibly glycated AGEs markedly increased connexin-43, and decreased caveolin-1 expression and actin cytoskeletal connectivity. Our data suggest that irreversibly glycated albumin and disturbed shear stress could promote CVD pathogenesis by enhancing EC inflammatory and thrombotic responses, and through the deterioration of the cytoskeletal organization.

Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels

PubMed Central

Zanotelli, Matthew R.; Ardalani, Hamisha; Zhang, Jue; Hou, Zhonggang; Nguyen, Eric H.; Swanson, Scott; Nguyen, Bao Kim; Bolin, Jennifer; Elwell, Angela; Bischel, Lauren L.; Xie, Angela W.; Stewart, Ron; Beebe, David J.; Thomson, James A.; Schwartz, Michael P.; Murphy, William L.

2016-01-01

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14 days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. PMID:26945632

MicroRNA-424/E2F6 feedback loop modulates cell invasion, migration and EMT in endometrial carcinoma

PubMed Central

Lu, Zheng; Nian, Zhou; Jingjing, Zhang; Tao, Luo; Quan, Li

2017-01-01

Our previous study explored the roles of microRNA-424 (miR-424) in the development of endometrial carcinoma (EC) and analyzed the miR-424/E2F7 axis in EC cell growth. In this study, we investigated the status of miR-424 in human endometrial cancer tissues, which were collected from a cohort of Zunyi patients. We found that the expression level of miR-424 was associated with clinical tumor stage, cell differentiation, lymph node metastasis and cell migration ability. Cell function experiments demonstrated that miR-424 overexpression suppressed the invasion and migration abilities of endometrial carcinoma cells in vitro. Bioinformatic predictions and dual-luciferase reporter assays suggested E2F6 as a possible target of miR-424. RT-PCR and western blot assays demonstrated that miR-424 transfection reduced the expression level of E2F6, while inhibiting miR-424 with ASO-miR-424 (antisense oligonucleotides of miR-424) increased the expression level of E2F6. Cell function experiments indicated that E2F6 transfection rescued the EC cell phenotype induced by miR-424. In addition, we also found that E2F6 negatively regulated miR-424 expression in EC cells. In summary, our results demonstrated that the miR-424/E2F6 feedback loop modulates cell invasion, migration and EMT in EC and that the miR-424/E2Fs regulation network may serve as a new and potentially important therapeutic target in EC. PMID:29371986

Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene

PubMed Central

Seandel, Marco; Butler, Jason M.; Kobayashi, Hideki; Hooper, Andrea T.; White, Ian A.; Zhang, Fan; Vertes, Eva L.; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V.; Hackett, Neil R.; Rabbany, Sina; Boyer, Julie L.; Rafii, Shahin

2008-01-01

Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1+ ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1+ ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1+ ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1+ ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1+ ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-κB pathways. Therefore, E4ORF1+ ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis. PMID:19036927

Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.

PubMed

Seandel, Marco; Butler, Jason M; Kobayashi, Hideki; Hooper, Andrea T; White, Ian A; Zhang, Fan; Vertes, Eva L; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V; Hackett, Neil R; Rabbany, Sina; Boyer, Julie L; Rafii, Shahin

2008-12-09

Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.

CD8(+) T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types.

PubMed

Migueles, Stephen A; Mendoza, Daniel; Zimmerman, Matthew G; Martins, Kelly M; Toulmin, Sushila A; Kelly, Elizabeth P; Peterson, Bennett A; Johnson, Sarah A; Galson, Eric; Poropatich, Kate O; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A; Jones, Sara; Hallahan, Claire W; Follmann, Dean A; Connors, Mark

2015-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8(+) T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8(+) T-cell specificity and function of B*27/57(neg) LTNP/EC (n = 23), B*27/57(pos) LTNP/EC (n = 23) and B*27/57(neg) progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57(neg) LTNP/EC did not target more highly conserved epitopes, their CD8(+) T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57(pos) LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8(+) T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people.

CD8+ T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types

PubMed Central

Migueles, Stephen A.; Mendoza, Daniel; Zimmerman, Matthew G.; Martins, Kelly M.; Toulmin, Sushila A.; Kelly, Elizabeth P.; Peterson, Bennett A.; Johnson, Sarah A.; Galson, Eric; Poropatich, Kate O.; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A.; Jones, Sara; Hallahan, Claire W.; Follmann, Dean A.; Connors, Mark

2014-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8+ T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8+ T-cell specificity and function of B*27/57neg LTNP/EC (n = 23), B*27/57pos LTNP/EC (n = 23) and B*27/57neg progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57neg LTNP/EC did not target more highly conserved epitopes, their CD8+ T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57pos LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8+ T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people. PMID:26137533

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

PubMed

Lau, Skadi; Eicke, Dorothee; Carvalho Oliveira, Marco; Wiegmann, Bettina; Schrimpf, Claudia; Haverich, Axel; Blasczyk, Rainer; Wilhelmi, Mathias; Figueiredo, Constança; Böer, Ulrike

2018-03-01

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism

PubMed Central

Gu, Mingxia; Mordwinkin, Nicholas M.; Kooreman, Nigel G.; Lee, Jaecheol; Wu, Haodi; Hu, Shijun; Churko, Jared M.; Diecke, Sebastian; Burridge, Paul W.; He, Chunjiang; Barron, Frances E.; Ong, Sang-Ging; Gold, Joseph D.; Wu, Joseph C.

2015-01-01

Aims High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays. Methods and results Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, Nω-nitro-l-arginine methyl ester. Conclusion This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population. PMID:25368203

Laminar shear stress promotes mitochondrial homeostasis in endothelial cells.

PubMed

Wu, Li-Hong; Chang, Hao-Chun; Ting, Pei-Ching; Wang, Danny L

2018-06-01

Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress that is crucial for endothelial functions. Laminar shear stress (LSS) exerts atheroprotection to ECs. Mitochondrial homeostasis is essential for cellular survival. However, the effects of LSS on mitochondrial homeostasis in ECs remain unclear. Mitochondrial homeostasis in ECs exposed to LSS was examined. Cultured human umbilical vein ECs were subjected to LSS (12 dynes/cm 2 ) generated by a parallel-plate flow chamber system. ECs subjected to LSS demonstrated an increment of mitochondria in tubular form coupled with the increase of fusion proteins (Mfn2, OPA1) and the decrease of fission protein (Fis1). An increase of both long- and short- OPA1 along with a higher protease YME1L level were observed. LSS triggered a rapid phosphorylation on S637 but a decrease on S616 of fission-controlled protein Drp1. Consistently, Drp1 translocation to mitochondria was decreased in sheared ECs, suggesting that LSS promotes mitochondrial fusion. Enhanced mitochondrial biogenesis in sheared ECs was shown by the increase of mitochondrial mass and its regulatory proeins (PGC1α, TFAM, Nrf1). LSS enhances the expression of mitochondrial antioxidant enzymes and improves mitochondrial functions indicated by the increase of mitochondrial membrane potential (ΔΨm) and ATP generation. TNFα treatment decreased mitochondrial tubular network and its functions in ECs. LSS mitigated TNFα-induced mitochondrial impairments in ECs. Our results clearly indicate that LSS promotes mitochondrial homeostasis and attenuates inflammation-induced mitochondrial impairments in ECs. Our results provide novel insights into the manner of mitochondrial dynamics and functions modulated by LSS that contribute to endothelial integrity. © 2017 Wiley Periodicals, Inc.

Enhanced Skeletal Muscle Expression of EcSOD Mitigates Streptozotocin-Induced Diabetic Cardiomyopathy by Reducing Oxidative Stress and Aberrant Cell Signaling

PubMed Central

Call, Jarrod A.; Chain, Kristopher H.; Martin, Kyle S.; Lira, Vitor A.; Okutsu, Mitsuharu; Zhang, Mei; Yan, Zhen

2015-01-01

Background Exercise training enhances extracellular superoxide dismutase (EcSOD) expression in skeletal muscle and elicits positive health outcomes in individuals with diabetes. The goal of this study was to determine if enhanced skeletal muscle expression of EcSOD is sufficient to mitigate streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM). Methods and Results Exercise training promotes EcSOD expression in skeletal muscle and provides protection against DCM; however, it is not known if enhanced EcSOD expression in skeletal muscle plays a functional role in this protection. Here, we show that skeletal muscle-specific EcSOD transgenic mice (TG) are protected from cardiac hypertrophy, fibrosis and dysfunction under the condition of type-1 diabetes induced by STZ injection. We also show that both exercise training and muscle-specific transgenic expression of EcSOD result in elevated EcSOD protein in the blood and heart without increased transcription in the heart, suggesting enhanced expression of EcSOD from skeletal muscle redistributes to the heart. Importantly, cardiac tissue in TG mice displayed significantly reduced oxidative stress, aberrant cell signaling and inflammatory cytokine expression compared with wild type mice under the same diabetic condition. Conclusions Enhanced expression of EcSOD in skeletal muscle is sufficient to mitigate STZ-induced DCM through attenuation of oxidative stress, aberrant cell signaling and inflammation, suggesting a cross-organ mechanism by which exercise training improves cardiac function in diabetes. PMID:25504759

Concurrent generation of functional smooth muscle and endothelial cells via a vascular progenitor.

PubMed

Marchand, Melanie; Anderson, Erica K; Phadnis, Smruti M; Longaker, Michael T; Cooke, John P; Chen, Bertha; Reijo Pera, Renee A

2014-01-01

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately, with low efficiencies, from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model, elucidate, and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media, these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells, respectively. Furthermore, we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations.

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

PubMed Central

Fang, Shentong; Wei, Jing; Pentinmikko, Nalle; Leinonen, Hannele; Salven, Petri

2012-01-01

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth. PMID:23091420

Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

PubMed

Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

2017-11-01

The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2.

PubMed

Ghilardi, Carmen; Silini, Antonietta; Figini, Sara; Anastasia, Alessia; Lupi, Monica; Fruscio, Robert; Giavazzi, Raffaella; Bani, Maria Rosa

2015-09-29

Proteases contribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy. We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix. Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy.

Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2

PubMed Central

Ghilardi, Carmen; Silini, Antonietta; Figini, Sara; Anastasia, Alessia; Lupi, Monica; Fruscio, Robert; Giavazzi, Raffaella; Bani, MariaRosa

2015-01-01

Proteasescontribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy. We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix. Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy. PMID:26318044

Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ko, Jae Hyung; Kim, Yang Hee; Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul

Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3more » dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.« less

Tissue oxygen demand in regulation of the behavior of the cells in the vasculature.

PubMed

Barvitenko, Nadezhda N; Aslam, Muhammad; Filosa, Jessica; Matteucci, Elena; Nikinmaa, Mikko; Pantaleo, Antonella; Saldanha, Carlota; Baskurt, Oguz K

2013-08-01

The control of arteriolar diameters in microvasculature has been in the focus of studies on mechanisms matching oxygen demand and supply at the tissue level. Functionally, important vascular elements include EC, VSMC, and RBC. Integration of these different cell types into functional units aimed at matching tissue oxygen supply with tissue oxygen demand is only achieved when all these cells can respond to the signals of tissue oxygen demand. Many vasoactive agents that serve as signals of tissue oxygen demand have their receptors on all these types of cells (VSMC, EC, and RBC) implying that there can be a coordinated regulation of their behavior by the tissue oxygen demand. Such functions of RBC as oxygen carrying by Hb, rheology, and release of vasoactive agents are considered. Several common extra- and intracellular signaling pathways that link tissue oxygen demand with control of VSMC contractility, EC permeability, and RBC functioning are discussed. © 2013 John Wiley & Sons Ltd.

CD146(+) cells are essential for kidney vasculature development.

PubMed

Halt, Kimmo J; Pärssinen, Heikki E; Junttila, Sanna M; Saarela, Ulla; Sims-Lucas, Sunder; Koivunen, Peppi; Myllyharju, Johanna; Quaggin, Susan; Skovorodkin, Ilya N; Vainio, Seppo J

2016-08-01

The kidney vasculature is critical for renal function, but its developmental assembly mechanisms remain poorly understood and models for studying its assembly dynamics are limited. Here, we tested whether the embryonic kidney contains endothelial cells (ECs) that are heterogeneous with respect to VEGFR2/Flk1/KDR, CD31/PECAM, and CD146/MCAM markers. Tie1Cre;R26R(YFP)-based fate mapping with a time-lapse in embryonic kidney organ culture successfully depicted the dynamics of kidney vasculature development and the correlation of the process with the CD31(+) EC network. Depletion of Tie1(+) or CD31(+) ECs from embryonic kidneys, with either Tie1Cre-induced diphtheria toxin susceptibility or cell surface marker-based sorting in a novel dissociation and reaggregation technology, illustrated substantial EC network regeneration. Depletion of the CD146(+) cells abolished this EC regeneration. Fate mapping of green fluorescent protein (GFP)-marked CD146(+)/CD31(-) cells indicated that they became CD31(+) cells, which took part in EC structures with CD31(+) wild-type ECs. EC network development depends on VEGF signaling, and VEGF and erythropoietin are expressed in the embryonic kidney even in the absence of any external hypoxic stimulus. Thus, the ex vivo embryonic kidney culture models adopted here provided novel ways for targeting renal EC development and demonstrated that CD146(+) cells are critical for kidney vasculature development. Copyright © 2016 International Society of Nephrology. All rights reserved.

Endothelial cells promote the proliferation of lymphocytes partly through the Wnt pathway via LEF-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Shu-Hong; Nan, Ke-Jun, E-mail: nankj@163.com; Wang, Yao-Chun

The function of T cells and B cells is to recognize specific 'non-self' antigens, during a process known as antigen presentation. Once they have identified an invader, the cells generate specific responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells. Endothelial cells (ECs) can trigger the activation of T cells through their class I and class II MHC molecules. In this study, we examined the effect of ECs on the proliferation of lymphocytes. We report that the proliferation of T and B cells can be improved by interaction with ECs. LEF-1 is one of the main molecularmore » mediators in this process, and the inhibition of LEF-1 induces apoptosis. These results suggest that LEF-1 modulates positively the proliferation of lymphocytes induced by their interaction with ECs.« less

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

PubMed

Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael; Nolan, Daniel J; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M; Prusky, Glen T; Llanos, Pierre; Rabbany, Sina Y; Maminishkis, Arvydas; Miller, Sheldon S; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-05-19

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

PubMed Central

Benedicto, Ignacio; Lehmann, Guillermo L.; Ginsberg, Michael; Nolan, Daniel J.; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M.; Prusky, Glen T.; Llanos, Pierre; Rabbany, Sina Y.; Maminishkis, Arvydas; Miller, Sheldon S.; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-01-01

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease. PMID:28524846

Differential signal pathway activation and 5-HT function: the role of gut enterochromaffin cells as oxygen sensors.

PubMed

Haugen, Martin; Dammen, Rikard; Svejda, Bernhard; Gustafsson, Bjorn I; Pfragner, Roswitha; Modlin, Irvin; Kidd, Mark

2012-11-15

The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O(2). Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1α (HIF-1α)-dependent pathway, we also hypothesized that increasing O(2) would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1α, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O(2) levels (0.5%). Reducing O(2) also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1α (1.7-3-fold). Increasing O(2) to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1α levels (∼75% of control). NF-κB signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O(2) levels differentially activate HIF-1α and tryptophan hydroxylase 1, as well as NF-κB signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing.

Young endothelial cells revive aging blood.

PubMed

Chang, Vivian Y; Termini, Christina M; Chute, John P

2017-11-01

The hematopoietic system declines with age, resulting in decreased hematopoietic stem cell (HSC) self-renewal capacity, myeloid skewing, and immune cell depletion. Aging of the hematopoietic system is associated with an increased incidence of myeloid malignancies and a decline in adaptive immunity. Therefore, strategies to rejuvenate the hematopoietic system have important clinical implications. In this issue of the JCI, Poulos and colleagues demonstrate that infusions of bone marrow (BM) endothelial cells (ECs) from young mice promoted HSC self-renewal and restored immune cell content in aged mice. Additionally, delivery of young BM ECs along with HSCs following total body irradiation improved HSC engraftment and enhanced survival. These results suggest an important role for BM endothelial cells (ECs) in regulating hematopoietic aging and support further research to identify the rejuvenating factors elaborated by BM ECs that restore HSC function and the immune repertoire in aged mice.

The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Yang; Gao Yu; Wang, Hong

Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted themore » proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs.« less

Mitochondrial biogenesis and energy production in differentiating murine stem cells: a functional metabolic study.

PubMed

Han, Sungwon; Auger, Christopher; Thomas, Sean C; Beites, Crestina L; Appanna, Vasu D

2014-02-01

The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells.

Endothelial cells (ECs) for vascular tissue engineering: venous ECs are less thrombogenic than arterial ECs.

PubMed

Geenen, I L A; Molin, D G M; van den Akker, N M S; Jeukens, F; Spronk, H M; Schurink, G W H; Post, M J

2015-05-01

Primary endothelial cells (ECs) are the preferred cellular source for luminal seeding of tissue-engineered (TE) vascular grafts. Research into the potential of ECs for vascular TE has focused particularly on venous rather than arterial ECs. In this study we evaluated the functional characteristics of arterial and venous ECs, relevant for vascular TE. Porcine ECs were isolated from femoral artery (PFAECs) and vein (PFVECs). The proliferation rate was comparable for both EC sources, whereas migration, determined through a wound-healing assay, was less profound for PFVECs. EC adhesion was lower for PFVECs on collagen I, measured after 10 min of arterial shear stress. Gene expression was analysed by qRT-PCR for ECs cultured under static conditions and after exposure to arterial shear stress and revealed differences in gene expression, with lower expression of EphrinB2 and VCAM-1 and higher levels of vWF and COUP-TFII in PFVECs than in PFAECs. PFVECs exhibited diminished platelet adhesion under flow and cell-based thrombin generation was delayed for PFVECs, indicating diminished tissue factor (TF) activity. After stimulation, prostacyclin secretion, but not nitric oxide (NO), was lower in PFVECs. Our data support the use of venous ECs for TE because of their beneficial antithrombogenic profile. Copyright © 2012 John Wiley & Sons, Ltd.

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state.

PubMed

Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

2011-05-05

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

Stem Cells Derived from Tooth Periodontal Ligament Enhance Functional Angiogenesis by Endothelial Cells

PubMed Central

Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J.; Tarle, Susan A.

2014-01-01

In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential, which could make them a very attractive cell population for utilization in regenerative cell therapies. PMID:24147894

Myocardial protective effect of extracellular superoxide dismutase gene modified bone marrow mesenchymal stromal cells on infarcted mice hearts.

PubMed

Pan, Qiao; Qin, Xing; Ma, Sai; Wang, Haichang; Cheng, Kang; Song, Xinxing; Gao, Haokao; Wang, Qiang; Tao, Rannie; Wang, Yabin; Li, Xiujuan; Xiong, Lize; Cao, Feng

2014-01-01

Extracellular superoxide dismutase (ecSOD) is a unique scavenger of superoxide anions and a promising target of gene therapy for ischemia/reperfusion injury (I/R). However, conventional gene therapies have limitation in effectiveness and efficiency. This study aimed to investigate the protective effects of ecSOD gene modified bone marrow mesenchymal stromal cells (BMSCs) on cardiac function improvement in mice infarcted heart. BMSCs were isolated from Fluc(+) transgenic mice (Tg FVB[Fluc(+)]) and transfected by adenovirus combined with human ecSOD gene. ELISA was performed to determine ecSOD protein level. Female syngeneic FVB mice were randomized into 5 groups: (1) Sham group (sham); (2) MI group (MI); (3) MI+BMSCs group (BMSC); (4) MI+BMSCs-vector group (BMSC-vector); (5) MI+ BMSCs-ecSOD group (BMSC-ecSOD). MI was accomplished by ligation of the left anterior descending artery. BMSCs (2 x 10(6)) were injected into the border zone of infarction. In vivo bioluminescence imaging (BLI) was performed to monitor transplanted BMSCs viability. Echocardiography and histological staining revealed that BMSCs-ecSOD significantly reduced myocardial infarction size and improved cardiac function. Lucigenin chemiluminescence, DHE and TUNEL staining demonstrated that BMSCs-ecSOD delivery reduced ROS level and cell apoptosis both in vivo and in vitro. Western blot assay revealed that ecSOD supplementation increased FoxO3a phosphorylation in cardiomyocytes. Moreover, quantitative real-time PCR showed that pro-apoptotic factors (bim and bax) were decreased while the anti-apoptotic factor mir-21 expression was increased after ecSOD intervention. Intra-myocardial transplantation of adenovirus-ecSOD transfected BMSCs could exert potential cardiac protection against MI, which may be partly through reduction of oxidative stress and improvement of BMSCs survival.

THE ENDOTHELIUM IN SEPSIS

PubMed Central

Ince, Can; Mayeux, Philip R.; Nguyen, Trung; Gomez, Hernando; Kellum, John A.; Ospina-Tascón, Gustavo A.; Hernandez, Glenn; Murray, Patrick; De Backer, Daniel

2017-01-01

Sepsis affects practically all aspects of endothelial cell (EC) function and is thought to be the key factor in the progression from sepsis to organ failure. Endothelial functions affected by sepsis include vasoregulation, barrier function, inflammation, and hemostasis. These are among other mechanisms often mediated by glycocalyx shedding, such as abnormal nitric oxide metabolism, up-regulation of reactive oxygen species generation due to down-regulation of endothelial-associated antioxidant defenses, transcellular communication, proteases, exposure of adhesion molecules, and activation of tissue factor. This review covers current insight in EC-associated hemostatic responses to sepsis and the EC response to inflammation. The endothelial cell lining is highly heterogeneous between different organ systems and consequently also in its response to sepsis. In this context, we discuss the response of the endothelial cell lining to sepsis in the kidney, liver, and lung. Finally, we discuss evidence as to whether the EC response to sepsis is adaptive or maladaptive. This study is a result of an Acute Dialysis Quality Initiative XIV Sepsis Workgroup meeting held in Bogota, Columbia, between October 12 and 15, 2014. PMID:26871664

The ICAM-1 expression level determines the susceptibility of human endothelial cells to simulated microgravity.

PubMed

Buravkova, Ludmila B; Rudimov, Eugene G; Andreeva, Elena R; Grigoriev, Anatoly I

2018-03-01

Microgravity is a principal risk factor hampering human cardiovascular regulation during space flights. Endothelial dysfunction associated with the impaired integrity and uniformity of the monolayer represents a potential trigger for vascular damage. We characterized the expression profile of the multi-step cascade of adhesion molecules (ICAM-1, VCAM-1, E-selectin, VE-cadherin) in umbilical cord endothelial cells (ECs) after 24 h of exposure to simulated microgravity (SMG), pro-inflammatory cytokine TNF-α, and the combination of the two. Random Positioning Machine (RPM)-mediated SMG was used to mimic microgravity effects. SMG stimulated the expression of E-selectin, which is known to be involved in slowing leukocyte rolling. Primary ECs displayed heterogeneity with respect to the proportion of ICAM-1-positive cells. ECs were divided into two groups: pre-activated ECs displaying high proportion of ICAM-1 + -cells (ECs-1) (greater than 50%) and non-activated ECs with low proportion of ICAM-1 + -cells (ECs-2) (less than 25%). Only non-activated ECs-2 responded to SMG by elevating gene transcription and increasing ICAM-1 and VE-cadherin expression. This effect was enhanced after cumulative SMG-TNF-α exposure. ECs-1 displayed an unexpected decrease in number of E-selectin- and ICAM-1-positive ECs and pronounced up-regulation of VCAM1 upon activation of inflammation, which was partially abolished by SMG. Thus, non-activated ECs-2 are quite resistant to the impacts of microgravity and even exhibited an elevation of the VE-cadherin gene and protein expression, thus improving the integrity of the endothelial monolayer. Pre-activation of ECs with inflammatory stimuli may disturb the EC adhesion profile, attenuating its barrier function. These alterations may be among the mechanisms underlying cardiovascular dysregulation in real microgravity conditions. © 2017 Wiley Periodicals, Inc.

Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nintasen, Rungrat; Multidisciplinary Cardiovascular Research Center; Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University

2012-04-20

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protectivemore » effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-{alpha} induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.« less

Cellular uptake of poly(allylamine hydrochloride) microcapsules with different deformability and its influence on cell functions.

PubMed

Yu, Wei; Zhang, Wenbo; Chen, Ying; Song, Xiaoxue; Tong, Weijun; Mao, Zhengwei; Gao, Changyou

2016-03-01

It is important to understand the safety issue and cell interaction pattern of polyelectrolyte microcapsules with different deformability before their use in biomedical applications. In this study, SiO2, poly(sodium-p-styrenesulfonate) (PSS) doped CaCO3 and porous CaCO3 spheres, all about 4μm in diameter, were used as templates to prepare microcapsules with different inner structure and subsequent deformability. As a result, three kinds of covalently assembled poly(allylaminehydrochloride)/glutaraldehyde (PAH/GA) microcapsules with similar size but different deformability under external osmotic pressure were prepared. The impact of different microcapsules on cell viability and functions are studied using smooth muscle cells (SMCs), endothelial cells (ECs) and HepG2 cells. The results demonstrated that viabilities of SMCs, ECs and HepG2 cells were not significantly influenced by either of the three kinds of microcapsules. However, the adhesion ability of SMCs and ECs as well as the mobility of SMCs, ECs and HepG2 cells were significantly impaired after treatment with microcapsules in a deformability dependent manner, especially the microcapsules with lower deformability caused higher impairment on cell functions. The cellular uptake kinetics, uptake pathways, intracellular distribution of microcapsules are further investigated in SMCs to reveal the potential mechanism. The SMCs showed faster uptake rate and exocytosis rate of microcapsules with lower deformability (Cap@CaCO3/PSS and Cap@CaCO3), leading to higher intracellular accumulation of microcapsules with lower deformability and possibly larger retardation of cell functions. The results pointed out that the deformability of microcapsules is an important factor governing the biological performance of microcapsules, which requires careful adjustment for further biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

Bacteria attenuation by iron electrocoagulation governed by interactions between bacterial phosphate groups and Fe(III) precipitates.

PubMed

Delaire, Caroline; van Genuchten, Case M; Amrose, Susan E; Gadgil, Ashok J

2016-10-15

Iron electrocoagulation (Fe-EC) is a low-cost process in which Fe(II) generated from an Fe(0) anode reacts with dissolved O2 to form (1) Fe(III) precipitates with an affinity for bacterial cell walls and (2) bactericidal reactive oxidants. Previous work suggests that Fe-EC is a promising treatment option for groundwater containing arsenic and bacterial contamination. However, the mechanisms of bacteria attenuation and the impact of major groundwater ions are not well understood. In this work, using the model indicator Escherichia coli (E. coli), we show that physical removal via enmeshment in EC precipitate flocs is the primary process of bacteria attenuation in the presence of HCO3(-), which significantly inhibits inactivation, possibly due to a reduction in the lifetime of reactive oxidants. We demonstrate that the adhesion of EC precipitates to cell walls, which results in bacteria encapsulation in flocs, is driven primarily by interactions between EC precipitates and phosphate functional groups on bacteria surfaces. In single solute electrolytes, both P (0.4 mM) and Ca/Mg (1-13 mM) inhibited the adhesion of EC precipitates to bacterial cell walls, whereas Si (0.4 mM) and ionic strength (2-200 mM) did not impact E. coli attenuation. Interestingly, P (0.4 mM) did not affect E. coli attenuation in electrolytes containing Ca/Mg, consistent with bivalent cation bridging between bacterial phosphate groups and inorganic P sorbed to EC precipitates. Finally, we found that EC precipitate adhesion is largely independent of cell wall composition, consistent with comparable densities of phosphate functional groups on Gram-positive and Gram-negative cells. Our results are critical to predict the performance of Fe-EC to eliminate bacterial contaminants from waters with diverse chemical compositions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioprinting of a functional vascularized mouse thyroid gland construct.

PubMed

Bulanova, Elena A; Koudan, Elizaveta V; Degosserie, Jonathan; Heymans, Charlotte; Pereira, Frederico DAS; Parfenov, Vladislav A; Sun, Yi; Wang, Qi; Akhmedova, Suraya A; Sviridova, Irina K; Sergeeva, Natalia S; Frank, Georgy A; Khesuani, Yusef D; Pierreux, Christophe E; Mironov, Vladimir A

2017-08-18

Bioprinting can be defined as additive biofabrication of three-dimensional (3D) tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. The thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of a functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS) as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modeling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within a collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, EC from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of a capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of the thyroid gland construct, we depleted endogenous EC from TS before bioprinting. EC from AS completely revascularized depleted thyroid tissue. The cultured bioprinted construct was functional as it could normalize blood thyroxine levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents a further advance in bioprinting technology, exploring the self-assembling properties of tissue spheroids.

Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells

PubMed Central

Butler, Jason M.; Nolan, Daniel J.; L.Vertes, Eva; Varnum-Finney, Barbara; Kobayashi, Hideki; Hooper, Andrea T.; Seandel, Marco; Shido, Koji; White, Ian A.; Kobayashi, Mariko; Witte, Larry; May, Chad; Shawber, Carrie; Kimura, Yuki; Kitajewski, Jan; Rosenwaks, Zev; Bernstein, Irwin D.; Rafii, Shahin

2010-01-01

Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long term-hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free co-cultures, ECs through direct cellular contact, stimulated incremental expansion of repopulating CD34−Flt3−cKit+Lineage−Sca1+ LT-HSCs, which retained their self-renewal ability, as determined by single cell and serial transplantation assays. Angiocrine expression of Notch-ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2 deficient mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp+ LT-HSCs were detected in cellular contact with sinusoidal ECs and interfering with angiocrine, but not perfusion function, of SECs impaired repopulation of TNR.Gfp+ LT-HSCs. ECs establish an instructive vascular niche for clinical scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens. PMID:20207228

Direct conversion of human amniotic cells into endothelial cells without transitioning through a pluripotent state

PubMed Central

Ginsberg, Michael; Schachterle, William; Shido, Koji; Rafii, Shahin

2016-01-01

Endothelial cells (ECs) have essential roles in organ development and regeneration, and therefore they could be used for regenerative therapies. However, generation of abundant functional endothelium from pluripotent stem cells has been difficult because ECs generated by many existing strategies have limited proliferative potential and display vascular instability. The latter difficulty is of particular importance because cells that lose their identity over time could be unsuitable for therapeutic use. Here, we describe a 3-week platform for directly converting human mid-gestation lineage-committed amniotic fluid–derived cells (ACs) into a stable and expandable population of vascular ECs (rAC-VECs) without using pluripotency factors. By transient expression of the ETS transcription factor ETV2 for 2 weeks and constitutive expression the ETS transcription factors FLI1 and ERG1, concomitant with TGF-β inhibition for 3 weeks, epithelial and mesenchymal ACs are converted, with high efficiency, into functional rAC-VECs. These rAC-VECs maintain their vascular repertoire and morphology over numerous passages in vitro, and they form functional vessels when implanted in vivo. rAC-VECs can be detected in recipient mice months after implantation. Thus, rAC-VECs can be used to establish a cellular platform to uncover the molecular determinants of vascular development and heterogeneity and potentially represent ideal ECs for the treatment of regenerative disorders. PMID:26540589

Physiological roles of Kv2 channels in entorhinal cortex layer II stellate cells revealed by Guangxitoxin‐1E

PubMed Central

Hönigsperger, Christoph; Nigro, Maximiliano J.

2016-01-01

Key points Kv2 channels underlie delayed‐rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia.Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space‐representing grid cells. We used the new Kv2 blocker Guangxitoxin‐1E (GTx) to study Kv2 functions in these neurons.Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed‐rectifier K+ current but not transient A‐type current.In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after‐depolarizations; (iii) reducing the fast and medium after‐hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering.GTx is a useful tool for studying Kv2 channels and their functions in neurons. Abstract The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin‐1E (GTx; 10–100 nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond –30 mV but not transient A‐type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after‐depolarization (ADP); (iii) reduced fast and medium after‐hyperpolarizations (AHPs); (iv) strongly enhanced burst firing and increased excitability at moderate spike rates but reduced spiking at high rates; and (v) reduced spike clustering and rebound potentials. The changes in bursting and excitability were related to the altered ADPs and AHPs. Kv2 channels strongly shape the activity of mEC SCs by affecting spike repolarization, after‐potentials, excitability and spike patterns. GTx is a useful tool and may serve to further clarify Kv2 channel functions in neurons. We conclude that Kv2 channels in mEC SCs are important determinants of intrinsic properties that allow these neurons to produce spatial representation. The results of the present study may also be important for the accurate modelling of grid cells. PMID:27562026

Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

PubMed

Abrahimi, Parwiz; Chang, William G; Kluger, Martin S; Qyang, Yibing; Tellides, George; Saltzman, W Mark; Pober, Jordan S

2015-07-03

The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression. CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering. © 2015 American Heart Association, Inc.

Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis

PubMed Central

Cao, Zhongwei; Ye, Tinghong; Sun, Yue; Ji, Gaili; Shido, Koji; Chen, Yutian; Luo, Lin; Na, Feifei; Li, Xiaoyan; Huang, Zhen; Ko, Jane L.; Mittal, Vivek; Qiao, Lina; Chen, Chong; Martinez, Fernando J.; Rafii, Shahin; Ding, Bi-Sen

2017-01-01

The regenerative capacity of lung and liver is sometimes impaired by chronic or overwhelming injury. Orthotopic transplantation of parenchymal stem cells to damaged organs might reinstate their self-repair ability. However, parenchymal cell engraftment is frequently hampered by the microenvironment in diseased recipient organs. Here, we show that targeting both the vascular niche and perivascular fibroblasts establishes “hospitable soil” to foster incorporation of “seed”, in this case the engraftment of parenchymal cells in injured organs. Specifically, ectopic induction of endothelial cell (EC)-expressed paracrine/angiocrine hepatocyte growth factor (HGF) and inhibition of perivascular NADPH Oxidase 4 (NOX4) synergistically enabled reconstitution of mouse and human parenchymal cells in damaged organs. Reciprocally, genetic knockout of Hgf in mouse ECs (HgfiΔEC/iΔEC) aberrantly upregulated perivascular NOX4 during liver and lung regeneration. Dysregulated HGF and NOX4 pathways subverted the function of vascular and perivascular cells from an epithelially-inductive niche to a microenvironment that inhibited parenchymal reconstitution. Perivascular NOX4 induction in HgfiΔEC/iΔEC mice recapitulated the phenotype of human and mouse fibrotic livers and lungs. Consequently, EC-directed HGF and NOX4 inhibitor GKT137831 stimulated regenerative integration of mouse and human parenchymal cells in chronically injured lung and liver. Our data suggest that targeting dysfunctional perivascular and vascular cells in diseased organs can bypass fibrosis and enable reparative cell engraftment to reinstate lung and liver regeneration. PMID:28855398

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

PubMed

Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

2015-03-02

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.

The Impact of Microgravity and Hypergravity on Endothelial Cells

PubMed Central

Maier, Jeanette A. M.

2015-01-01

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature. PMID:25654101

Endothelial cells: From innocent bystanders to active participants in immune responses.

PubMed

Al-Soudi, A; Kaaij, M H; Tas, S W

2017-09-01

The endothelium is crucially important for the delivery of oxygen and nutrients throughout the body under homeostatic conditions. However, it also contributes to pathology, including the initiation and perpetuation of inflammation. Understanding the function of endothelial cells (ECs) in inflammatory diseases and molecular mechanisms involved may lead to novel approaches to dampen inflammation and restore homeostasis. In this article, we discuss the various functions of ECs in inflammation with a focus on pathological angiogenesis, attraction of immune cells, antigen presentation, immunoregulatory properties and endothelial-to-mesenchymal transition (EndMT). We also review the current literature on approaches to target these processes in ECs to modulate immune responses and advance anti-inflammatory therapies. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

The impact of microgravity and hypergravity on endothelial cells.

PubMed

Maier, Jeanette A M; Cialdai, Francesca; Monici, Monica; Morbidelli, Lucia

2015-01-01

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

PubMed

Rossman, Matthew J; Kaplon, Rachelle E; Hill, Sierra D; McNamara, Molly N; Santos-Parker, Jessica R; Pierce, Gary L; Seals, Douglas R; Donato, Anthony J

2017-11-01

Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P < 0.05), respectively, in ECs obtained from antecubital veins of older sedentary (60 ± 1 yr, n = 12) versus young sedentary (22 ± 1 yr, n = 9) adults. These age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r  = -0.49, P = 0.003), p21 ( r  = -0.38, P = 0.03), and p16 ( r  = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P < 0.05), respectively, in ECs sampled from brachial arteries of healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed ( P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age. NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function. Copyright © 2017 the American Physiological Society.

Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide.

PubMed

Parkes, Christina; Kamal, Areege; Valentijn, Anthony J; Alnafakh, Rafah; Gross, Stephane R; Barraclough, Roger; Moss, Diana; Kirwan, John; Hapangama, Dharani K

2018-01-01

Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models. Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model. Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data. We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

Plasmodium falciparum-infected erythrocytes induce Tissue Factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes

PubMed Central

Francischetti, Ivo MB; Seydel, Karl B; Monteiro, Robson Q; Whitten, Richard O; Erexson, Cindy R; Noronha, Almério LL; Ostera, Graciela R.; Kamiza, Steve B; Molyneux, Malcolm E; Ward, Jerrold M; Taylor, Terrie E

2010-01-01

Summary Background Plasmodium falciparum malaria infects 300–500 million people every year causing 1–2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. Objectives We have asked whether parasitized red blood cells (pRBC) interaction with EC induces Tissue Factor expression in vitro and in vivo. The potential of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated. Results We demonstrate that mature forms of pRBC induce functional expression of tissue factor (TF) by endothelial cells (EC) in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, postmortem brain sections obtained from P. falciparum-infected children who died from Cerebral Malaria and other causes display a consistent staining for TF in the EC. Conclusions These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications. PMID:17002660

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer.

PubMed

Eritja, Núria; Chen, Bo-Juen; Rodríguez-Barrueco, Ruth; Santacana, Maria; Gatius, Sònia; Vidal, August; Martí, Maria Dolores; Ponce, Jordi; Bergadà, Laura; Yeramian, Andree; Encinas, Mario; Ribera, Joan; Reventós, Jaume; Boyd, Jeff; Villanueva, Alberto; Matias-Guiu, Xavier; Dolcet, Xavier; Llobet-Navàs, David

2017-03-04

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.

Lipocalin-type prostaglandin D synthase-derived PGD2 attenuates malignant properties of tumor endothelial cells.

PubMed

Omori, Keisuke; Morikawa, Teppei; Kunita, Akiko; Nakamura, Tatsuro; Aritake, Kosuke; Urade, Yoshihiro; Fukayama, Masashi; Murata, Takahisa

2018-01-01

Endothelial cells (ECs) are a key component of the tumor microenvironment. They have abnormal characteristics compared to the ECs in normal tissues. Here, we found a marked increase in lipocalin-type prostaglandin D synthase (L-PGDS) mRNA (Ptgds) expression in ECs isolated from mouse melanoma. Immunostaining of mouse melanoma revealed expression of L-PGDS protein in the ECs. In situ hybridization also showed L-PGDS (PTGDS) mRNA expression in the ECs of human melanoma and oral squamous cell carcinoma. In vitro experiments showed that stimulation with tumor cell-derived IL-1 and TNF-α increased L-PGDS mRNA expression and its product prostaglandin D 2 (PGD 2 ) in human normal ECs. We also investigated the contribution of L-PGDS-PGD 2 to tumor growth and vascularization. Systemic or EC-specific deficiency of L-PGDS accelerated the growth of melanoma in mice, whereas treatment with an agonist of the PGD 2 receptor, DP1 (BW245C, 0.1 mg/kg, injected intraperitoneally twice daily), attenuated it. Morphological and in vivo studies showed that endothelial L-PGDS deficiency resulted in functional changes of tumor ECs such as accelerated vascular hyperpermeability, angiogenesis, and endothelial-to-mesenchymal transition (EndMT) in tumors, which in turn reduced tumor cell apoptosis. These observations suggest that tumor cell-derived inflammatory cytokines increase L-PGDS expression and subsequent PGD 2 production in the tumor ECs. This PGD 2 acts as a negative regulator of the tumorigenic changes in tumor ECs. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

PubMed Central

Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

2011-01-01

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel ‘RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo. PMID:21544092

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

2013-10-29

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

PubMed

Lis, Raphael; Karrasch, Charles C; Poulos, Michael G; Kunar, Balvir; Redmond, David; Duran, Jose G Barcia; Badwe, Chaitanya R; Schachterle, William; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy A; Butler, Jason M; Scandura, Joseph M; Rafii, Shahin

2017-05-25

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells

PubMed Central

Lis, Raphael; Karrasch, Charles C.; Poulos, Michael G.; Kunar, Balvir; Redmond, David; Barcia Duran, Jose G.; Badwe, Chaitanya R.; Schachterle, Will; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy; Butler, Jason M.; Scandura, Joseph M.; Rafii, Shahin

2018-01-01

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully converting adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of genes encoding the transcription factors Fosb, Gfi1, Runx1, and Spi1 (also known as Fgrs) and vascular-niche-derived angiocrine factors. The induction phase (day 0–8) of conversion is initiated by expression of Fgrs in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (day 8–20), Runx1+ Fgrs-transduced endothelial cells commit to a haematopoietic fate yielding rEC-HSCs that no longer require Fgrs expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (at day 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, are competent for clonal engraftment and serial primary and secondary multi-lineage reconstituting potential, including antigen-dependent adaptive immune function. Inhibition of TGF-β and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. PMID:28514438

Direct Isolation, Culture and Transplant of Mouse Skeletal Muscle Derived Endothelial Cells with Angiogenic Potential

PubMed Central

Ieronimakis, Nicholas; Balasundaram, Gayathri; Reyes, Morayma

2008-01-01

Background Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study. Methodology By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1+, CD31+, CD34dim and CD45− cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature. Conclusion This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications. PMID:18335025

Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis

PubMed Central

Cochrane, Amy; Kelaini, Sophia; Tsifaki, Marianna; Bojdo, James; Vilà‐González, Marta; Drehmer, Daiana; Caines, Rachel; Magee, Corey; Eleftheriadou, Magdalini; Hu, Yanhua; Grieve, David; Stitt, Alan W.; Zeng, Lingfang; Xu, Qingbo

2017-01-01

Abstract The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA‐binding protein Quaking isoform 5 (QKI‐5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA‐binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient‐specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI‐5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI‐5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3′ UTR of STAT3. Importantly, mouse iPS‐ECs overexpressing QKI‐5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI‐5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI‐5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI‐5 is induced during EC differentiation from iPSCs. RNA binding protein QKI‐5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI‐5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm). stem cells 2017 Stem Cells 2017;35:952–966 PMID:28207177

Endothelial mineralocorticoid receptor ablation does not alter blood pressure, kidney function or renal vessel contractility

PubMed Central

Laursen, Sidsel B.; Finsen, Stine; Marcussen, Niels; Quaggin, Susan E.

2018-01-01

Aldosterone blockade confers substantial cardiovascular and renal protection. The effects of aldosterone on mineralocorticoid receptors (MR) expressed in endothelial cells (EC) within the renal vasculature have not been delineated. We hypothesized that lack of MR in EC may be protective in renal vasculature and examined this by ablating the Nr3c2 gene in endothelial cells (EC-MR) in mice. Blood pressure, heart rate and PAH clearance were measured using indwelling catheters in conscious mice. The role of the MR in EC on contraction and relaxation was investigated in the renal artery and in perfused afferent arterioles. Urinary sodium excretion was determined by use of metabolic cages. EC-MR transgenics had markedly decreased MR expression in isolated aortic endothelial cells as compared to littermates (WT). Blood pressure and effective renal plasma flow at baseline and following AngII infusion was similar between groups. No differences in contraction and relaxation were observed between WT and EC-MR KO in isolated renal arteries during baseline or following 2 or 4 weeks of AngII infusion. The constriction or dilatations of afferent arterioles between genotypes were not different. No changes were found between the groups with respect to urinary excretion of sodium after 4 weeks of AngII infusion, or in urinary albumin excretion and kidney morphology. In conclusion, deletion of the EC-MR does not confer protection towards the development of hypertension, endothelial dysfunction of renal arteries or renal function following prolonged AngII-infusion. PMID:29466427

Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

PubMed

Shen, S Q; Wang, R; Huang, S G

2017-03-08

Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P < 0.01). There were no significant differences in CD334-SCF double-positive FB and CD31-SCF double-positive EC levels between the two periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P < 0.01). FB, EC, and MP levels were significantly high and densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs improved considerably in chronic periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

Fatty acids rather than hormones restore in vitro angiogenesis in human male and female endothelial cells cultured in charcoal-stripped serum

PubMed Central

Vanetti, Claudia; Bifari, Francesco; Vicentini, Lucia M.

2017-01-01

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-β estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS. PMID:29232396

Islet graft survival and function: concomitant culture and transplantation with vascular endothelial cells in diabetic rats.

PubMed

Pan, Xiaoming; Xue, Wujun; Li, Yang; Feng, Xinshun; Tian, Xiaohui; Ding, Chenguang

2011-12-15

Human islet transplantation is a great potential therapy for type I diabetes. To investigate islet graft survival and function, we recently showed the improved effects after co-culture and co-transplantation with vascular endothelial cells (ECs) in diabetic rats. ECs were isolated, and the viability of isolated islets was assessed in two groups (standard culture group and co-culture group with ECs). Then streptozotocin-induced diabetic rats were divided into four groups before islet transplantation as follows: group A with infusion of islet grafts; group B with combined vascular ECs and islet grafts; groups C and D as controls with single ECs infusion and phosphate-buffered saline injection, respectively. Blood glucose and insulin concentrations were measured daily. Expression of vascular endothelial growth factor was investigated by immunohistochemical staining. The mean microvascular density was also calculated. More than 90% of acridine orange-propidium iodide staining positive islets demonstrated normal morphology while co-cultured with ECs for 7 days. Compared with standard control, insulin release assays showed a significantly higher simulation index in co-culture group except for the first day (P<0.05). After transplantation, there was a significant difference in concentrations of blood glucose and insulin among these groups after 3 days (P<0.05). The mean microvascular density in co-culture group was significantly higher than that in single islet group (P=0.04). Co-culture with ECs in vitro could improve the survival and function of isolated rat islet, and co-transplantation of islets with ECs could effectively prolong the islet graft survival in diabetic rats.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

PubMed

Vlasova, Anastasia N; Shao, Lulu; Kandasamy, Sukumar; Fischer, David D; Rauf, Abdul; Langel, Stephanie N; Chattha, Kuldeep S; Kumar, Anand; Huang, Huang-Chi; Rajashekara, Gireesh; Saif, Linda J

2016-10-01

Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4 + mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172 + MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TGFβ Triggers miR-143/145 Transfer From Smooth Muscle Cells to Endothelial Cells, Thereby Modulating Vessel Stabilization.

PubMed

Climent, Montserrat; Quintavalle, Manuela; Miragoli, Michele; Chen, Ju; Condorelli, Gianluigi; Elia, Leonardo

2015-05-22

The miR-143/145 cluster is highly expressed in smooth muscle cells (SMCs), where it regulates phenotypic switch and vascular homeostasis. Whether it plays a role in neighboring endothelial cells (ECs) is still unknown. To determine whether SMCs control EC functions through passage of miR-143 and miR-145. We used cocultures of SMCs and ECs under different conditions, as well as intact vessels to assess the transfer of miR-143 and miR-145 from one cell type to another. Imaging of cocultured cells transduced with fluorescent miRNAs suggested that miRNA transfer involves membrane protrusions known as tunneling nanotubes. Furthermore, we show that miRNA passage is modulated by the transforming growth factor (TGF) β pathway because both a specific transforming growth factor-β (TGFβ) inhibitor (SB431542) and an shRNA against TGFβRII suppressed the passage of miR-143/145 from SMCs to ECs. Moreover, miR-143 and miR-145 modulated angiogenesis by reducing the proliferation index of ECs and their capacity to form vessel-like structures when cultured on matrigel. We also identified hexokinase II (HKII) and integrin β 8 (ITGβ8)-2 genes essential for the angiogenic potential of ECs-as targets of miR-143 and miR-145, respectively. The inhibition of these genes modulated EC phenotype, similarly to miR-143 and miR-145 overexpression in ECs. These findings were confirmed by ex vivo and in vivo approaches, in which it was shown that TGFβ and vessel stress, respectively, triggered miR-143/145 transfer from SMCs to ECs. Our results demonstrate that miR-143 and miR-145 act as communication molecules between SMCs and ECs to modulate the angiogenic and vessel stabilization properties of ECs. © 2015 American Heart Association, Inc.

Expression of Eph receptor tyrosine kinases and their ligands in human Granulosa lutein cells and human umbilical vein endothelial cells.

PubMed

Xu, Y; Zagoura, D; Keck, C; Pietrowski, D

2006-11-01

Corpus luteum development is regulated by gonadotropins and accompanied by extremely rapid vascularization of the avascular granulosa cell compartiment by endothelial cells (EC). The proliferation of Granulosa cells (GC) and EC is a complex interplay and takes place in a spatially and temporarily coordinated manner. The erythropoietin-producing hepatoma amplified sequence (Eph) receptors and their ligands-the ephrins- are a recently detected family of membrane located protein tyrosine kinases which play a crucial role in the growth and development of nerve and blood vessel network. We report about the mRNA expression pattern of Ephs and their ligands in human GC, in human EC, and in carcinoma cell lines OvCar-3 and Hela. The mRNA of EphA4, EphA7, ephrinA4, ephrinB1 and ephrinB2 was detected in GC and EC, while EphA2 was expressed only in GC. The expression of various Ephs and ephrins did not change in GC after stimulation with human chorion gonadotropin. Our study analyzes for the first time the expression of the complete human Eph/ephriny-system in GC and in EC. The remarkable similarity between these two cell types supports the theory of a functional relationship of EC and GC. In addition, it was shown that hCG is not a major determinant of Eph/ephrin regulation in GC.

CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule.

PubMed Central

Latza, U.; Foss, H. D.; Dürkop, H.; Eitelbach, F.; Dieckmann, K. P.; Loy, V.; Unger, M.; Pizzolo, G.; Stein, H.

1995-01-01

The expression, serological detection, and possible functional role of the CD30 antigen in Hodgkin's disease and anaplastic large cell lymphoma is well documented. In embryonal carcinoma (EC), the expression of this cytokine receptor has been demonstrated only by immunohistology. Because the CD30 monoclonal antibody Ki-1 was found to cross-react with an unrelated molecule, we examined by in situ hybridization testicular germ cell neoplasms for the presence of CD30-specific transcripts. CD30 mRNA was detectable in the tumor cells of 9 of 9 cases of EC or mixed germ cell tumors with an EC component but in no other nonlymphoid tumors. Thus, the CD30 transcript expression pattern proved to be identical to the immunostaining pattern seen with the CD30-specific monoclonal antibody Ber-H2. By Northern blot analysis, CD30 transcripts could be demonstrated in the EC cell line Tera-2. Employing a highly sensitive second generation sandwich enzyme-linked immunosorbent assay, we could detect the soluble CD30 molecule in 8 of 8 sera from patients with a diagnosis of EC but not in 8 of 10 sera from patients with other testicular germ cell tumors. In fetal tissue, no CD30-expressing germ cells or epithelial cells could be observed. Thus, the cellularly expressed CD30 marker for testicular neoplasms of EC type. Moreover, the serum levels of soluble CD30 antigen seem to be a promising parameter for monitoring patients with EC. Images Figure 1 Figure 2 PMID:7856755

MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Martina; Baker, Meredith B.; Moore, Jeffrey P.

Mechanical forces associated with blood flow play an important role in regulating vascular signaling and gene expression in endothelial cells (ECs). MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. miRNAs are known to have an important role in modulating EC biology, but their expression and functions in cells subjected to shear stress conditions are unknown. We sought to determine the miRNA expression profile in human ECs subjected to unidirectional shear stress and define the role of miR-21 in shear stress-induced changes inmore » EC function. TLDA array and qRT-PCR analysis performed on HUVECs exposed to prolonged unidirectional shear stress (USS, 24 h, 15 dynes/cm{sup 2}) identified 13 miRNAs whose expression was significantly upregulated (p < 0.05). The miRNA with the greatest change was miR-21; it was increased 5.2-fold (p = 0.002) in USS-treated versus control cells. Western analysis demonstrated that PTEN, a known target of miR-21, was downregulated in HUVECs exposed to USS or transfected with pre-miR-21. Importantly, HUVECs overexpressing miR-21 had decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO{sup {center_dot}}) production. These data demonstrate that shear stress forces regulate the expression of miRNAs in ECs, and that miR-21 influences endothelial biology by decreasing apoptosis and activating the NO{sup {center_dot}} pathway. These studies advance our understanding of the mechanisms by which shear stress forces modulate vascular homeostasis.« less

Protection of dopaminergic neurons by electroconvulsive shock in an animal model of Parkinson's disease.

PubMed

Anastasia, Agustín; de Erausquin, Gabriel A; Wojnacki, José; Mascó, Daniel H

2007-11-01

Electroconvulsive shock (ECS) improves motor function in Parkinson's disease. In rats, ECS stimulates the expression of various factors some of which have been proposed to exert neuroprotective actions. We have investigated the effects of ECS on 6-hydroxydopamine (6-OHDA)-injected rats. Three weeks after a unilateral administration of 6-OHDA, 85-95% nigral dopaminergic neurons are lost. Chronic ECS prevented this cell loss, protect the nigrostriatal pathway (assessed by FloroGold retrograde labeling) and reduce motor impairment in 6-OHDA-treated animals. Injection of 6-OHDA caused loss of expression of glial cell-line derived neurotrophic factor (GDNF) in the substantia nigra. Chronic ECS completely prevented this loss of GDNF expression in 6-OHDA-treated animals. We also found that protected dopaminergic neurons co-express GDNF receptor proteins. These results strongly suggest that endogenous changes in GDNF expression may participate in the neuroprotective mechanism of ECS against 6-OHDA induced toxicity.

[Knockdown of NEDD9 inhibits the proliferation, invasion and migration of esophageal carcinoma EC109 cells].

PubMed

Zhang, Wen; Li, Shaojun; Zhao, Yunlong; Guo, Nannan; Li, Yingjie

2016-12-01

Objective To observe the expression of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) in esophageal cancer, to investigate the impact of decreased expression of NEDD9 on invasion and migration, and to explicit the function of NEDD9 in EC109 human esophageal cancer cell line. Methods Immunohistochemical staining was used to detect the expression of NEDD9 in human esophageal cancer tissues and paracancerous normal tissues. RNA interfering (RNAi) was used to knockdown NEDD9 in EC109 cells. The interference efficiency was detected by reverse transcription PCR (RT-PCR) and Western blot analysis. Cell proliferation was determined by MTT assay and the invasion and migration abilities of EC109 cells were monitored by Transwell TM assay. The protein levels of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 were tested by Western blotting. Results The positive expression rate of NEDD9 in esophageal carcinoma tissues was significantly higher compared with that in the paracancerous tissues. After NEDD9 expression was successfully downregulated in EC109 cells by siRNA, the proliferation, invasion and migration rates in transfection group were significantly lower than those in control group; meanwhile, the expression of Bcl-2 was reduced and Bax expression was enhanced. Conclusion The protein expression level of NEDD9 is higher in esophageal carcinoma tissues than that in adjacent normal tissues. Knockdown of NEDD9 expression can restrain the proliferation, invasion and migration of EC109 cells.

Preparation of an Arg-Glu-Asp-Val Peptide Density Gradient on Hyaluronic Acid-Coated Poly(ε-caprolactone) Film and Its Influence on the Selective Adhesion and Directional Migration of Endothelial Cells.

PubMed

Yu, Shan; Gao, Ying; Mei, Xu; Ren, Tanchen; Liang, Su; Mao, Zhengwei; Gao, Changyou

2016-11-02

Selective adhesion and migration of endothelial cells (ECs) over smooth muscle cells (SMCs) is very important in the rapid endothelialization of blood-contacting implants to prevent vascular restenosis. In this study, a uniform cell-resistant layer of methacrylate-functionalized hyaluronic acid (HA) was first immobilized on a poly(ε-caprolactone) (PCL) film via polydopamine coupling. Then, a density gradient of thiol-functionalized Arg-Glu-Asp-Val (REDV) peptide was prepared on the HA layer via thiol-ene click chemistry and the continuous injection method. The REDV gradient selectively enhanced EC adhesion and preferential directional migration toward the region of higher REDV density, reaching 86% directionality in the middle of the gradient. The migration rate of ECs was also significantly enhanced twofold compared with that on tissue culture polystyrene (TCPS). In contrast, the gradient significantly weakened the adhesion of SMCs to 25% of that on TCPS but had no obvious impact on the migration rate and directionality. Successful modulation of the selective adhesion and directional migration of ECs over SMCs on biodegradable polymers serves as an important step toward practical applications for guided tissue regeneration.

6-Methylsulfinylhexyl isothiocyanate modulates endothelial cell function and suppresses leukocyte adhesion.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Nagai, Masashi; Hayashi, Tatsuya; Suzuki, Koji

2014-01-01

6-Methylsulfinylhexyl isothiocyanate (6-MSITC) is an active compound in wasabi (Wasabia japonica Matsum.), which is one of the most popular spices in Japan. 6-MSITC suppresses lipopolysaccharide-induced macrophage activation, arachidonic- or adenosine diphosphate-induced platelet activation, and tumor cell proliferation. These data indicate that 6-MSITC has several biological activities involving anti-inflammatory, anti-coagulant, and anti-apoptosis properties. Endothelial cells (ECs) maintain vascular homeostasis and play crucial roles in crosstalk between blood coagulation and vascular inflammation. In this study, we determined the anti-coagulant and anti-inflammatory effects of 6-MSITC on human umbilical vein endothelial cells (HUVECs). 6-MSITC slightly reduced tissue factor expression, but did not alter von Willebrand factor release in activated HUVECs. 6-MSITC modulated the generation of activated protein C, which is essential for negative regulation of blood coagulation, on normal ECs. In addition, 6-MSITC reduced tumor necrosis factor-α (TNF-α)-induced interleukin-6 and monocyte chemoattractant protein-1 expression. 6-MSITC markedly attenuated TNF-α-induced adhesion of human monoblast U937 cells to HUVECs and reduced vascular cell adhesion molecule-1 and E-selectin mRNA expression in activated ECs. These results showed that 6-MSITC modulates EC function and suppresses cell adhesion. This study provides new insight into the mechanism of the anti-inflammatory effect of 6-MSITC, suggesting that 6-MSITC has therapeutic potential as a treatment for vasculitis and vascular inflammation.

Upregulation of TrkB Promotes Epithelial-Mesenchymal Transition and Anoikis Resistance in Endometrial Carcinoma

PubMed Central

Bao, Wei; Qiu, Haifeng; Yang, Tingting; Luo, Xin; Zhang, Huijuan; Wan, Xiaoping

2013-01-01

Mechanisms governing the metastasis of endometrial carcinoma (EC) are poorly defined. Recent data support a role for the cell surface receptor tyrosine kinase TrkB in the progression of several human tumors. Here we present evidence for a direct role of TrkB in human EC. Immunohistochemical analysis revealed that TrkB and its secreted ligand, brain-derived neurotrophic factor (BDNF), are more highly expressed in EC than in normal endometrium. High TrkB levels correlated with lymph node metastasis (p<0.05) and lymphovascular space involvement (p<0.05) in EC. Depletion of TrkB by stable shRNA-mediated knockdown decreased the migratory and invasive capacity of cancer cell lines in vitro and resulted in anoikis in suspended cells. Conversely, exogenous expression of TrkB increased cell migration and invasion and promoted anoikis resistance in suspension culture. Furthermore, over-expression of TrkB or stimulation by BDNF resulted in altered the expression of molecular mediators of the epithelial-to-mesenchymal transition (EMT). RNA interference (RNAi)-mediated depletion of the downstream regulator, Twist, blocked TrkB-induced EMT-like transformation. The use of in vivo models revealed decreased peritoneal dissemination in TrkB-depleted EC cells. Additionally, TrkB-depleted EC cells underwent mesenchymal-to-epithelial transition and anoikis in vivo. Our data support a novel function for TrkB in promoting EMT and resistance to anoikis. Thus, TrkB may constitute a potential therapeutic target in human EC. PMID:23936232

The impact of 27-hydroxycholesterol on endometrial cancer proliferation.

PubMed

Gibson, Douglas A; Collins, Frances; Cousins, Fiona L; Esnal Zufiaurre, Arantza; Saunders, Philippa T K

2018-04-01

Endometrial cancer (EC) is the most common gynaecological malignancy. Obesity is a major risk factor for EC and is associated with elevated cholesterol. 27-hydroxycholesterol (27HC) is a cholesterol metabolite that functions as an endogenous agonist for Liver X receptor (LXR) and a selective oestrogen receptor modulator (SERM). Exposure to oestrogenic ligands increases risk of developing EC; however, the impact of 27HC on EC is unknown. Samples of stage 1 EC ( n  = 126) were collected from postmenopausal women undergoing hysterectomy. Expression of LXRs ( NR1H3 , LXRα; NR1H2 , LXRβ) and enzymes required for the synthesis ( CYP27A1 ) or breakdown ( CYP7B1 ) of 27HC were detected in all grades of EC. Cell lines originating from well-, moderate- and poorly-differentiated ECs (Ishikawa, RL95, MFE 280 respectively) were used to assess the impact of 27HC or the LXR agonist GW3965 on proliferation or expression of a luciferase reporter gene under the control of LXR- or ER-dependent promoters (LXRE, ERE). Incubation with 27HC or GW3965 increased transcription via LXRE in Ishikawa, RL95 and MFE 280 cells ( P  < 0.01). 27HC selectively activated ER-dependent transcription ( P  < 0.001) in Ishikawa cells and promoted proliferation of both Ishikawa and RL95 cells ( P  < 0.001). In MFE 280 cells, 27HC did not alter proliferation but selective targeting of LXR with GW3965 significantly reduced cell proliferation ( P  < 0.0001). These novel results suggest that 27HC can contribute to risk of EC by promoting proliferation of endometrial cancer epithelial cells and highlight LXR as a potential therapeutic target in the treatment of advanced disease. © 2018 The authors.

Prorenin induces ERK activation in endothelial cells to enhance neovascularization independently of the renin-angiotensin system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uraoka, Maki; Ikeda, Koji, E-mail: ikedak@koto.kpu-m.ac.jp; Nakagawa, Yusuke

Prorenin is an enzymatically inactive precursor of renin, and its biological function in endothelial cells (ECs) is unknown despite its relevance with the incidence of diabetic microvascular complications. Recently, (pro)renin receptor was identified, and the receptor-associated prorenin system has been discovered, whereas its expression as well as function in ECs remain unclear. In the present study, we found that ECs express the (pro)renin receptor, and that prorenin provoked ERK activation through (pro)renin receptor independently of the renin-angiotensin system (RAS). Prorenin stimulated the proliferation, migration and tube-formation of ECs, while it inhibited endothelial apoptosis induced by serum and growth factor depletion.more » MEK inhibitor abrogated these proangiogenic effects of prorenin, while AT1 receptor antagonist or angiotensin-converting enzyme inhibitor failed to block them. In vivo neovascularization in the Matrigel-plugs implanted into mouse flanks was significantly enhanced by prorenin, in which significant ERK activation was detected in ECs. Furthermore, tumor xenografts stably transfected with prorenin demonstrated the significantly accelerated growth rate concomitantly with enhanced intratumoral neovascularization. Our data demonstrated that the RAS-independent (pro)renin receptor-mediated signal transduction plays a pivotal role in the regulation of ECs function as well as in the neovascularization, and thus prorenin is potentially involved in the pathophysiology of diabetic microvascular complications as well as cancers.« less

Long Noncoding RNA-GAS5: A Novel Regulator of Hypertension-Induced Vascular Remodeling.

PubMed

Wang, Yang-Ning-Zhi; Shan, Kun; Yao, Mu-Di; Yao, Jin; Wang, Jia-Jian; Li, Xiang; Liu, Ban; Zhang, Yang-Yang; Ji, Yong; Jiang, Qin; Yan, Biao

2016-09-01

Vascular remodeling is an important pathological feature of hypertension, leading to increased vascular resistance and reduced compliance. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of EC and VSMC function. Herein, we determined whether long noncoding RNA-growth arrest-specific 5 (GAS5) is involved in hypertension-related vascular remodeling. We revealed that GAS5 knockdown aggravated hypertension-induced microvascular dysfunction as shown by increased retinal neovascularization and capillary leakage. GAS5 regulated the remodeling of arteries, including caudal arteries, carotid arteries, renal arteries, and thoracic arteries. GAS5 was mainly expressed in ECs and VSMCs, and its expression was significantly downregulated in hypertension. GAS5 knockdown affected endothelial activation, endothelial proliferation, VSMC phenotypic conversion, and EC-VSMC communication in vivo and in vitro. Mechanistically, GAS5 regulated EC and VSMC function through β-catenin signaling. This study identified GAS5 as a critical regulator in hypertension and demonstrated the potential of gene therapy and drug development for treating hypertension. © 2016 American Heart Association, Inc.

Mechanical Adaptability of the MMP-Responsive Film Improves the Functionality of Endothelial Cell Monolayer.

PubMed

Hu, Mi; Chang, Hao; Zhang, He; Wang, Jing; Lei, Wen-Xi; Li, Bo-Chao; Ren, Ke-Feng; Ji, Jian

2017-07-01

Extracellular matrix and cells are inherent in coordinating and adapting to each other during all physiological and pathological processes. Synthetic materials, however, show rarely reciprocal and spatiotemporal responses to cells, and lacking self-adapting properties as well. Here, a mechanical adaptability based on the matrix metalloproteinase (MMPs) sensitive polyelectrolyte film is reported. Poly-lysine (PLL) and methacrylated hyaluronic acid (HA-MA) nanolayers are employed to build the thin film through the layer-by-layer assembly, and it is further crosslinked using MMP sensitive peptides, which endows the films with changeable mechanical properties in response to MMPs. It is demonstrated that stiffness of the (PLL/HA-MA) films increases with the crosslinking, and then decreases in response to a treatment of enzyme. Consequently, the crosslinked (PLL/HA-MA) films reveal effective growth of endothelial cells (ECs), leading to fast formation of EC monolayer. Importantly, significantly improved endothelial function of the EC monolayer, which is characterized by integrity, biomolecules release, expression of function related gene, and antithrombotic properties, is achieved along with the decrosslinking of the film because of EC-secreted MMPs. These results suggest that mechanical adaptability of substrate in Young's modulus plays a significant role in endothelial progression, which shows great application potential in tissue engineering, regenerative medicine, and organ-on-a-chip. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis.

PubMed

Cao, Zhongwei; Ye, Tinghong; Sun, Yue; Ji, Gaili; Shido, Koji; Chen, Yutian; Luo, Lin; Na, Feifei; Li, Xiaoyan; Huang, Zhen; Ko, Jane L; Mittal, Vivek; Qiao, Lina; Chen, Chong; Martinez, Fernando J; Rafii, Shahin; Ding, Bi-Sen

2017-08-30

The regenerative capacity of lung and liver is sometimes impaired by chronic or overwhelming injury. Orthotopic transplantation of parenchymal stem cells to damaged organs might reinstate their self-repair ability. However, parenchymal cell engraftment is frequently hampered by the microenvironment in diseased recipient organs. We show that targeting both the vascular niche and perivascular fibroblasts establishes "hospitable soil" to foster the incorporation of "seed," in this case, the engraftment of parenchymal cells in injured organs. Specifically, ectopic induction of endothelial cell (EC)-expressed paracrine/angiocrine hepatocyte growth factor (HGF) and inhibition of perivascular NOX4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 4] synergistically enabled reconstitution of mouse and human parenchymal cells in damaged organs. Reciprocally, genetic knockout of Hgf in mouse ECs ( Hgf iΔEC/iΔEC ) aberrantly up-regulated perivascular NOX4 during liver and lung regeneration. Dysregulated HGF and NOX4 pathways subverted the function of vascular and perivascular cells from an epithelially inductive niche to a microenvironment that inhibited parenchymal reconstitution. Perivascular NOX4 induction in Hgf iΔEC/iΔEC mice recapitulated the phenotype of human and mouse liver and lung fibrosis. Consequently, EC-directed HGF and NOX4 inhibitor GKT137831 stimulated regenerative integration of mouse and human parenchymal cells in chronically injured lung and liver. Our data suggest that targeting dysfunctional perivascular and vascular cells in diseased organs can bypass fibrosis and enable reparative cell engraftment to reinstate lung and liver regeneration. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

PubMed Central

Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

2006-01-01

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

Intra-islet endothelial cell and β-cell crosstalk: Implication for islet cell transplantation

PubMed Central

Narayanan, Siddharth; Loganathan, Gopalakrishnan; Dhanasekaran, Maheswaran; Tucker, William; Patel, Ankit; Subhashree, Venugopal; Mokshagundam, SriPrakash; Hughes, Michael G; Williams, Stuart K; Balamurugan, Appakalai N

2017-01-01

The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of β-cells with endothelial cells (ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation. These cells also tend to behave as “guardians”, controlling the expression and movement of a number of important immune mediators, thereby strongly contributing to the physiology of islets. This review will focus on the molecular signalling and crosstalk between the intra-islet ECs and β-cells and how their relationship can be a potential target for intervention strategies in islet pathology and islet transplantation. PMID:28507914

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell-cell interaction with activated cells.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Shimaoka, Motomu; Suzuki, Koji

2014-10-01

Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion.

PubMed

Lacal, Pedro Miguel; Petrillo, Maria Grazia; Ruffini, Federica; Muzi, Alessia; Bianchini, Rodolfo; Ronchetti, Simona; Migliorati, Graziella; Riccardi, Carlo; Graziani, Grazia; Nocentini, Giuseppe

2013-10-01

The interaction of glucocorticoid-induced tumor necrosis factor receptor-family related (GITR) protein with its ligand (GITRL) modulates different functions, including immune/inflammatory response. These effects are consequent to intracellular signals activated by both GITR and GITRL. Previous results have suggested that lack of GITR expression in GITR(-/-) mice decreases the number of leukocytes within inflamed tissues. We performed experiments to analyze whether the GITRL/GITR system modulates leukocyte adhesion and extravasation. For that purpose, we first evaluated the capability of murine splenocytes to adhere to endothelial cells (EC). Our results indicated that adhesion of GITR(-/-) splenocytes to EC was reduced as compared with wild-type cells, suggesting that GITR plays a role in adhesion and that this effect may be due to GITRL-GITR interaction. Moreover, adhesion was increased when EC were pretreated with an agonist GITR-Fc fusion protein, thus indicating that triggering of GITRL plays a role in adhesion by EC regulation. In a human in vitro model, the adhesion to human EC of HL-60 cells differentiated toward the monocytic lineage was increased by EC pretreatment with agonist GITR-Fc. Conversely, antagonistic anti-GITR and anti-GITRL Ab decreased adhesion, thus further indicating that GITRL triggering increases the EC capability to support leukocyte adhesion. EC treatment with GITR-Fc favored extravasation, as demonstrated by a transmigration assay. Notably, GITRL triggering increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and anti-ICAM-1 and anti-VCAM-1 Abs reversed GITR-Fc effects. Our study demonstrates that GITRL triggering in EC increases leukocyte adhesion and transmigration, suggesting new anti-inflammatory therapeutic approaches based on inhibition of GITRL-GITR interaction.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: implications for coronary artery stent

PubMed Central

Wang, Xintong; Zachman, Angela L.; Chun, Young Wook; Shen, Fang-Wen; Hwang, Yu-Shik; Sung, Hak-Joon

2014-01-01

Background Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. Methods Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. Results We demonstrated poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule-1 (VCAM) along with decreased nitric oxide production, indicating ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced release of elastase or elastase-like protease, which further accelerated polymer degradation. Conclusions This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis. PMID:24820736

Plasma functionalization of polycarbonaturethane to improve endothelialization--Effect of shear stress as a critical factor for biocompatibility control.

PubMed

Lukas, Karin; Thomas, Ulrich; Gessner, André; Wehner, Daniel; Schmid, Thomas; Schmid, Christof; Lehle, Karla

2016-04-01

Medical devices made of polycarbonaturethane (PCU) combine excellent mechanical properties and little biological degradation, but restricted hemocompatibility. Modifications of PCU might reduce platelet adhesion and promote stable endothelialization. PCU was modified using gas plasma treatment, binding of hydrogels, and coupling of cell-active molecules (modified heparin, anti-thrombin III (ATIII), argatroban, fibronectin, laminin-nonapeptide, peptides with integrin-binding arginine-glycine-aspartic acid (RGD) motif). Biocompatibility was verified with static and dynamic cell culture techniques. Blinded analysis focused on improvement in endothelial cell (EC) adhesion/proliferation, anti-thrombogenicity, reproducible manufacturing process, and shear stress tolerance of ECs. EC adhesion and antithrombogenicity were achieved with 9/35 modifications. Additionally, 6/9 stimulated EC proliferation and 3/6 modification processes were highly reproducible for endothelialization. The latter modifications comprised immobilization of ATIII (A), polyethyleneglycole-diamine-hydrogel (E) and polyethylenimine-hydrogel connected with modified heparin (IH). Under sheer stress, only the IH modification improved EC adhesion within the graft. However, ECs did not arrange in flow direction and cell anchorage was restricted. Despite large variation in surface modification chemistry and improved EC adhesion under static culture conditions, additional introduction of shear stress foiled promising preliminary data. Therefore, biocompatibility testing required not only static tests but also usage of physiological conditions such as shear stress in the case of vascular grafts. © The Author(s) 2016.

Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources.

PubMed

Reed, Daniel M; Foldes, Gabor; Kirkby, Nicholas S; Ahmetaj-Shala, Blerina; Mataragka, Stefania; Mohamed, Nura A; Francis, Catherine; Gara, Edit; Harding, Sian E; Mitchell, Jane A

2014-12-12

Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

Antiviral function of grouper MDA5 against iridovirus and nodavirus.

PubMed

Huang, Youhua; Yu, Yepin; Yang, Ying; Yang, Min; Zhou, Linli; Huang, Xiaohong; Qin, Qiwei

2016-07-01

Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines. Copyright © 2016 Elsevier Ltd. All rights reserved.

CD146 coordinates brain endothelial cell–pericyte communication for blood–brain barrier development

PubMed Central

Chen, Jianan; Luo, Yongting; Hui, Hui; Cai, Tanxi; Huang, Hongxin; Yang, Fuquan; Feng, Jing; Zhang, Jingjing; Yan, Xiyun

2017-01-01

The blood–brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte–EC interaction during BBB development, the molecular mechanisms coordinating the pericyte–EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-β to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-β1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB. PMID:28827364

Evaluation of Fuel Cell Operation and Degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, Mark; Gemmen, Randall; Richards, George

The concepts of area specific resistance (ASR) and degradation are developed for different fuel cell operating modes. The concepts of exergetic efficiency and entropy production were applied to ASR and degradation. It is shown that exergetic efficiency is a time-dependent function useful describing the thermal efficiency of a fuel cell and the change in thermal efficiency of a degrading fuel cell. Entropy production was evaluated for the cases of constant voltage operation and constant current operation of the fuel cell for a fuel cell undergoing ohmic degradation. It was discovered that the Gaussian hypergeometric function describes the cumulative entropy andmore » electrical work produced by fuel cells operating at constant voltage. The Gaussian hypergeometric function is found in many applications in modern physics. This paper builds from and is an extension of several papers recently published by the authors in the Journal of The Electrochemical Society (ECS), ECS Transactions, Journal of Power Sources, and the Journal of Fuel Cell Science and Technology.« less

MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Bing; Xiao, Bo; Liang, Desheng

Highlights: {yields} We evaluated the role of miRNAs in ox-LDL induced apoptosis in ECs. {yields} We found 4 up-regulated and 11 down-regulated miRNAs in apoptotic ECs. {yields} Target genes of the dysregulated miRNAs regulate ECs apoptosis and atherosclerosis. {yields} MiR-365 promotes ECs apoptosis via suppressing Bcl-2 expression. {yields} MiR-365 inhibitor alleviates ECs apoptosis induced by ox-LDL. -- Abstract: Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth,more » proliferation, and apoptosis. However, whether miRNAs are associated with ox-LDL induced apoptosis and their effect on ECs is still unknown. Therefore, this study evaluated potential miRNAs and their involvement in ECs apoptosis in response to ox-LDL stimulation. Microarray and qRT-PCR analysis performed on human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL identified 15 differentially expressed (4 up- and 11 down-regulated) miRNAs. Web-based query tools were utilized to predict the target genes of the differentially expressed miRNAs, and the potential target genes were classified into different function categories with the gene ontology (GO) term and KEGG pathway annotation. In particular, bioinformatics analysis suggested that anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) is a target gene of miR-365, an apoptomir up-regulated by ox-LDL stimulation in HUVECs. We further showed that transfection of miR-365 inhibitor partly restored Bcl-2 expression at both mRNA and protein levels, leading to a reduction of ox-LDL-mediated apoptosis in HUVECs. Taken together, our findings indicate that miRNAs participate in ox-LDL-mediated apoptosis in HUVECs. MiR-365 potentiates ox-LDL-induced ECs apoptosis by regulating the expression of Bcl-2, suggesting potential novel therapeutic targets for atherosclerosis.« less

Molecular identification of venous progenitors in the dorsal aorta reveals an aortic origin for the cardinal vein in mammals

PubMed Central

Lindskog, Henrik; Kim, Yung Hae; Jelin, Eric B.; Kong, Yupeng; Guevara-Gallardo, Salvador; Kim, Tyson N.; Wang, Rong A.

2014-01-01

Coordinated arterial-venous differentiation is crucial for vascular development and function. The origin of the cardinal vein (CV) in mammals is unknown, while conflicting theories have been reported in chick and zebrafish. Here, we provide the first molecular characterization of endothelial cells (ECs) expressing venous molecular markers, or venous-fated ECs, within the emergent dorsal aorta (DA). These ECs, expressing the venous molecular markers Coup-TFII and EphB4, cohabited the early DA with ECs expressing the arterial molecular markers ephrin B2, Notch and connexin 40. These mixed ECs in the early DA expressed either the arterial or venous molecular marker, but rarely both. Subsequently, the DA exhibited uniform arterial markers. Real-time imaging of mouse embryos revealed EC movement from the DA to the CV during the stage when venous-fated ECs occupied the DA. We analyzed mutants for EphB4, which encodes a receptor tyrosine kinase for the ephrin B2 ligand, as we hypothesized that ephrin B2/EphB4 signaling may mediate the repulsion of venous-fated ECs from the DA to the CV. Using an EC quantification approach, we discovered that venous-fated ECs increased in the DA and decreased in the CV in the mutants, whereas the rest of the ECs in each vessel were unaffected. This result suggests that the venous-fated ECs were retained in the DA and missing in the CV in the EphB4 mutant, and thus that ephrin B2/EphB4 signaling normally functions to clear venous-fated ECs from the DA to the CV by cell repulsion. Therefore, our cellular and molecular evidence suggests that the DA harbors venous progenitors that move to participate in CV formation, and that ephrin B2/EphB4 signaling regulates this aortic contribution to the mammalian CV. PMID:24550118

Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity.

PubMed

Wang, Yanshu; Rattner, Amir; Zhou, Yulian; Williams, John; Smallwood, Philip M; Nathans, Jeremy

2012-12-07

Norrin/Frizzled4 (Fz4) signaling activates the canonical Wnt pathway to control retinal vascular development. Using genetically engineered mice, we show that precocious Norrin production leads to premature retinal vascular invasion and delayed Norrin production leads to characteristic defects in intraretinal vascular architecture. In genetic mosaics, wild-type endothelial cells (ECs) instruct neighboring Fz4(-/-) ECs to produce an architecturally normal mosaic vasculature, a cell nonautonomous effect. However, over the ensuing weeks, Fz4(-/-) ECs are selectively eliminated from the mosaic vasculature, implying the existence of a quality control program that targets defective ECs. In the adult retina and cerebellum, gain or loss of Norrin/Fz4 signaling results in a cell-autonomous gain or loss, respectively, of blood retina barrier and blood brain barrier function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure. Copyright © 2012 Elsevier Inc. All rights reserved.

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier

PubMed Central

Mooren, Olivia L.; Li, Jinmei; Nawas, Julie; Cooper, John A.

2014-01-01

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells. PMID:25355948

Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

PubMed

Babae, Negar; Bourajjaj, Meriem; Liu, Yijia; Van Beijnum, Judy R; Cerisoli, Francesco; Scaria, Puthupparampil V; Verheul, Mark; Van Berkel, Maaike P; Pieters, Ebel H E; Van Haastert, Rick J; Yousefi, Afrouz; Mastrobattista, Enrico; Storm, Gert; Berezikov, Eugene; Cuppen, Edwin; Woodle, Martin; Schaapveld, Roel Q J; Prevost, Gregoire P; Griffioen, Arjan W; Van Noort, Paula I; Schiffelers, Raymond M

2014-08-30

Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

Overexpression of miR-133 decrease primary endothelial cells proliferation and migration via FGFR1 targeting.

PubMed

Zomorrod, Mina Soufi; Kouhkan, Fatemeh; Soleimani, Masoud; Aliyan, Amir; Tasharrofi, Nooshin

2018-03-30

Angiogenesis is one of the essential hallmarks of cancer that is controlled by the balance between positive and negative regulators. FGFR1 signaling is crucial for the execution of bFGF-induced proliferation, migration, and tube formation of endothelial cells (ECs) and onset of angiogenesis on tumors. The purpose of this study is to identify whether or not miR-133 regulates FGFR1 expression and accordingly hypothesize if it plays a crucial role in modulating bFGF/FGFR1 activity in ECs and blocking tumor angiogenesis through targeting FGFR1. The influences of miR-133 overexpression on bFGF stimulated endothelial cells were assessed by cell growth curve, MTT assaying, tube formation, and migration assays. Forced expression of miR-133 caused significant reductions in bFGF-induced proliferation and migratory ability of ECs. MiR-133 Expression was negatively correlated with both mRNA and protein levels of FGFR1 in the transfected ECs isolated from peripheral blood. Moreover, overexpression of miR-133 drastically reduced the rate of cell division and disturbed capillary network formation of transfected ECs. These findings suggest that miR-133 plays an important function in bFGF-induced angiogenesis processes in ECs and provides a rationale for new therapeutic approaches to suppress tumor angiogenesis and cancer. Copyright © 2018. Published by Elsevier Inc.

Interleukin-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

PubMed Central

Liu, Rebecca; Lauridsen, Holly M.; Amezquita, Robert A.; Pierce, Richard W.; Jane-wit, Dan; Fang, Caodi; Pellowe, Amanda S.; Kirkiles-Smith, Nancy C.; Gonzalez, Anjelica L.; Pober, Jordan S.

2016-01-01

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multi-step process that involves sequential cell-cell interactions of circulating leukocytes with interleukin (IL)-1- or tumor necrosis factor-α (TNF)-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a pro-inflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, while neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA-seq analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media (CM) from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and also stimulate neutrophil production of pro-inflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17-activated PCs but not ECs can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondria outer membrane permeabilization and caspase 9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by CM from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space. PMID:27534549

Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

PubMed Central

Huang, Ngan F.; Fleissner, Felix; Sun, John

2010-01-01

Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (l-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, l-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of l-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of l-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by l-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function. PMID:20064011

Cathepsin L is required for endothelial progenitor cell-induced neovascularization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra

Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sitesmore » of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.« less

c-Met–mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma

PubMed Central

Huang, Menggui; Liu, Tianrun; Ma, Peihong; Mitteer, R. Alan; Zhang, Zhenting; Kim, Hyun Jun; Yeo, Eujin; Zhang, Duo; Cai, Peiqiang; Li, Chunsheng; Zhang, Lin; Zhao, Botao; Roccograndi, Laura; O’Rourke, Donald M.; Dahmane, Nadia; Gong, Yanqing; Koumenis, Constantinos

2016-01-01

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase–14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor–derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors. PMID:27043280

Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

PubMed

Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

2016-11-01

Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

Tailoring of TiO2 films by H2SO4 treatment and UV irradiation to improve anticoagulant ability and endothelial cell compatibility.

PubMed

Liao, Yuzhen; Li, Linhua; Chen, Jiang; Yang, Ping; Zhao, Ansha; Sun, Hong; Huang, Nan

2017-07-01

Surfaces with dual functions that simultaneously exhibit good anticoagulant ability and endothelial cell (EC) compatibility are desirable for blood contact materials. However, these dual functions have rarely been achieved by inorganic materials. In this study, titanium dioxide (TiO 2 ) films were treated by sulphuric acid (H 2 SO 4 ) and ultraviolet (UV) irradiation successively (TiO 2 H 2 SO 4 -UV), resulting in good anticoagulant ability and EC compatibility simultaneously. We found that UV irradiation improved the anticoagulant ability of TiO 2 films significantly while enhancing EC compatibility, though not significantly. The enhanced anticoagulant ability could be related to the oxidation of surface-adsorbed hydrocarbons and increased hydrophilicity. The H 2 SO 4 treatment improved the anticoagulant ability of TiO 2 films slightly, while UV irradiation improved the anticoagulant ability strongly. The enhanced EC compatibility could be related to the increased surface roughness and positive charges on the surface of the TiO 2 films. Furthermore, the time-dependent degradation of the enhanced EC compatibility and anticoagulant ability of TiO 2 H 2 SO 4 -UV was observed. In summary, TiO 2 H 2 SO 4 -UV expressed both excellent anticoagulant ability and good EC compatibility at the same time, which could be desirable for blood contact materials. However, the compatibility of TiO 2 H 2 SO 4 -UV with smooth muscle cells (SMCs) and macrophages was also improved. More effort is still needed to selectively improve EC compatibility on TiO 2 films for better re-endothelialization. Copyright © 2017 Elsevier B.V. All rights reserved.

Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16INK4a during immortalization

PubMed Central

Kan, C-Y; Wen, V W; Pasquier, E; Jankowski, K; Chang, M; Richards, L A; Kavallaris, M; MacKenzie, K L

2012-01-01

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16INK4a and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16INK4a on EC function and to identify molecular changes associated with repression of p16INK4a. In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16INK4a showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16INK4a-negative and a p16INK4a-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16INK4a expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16INK4a expression. This corresponded with aberrant cytoskeletal architectures among p16INK4a-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16INK4a-positive cells. A direct link between p16INK4a repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16INK4a. siRNA-mediated repression of p16INK4a significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16INK4a are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies. PMID:22310292

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

PubMed

Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

2018-01-01

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

Functional interplay between endothelial nitric oxide synthase and membrane type 1–matrix metalloproteinase in migrating endothelial cells

PubMed Central

Genís, Laura; Gonzalo, Pilar; Tutor, Antonio S.; Gálvez, Beatriz G.; Martínez-Ruiz, Antonio; Zaragoza, Carlos; Lamas, Santiago; Tryggvason, Karl; Apte, Suneel S.

2007-01-01

Nitric oxide (NO) is essential for vascular homeostasis and is also a critical modulator of angiogenesis; however, the molecular mechanisms of NO action during angiogenesis remain elusive. We have investigated the potential relationship between NO and membrane type 1–matrix metalloproteinase (MT1-MMP) during endothelial migration and capillary tube formation. Endothelial NO synthase (eNOS) colocalizes with MT1-MMP at motility-associated structures in migratory human endothelial cells (ECs); moreover, NO is produced at these structures and is released into the medium during EC migration. We have therefore addressed 2 questions: (1) the putative regulation of MT1-MMP by NO in migratory ECs; and (2) the requirement for MT1-MMP in NO-induced EC migration and tube formation. NO upregulates MT1-MMP membrane clustering on migratory human ECs, and this is accompanied by increased degradation of type I collagen substrate. MT1-MMP membrane expression and localization are impaired in lung ECs from eNOS-deficient mice, and these cells also show impaired migration and tube formation in vitro. Inhibition of MT1-MMP with a neutralizing antibody impairs NOinduced tube formation by human ECs, and NO-induced endothelial migration and tube formation are impaired in lung ECs from mice deficient in MT1-MMP. MT1-MMP thus appears to be a key molecular effector of NO during the EC migration and angiogenic processes, and is a potential therapeutic target for NO-associated vascular disorders. PMID:17606763

The impact of simulated microgravity on purinergic signaling in an endothelial and smooth muscle cell co-culture model

NASA Astrophysics Data System (ADS)

Zhang, Yu; Hemmersbach, Ruth; Lau, Patrick; Pansky, Andreas; Kassack, Matthias; Tobiasch, Edda

Astronauts suffer from cardiovascular deconditioning when they are exposed to microgravity conditions during space missions. Thus, current research focuses on the identification of the underlying mechanism also with respect to therapy and countermeasures. Endothelial cells (ECs) and smooth muscle cells (SMCs) play a key role in a variety of vascular functions. Gene expression, cytoskeleton morphology and apoptosis in both, ECs and SMCs, have shown alterations under simulated and real microgravity condition. However, all these data were observed during single culturing of either ECs or SMCs under microgravity conditions, which is different from the in vivo situation. Purinergic 2 (P2) receptors bind extracellular nucleotides and can regulate the vascular tone and vascular cell proliferation, migration and apoptosis. In this study primary ECs and SMCs were obtained from bovine aorta and characterized using specific markers. Here we show for the first time that the P2-receptor expressions pattern in ECs and in SMCs is altered after 24h in simulated microgravity. Specific receptors are down- or up-regulated on the gene and protein level. In addition the supernatant of ECs during culture was used as conditioned medium for SMCs and vice visa to investigate the influence of either cell type on the other. ECs and SMCs secret cytokines which induce pathogenic proliferation and an altered migration behavior under simulated microgravity conditions. Interestingly, co-culturing with condition medium could compensate this change. In detail, P2X7 was down-regulated in ECs after 24h clinorotation but recovered to the 1 g level when cultured with conditioned medium from SMCs collected under normal gravity. In conclusion, our data indicate that the paracrine effect between ECs and SMCs is an important regulator of cell behavior, also under altered gravity conditions. P2-receptor gene and protein expression were altered during microgravity. Since several P2-receptor artificial ligands are already established as drugs, P2-receptors might be a reasonable candidate for drug development for astronaut treatment of vascular deconditioning in the future. Keywords: simulated microgravity, purinergic signaling, endothelial cells, smooth muscle cells, co-culture, clinostat

NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology

PubMed Central

Ushio-Fukai, Masuko; Malik, Asrar B.

2009-01-01

Abstract Reactive oxygen species (ROS) including superoxide (O2·−) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, “oxidant signaling,” has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791–810. PMID:18783313

Genomic organization and chromosomal localization of the gene TCF15 encoding the early mesodermal basic helix-loop-helix factor bHLH-EC2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hidai, H.; Quertermous, E.E.; Quertermous, T.

1995-12-10

bHLH-EC2 is a recently characterized member of a growing family of basic helix-loop-helix transcription factors. This family includes bHLH factors such as twist, which appear to be primarily involved in early mesodermal differentiation, and bHLH factors such as TAL-1, which have been characterized through their association with chromosomal breakpoints associated with T-cell leukemias. To provide for studies aimed at understanding the genetic regulation of bHLH-EC2, we have characterized the organization of this gene and conducted preliminary studies of the transcriptional activity of the upstream promoter region. The mouse bHLH-EC2 gene was found to consist of two exons separated by amore » 5-kb intron, an organization pattern similar to the mouse twist gene. The transcription initiation site was identified by RNase protection assay and primer extension analysis. Linked promoter-reporter gene transfection experiments in cultured cells indicated that while the identified upstream sequence can function to promote transcription, it does not function in a cell-specific fashion. To investigate the possible association of bHLH-EC2 with hematological malignancy, the chromosomal location of this gene in the human was mapped by fluorescence in situ hybridization and assigned to chromosome band 20p13. 16 refs., 3 figs.« less

Roles of endothelial A-type lamins in migration of T cells on and under endothelial layers

NASA Astrophysics Data System (ADS)

Song, Kwang Hoon; Lee, Jaehyun; Park, Hyoungjun; Kim, Hye Mi; Park, Jeehun; Kwon, Keon Woo; Doh, Junsang

2016-03-01

Stiff nuclei in cell-dense microenvironments may serve as distinct biomechanical cues for cell migration, but such a possibility has not been tested experimentally. As a first step addressing this question, we altered nuclear stiffness of endothelial cells (ECs) by reducing the expression of A-type lamins using siRNA, and investigated the migration of T cells on and under EC layers. While most T cells crawling on control EC layers avoided crossing over EC nuclei, a significantly higher fraction of T cells on EC layers with reduced expression of A-type lamins crossed over EC nuclei. This result suggests that stiff EC nuclei underlying T cells may serve as “duro-repulsive” cues to direct T cell migration toward less stiff EC cytoplasm. During subendothelial migration under EC layers with reduced expression of A-type lamins, T cells made prolonged contact and substantially deformed EC nuclei, resulting in reduced speed and directional persistence. This result suggests that EC nuclear stiffness promotes fast and directionally persistent subendothelial migration of T cells by allowing minimum interaction between T cells and EC nuclei.

Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

PubMed Central

Chen, Yanling; Reese, David H.

2016-01-01

Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), which controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. The surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of Corexit-EC9527A in the cell. PMID:27684493

ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

PubMed

Abbruzzese, Genevieve; Becker, Sarah F; Kashef, Jubin; Alfandari, Dominique

2016-07-15

The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. Copyright © 2015. Published by Elsevier Inc.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

Engineered myocardium model to study the roles of HIF-1α and HIF1A-AS1 in paracrine-only signaling under pathological level oxidative stress.

PubMed

Acun, Aylin; Zorlutuna, Pinar

2017-08-01

Studying heart tissue is critical for understanding and developing treatments for cardiovascular diseases. In this work, we fabricated precisely controlled and biomimetic engineered model tissues to study how cell-cell and cell-matrix interactions influence myocardial cell survival upon exposure to pathological level oxidative stress. Specifically, the interactions of endothelial cells (ECs) and cardiomyocytes (CMs), and the role of hypoxia inducible factor-1α (HIF-1α), with its novel alternative regulator, HIF-1α antisense RNA1 (HIF1A-AS1), in these interactions were investigated. We encapsulated CMs in photo-crosslinkable, biomimetic hydrogels with or without ECs, then exposed to oxidative stress followed by normoxia. With precisely controlled microenvironment provided by the model tissues, cell-cell interactions were restricted to be solely through the secreted factors. CM survival after oxidative stress was significantly improved, in the presence of ECs, when cells were in the model tissues that were functionalized with cell attachment motifs. Importantly, the cardioprotective effect of ECs was reduced when HIF-1α expression was knocked down suggesting that HIF-1α is involved in cardioprotection from oxidative damage, provided through secreted factors conferred by the ECs. Using model tissues, we showed that cell survival increased with increased cell-cell communication and enhanced cell-matrix interactions. In addition, whole genome transcriptome analysis showed, for the first time to our knowledge, a possible role for HIF1A-AS1 in oxidative regulation of HIF-1α. We showed that although HIF1A-AS1 knockdown helps CM survival, its effect is overridden by CM-EC bidirectional interactions as we showed that the conditioned media taken from the CM-EC co-cultures improved CM survival, regardless of HIF1A-AS1 expression. Cardiovascular diseases, most of which are associated with oxidative stress, is the most common cause of death worldwide. Thus, understanding the molecular events as well as the role of intercellular communication under oxidative stress is upmost importance in its prevention. In this study we used 3D engineered tissue models to investigate the role of HIF-1α and its regulation in EC-mediated cardioprotection. We showed that EC-mediated protection is only possible when there is a bidirectional crosstalk between ECs and CMs even without physical cell-cell contact. In addition, this protective effect is at least partially related to cell-ECM interactions and HIF-1α, which is regulated by HIF1A-AS1 under oxidative stress. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Attenuation of tumor necrosis factor-induced endothelial cell cytotoxicity and neutrophil chemiluminescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, H.; Crowley, J.J.; Chan, J.C.

Our laboratory has previously shown that the administration of tumor necrosis factor (TNF), a cytokine produced by activated mononuclear cells, to guinea pigs produces a syndrome similar to gram-negative sepsis or ARDS. Pentoxifylline (PTX), a methylxanthine, protects against TNF-induced and sepsis-induced acute lung injury in vivo. We now report on in vitro cellular studies of PMN-mediated cellular injury and its attenuation. We studied TNF-induced bovine pulmonary artery endothelial cell (EC) cytotoxicity both with and without PMN. A 51Cr release assay was used to measure EC damage. Further, we investigated PMN function in response to TNF by measuring chemiluminescence. Agents thatmore » attenuate EC damage and PMN activation were evaluated in the above assays. Results revealed that TNF causes EC injury (p less than 0.05) and PMN increase TNF-induced EC injury. Furthermore, PTX, aminophylline (AMPH), caffeine, and forskolin attenuate TNF-induced EC cytotoxicity only in the presence of PMN (p less than 0.05). Of interest, dibutyryl cAMP (DBcAMP) protects EC from TNF-induced injury both with and without PMN. Agents that may increase cAMP levels in PMN (PTX, DBcAMP, forskolin, isobutyl methylxanthine, and terbutaline) significantly attenuate TNF-induced PMN chemiluminescence (p less than 0.05). We conclude that TNF causes EC damage and PMN increase this damage. Furthermore, PTX, AMPH, caffeine, and forskolin can attenuate TNF-induced EC injury in the presence of PMN, whereas DBcAMP attenuates TNF-induced EC injury with and without PMN. In addition, agents that may increase intracellular cAMP levels in PMN can attenuate TNF-induced PMN chemiluminescence. Thus, these agents likely attenuate TNF-induced PMN-mediated EC injury through their inhibitory effects on PMN.« less

Human iPSC-derived myocardium-on-chip with capillary-like flow for personalized medicine.

PubMed

Ellis, Bradley W; Acun, Aylin; Can, U Isik; Zorlutuna, Pinar

2017-03-01

The heart wall tissue, or the myocardium, is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally, current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells, lack of a 3D biomimetic environment, and the use of non-human cells. In this study, we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels, to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining, genetic, and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality, and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment, and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days, and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line, essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner.

Human iPSC-derived myocardium-on-chip with capillary-like flow for personalized medicine

PubMed Central

Ellis, Bradley W.; Acun, Aylin; Can, U. Isik; Zorlutuna, Pinar

2017-01-01

The heart wall tissue, or the myocardium, is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally, current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells, lack of a 3D biomimetic environment, and the use of non-human cells. In this study, we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels, to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining, genetic, and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality, and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment, and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days, and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line, essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner. PMID:28396709

Extracellular Superoxide Dismutase Enhances Recruitment of Immature Neutrophils to the Liver

PubMed Central

Break, Timothy J.; Witter, Alexandra R.; Indramohan, Mohanalaxmi; Mummert, Mark E.; Dory, Ladislav

2016-01-01

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes spontaneous abortion in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals. Although L. monocytogenes can usually be effectively treated with antibiotics, there is still around a 25% mortality rate with individuals who develop clinical listeriosis. Neutrophils are innate immune cells required for the clearance of pathogenic organisms, including L. monocytogenes. The diverse roles of neutrophils during both infectious and noninfectious inflammation have recently gained much attention. However, the impact of reactive oxygen species, and the enzymes that control their production, on neutrophil recruitment and function is not well understood. Using congenic mice with varying levels of extracellular superoxide dismutase (ecSOD) activity, we have recently shown that the presence of ecSOD decreases clearance of L. monocytogenes while increasing the recruitment of neutrophils that are not protective in the liver. The data presented here show that ecSOD activity does not lead to a cell-intrinsic increase in neutrophil-homing potential or a decrease in protection against L. monocytogenes. Instead, ecSOD activity enhances the production of neutrophil-attracting factors and protects hyaluronic acid (HA) from damage. Furthermore, neutrophils from the livers of ecSOD-expressing mice have decreased intracellular and surface-bound myeloperoxidase, are less capable of killing phagocytosed L. monocytogenes, and have decreased oxidative burst. Collectively, our data reveal that ecSOD activity modulates neutrophil recruitment and function in a cell-extrinsic fashion, highlighting the importance of the enzyme in protecting tissues from oxidative damage. PMID:27600509

Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting

PubMed Central

Davis, George E.; Stratman, Amber N.; Sacharidou, Anastasia; Koh, Wonshill

2013-01-01

Many studies reveal a fundamental role for extracellular matrix-mediated signaling through integrins and Rho GTPases as well as matrix metalloproteinases (MMPs) in the molecular control of vascular tube morphogenesis in three-dimensional (3D) tissue environments. Recent work has defined an EC lumen signaling complex of proteins that controls these vascular morphogenic events. These findings reveal a signaling interdependence between Cdc42 and MT1-MMP to control the 3D matrix-specific process of EC tubulogenesis. The EC tube formation process results in the creation of a network of proteolytically-generated vascular guidance tunnels in 3D matrices that are utilized to remodel EC-lined tubes through EC motility and could facilitate processes such as flow-induced remodeling and arteriovenous EC sorting and differentiation. Within vascular guidance tunnels, key dynamic interactions occur between endothelial cells (ECs) and pericytes to affect vessel remodeling, diameter, and vascular basement membrane matrix assembly, a fundamental process necessary for endothelial tube maturation and stabilization. Thus, the EC lumen and tube formation mechanism coordinates the concomitant establishment of a network of vascular tubes within tunnel spaces to allow for flow responsiveness, EC-mural cell interactions, and vascular extracellular matrix assembly to control the development of the functional microcirculation. PMID:21482411

ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site

PubMed Central

Kashef, Jubin; Alfandari, Dominique

2015-01-01

The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell–cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. PMID:26206614

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis.

PubMed

Poulos, Michael G; Ramalingam, Pradeep; Gutkin, Michael C; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J P; Elemento, Olivier; Levine, Ross L; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B; Shim, Jae-Hyuck; Butler, Jason M

2016-12-21

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

PubMed Central

Poulos, Michael G.; Ramalingam, Pradeep; Gutkin, Michael C.; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J. P.; Elemento, Olivier; Levine, Ross L.; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B.; Shim, Jae-Hyuck; Butler, Jason M.

2016-01-01

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens. PMID:28000664

Engineering Robust and Functional Vascular Networks in Vivo with Human Adult and Cord Blood-Derived Progenitor Cells

DTIC Science & Technology

2008-12-01

for other sources of ECs such as those derived from embryonic and adult progenitor cells ( Rafii ; Lyden 2003). For example, human ES-derived...functional endothelial precursors. Blood, 95, 952-958. Rafii , S., and D. Lyden, 2003: Therapeutic stem and progenitor cell transplantation for

Metformin improves circulating endothelial cells and endothelial progenitor cells in type 1 diabetes: MERIT study.

PubMed

Ahmed, Fahad W; Rider, Rachel; Glanville, Michael; Narayanan, Kilimangalam; Razvi, Salman; Weaver, Jolanta U

2016-08-26

Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45(dim)CD34(+)VEGFR-2(+) and CD45(dim)CD133(-)CD34(+)CD144(+) respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill's colonies). At baseline TG had lower cEPCs, PACs, CFU-Hills' colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill's colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill's colonies with cECs. Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill's colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132.

Cocoa flavonoids protect hepatic cells against high-glucose-induced oxidative stress: relevance of MAPKs.

PubMed

Cordero-Herrera, Isabel; Martín, María Angeles; Goya, Luis; Ramos, Sonia

2015-04-01

Oxidative stress plays a main role in the pathogenesis of type 2 diabetes mellitus. Cocoa and (-)-epicatechin (EC), a main cocoa flavanol, have been suggested to exert beneficial effects in type 2 diabetes mellitus because of their protective effects against oxidative stress and insulin-like properties. In this study, the protective effect of EC and a cocoa phenolic extract (CPE) against oxidative stress induced by a high-glucose challenge, which causes insulin resistance, was investigated on hepatic HepG2 cells. Oxidative status, phosphorylated mitogen-activated protein kinases (MAPKs), nuclear factor E2 related factor 2 (Nrf2) and p-(Ser)-IRS-1 expression, and glucose uptake were evaluated. EC and CPE regulated antioxidant enzymes and activated extracellular-regulated kinase and Nrf2. EC and CPE pre-treatment prevented high-glucose-induced antioxidant defences and p-MAPKs, and maintained Nrf2 stimulation. The presence of selective MAPK inhibitors induced changes in redox status, glucose uptake, p-(Ser)- and total IRS-1 levels that were observed in CPE-mediated protection. EC and CPE recovered redox status of insulin-resistant HepG2 cells, suggesting that the functionality in EC- and CPE-treated cells was protected against high-glucose-induced oxidative insult. CPE beneficial effects on redox balance and insulin resistance were mediated by targeting MAPKs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

PubMed Central

Xiang, Wei; Ke, Zhiyuan; Zhang, Yong; Ho-Yuet Cheng, Grace; Irwan, Ishak Darryl; Sulochana, K N; Potturi, Padma; Wang, Zhengyuan; Yang, He; Wang, Jingyu; Zhuo, Lang; Kini, R Manjunatha; Ge, Ruowen

2011-01-01

Abstract Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen-tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis. PMID:19874420

Posttranscriptional Control of PD-L1 Expression by 17β-Estradiol via PI3K/Akt Signaling Pathway in ERα-Positive Cancer Cell Lines.

PubMed

Yang, Lingyun; Huang, Feng; Mei, Jiandong; Wang, Xun; Zhang, Qiuyang; Wang, Hongjing; Xi, Mingrong; You, Zongbing

2017-02-01

Estrogen is a well-known oncogenic driver in endometrial (ECs) and breast cancers (BCs). Programmed cell death protein 1 (PD-1) and its ligands PD-1 Ligand 1 (PD-L1) and PD-L2 have been shown to mediate immune evasion of the tumor cells. The purpose of the present study was to assess the effects of estrogen on PD-L1 and PD-L2 expression in EC and BC cell lines. 17β-Estradiol (E2)-induced expression of PD-L1 and PD-L2 and possible signaling pathway were investigated in EC and BC cells. Coculture of T cells and cancer cells with E2 stimulation was performed to assess the functions of T cells. We found that E2 increased expression of PD-L1, but not PD-L2, protein via activation of phosphoinositide 3-kinase (PI3K)/Akt pathway in Ishikawa and Michigan Cancer Foundation-7 (MCF-7) cells. Phosphoinositide 3-kinase and Akt inhibitors could block E2's effects. 17β-Estradiol did not increase PD-L1 mRNA transcription, but stabilized PD-L1 mRNA. 17β-Estradiol's effects were only observed in estrogen receptor α (ERα)-positive Ishikawa and MCF-7 cells, but not in ERα-negative MDA-MB-231 cells. Coculture of Ishikawa or MCF-7 cells with T cells inhibited expression of interferon-γ and interleukin-2 and increased BCL-2-interacting mediator of cell death expression in the presence of E2. This study provides the first evidence that estrogen upregulates PD-L1 protein expression in ERα-positive EC and BC cells to suppress immune functions of T cells in the tumor microenvironment, demonstrating a new mechanism of how estrogen drives cancer progression.

Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion.

PubMed

Chan, Bernard P; Reichert, William M; Truskey, George A

2004-08-01

The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.

Mathematical model of depolarization mechanism of conducted vasoreactivity

NASA Astrophysics Data System (ADS)

Neganova, Anastasiia Y.; Stiukhina, Elena S.; Postnov, Dmitry E.

2015-03-01

We address the problem of conducted vasodilation, the phenomenon which is also known as functional hyperemia. Specifically, we test the mechanism of nondecremental propagation of electric signals along endothelial cell layer recently hypothesized by Figueroa et al. By means of functional modeling we focus on possible nonlinear mechanisms that can underlie such regenerative pulse transmission (RPT). Since endothelial cells (EC) are generally known as electrically inexcitable, the possible role of ECs in RPT mechanisms is not evident. By means of mathematical modeling we check the dynamical self-consistency of Figueroa's hypothesis, as well as estimate the possible contribution of specific ionic currents to the suggested RPT mechanism.

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo.

PubMed

Shaye, Daniel D; Greenwald, Iva

2016-11-15

The C. elegans excretory cell (EC) is a powerful model for tubulogenesis, a conserved process that requires precise cytoskeletal regulation. EXC-6, an ortholog of the disease-associated formin INF2, coordinates cell outgrowth and lumen formation during EC tubulogenesis by regulating F-actin at the tip of the growing canal and the dynamics of basolateral microtubules. EXC-6 functions in parallel with EXC-5/FGD, a predicted activator of the Rho GTPase Cdc42. Here, we identify the parallel pathway: EXC-5 functions through CDC-42 to regulate two other formins: INFT-2, another INF2 ortholog, and CYK-1, the sole ortholog of the mammalian diaphanous (mDia) family of formins. We show that INFT-2 promotes F-actin accumulation in the EC, and that CYK-1 inhibits INFT-2 to regulate F-actin levels and EXC-6-promoted outgrowth. As INF2 and mDia physically interact and cross-regulate in cultured cells, our work indicates that a conserved EXC-5-CDC-42 pathway modulates this regulatory interaction and that it is functionally important in vivo during tubulogenesis. © 2016. Published by The Company of Biologists Ltd.

Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism.

PubMed

Qi, Jian Hua; Anand-Apte, Bela

2015-04-01

Tissue inhibitor of metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in ECs in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in ECs expressing KDR (PAE/KDR), but not in ECs expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway.

Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes.

PubMed

Araujo, Thaís L S; Zeidler, Julianna D; Oliveira, Percíllia V S; Dias, Matheus H; Armelin, Hugo A; Laurindo, Francisco R M

2017-02-01

Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions. Copyright © 2016 Elsevier Inc. All rights reserved.

IGF-1 promotes angiogenesis in endothelial cells/adipose-derived stem cells co-culture system with activation of PI3K/Akt signal pathway.

PubMed

Lin, Shiyu; Zhang, Qi; Shao, Xiaoru; Zhang, Tao; Xue, Changyue; Shi, Sirong; Zhao, Dan; Lin, Yunfeng

2017-12-01

The aim of this study was to investigate the role of insulin-like growth factor-1 (IGF-1) and crosstalk between endothelial cells (ECs) and adipose-derived stem cells (ASCs) in the process of angiogenesis. A three-dimensional collagen gel used to culture mouse ASCs and mouse ECs in vitro was established. The effects of angiogenesis after exposure to IGF-1 were observed by confocal laser scanning microscopy. Western blotting and qPCR were performed to elucidate the underlying mechanisms. IGF-1 treatment promoted the formation of vessel-like structures and the recruitment of ASCs in the three-dimensional collagen gel. The angiogenic genes and proteins in ECs were up-regulated by IGF-1 and in co-culture. Similar changes in the genes and in the proteins were detected in ASCs after exposure to IGF-1 and co-culture. p-Akt expression levels were high in ECs and ASCs after exposure to IGF-1 and co-culture. IGF-1 and co-culture between cells facilitate the process of angiogenesis via the PI3-kinase/Akt signalling pathway. In ECs, IGF-1 stimulates the expression of angiogenesis-related growth factors with the activation of the PI3-kinase/Akt signalling pathway. Co-cultured ECs exposed to excess VEGF-A and other angiogenesis-related growth factors para-secreted from ASCs exhibit high expression of angiogenesis-related genes and proteins. In ASCs, IGF-1 induces the recruitment and function of ASCs by up-regulating the expression of PDGFB, MMPs and α-SMA. Crosstalk with ECs further facilitates changes in ASCs. © 2017 John Wiley & Sons Ltd.

Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

PubMed

Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

1995-06-01

We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

Mapping cell-specific functional connections in the mouse brain using ChR2-evoked hemodynamics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Bauer, Adam Q.; Kraft, Andrew; Baxter, Grant A.; Bruchas, Michael; Lee, Jin-Moo; Culver, Joseph P.

2017-02-01

Functional magnetic resonance imaging (fMRI) has transformed our understanding of the brain's functional organization. However, mapping subunits of a functional network using hemoglobin alone presents several disadvantages. Evoked and spontaneous hemodynamic fluctuations reflect ensemble activity from several populations of neurons making it difficult to discern excitatory vs inhibitory network activity. Still, blood-based methods of brain mapping remain powerful because hemoglobin provides endogenous contrast in all mammalian brains. To add greater specificity to hemoglobin assays, we integrated optical intrinsic signal(OIS) imaging with optogenetic stimulation to create an Opto-OIS mapping tool that combines the cell-specificity of optogenetics with label-free, hemoglobin imaging. Before mapping, titrated photostimuli determined which stimulus parameters elicited linear hemodynamic responses in the cortex. Optimized stimuli were then scanned over the left hemisphere to create a set of optogenetically-defined effective connectivity (Opto-EC) maps. For many sites investigated, Opto-EC maps exhibited higher spatial specificity than those determined using spontaneous hemodynamic fluctuations. For example, resting-state functional connectivity (RS-FC) patterns exhibited widespread ipsilateral connectivity while Opto-EC maps contained distinct short- and long-range constellations of ipsilateral connectivity. Further, RS-FC maps were usually symmetric about midline while Opto-EC maps displayed more heterogeneous contralateral homotopic connectivity. Both Opto-EC and RS-FC patterns were compared to mouse connectivity data from the Allen Institute. Unlike RS-FC maps, Thy1-based maps collected in awake, behaving mice closely recapitulated the connectivity structure derived using ex vivo anatomical tracer methods. Opto-OIS mapping could be a powerful tool for understanding cellular and molecular contributions to network dynamics and processing in the mouse brain.

Modulation of Gardos channel activity by cytokines in sickle erythrocytes.

PubMed

Rivera, Alicia; Jarolim, Petr; Brugnara, Carlo

2002-01-01

It has recently been shown that the Gardos channel activity of mouse erythrocytes can be modified by endothelins, suggesting a functional linkage between endothelin receptors and the Gardos channel. Using (86)Rubidium ((86)Rb) influx, effects were estimated of proinflammatory molecules such as platelet activator factor (PAF), endothelin-1 (ET-1), interleukin-10 (IL-10), and regulated on activation normal T cells expressed and secreted (RANTES) on the Gardos channel activity in human normal and sickle red cells. It was found that PAF (EC(50): 15 +/- 7 nM), RANTES (EC(50), 9 +/- 6 ng/mL [1.2 +/- 0.8 nM]), IL-10 (EC(50), 11 +/- 8 ng/mL [204 +/- 148 nM]), and ET-1 (EC(50), 123 +/- 34 nM) induce a significant increase in Gardos channel activity-between 28% and 84%-over the control. In addition, these agents modify the Gardos channel affinity for internal Ca(++) (K(0.5)) by 2- to 6-fold. Biochemical evidence is provided for the presence of ET receptor subtype B in sickle and normal red cells. Furthermore, it was found that ET-1, PAF, RANTES, and IL-10 induce a significant increase in red cell density (P <.05). These data suggest that activation of the Gardos channel is functionally coupled to receptor motifs such as C-X-C (PAF), C-C (RANTES), and ET receptor subtype B. Thus, cell volume regulation or erythrocyte hydration states might be altered by activation of the Gardos channel by cytokines in vivo. The role of these mediators in promoting sickle cell dehydration in vivo is under investigation.

Raised erythrocyte creatine in patients with pulmonary arterial hypertension--evidence for subclinical hemolysis.

PubMed

Fox, Benjamin D; Okumiya, Toshika; Attas-Fox, Liat; Kassirer, Michael; Raviv, Yael; Kramer, Mordechai R

2012-04-01

Pulmonary arterial hypertension (PAH) has been associated with hemolytic conditions such as sickle cell disease but the possible role of hemolysis in the pathogenesis or pathophysiology of other forms of PAH has not been studied. Erythrocyte lifespan is the gold-standard test of hemolysis and may be measured by assaying erythrocyte creatine (EC) levels. EC decreases as the erythrocyte ages, so patients with hemolysis have high EC levels. We measured EC and other parameters of hemolysis in patients with idiopathic and connective tissue associated PAH and normal controls. In patients with PAH (n = 40), EC levels were higher than in controls n = 30 (patients EC 1.72 mcmol/g HgB 95%CI[1.51, 1.96], controls EC 1.05 mcmol/g HgB [0.93, 1.19], p < 0.0001). High levels of EC correlated with worse 6 min walk (r = -0.42, p < 0.0001) and worse functional class (p = 0.002). Other indirect indices of hemolysis (total lactate dehydrogenase, red cell distribution width) were also increased in patients with PAH relative to controls. There is evidence of subclinical hemolysis in patients with PAH, and higher levels of hemolysis are associated with poorer exercise capacity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Blackberry subjected to in vitro gastrointestinal digestion affords protection against Ethyl Carbamate-induced cytotoxicity.

PubMed

Chen, Wei; Xu, Yang; Zhang, Lingxia; Su, Hongming; Zheng, Xiaodong

2016-12-01

Ethyl Carbamate (EC) was detected in many fermented foods. Previous studies indicated that frequent exposure to ethyl carbamate may increase the risk to suffer from cancers. Blackberry is rich in polyphenols and possesses potent antioxidant activity. This study aims to investigate the protective effect of blackberry homogenates produced before (BH) and after in vitro simulated gastrointestinal digestion (BD) on EC-induced toxicity in Caco-2 cells. Our results showed that blackberry homogenates after digestion (BD) was more effective than that before digestion (BH) in ameliorating EC-induced toxicity in Caco-2 cells. Further investigation revealed that BD remarkably attenuated EC-induced toxicity through restoring mitochondrial function, inhibiting glutathione depletion and decreasing overproduction of intracellular reactive oxygen species. Additionally, LC-MS result implied that the better protective capacity of BD may be related to the increased content of two anthocyanins (cyanidin-3-glucoside and cyanidin-3-dioxalyglucoside). Overall, the present study may give implication to prevent EC-induced health problem. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure and Sequence Analyses of Clustered Protocadherins Reveal Antiparallel Interactions that Mediate Homophilic Specificity.

PubMed

Nicoludis, John M; Lau, Sze-Yi; Schärfe, Charlotta P I; Marks, Debora S; Weihofen, Wilhelm A; Gaudet, Rachelle

2015-11-03

Clustered protocadherin (Pcdh) proteins mediate dendritic self-avoidance in neurons via specific homophilic interactions in their extracellular cadherin (EC) domains. We determined crystal structures of EC1-EC3, containing the homophilic specificity-determining region, of two mouse clustered Pcdh isoforms (PcdhγA1 and PcdhγC3) to investigate the nature of the homophilic interaction. Within the crystal lattices, we observe antiparallel interfaces consistent with a role in trans cell-cell contact. Antiparallel dimerization is supported by evolutionary correlations. Two interfaces, located primarily on EC2-EC3, involve distinctive clustered Pcdh structure and sequence motifs, lack predicted glycosylation sites, and contain residues highly conserved in orthologs but not paralogs, pointing toward their biological significance as homophilic interaction interfaces. These two interfaces are similar yet distinct, reflecting a possible difference in interaction architecture between clustered Pcdh subfamilies. These structures initiate a molecular understanding of clustered Pcdh assemblies that are required to produce functional neuronal networks. Copyright © 2015 Elsevier Ltd. All rights reserved.

Wild Raspberry Subjected to Simulated Gastrointestinal Digestion Improves the Protective Capacity against Ethyl Carbamate-Induced Oxidative Damage in Caco-2 Cells

PubMed Central

Chen, Wei; Xu, Yang; Zhang, Lingxia; Li, Ya; Zheng, Xiaodong

2016-01-01

Ethyl carbamate (EC), a probable human carcinogen, occurs widely in many fermented foods. Previous studies indicated that EC-induced cytotoxicity was associated with oxidative stress. Wild raspberries are rich in polyphenolic compounds, which possess potent antioxidant activity. This study was conducted to investigate the protective effect of wild raspberry extracts produced before (RE) and after in vitro simulated gastrointestinal digestion (RD) on EC-induced oxidative damage in Caco-2 cells. Our primary data showed that ethyl carbamate could result in cytotoxicity and genotoxicity in Caco-2 cells and raspberry extract after digestion (RD) may be more effective than that before digestion (RE) in attenuating toxicity caused by ethyl carbamate. Further investigation by fluorescence microscope revealed that RD may significantly ameliorate EC-induced oxidative damage by scavenging the overproduction of intracellular reactive oxygen species (ROS), maintaining mitochondrial function and preventing glutathione (GSH) depletion. In addition, HPLC-ESI-MS results showed that the contents of identified polyphenolic compounds (esculin, kaempferol O-hexoside, and pelargonidin O-hexoside) were remarkably increased after digestion, which might be related to the better protective effect of RD. Overall, our results demonstrated that raspberry extract undergoing simulated gastrointestinal digestion may improve the protective effect against EC-induced oxidative damage in Caco-2 cells. PMID:26788245

Activated ovarian endothelial cells promote early follicular development and survival.

PubMed

Kedem, Alon; Aelion-Brauer, Anate; Guo, Peipei; Wen, Duancheng; Ding, Bi-Sen; Lis, Raphael; Cheng, Du; Sandler, Vladislav M; Rafii, Shahin; Rosenwaks, Zev

2017-09-19

New data suggests that endothelial cells (ECs) elaborate essential "angiocrine factors". The aim of this study is to investigate the role of activated ovarian endothelial cells in early in-vitro follicular development. Mouse ovarian ECs were isolated using magnetic cell sorting or by FACS and cultured in serum free media. After a constitutive activation of the Akt pathway was initiated, early follicles (50-150 um) were mechanically isolated from 8-day-old mice and co-cultured with these activated ovarian endothelial cells (AOEC) (n = 32), gel (n = 24) or within matrigel (n = 27) in serum free media for 14 days. Follicular growth, survival and function were assessed. After 6 passages, flow cytometry showed 93% of cells grown in serum-free culture were VE-cadherin positive, CD-31 positive and CD 45 negative, matching the known EC profile. Beginning on day 4 of culture, we observed significantly higher follicular and oocyte growth rates in follicles co-cultured with AOECs compared with follicles on gel or matrigel. After 14 days of culture, 73% of primary follicles and 83% of secondary follicles co-cultured with AOEC survived, whereas the majority of follicles cultured on gel or matrigel underwent atresia. This is the first report of successful isolation and culture of ovarian ECs. We suggest that co-culture with activated ovarian ECs promotes early follicular development and survival. This model is a novel platform for the in vitro maturation of early follicles and for the future exploration of endothelial-follicular communication. In vitro development of early follicles necessitates a complex interplay of growth factors and signals required for development. Endothelial cells (ECs) may elaborate essential "angiocrine factors" involved in organ regeneration. We demonstrate that co-culture with ovarian ECs enables culture of primary and early secondary mouse ovarian follicles.

CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells *

PubMed Central

Sun, Run-Lu; Huang, Can-Xia; Bao, Jin-Lan; Jiang, Jie-Yu; Zhang, Bo; Zhou, Shu-Xian; Cai, Wei-Bin; Wang, Hong; Wang, Jing-Feng; Zhang, Yu-Ling

2016-01-01

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [3H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD. PMID:27458015

Identification of an endocannabinoid system in the rat pars tuberalis-a possible interface in the hypothalamic-pituitary-adrenal system?

PubMed

Jafarpour, Arsalan; Dehghani, Faramarz; Korf, Horst-Werner

2017-04-01

Endocannabinoids (ECs) are ubiquitous endogenous lipid derivatives and play an important role in intercellular communication either in an autocrine/paracrine or in an endocrine fashion. Recently, an intrinsic EC system has been discovered in the hypophysial pars tuberalis (PT) of hamsters and humans. In hamsters, this EC system is under photoperiodic control and appears to influence the secretion of hormones such as prolactin from the adenohypophysis. We investigate the EC system in the PT of the rat, a frequently used species in endocrine research. By means of immunocytochemistry, enzymes involved in EC biosynthesis, e.g., N-arachidonoyl-phosphatidylethanolamine-phospholipase D (NAPE-PLD) and diacylglycerol lipase α (DAGLα) and enzymes involved in EC degradation, e.g., fatty acid amide hydrolase (FAAH) and cyclooxygenase-2 (COX-2), were demonstrated in PT cells of the rat. Immunoreactions (IR) for FAAH and for the cannabinoid receptor CB 1 were observed in corticotrope cells of the rat adenohypophysis; these cells were identified by antibodies against proopiomelanocortin (POMC) or adrenocorticotrophic hormone (ACTH). In the outer zone of the median eminence, numerous nerve fibers and terminals displayed CB 1 IR. The majority of these were also immunolabeled by an antibody against corticotropin-releasing factor (CRF). These results suggest that the EC system at the hypothalamo-hypophysial interface affects both the CRF-containing nerve fibers and the corticotrope cells in the adenohypophysis. Our data give rise to the hypothesis that, in addition to its well-known role in the reproductive axis, the PT might influence adrenal functions and, thus, the stress response and immune system.

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism

PubMed Central

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-01-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. PMID:26081516

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism.

PubMed

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-09-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE-/- mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Expression and Functional Significance of HtrA1 Loss in Endometrial Cancer

PubMed Central

Mullany, Sally A.; Moslemi-Kebria, Mehdi; Rattan, Ramandeep; Khurana, Ashwani; Clayton, Amy; Ota, Takayo; Mariani, Andrea; Podratz, Karl C.; Chien, Jeremy; Shridhar, Viji

2010-01-01

Purpose The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines. Experimental design Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA-1 knocked down clones. Results Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all 7 papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a 3-4 fold increase in the invasive potential. Exogenous expression of HtrA1 in Ark 1 and Ark 2 cells resulted in 3-4 fold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark 2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark 2 cells. Immunohistochemical (IHC) analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (p=0.016). Conclusions Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. PMID:21098697

The endogenous zinc finger transcription factor, ZNF24, modulates the angiogenic potential of human microvascular endothelial cells

PubMed Central

Jia, Di; Huang, Lan; Bischoff, Joyce; Moses, Marsha A.

2015-01-01

We have previously identified a zinc finger transcription factor, ZNF24 (zinc finger protein 24), as a novel inhibitor of tumor angiogenesis and have demonstrated that ZNF24 exerts this effect by repressing the transcription of VEGF in breast cancer cells. Here we focused on the role of ZNF24 in modulating the angiogenic potential of the endothelial compartment. Knockdown of ZNF24 by siRNA in human primary microvascular endothelial cells (ECs) led to significantly decreased cell migration and invasion compared with control siRNA. ZNF24 knockdown consistently led to significantly impaired VEGF receptor 2 (VEGFR2) signaling and decreased levels of matrix metalloproteinase-2 (MMP-2), with no effect on levels of major regulators of MMP-2 activity such as the tissue inhibitors of metalloproteinases and MMP-14. Moreover, silencing ZNF24 in these cells led to significantly decreased EC proliferation. Quantitative PCR array analyses identified multiple cell cycle regulators as potential ZNF24 downstream targets which may be responsible for the decreased proliferation in ECs. In vivo, knockdown of ZNF24 specifically in microvascular ECs led to significantly decreased formation of functional vascular networks. Taken together, these results demonstrate that ZNF24 plays an essential role in modulating the angiogenic potential of microvascular ECs by regulating the proliferation, migration, and invasion of these cells.— Jia, D., Huang, L., Bischoff, J., Moses, M. A. The endogenous zinc finger transcription factor, ZNF24, modulates the angiogenic potential of human microvascular endothelial cells. PMID:25550468

Inward rectifier potassium (Kir2.1) channels as end-stage boosters of endothelium-dependent vasodilators.

PubMed

Sonkusare, Swapnil K; Dalsgaard, Thomas; Bonev, Adrian D; Nelson, Mark T

2016-06-15

Increase in endothelial cell (EC) calcium activates calcium-sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium-dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium-dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium-dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. Endothelium-dependent vasodilators, such as acetylcholine, increase intracellular Ca(2+) through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca(2+) -sensitive intermediate and small conductance K(+) (IK and SK, respectively) channels. Although strong inward rectifier K(+) (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC-dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC-dependent vasodilatation of resistance-sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC-specific Kir2.1 knockdown (EC-Kir2.1(-/-) ) mice. Elevation of extracellular K(+) to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba(2+) , ML-133) or in the arteries from EC-Kir2.1(-/-) mice. Potassium-induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba(2+) , did not affect currents through TRPV4, IK or SK channels. Endothelial cell-dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC-Kir2.1(-/-) . In angiotensin II-induced hypertension, the Kir channel function was not altered, although the endothelium-dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca(2+) -dependent activation of IK and SK channels. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

DOE PAGES

Chen, Yanling; Reese, David H.; Kelly, Gregory M.

2016-09-29

Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), whichmore » controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. In conclusion, the surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of Corexit-EC9527A in the cell.« less

Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yanling; Reese, David H.; Kelly, Gregory M.

Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), whichmore » controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. In conclusion, the surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of Corexit-EC9527A in the cell.« less

Enteric neuromodulators and mucus discharge in a fish infected with the intestinal helminth Pomphorhynchus laevis.

PubMed

Bosi, Giampaolo; Shinn, Andrew Paul; Giari, Luisa; Sayyaf Dezfuli, Bahram

2015-07-08

In vertebrates, the presence of enteric worms can induce structural changes to the alimentary canal impacting on the neuroendocrine system, altering the proper functioning of the gastrointestinal tract and affecting the occurrence and relative density of endocrine cells (ECs). This account represents the first immunohistochemistry and ultrastructure-based study which documents the intimate relationship between the intestinal mucous cells and ECs in a fish-helminth system, investigating the potential effects of enteric neuromodulators on gut mucus secretion/discharge. A modified dual immunohisto- and histochemical staining technique was applied on intestinal sections from both infected and uninfected fish. Sections were incubated in antisera to a range of neuromodulators (i.e. leu-enkephalin, met-enkephalin, galanin and serotonin) and the glycoconjugate histochemistry of the mucous cells was determined using a subsequent alcian blue - periodic acid Schiff staining step. Dual fluorescent staining on sections prepared for confocal laser scanning microscopy and transmission electron microscopy were also used to document the relationship between ECs and mucous cells. From a total of 26 specimens of Squalius cephalus sampled from the River Paglia, 16 (i.e. 62 %) specimens were found to harbour an infection of the acanthocephalan Pomphorhynchus laevis (average intensity of infection 9.2 ± 0.8 parasites host(-1), mean ± standard error). When acanthocephalans were present, the numbers of mucous cells (most notably those containing acidic or mixed glycoconjugates) and ECs secreting leu-enkephalin, met-enkephalin, galanin, serotonin were significantly higher than those seen on sections from uninfected fish. The relationship between met-enkephalin-like or serotonin-like ECs and lectin DBA positive mucous cells was demonstrated through a dual fluorescent staining. The presence of tight connections and desmosomes between mucous and ECs in transmission electron micrographs provides further evidence of this intimate relationship. The presence of P. laevis induces an increase in the number of enteric ECs that are immunoreactive to leu- and met-enkephalin, galanin, and serotonin anti-sera. The mucous cells hyperplasia and enhanced mucus secretion in the helminth-infected intestines could be elicited by the increase in the number of ECs which release these regulatory substances.

Phase resolved and coherence gated en face reflection imaging of multilayered embryonal carcinoma cells

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Fukami, Tadashi; Iwai, Hidenao; Yamashita, Yutaka

2012-03-01

Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells, undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera. P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers. The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of cell properties including their pluripotency.

Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

PubMed

Rose, Jonathan A; Wanner, Nicholas; Cheong, Hoi I; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V; Erzurum, Serpil; Asosingh, Kewal

2016-01-01

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension

PubMed Central

Rose, Jonathan A.; Wanner, Nicholas; Cheong, Hoi I.; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V.; Erzurum, Serpil; Asosingh, Kewal

2016-01-01

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH. PMID:27270458

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

PubMed

Mooren, Olivia L; Li, Jinmei; Nawas, Julie; Cooper, John A

2014-12-15

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells. © 2014 Mooren et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Impact of Simulated Microgravity on Cytoskeleton and Viscoelastic Properties of Endothelial Cell

NASA Astrophysics Data System (ADS)

Janmaleki, M.; Pachenari, M.; Seyedpour, S. M.; Shahghadami, R.; Sanati-Nezhad, A.

2016-09-01

This study focused on the effects of simulated microgravity (s-μg) on mechanical properties, major cytoskeleton biopolymers, and morphology of endothelial cells (ECs). The structural and functional integrity of ECs are vital to regulate vascular homeostasis and prevent atherosclerosis. Furthermore, these highly gravity sensitive cells play a key role in pathogenesis of many diseases. In this research, impacts of s-μg on mechanical behavior of human umbilical vein endothelial cells were investigated by utilizing a three-dimensional random positioning machine (3D-RPM). Results revealed a considerable drop in cell stiffness and viscosity after 24 hrs of being subjected to weightlessness. Cortical rigidity experienced relatively immediate and significant decline comparing to the stiffness of whole cell body. The cells became rounded in morphology while western blot analysis showed reduction of the main cytoskeletal components. Moreover, fluorescence staining confirmed disorganization of both actin filaments and microtubules (MTs). The results were compared statistically among test and control groups and it was concluded that s-μg led to a significant alteration in mechanical behavior of ECs due to remodeling of cell cytoskeleton.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase C674 promotes ischemia- and hypoxia-induced angiogenesis via coordinated endothelial cell and macrophage function.

PubMed

Mei, Yu; Thompson, Melissa D; Shiraishi, Yasunaga; Cohen, Richard A; Tong, Xiaoyong

2014-11-01

Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its ECs show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and were blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice.

PubMed

Xiang, Wei; Ke, Zhiyuan; Zhang, Yong; Cheng, Grace Ho-Yuet; Irwan, Ishak Darryl; Sulochana, K N; Potturi, Padma; Wang, Zhengyuan; Yang, He; Wang, Jingyu; Zhuo, Lang; Kini, R Manjunatha; Ge, Ruowen

2011-02-01

Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ(5) integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts.

PubMed

Burger, Dylan; Montezano, Augusto C; Nishigaki, Nobuhiro; He, Ying; Carter, Anthony; Touyz, Rhian M

2011-08-01

Circulating microparticles are increased in cardiovascular disease and may themselves promote oxidative stress and inflammation. Molecular mechanisms underlying their formation and signaling are unclear. We investigated the role of reactive oxygen species (ROS), Rho kinase, and lipid rafts in microparticle formation and examined their functional significance in endothelial cells (ECs). Microparticle formation from angiotensin II (Ang II)-stimulated ECs and apolipoprotein E(-/-) mice was assessed by annexin V or by CD144 staining and electron microscopy. Ang II promoted microparticle formation and increased EC O(2)(-) generation and Rho kinase activity. Ang II-stimulated effects were inhibited by irbesartan (Ang II receptor type I blocker) and fasudil (Rho kinase inhibitor). Methyl-β-cyclodextrin and nystatin, which disrupt lipid rafts/caveolae, blocked microparticle release. Functional responses, assessed in microparticle-stimulated ECs, revealed increased O(2)(-) production, enhanced vascular cell adhesion molecule/platelet-EC adhesion molecule expression, and augmented macrophage adhesion. Inhibition of epidermal growth factor receptor blocked the prooxidative and proinflammatory effects of microparticles. In vitro observations were confirmed in apolipoprotein E(-/-) mice, which displayed vascular inflammation and high levels of circulating endothelial microparticles, effects that were reduced by apocynin. We demonstrated direct actions of Ang II on endothelial microparticle release, mediated through NADPH oxidase, ROS, and Rho kinase targeted to lipid rafts. Microparticles themselves stimulated endothelial ROS formation and inflammatory responses. Our findings suggest a feedforward system whereby Ang II promotes EC injury through its own endothelial-derived microparticles.

Increase in endothelial cell Ca2+ in response to mouse cremaster muscle contraction

PubMed Central

Duza, Tasmia; Sarelius, Ingrid H

2004-01-01

We addressed the role of endothelial cells (ECs) in metabolic dilatation of skeletal muscle arterioles in anaesthetized mice in situ. Electrical field stimulation was used to contract the cremaster muscle for 15 s at 30 Hz. Diameter was observed using bright field microscopy. In controls, muscle contraction produced a 15.7 ± 1.5 μm dilatation from a baseline of 17.4 ± 1.6 μm. Endothelial denudation (−EC) via intraluminal perfusion of air abolished this response (1.6 ± 1.2 μm in −EC, P < 0.05), identifying endothelium as the primary vascular cell type initiating the dilatation. To investigate the role of EC Ca2+ in metabolic dilatation, arteriolar ECs were loaded with Fluo-4 AM or BAPTA AM by intraluminal perfusion, after which blood flow was re-established. Ca2+ activity of individual ECs was monitored as a function of change from baseline fluorescence using confocal microscopy. In ECs, whole cell Ca2+ increased (>10%, P < 0.05) during muscle contraction, and localized Ca2+ transients were increased (>20%, P < 0.05) during the first minute after contraction. Chelation of EC Ca2+ abolished the dilatations in response to muscle contraction (1.1 ± 0.7 μm, P < 0.05). Inhibition of P1 purinergic receptors (with xanthine amine congener) did not alter the rate of onset of the dilatation (P > 0.05) but decreased its magnitude immediately post stimulation (7.1 ± 0.9 μm, P < 0.05) and during recovery. These findings demonstrate obligatory roles for endothelium and EC Ca2+ during metabolic dilatation in intact arterioles. Furthermore, they suggest that at least two separate pathways mediate the local response, one of which involves stimulation of endothelial P1 purinergic receptors via endogenous adenosine produced during muscle activity. PMID:14694141

Measuring Leukocyte Adhesion to (Primary) Endothelial Cells after Photon and Charged Particle Exposure with a Dedicated Laminar Flow Chamber

PubMed Central

Erbeldinger, Nadine; Rapp, Felicitas; Ktitareva, Svetlana; Wendel, Philipp; Bothe, Anna S.; Dettmering, Till; Durante, Marco; Friedrich, Thomas; Bertulat, Bianca; Meyer, Stephanie; Cardoso, M. C.; Hehlgans, Stephanie; Rödel, Franz; Fournier, Claudia

2017-01-01

The vascular endothelium interacts with all types of blood cells and is a key modulator of local and systemic inflammatory processes, for example, in the adhesion of blood leukocytes to endothelial cells (EC) and the following extravasation into the injured tissue. The endothelium is constantly exposed to mechanical forces caused by blood flow, and the resulting shear stress is essential for the maintenance of endothelial function. Changes in local hemodynamics are sensed by EC, leading to acute or persistent changes. Therefore, in vitro assessment of EC functionality should include shear stress as an essential parameter. Parallel-plate flow chambers with adjustable shear stress can be used to study EC properties. However, commercially available systems are not suitable for radiation experiments, especially with charged particles, which are increasingly used in radiotherapy of tumors. Therefore, research on charged-particle-induced vascular side effects is needed. In addition, α-particle emitters (e.g., radon) are used to treat inflammatory diseases at low doses. In the present study, we established a flow chamber system, applicable for the investigation of radiation induced changes in the adhesion of lymphocytes to EC as readout for the onset of an inflammatory reaction or the modification of a pre-existing inflammatory state. In this system, primary human EC are cultured under physiological laminar shear stress, subjected to a proinflammatory treatment and/or irradiation with X-rays or charged particles, followed by a coincubation with primary human lymphocytes (peripheral blood lymphocytes (PBL)). Analysis is performed by semiautomated quantification of fluorescent staining in microscopic pictures. First results obtained after irradiation with X-rays or helium ions indicate decreased adhesion of PBL to EC under laminar conditions for both radiation qualities, whereas adhesion of PBL under static conditions is not clearly affected by irradiation. Under static conditions, no radiation-induced changes in surface expression of adhesion molecules and activation of nuclear factor kappa B (NF-κB) signaling were observed after single cell-based high-throughput analysis. In subsequent studies, these investigations will be extended to laminar conditions. PMID:28620384

Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia.

PubMed

Barwinska, Daria; Traktuev, Dmitry O; Merfeld-Clauss, Stephanie; Cook, Todd G; Lu, Hongyan; Petrache, Irina; March, Keith L

2018-06-01

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS-exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856-867. © 2018 AlphaMed Press.

Enhanced Re-Endothelialization of Decellularized Rat Lungs

PubMed Central

Stabler, Collin T.; Caires, Luiz C.; Mondrinos, Mark J.; Marcinkiewicz, Cezary; Lazarovici, Philip; Wolfson, Marla R.

2016-01-01

Decellularized lung tissue has been recognized as a potential platform to engineer whole lung organs suitable for transplantation or for modeling a variety of lung diseases. However, many technical hurdles remain before this potential may be fully realized. Inability to efficiently re-endothelialize the pulmonary vasculature with a functional endothelium appears to be the primary cause of failure of recellularized lung scaffolds in early transplant studies. Here, we present an optimized approach for enhanced re-endothelialization of decellularized rodent lung scaffolds with rat lung microvascular endothelial cells (ECs). This was achieved by adjusting the posture of the lung to a supine position during cell seeding through the pulmonary artery. The supine position allowed for significantly more homogeneous seeding and better cell retention in the apex regions of all lobes than the traditional upright position, especially in the right upper and left lobes. Additionally, the supine position allowed for greater cell retention within large diameter vessels (proximal 100–5000 μm) than the upright position, with little to no difference in the small diameter distal vessels. EC adhesion in the proximal regions of the pulmonary vasculature in the decellularized lung was dependent on the binding of EC integrins, specifically α1β1, α2β1, and α5β1 integrins to, respectively, collagen type-I, type-IV, and fibronectin in the residual extracellular matrix. Following in vitro maturation of the seeded constructs under perfusion culture, the seeded ECs spread along the vascular wall, leading to a partial reestablishment of endothelial barrier function as inferred from a custom-designed leakage assay. Our results suggest that attention to cellular distribution within the whole organ is of paramount importance for restoring proper vascular function. PMID:26935764

Patterning vascular networks in vivo for tissue engineering applications.

PubMed

Chaturvedi, Ritika R; Stevens, Kelly R; Solorzano, Ricardo D; Schwartz, Robert E; Eyckmans, Jeroen; Baranski, Jan D; Stapleton, Sarah Chase; Bhatia, Sangeeta N; Chen, Christopher S

2015-05-01

The ultimate design of functionally therapeutic engineered tissues and organs will rely on our ability to engineer vasculature that can meet tissue-specific metabolic needs. We recently introduced an approach for patterning the formation of functional spatially organized vascular architectures within engineered tissues in vivo. Here, we now explore the design parameters of this approach and how they impact the vascularization of an engineered tissue construct after implantation. We used micropatterning techniques to organize endothelial cells (ECs) into geometrically defined "cords," which in turn acted as a template after implantation for the guided formation of patterned capillaries integrated with the host tissue. We demonstrated that the diameter of the cords before implantation impacts the location and density of the resultant capillary network. Inclusion of mural cells to the vascularization response appears primarily to impact the dynamics of vascularization. We established that clinically relevant endothelial sources such as induced pluripotent stem cell-derived ECs and human microvascular endothelial cells can drive vascularization within this system. Finally, we demonstrated the ability to control the juxtaposition of parenchyma with perfused vasculature by implanting cords containing a mixture of both a parenchymal cell type (hepatocytes) and ECs. These findings define important characteristics that will ultimately impact the design of vasculature structures that meet tissue-specific needs.

Local Oxidative Stress Expansion through Endothelial Cells – A Key Role for Gap Junction Intercellular Communication

PubMed Central

Feine, Ilan; Pinkas, Iddo; Salomon, Yoram; Scherz, Avigdor

2012-01-01

Background Major circulation pathologies are initiated by oxidative insult expansion from a few injured endothelial cells to distal sites; this possibly involves mechanisms that are important to understanding circulation physiology and designing therapeutic management of myocardial pathologies. We tested the hypothesis that a localized oxidative insult of endothelial cells (ECs) propagates through gap junction inter-cellular communication (GJIC). Methodology/Principal Findings Cultures comprising the bEnd.3 cell line, that have been established and recognized as suitable for examining communication among ECs, were used to study the propagation of a localized oxidative insult to remote cells. Spatially confined near infrared illumination of parental or genetically modified bEnd.3 cultures, pretreated with the photosensitizer WST11, generated O2•− and •OH radicals in the illuminated cells. Time-lapse fluorescence microscopy, utilizing various markers, and other methods, were used to monitor the response of non-illuminated bystander and remote cells. Functional GJIC among ECs was shown to be mandatory for oxidative insult propagation, comprising de-novo generation of reactive oxygen and nitrogen species (ROS and RNS, respectively), activation and nuclear translocation of c-Jun N-terminal kinase, followed by massive apoptosis in all bystander cells adjacent to the primarily injured ECs. The oxidative insult propagated through GJIC for many hours, over hundreds of microns from the primary photogeneration site. This wave is shown to be limited by intracellular ROS scavenging, chemical GJIC inhibition or genetic manipulation of connexin 43 (a key component of GJIC). Conclusion/Significance Localized oxidative insults propagate through GJIC between ECs, while stimulating de-novo generation of ROS and RNS in bystander cells, thereby driving the insult's expansion. PMID:22911831

MicroRNA-101 mediates the suppressive effect of laminar shear stress on mTOR expression in vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Kui; Fan, Wendong; Wang, Xing

Highlights: Black-Right-Pointing-Pointer Laminar shear stress upregulates miR-101 expression in vascular endothelial cells. Black-Right-Pointing-Pointer miR-101 represses mTOR expression through a specific 3 Prime UTR binding site. Black-Right-Pointing-Pointer Overexpression of miR-101 inhibits G1/S transition and endothelial cell proliferation. Black-Right-Pointing-Pointer Blockade of miR-101 attenuates the suppressive effect of laminar flow on mTOR expression. -- Abstract: Shear stress associated with blood flow plays an important role in regulating gene expression and cell function in endothelial cells (ECs). MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that negatively regulate the expression of target genes by binding to the mRNA 3 Prime -untranslated region (3 Primemore » UTR) at the posttranscriptional level involved in diverse cellular processes. This study demonstrates that microRNA-101 in response to laminar shear stress (LSS) is involved in the flow regulation of gene expression in ECs. qRT-PCR analysis showed that miR-101 expression was significantly upregulated in human umbilical vein endothelial cells (HUVECs) exposed to 12 dyn/cm{sup 2} laminar shear stress for 12 h. We found that transfection of miR-101 significantly decreased the luciferase activity of plasmid reporter containing the 3 Prime UTR of mammalian target of rapamycin (mTOR) gene. Western analysis revealed that the protein level of mTOR was significantly reduced in ECs transfected with miR-101. Furthermore, miR-101 overexpression induced cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation. Finally, transfection of miR-101 inhibitors attenuated the suppressive effects of LSS on mTOR expression, which identified the efficacy of loss-of-function of miR-101 in laminar flow-treated ECs. In conclusion, we have demonstrated that upregulation of miR-101 in response to LSS contributes to the suppressive effects of LSS on mTOR expression and EC proliferation. These studies advance our understanding of the posttranscriptional mechanisms by which shear stress modulates endothelial homeostasis.« less

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

PubMed Central

Sahara, Makoto; Hansson, Emil M; Wernet, Oliver; Lui, Kathy O; Später, Daniela; Chien, Kenneth R

2014-01-01

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. PMID:24810299

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells.

PubMed

Sun, Run-Lu; Huang, Can-Xia; Bao, Jin-Lan; Jiang, Jie-Yu; Zhang, Bo; Zhou, Shu-Xian; Cai, Wei-Bin; Wang, Hong; Wang, Jing-Feng; Zhang, Yu-Ling

2016-09-09

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [(3)H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The modulation of extracellular superoxide dismutase in the specifically enhanced cellular immune response against secondary challenge of Vibrio splendidus in Pacific oyster (Crassostrea gigas).

PubMed

Liu, Conghui; Zhang, Tao; Wang, Lingling; Wang, Mengqiang; Wang, Weilin; Jia, Zhihao; Jiang, Shuai; Song, Linsheng

2016-10-01

Extracellular superoxide dismutase (EcSOD) is a copper-containing glycoprotein playing an important role in antioxidant defense of living cells exposed to oxidative stress, and also participating in microorganism internalization and cell adhesion in invertebrates. EcSOD from oyster (designated CgEcSOD) had been previously reported to bind lipopolysaccharides (LPS) and act as a bridge molecule in Vibrio splendidus internalization. Its mRNA expression pattern, PAMP binding spectrum and microorganism binding capability were examined in the present study. The mRNA expression of CgEcSOD in hemocytes was significantly up-regulated at the initial phase and decreased sharply at 48 h post V. splendidus stimulation. The recombinant CgEcSOD protein (rCgEcSOD) could bind LPS, PGN and poly (I:C), as well as various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica at the presence of divalent metal ions Cu(2+). After the secondary V. splendidus stimulation, the mRNA and protein of CgEcSOD were both down-regulated significantly. The results collectively indicated that CgEcSOD could not only function in the immune recognition, but also might contribute to the immune priming of oyster by inhibiting the foreign microbe invasion through a specific down-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

PubMed

Garcia, Nahuel A; Moncayo-Arlandi, Javier; Sepulveda, Pilar; Diez-Juan, Antonio

2016-03-01

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Ping, E-mail: fanpinggoodluck@163.com; He, Lan; Pu, Dan

Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertolimore » cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-{gamma}-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-{alpha} induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.« less

EcR expression in the prothoracicotropic hormone-producing neurosecretory cells of the Bombyx mori brain.

PubMed

Hossain, Monwar; Shimizu, Sakiko; Fujiwara, Haruhiko; Sakurai, Sho; Iwami, Masafumi

2006-08-01

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the prothoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secretion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis.

A Contact-Based Method for Differentiation of Human Mesenchymal Stem Cells into an Endothelial Cell-Phenotype.

PubMed

Joddar, Binata; Kumar, Shweta Anil; Kumar, Alok

2018-06-01

Adult stem cells such as mesenchymal stem cells (MSC) are known to possess the ability to augment neovascularization processes and are thus widely popular as an autologous source of progenitor cells. However there is a huge gap in our current knowledge of mechanisms involved in differentiating MSC into endothelial cells (EC), essential for lining engineered blood vessels. To fill up this gap, we attempted to differentiate human MSC into EC, by culturing the former onto chemically fixed layers of EC or its ECM, respectively. We expected direct contact of MSC when cultured atop fixed EC or its ECM, would coax the former to differentiate into EC. Results showed that human MSC cultured atop chemically fixed EC or its ECM using EC-medium showed enhanced expression of CD31, a marker for EC, compared to other cases. Further in all human MSC cultured using EC-medium, typically characteristic cobble stone shaped morphologies were noted in comparison to cells cultured using MSC medium, implying that the differentiated cells were sensitive to soluble VEGF supplementation present in the EC-medium. Results will enhance and affect therapies utilizing autologous MSC as a cell source for generating vascular cells to be used in a variety of tissue engineering applications.

EGF and hydrocortisone as critical factors for the co-culture of adipogenic differentiated ASCs and endothelial cells.

PubMed

Volz, Ann-Cathrin; Huber, Birgit; Schwandt, Alina Maria; Kluger, Petra Juliane

In vitro composed vascularized adipose tissue is and will continue to be in great demand e.g. for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Up to date, the lack of adequate culture conditions, mainly a culture medium, decelerates further achievements. In our study, we evaluated the influence of epidermal growth factor (EGF) and hydrocortisone (HC), often supplemented in endothelial cell (EC) specific media, on the co-culture of adipogenic differentiated adipose-derived stem cells (ASCs) and microvascular endothelial cells (mvECs). In ASCs, EGF and HC are thought to inhibit adipogenic differentiation and have lipolytic activities. Our results showed that in indirect co-culture for 14 days, adipogenic differentiated ASCs further incorporated lipids and partly gained an univacuolar morphology when kept in media with low levels of EGF and HC. In media with high EGF and HC levels, cells did not incorporate further lipids, on the contrary, cells without lipid droplets appeared. Glycerol release, to measure lipolysis, also increased with elevated amounts of EGF and HC in the culture medium. Adipogenic differentiated ASCs were able to release leptin in all setups. MvECs were functional and expressed the cell specific markers, CD31 and von Willebrand factor (vWF), independent of the EGF and HC content as long as further EC specific factors were present. Taken together, our study demonstrates that adipogenic differentiated ASCs can be successfully co-cultured with mvECs in a culture medium containing low or no amounts of EGF and HC, as long as further endothelial cell and adipocyte specific factors are available. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

A fish model for the study of the relationship between neuroendocrine and immune cells in the intestinal epithelium: Silurus glanis infected with a tapeworm.

PubMed

Dezfuli, B Sayyaf; DePasquale, J A; Castaldelli, G; Giari, L; Bosi, G

2017-05-01

Immunohistochemical, immunofluorescence and ultrastructural studies were conducted on a sub-population of 20 wels catfish Silurus glanis from a tributary of the River Po (Northern Italy). Fish were examined for the presence of ecto- and endo-parasites; in the intestine of 5 fish, 11 specimens of cestode Glanitaenia osculata were noted and was the only helminth species encountered. The architecture of intestine and its cellular features were nearly identical in either the uninfected S. glanis or in those harboring G. osculata. Near the site of worm's attachment, mucous cells, several mast cells (MCs), few neutrophils and some endocrine cells (ECs) were found to co-occur within the intestinal epithelium. MCs and neutrophils were abundant also in the submucosa. Immunohistochemical staining revealed that enteric ECs were immunoreactive to met-enkephalin, galanin and serotonin anti-bodies. The numbers of ECs, mucous cells and MCs were significantly higher in infected wels catfish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the biotinylated lectin Sambucus nigra Agglutinin and the rabbit polyclonal anti-met-enkephalin or anti-serotonin, with parallel transmission electron microscopy, showed that ECs often made intimate contact with the mucous cells and epithelial MCs. The presence of numerous MCs in intestinal epithelium shows S. glanis to be an interesting model fish to study processes underlying intestinal inflammation elicited by an enteric worm. Immune cells, ECs and mucous cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions together will be discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhancer of zeste homolog 2 blockade by RNA interference is implicated with inhibited proliferation, invasion and promoted apoptosis in endometrial carcinoma.

PubMed

Wang, Juan; Ai, Zhihong; Chen, Jing; Teng, Yincheng; Zhu, Jieping

2018-06-01

Endometrial carcinoma is the most common gynecological malignancy of the female genital tract worldwide (2012). Enhancer of zeste homolog 2 (EZH2), a critical component of the polycomb repressive complex 2, has been found to be associated with multiple biological processes and is overexpressed in multiple types of cancer. Previous studies have demonstrated that EZH2 is associated with endometrial carcinoma. The present study investigated the expression and biology function of EZH2 in endometrial cancer (EC). It was found that EZH2 levels were markedly increased in endometrial cancer tissues compared with that in adjacent normal tissues. EZH2 was significantly overexpressed in 3 separate endometrial cancer cell lines (Ishikawa, RL95-2 and HEC1-A) when compared with the normal endometrial cell line ESC. Additionally, small interfering RNA was used to investigate the role of EZH2 in endometrial carcinoma cell proliferation, and the results showed that EZH2 knockdown suppressed the proliferation of endometrial carcinoma cells in vitro . Furthermore, EZH2 knockdown induced apoptosis of human EC cells by promoting the expression of pro-apoptosis protein caspase 3, caspase 9, BCL2 associated X and decreasing the expression of anti-apoptosis protein Bcl-2. Finally, the present study demonstrated that EZH2 knockdown suppressed the invasion of EC cells through downregulation of the epithelial-mesenchymal transition. Collectively, these data demonstrate that EZH2 is frequently overexpressed in EC cells and its overexpression is associated with promoting the proliferation and invasion and decreasing the apoptosis of EC cells, suggesting that EZH2 may provide potential therapeutic targets for treatment of endometrial carcinoma.

Serotonin-producing enterochromaffin (EC) cells of gastrointestinal mucosa in dexamethasone-treated rats.

PubMed

Glisić, Radmila; Koko, Vesna; Todorović, Vera; Drndarević, Neda; Cvijić, Gordana

2006-09-11

The aim of our study was to investigate the morphological, immunohistochemical and ultrastructural changes of rat serotonin-producing enterochromaffin (EC) cells of gastrointestinal mucosa in dexamethasone-treated rats (D). After 12-daily intraperitoneal administration of 2 mg/kg dexamethasone, rats developed diabetes similar to human diabetes type 2. Stomach, small and large intestines were examined. Large serotonin positive EC cells appeared in the corpus mucosa epithelium of D group of rats, although these cells were not present in control (C) rats. Both volume fraction and the number of EC cells per mm(2) of mucosa were significantly increased only in the duodenum. However, the number of EC cells per circular sections of both antrum and small intestine was increased, but reduced both in the ascending and descending colon in D group. The dexamethasone treatment caused a strong reduction in number of granules in the antral EC cells, while it was gradually increased beginning from the jejunum to descending colon. The mean granular content was reduced in the antral EC cells but increased in the jejunal EC cells in D group. In conclusion, the present study showed that morphological changes in gut serotonin-producing EC cells occurred in diabetic rats.

Shiga Toxin 1 Induces on Lipopolysaccharide-Treated Astrocytes the Release of Tumor Necrosis Factor-alpha that Alter Brain-Like Endothelium Integrity

PubMed Central

Landoni, Verónica I.; Schierloh, Pablo; de Campos Nebel, Marcelo; Fernández, Gabriela C.; Calatayud, Cecilia; Lapponi, María J.; Isturiz, Martín A.

2012-01-01

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS. PMID:22479186

A novel culture device for the evaluation of three-dimensional extracellular matrix materials.

PubMed

Akhyari, Payam; Ziegler, Heiko; Gwanmesia, Patricia; Barth, Mareike; Schilp, Soeren; Huelsmann, Joern; Hoffmann, Stefanie; Bosch, Julia; Kögler, Gesine; Lichtenberg, Artur

2014-09-01

Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM. Copyright © 2012 John Wiley & Sons, Ltd.

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

PubMed

Tan, W; Wang, J; Zhou, F; Gao, L; Yin, R; Liu, H; Sukanthanag, A; Wang, G; Mihm, M C; Chen, D-B; Nelson, J S

2017-12-01

Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133 + /CD166 + /EphB1 + /EfnB2 + , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures. © 2017 British Association of Dermatologists.

Beneficial Autoimmunity at Body Surfaces – Immune Surveillance and Rapid Type 2 Immunity Regulate Tissue Homeostasis and Cancer

PubMed Central

Dalessandri, Tim; Strid, Jessica

2014-01-01

Epithelial cells (ECs) line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation, or toxins cause activation of ECs with release of cytokines and chemokines as well as alterations in the expression of cell-surface ligands. Such display of epithelial stress is rapidly sensed by tissue-resident immunocytes, which can directly interact with self-moieties on ECs and initiate both local and systemic immune responses. ECs are thus key drivers of immune surveillance at body surface tissues. However, ECs have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here, we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis. PMID:25101088

Study on chemotherapeutic sensitizing effect of nimotuzumab on different human esophageal squamous carcinoma cells.

PubMed

Yang, Xiaoyu; Ji, Yinghua; Kang, Xiaochun; Chen, Meiling; Kou, Weizheng; Jin, Cailing; Lu, Ping

2016-02-01

Esophageal cancer is one of the leading causes of mortality worldwide. Although, surgery, radio- and chemotherapy are used to treat the disease, the identification of new drugs is crucial to increase the curative effect. The aim of the present study was to examine the chemotherapeutic sensitizing effect of nimotuzumab (h-R3) and cisplatin cytotoxic drugs cisplatin (DDP) and 5-fluorouracil (5-FU) on esophageal carcinoma cells with two different epidermal growth factor receptor (EGFR) expressions. The expression of EGFR was detected in the human EC1 or EC9706 esophageal squamous cell carcinoma cell line using immunohistochemistry. The inhibitory effect of DDP and 5-FU alone or combined with h-R3 on EC1 or EC9706 cell proliferation was detected using an MTT assay. Flow cytometry and the TUNEL assay were used to determine the effect of single or combined drug treatment on cell apoptosis. The results showed that the expression of EGFR was low in EC1 cells but high in EC9706 cells. The inhibitory effect of the single use of h-R3 on EC1 or EC9706 cell proliferation was decreased. The inhibitory effect between single use of h-R3 alone and combined use of the chemotherapy drugs showed no statistically significant difference (P>0.05) on the EC1 cell growth rate, but showed a statistically significant difference (a=0.05) on EC9706 cell growth rate. The results detected by flow cytometry and TUNEL assay showed that the difference between single use of h-R3 alone and the control group was statistically significant with regard to the EC1 apoptosis rate effect (P<0.05), but not statistically significant for EC9706 (P>0.05). However, statistically significant differences were identified in the apoptotic rate of EC9706 cells between the h-R3 combined chemotherapy group and single chemotherapy group (P<0.05), but not on in the EC1 chemotherapy group (P>0.05). In conclusion, the sensitization effect of h-R3 on chemotherapy drugs is associated with the expression level of EGFR in EC1 or EC9706 cells. The cell killing effect of the combined use of h-R3 with DDP and 5-FU showed no obvious synergistic effect compared to the single-drug group, but only an additive effect.

Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

PubMed Central

Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

2016-01-01

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells

PubMed Central

Wang, Chen; Yi, Tai; Qin, Lingfeng; Maldonado, Roberto A.; von Andrian, Ulrich H.; Kulkarni, Sanjay; Tellides, George; Pober, Jordan S.

2013-01-01

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4+ memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4+ memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4+CD25hiCD127loFoxP3+ cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection. PMID:23478407

Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells.

PubMed

Lefranc, G; Chung, Y T; Barrière, P; Pradal, G

1980-01-01

The thiocarbohydrazide-silver proteinate (TCH SP) method was applied to the study of cat, rabbit and mouse gastric mucosa endocrine cells. After 24-h treatment with thiocarbohydrazide (TCH), glycogen was seen in the hyaloplasm of X, D, P, A and O cells but not in EC, EC-like or D1 cells. With flotation times as short as 30 to 40 min glycogen was readily detected in X cells. Secretory granules of EC cells were constantly stained, while those of D1 cells failed to react. In most experiments granules of X, A and O cells showed peripheral "staining", while in others staining of variable intensity affected the entire granular cross-section in X, D and P cells. With 72-h exposure to TCH, EC and EC-like cells showed particles resembling glycogen, even staining or only peripheral staining of certain EC cell granules. From the results of this and previous studies, EC cell staining is believed to be due wholly or partly, according to exposure times, to the action of silver proteinate, while that of certain non-EC cells is probably a specific indicator of complexed carbohydrates.

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

PubMed Central

Huang, Xianliang; Shen, Yang; Zhang, Yi; Wei, Lin; Lai, Yi; Wu, Jiang; Liu, Xiaojing; Liu, Xiaoheng

2013-01-01

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm2). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm2) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases. PMID:24430179

Tc-99m Ethylenedicysteine and Tc-99m Dimercaptosuccinic Acid Scintigraphy-Comparison of the Two for Detection of Scarring and Differential Cortical Function.

PubMed

Dharmalingam, Anitha; Pawar, Shwetal U; Parelkar, Sandesh V; Shetye, Suruchi S; Ghorpade, Mangala K; Tilve, Gundu H

2017-01-01

The differential cortical function obtained by Tc-99m EC is comparable to that of Tc-99m DMSA. However, identification of scars on Tc-99m EC images needs to be studied. The aim of the study is to evaluate role of Tc-99m EC for detection of scarring and differential cortical function by comparing with Tc-99m DMSA. Prospective observational study of recurrent UTI; minimum 6 weeks after acute episode; when urine examination is negative for pus cells. Forty-seven children with normal positioned kidneys underwent Tc-99m EC and DMSA scintigraphy. The DRF and cortical phase images of both studies in the same image matrix size were evaluated by two independent observers for scarring; Tc-99m DMSA was considered as the gold standard. MS Excel 2007 and GraphPad Instat V3.1 and ROC analysis. There was no significant difference in the detection of scarring using two studies with Cohen's kappa coefficient (κ) 0.932. The sensitivity and specificity of Tc-99m EC for detection of scarring was 98.75% and 99.15%, respectively. There was good agreement between the differential cortical function calculated using two studies. The summed Tc-99m EC images with an acceptable high image contrast allow detection of cortical scarring in patients with normal kidney positions. It is an excellent single-modality comprehensive investigational agent for renal parenchymal defects, function, and excretion evaluation with the added advantages of lower cost, convenience, and low radiation exposure to the child.

Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

NASA Technical Reports Server (NTRS)

Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

1987-01-01

Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

Generating induced pluripotent stem cell derived endothelial cells and induced endothelial cells for cardiovascular disease modelling and therapeutic angiogenesis.

PubMed

Clayton, Z E; Sadeghipour, S; Patel, S

2015-10-15

Standard therapy for atherosclerotic coronary and peripheral arterial disease is insufficient in a significant number of patients because extensive disease often precludes effective revascularization. Stem cell therapy holds promise as a supplementary treatment for these patients, as pre-clinical and clinical research has shown transplanted cells can promote angiogenesis via direct and paracrine mechanisms. Induced pluripotent stem cells (iPSCs) are a novel cell type obtained by reprogramming somatic cells using exogenous transcription factor cocktails, which have been introduced to somatic cells via viral or plasmid constructs, modified mRNA or small molecules. IPSCs are now being used in disease modelling and drug testing and are undergoing their first clinical trial, but despite recent advances, the inefficiency of the reprogramming process remains a major limitation, as does the lack of consensus regarding the optimum transcription factor combination and delivery method and the uncertainty surrounding the genetic and epigenetic stability of iPSCs. IPSCs have been successfully differentiated into vascular endothelial cells (iPSC-ECs) and, more recently, induced endothelial cells (iECs) have also been generated by direct differentiation, which bypasses the pluripotent intermediate. IPSC-ECs and iECs demonstrate endothelial functionality in vitro and have been shown to promote neovessel growth and enhance blood flow recovery in animal models of myocardial infarction and peripheral arterial disease. Challenges remain in optimising the efficiency, safety and fidelity of the reprogramming and endothelial differentiation processes and establishing protocols for large-scale production of clinical-grade, patient-derived cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically

PubMed Central

Szulcek, Robert; van Bezu, Jan; Boonstra, Johannes; van Loon, Jack J. W. A.; van Nieuw Amerongen, Geerten P.

2015-01-01

Background Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to ‘classical’ biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied. Methods In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces. Results Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011). Conclusion In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion. PMID:26637177

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression

PubMed Central

Jacobs, Evan S.; Abdel-Mohsen, Mohamed; Gibb, Stuart L.; Heitman, John W.; Inglis, Heather C.; Martin, Jeffrey N.; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C.; Young, Mary; Greenblatt, Ruth M.; Landay, Alan L.; Gange, Stephen J.; Deeks, Steven G.; Golub, Elizabeth T.; Pillai, Satish K.

2017-01-01

ABSTRACT A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies. IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells. PMID:28053103

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression.

PubMed

Jacobs, Evan S; Keating, Sheila M; Abdel-Mohsen, Mohamed; Gibb, Stuart L; Heitman, John W; Inglis, Heather C; Martin, Jeffrey N; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C; Young, Mary; Greenblatt, Ruth M; Landay, Alan L; Gange, Stephen J; Deeks, Steven G; Golub, Elizabeth T; Pillai, Satish K; Norris, Philip J

2017-03-15

A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4 + T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4 + T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4 + T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies. IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4 + T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells. Copyright © 2017 American Society for Microbiology.

Rapamycin inhibits the proliferation of endothelial cells in hemangioma by blocking the mTOR-FABP4 pathway.

PubMed

Wang, Ying; Chen, Jiarui; Tang, Weiqing; Zhang, Yanping; Li, Xiaoyan

2017-01-01

FABP4 is widely expressed in both normal and pathologic tissues. It promotes cell proliferation, survival and migration of endothelial cells, and therefore, angiogenesis. However, the role of FABP4 in hemangioma or hemangioma endothelial cells (HemECs) has not been explored. In this study, we investigated whether FABP4 directly regulates the proliferation of HemECs. The expression of cell cycle checkpoint genes was analyzed with the microarray data of human dermal microvascular endothelial cells (HDVECs) and infantile hemangioma endothelial cells. Real-time RT-PCR and western blotting were used to examine the expression of FABP4 in HemECs. Next, the FABP4 expression was inhibited in HemECs using siRNA or rapamycin and upregulated using retroviral transduction of HemECs to assess its influence on proliferation of HemECs. The microarray data showed that cell cycle checkpoint genes were upregulated in HemECs. Moreover, HemECs showed significantly higher proliferation rates than HDVECs. The expression of FABP4 and mTOR was increased in the HemECs. While FABP4 knockdown reduced the BrdU incorporation and cell number of HemECs as expected, cell proliferation was accelerated by FABP4 over-expression. Moreover, rapamycin (10nM) inhibited mTOR-FABP4 signaling and HemEC proliferation. Taken together, these results indicated that mTOR signaling pathway-activated FABP4 directly regulates the proliferation of endothelial cells in hemangioma. Rapamycin and inhibitors of FABP4 have therapeutic potential for treating infantile hemangiomas. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Overexpression of SKP2 Inhibits the Radiation-Induced Bystander Effects of Esophageal Carcinoma.

PubMed

Wang, Xiao-Chun; Zhang, Tie-Jun; Guo, Zi-Jian; Xiao, Chang-Yan; Ding, Xiao-Wen; Fang, Fang; Sheng, Wen-Tao; Shu, Xu; Li, Jue

2017-02-06

To investigate the effects of S-phase kinase protein 2 (SKP2) expression on the radiation induced bystander effect (RIBE) in esophageal cancer (EC) cells. Western blot was used to detect the levels of SKP2, Rad51, and Ku70 in EC cells. Positive transfection, RNAi, micronucleus (MN), and γ-H2AX focus formation assay were used to investigate the effects of SKP2 on RIBE induced by irradiated cells. We found a significant negative correlation between SKP2 expression and MN frequency ( p < 0.05) induced by RIBE. The results were further confirmed by positive transfection, RNAi, and rescue experiments.γ-H2AX focus formation assay results indicated that overexpression of SKP2 in the irradiated cells inhibited the DNA damage of RIBE cells. However, when SKP2 expression decreased in irradiated cells, the DNA damage of RIBE cells increased. Increased or decreased expression levels of SKP2 had effects on Rad51 expression under the conditions of RIBE. These results showed, for the first time, that SKP2 expression can inhibit RIBE of EC cells. The mechanism may function, at least partly, through the regulation of Rad51 in the ability to repair DNA damage.

Autologous osteoblast transplantation, an innovative method of bone defect treatment: role of a tissue and cell bank in the process.

PubMed

Olender, E; Brubaker, S; Uhrynowska-Tyszkiewicz, I; Wojtowicz, A; Kaminski, A

2014-10-01

The idea of cell treatment of various diseases and medical conditions has become very popular. Some procedures are well established, as is autologous chondrocyte implantation, whereas others are still in the process of early development, laboratory experiments, and some clinical trials. This report is devoted to an example of an emerging cell treatment: bone augmentation with the use of autologous cells and its legal and technical background. Various requirements set by law must be met by tissue banks performing cell seeding of grafts. In Europe, the requirements are described in directives 2004/23/EC, 2006/17/EC, 2006/86/EC, and in the regulation 2007/1394/EC. Revitalization of biostatic allografts gives new, promising tools for creation of functional parts of organs; brings the methodology used in tissue banks closer to tissue engineering; places the enterprise in the mainstream of advanced biotechnology; allows the full potential of tissue allografts; and opens a new, large area for clinical and laboratory research. Cell and tissue processing also have a financial impact on the treatment: it produces additional expenditures. Clinical effectiveness will be the most decisive factor of whether this innovative treatment will be applied in a particular type of medical condition. From a tissue establishment perspective, the most important issue is to develop a procedure that ensures safety for the patient in graft quality terms.

Histone Acetylation Regulates the Cell-Specific and Interferon-γ–Inducible Expression of Extracellular Superoxide Dismutase in Human Pulmonary Arteries

PubMed Central

Stepp, Marcus W.; Vorst, Alan L.; Folz, Rodney J.

2011-01-01

Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme present in the vascular wall, and is responsible for both the protection of vessels from oxidative stress and for the modulation of vascular tone. Concentrations of EC-SOD in human pulmonary arteries are very high relative to other tissues, and the expression of EC-SOD appears highly restricted to smooth muscle. The molecular basis for this smooth muscle–specific expression of EC-SOD is not known. Here we assessed the role of epigenetic factors in regulating the cell-specific and IFN-γ–inducible expression of EC-SOD in human pulmonary artery cells. The analysis of CpG site methylation within the promoter and coding regions of the EC-SOD gene demonstrated higher levels of DNA methylation within the distal promoter region in endothelial cells compared with smooth muscle cells. Exposure of both cell types to DNA demethylation agents reactivated the transcription of EC-SOD in endothelial cells alone. However, exposure to the histone deacetylase inhibitor trichostatin A (TSA) significantly induced EC-SOD gene expression in both endothelial cells and smooth muscle cells. Concentrations of EC-SOD mRNA were also induced up to 45-fold by IFN-γ in smooth muscle cells, but not in endothelial cells. The IFN-γ–dependent expression of EC-SOD was regulated by the Janus tyrosine kinase/signal transducers and activators of transcription proteins signaling pathway. Simultaneous exposure to TSA and IFN-γ produced a synergistic effect on the induction of EC-SOD gene expression, but only in endothelial cells. These findings provide strong evidence that EC-SOD cell-specific and IFN-γ–inducible expression in pulmonary artery cells is regulated, to a major degree, by epigenetic mechanisms that include histone acetylation and DNA methylation. PMID:21493784

Cardiac Endothelial Cell Transcriptome.

PubMed

Lother, Achim; Bergemann, Stella; Deng, Lisa; Moser, Martin; Bode, Christoph; Hein, Lutz

2018-03-01

Endothelial cells (ECs) are a highly specialized cell type with marked diversity between different organs or vascular beds. Cardiac ECs are an important player in cardiac physiology and pathophysiology but are not sufficiently characterized yet. Thus, the aim of the present study was to analyze the cardiac EC transcriptome. We applied fluorescence-assisted cell sorting to isolate pure ECs from adult mouse hearts. RNAseq revealed 1288 genes predominantly expressed in cardiac ECs versus heart tissue including several transcription factors. We found an overrepresentation of corresponding transcription factor binding motifs within the promotor region of EC-enriched genes, suggesting that they control the EC transcriptome. Cardiac ECs exhibit a distinct gene expression profile when compared with renal, cerebral, or pulmonary ECs. For example, we found the Meox2 / Tcf15, Fabp4 , and Cd36 signaling cascade higher expressed in cardiac ECs which is a key regulator of fatty acid uptake and involved in the development of atherosclerosis. The results from this study provide a comprehensive resource of gene expression and transcriptional control in cardiac ECs. The cardiac EC transcriptome exhibits distinct differences in gene expression compared with other cardiac cell types and ECs from other organs. We identified new candidate genes that have not been investigated in ECs yet as promising targets for future evaluation. © 2018 American Heart Association, Inc.

Angiotensin II promotes iron accumulation and depresses PGI₂ and NO synthesis in endothelial cells: effects of losartan and propranolol analogs.

PubMed

Mak, I Tong; Landgraf, Kenneth M; Chmielinska, Joanna J; Weglicki, William B

2012-10-01

Angiotensin may promote endothelial dysfunction through iron accumulation. To research this, bovine endothelial cells (ECs) were incubated with iron (30 µmol·L⁻¹) with or without angiotensin II (100 nmol·L⁻¹). After incubation for 6 h, it was observed that the addition of angiotensin enhanced EC iron accumulation by 5.1-fold compared with a 1.8-fold increase for cells incubated with iron only. This enhanced iron uptake was attenuated by losartan (100 nmol·L⁻¹), d-propranolol (10 µmol·L⁻¹), 4-HO-propranolol (5 µmol·L⁻¹), and methylamine, but not by vitamin E or atenolol. After 6 h of incubation, angiotensin plus iron provoked intracellular oxidant formation (2'7'-dichlorofluorescein diacetate (DCF-DA) fluorescence) and elevated oxidized glutathione; significant loss of cell viability occurred at 48 h. Stimulated prostacyclin release decreased by 38% (6 h) and NO synthesis was reduced by 41% (24 h). Both oxidative events and functional impairment were substantially attenuated by losartan or d-propranolol. It is concluded that angiotensin promoted non-transferrin-bound iron uptake via AT-1 receptor activation, leading to EC oxidative functional impairment. The protective effects of d-propranolol and 4-HO-propranolol may be related to their lysosomotropic properties.

Structure-Based Annotation of a Novel Sugar Isomerase from the Pathogenic E. coli O157:H7

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Staalduinen, L.; Park, C; Yeom, S

2010-01-01

Prokaryotes can use a variety of sugars as carbon sources in order to provide a selective survival advantage. The gene z5688 found in the pathogenic Escherichia coli O157:H7 encodes a 'hypothetical' protein of unknown function. Sequence analysis identified the gene product as a putative member of the cupin superfamily of proteins, but no other functional information was known. We have determined the crystal structure of the Z5688 protein at 1.6 {angstrom} resolution and identified the protein as a novel E. coli sugar isomerase (EcSI) through overall fold analysis and secondary-structure matching. Extensive substrate screening revealed that EcSI is capable ofmore » acting on D-lyxose and D-mannose. The complex structure of EcSI with fructose allowed the identification of key active-site residues, and mutagenesis confirmed their importance. The structure of EcSI also suggested a novel mechanism for substrate binding and product release in a cupin sugar isomerase. Supplementation of a nonpathogenic E. coli strain with EcSI enabled cell growth on the rare pentose d-lyxose.« less

VEGF-Induced Expression of miR-17–92 Cluster in Endothelial Cells Is Mediated by ERK/ELK1 Activation and Regulates Angiogenesis

PubMed Central

Chamorro-Jorganes, Aránzazu; Lee, Monica Y.; Araldi, Elisa; Landskroner-Eiger, Shira; Fernández-Fuertes, Marta; Sahraei, Mahnaz; Quiles del Rey, Maria; van Solingen, Coen; Yu, Jun; Fernández-Hernando, Carlos; Sessa, William C.

2016-01-01

Rationale: Several lines of evidence indicate that the regulation of microRNA (miRNA) levels by different stimuli may contribute to the modulation of stimulus-induced responses. The miR-17–92 cluster has been linked to tumor development and angiogenesis, but its role in vascular endothelial growth factor–induced endothelial cell (EC) functions is unclear and its regulation is unknown. Objective: The purpose of this study was to elucidate the mechanism by which VEGF regulates the expression of miR-17–92 cluster in ECs and determine its contribution to the regulation of endothelial angiogenic functions, both in vitro and in vivo. This was done by analyzing the effect of postnatal inactivation of miR-17–92 cluster in the endothelium (miR-17–92 iEC-KO mice) on developmental retinal angiogenesis, VEGF-induced ear angiogenesis, and tumor angiogenesis. Methods and Results: Here, we show that Erk/Elk1 activation on VEGF stimulation of ECs is responsible for Elk-1-mediated transcription activation (chromatin immunoprecipitation analysis) of the miR-17–92 cluster. Furthermore, we demonstrate that VEGF-mediated upregulation of the miR-17–92 cluster in vitro is necessary for EC proliferation and angiogenic sprouting. Finally, we provide genetic evidence that miR-17–92 iEC-KO mice have blunted physiological retinal angiogenesis during development and diminished VEGF-induced ear angiogenesis and tumor angiogenesis. Computational analysis and rescue experiments show that PTEN (phosphatase and tensin homolog) is a target of the miR-17–92 cluster and is a crucial mediator of miR-17-92–induced EC proliferation. However, the angiogenic transcriptional program is reduced when miR-17–92 is inhibited. Conclusions: Taken together, our results indicate that VEGF-induced miR-17–92 cluster expression contributes to the angiogenic switch of ECs and participates in the regulation of angiogenesis. PMID:26472816

Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

PubMed Central

Strohbach, Anne; Begunk, Robert; Petersen, Svea; Felix, Stephan B.; Sternberg, Katrin; Busch, Raila

2016-01-01

Drug-eluting stents (DES) have reduced in-stent-restenosis drastically. Yet, the stent surface material directly interacts with cascades of biological processes leading to an activation of cellular defense mechanisms. To prevent adverse clinical implications, to date almost every patient with a coronary artery disease is treated with statins. Besides their clinical benefit, statins exert a number of pleiotropic effects on endothelial cells (ECs). Since maintenance of EC function and reduction of uncontrolled smooth muscle cell (SMC) proliferation represents a challenge for new generation DES, we investigated the effect of atorvastatin (ATOR) on human coronary artery cells grown on biodegradable polymers. Our results show a cell type-dependent effect of ATOR on ECs and SMCs. We observed polymer-dependent changes in IC50 values and an altered ATOR-uptake leading to an attenuation of statin-mediated effects on SMC growth. We conclude that the selected biodegradable polymers negatively influence the anti-proliferative effect of ATOR on SMCs. Hence, the process of developing new polymers for DES coating should involve the characterization of material-related changes in mechanisms of drug actions. PMID:26805825

Le(x) glycan mediates homotypic adhesion of embryonal cells independently from E-cadherin: a preliminary note.

PubMed

Handa, Kazuko; Takatani-Nakase, Tomoka; Larue, Lionel; Stemmler, Marc P; Kemler, Rolf; Hakomori, Sen-itiroh

2007-06-22

Le(x) glycan and E-cadherin (Ecad) are co-expressed at embryonal stem (ES) cells and embryonal carcinoma (EC) cells. While the structure and function of Ecad mediating homotypic adhesion of these cells have been well established, evidence that Le(x) glycan also mediates such adhesion is weak, despite the fact that Le(x) oligosaccharide inhibits the compaction process. To provide stronger evidence, we knocked out Ecad gene in EC and ES cells to establish F9 Ecad (-/-) and D3M Ecad (-/-) cells, which highly express Le(x) glycan but do not express Ecad at all. Both F9 Ecad (-/-) and D3M Ecad (-/-) cells displayed strong autoaggregation in the presence of Ca(2+), while PYS-2 cells, which express trace amount of Ecad and undetectable level of Le(x) glycan, did not display autoaggregation. In addition, F9 Ecad (-/-) and D3M Ecad (-/-) cells displayed strong adhesion to plates coated with Le(x) glycosphingolipid (III(3)FucnLc4Cer), in dose-dependent manner, in the presence of Ca(2+). Thus, ES or EC cells display autoaggregation and strong adhesion to Le(x)-coated plates in the absence of Ecad, further supporting the notion of Le(x) self-recognition (i.e., Le(x)-to-Le(x) interaction) in cell adhesion.

Interleukin-27 induces the endothelial differentiation in Sca-1+ cardiac resident stem cells.

PubMed

Tanaka, Tomohiro; Obana, Masanori; Mohri, Tomomi; Ebara, Masaki; Otani, Yuta; Maeda, Makiko; Fujio, Yasushi

2015-10-01

Cytokines play important roles in cardiac repair and regeneration. Recently, we demonstrated that interleukin (IL)-6 family cytokines induce the endothelial differentiation of Sca-1+ cardiac resident stem cells through STAT3/Pim-1 signaling pathway. In contrast, the biological functions of IL-12 family cytokines in heart remain to be elucidated, though they show structural homology with IL-6. In the present study, we examined the effects of IL-12 family cytokines on the transdifferentiation of cardiac Sca-1+ cells into cardiac cells. RT-PCR analyses revealed that IL-27 receptor α (IL-27Rα), but not IL-12R or IL-23R, was expressed in cardiac Sca-1+ cells. The transcript expression of IL-27 was elevated in murine hearts in cardiac injury models. Intriguingly, IL-27 stimulation for 14 days induced the endothelial cell (EC) marker genes, such as CD-31 and VE-cadherin. Immunoblot analyses clarified that IL-27 treatment rapidly phosphorylated STAT3. IL-27 upregulated the expression of Pim-1, but the overexpression of dominant negative STAT3 abrogated the induction of Pim-1 by IL-27. Finally, adenoviral transfection of dominant negative Pim-1 inhibited IL-27-induced EC differentiation of cardiac Sca-1+ cells. These findings demonstrated that IL-27 promoted the commitment of cardiac stem cells into the EC lineage, possibly leading to neovascularization as a novel biological function. IL-27 could not only regulate the inflammation but also contribute to the maintenance of the tissue homeostasis through stem cell differentiation at inflammatory sites. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Decrease in Glomerular Endothelial Cells and Endothelial-mesenchymal Transition during Glomerulosclerosis in the Tensin2-deficient Mice (ICGN strain).

PubMed

Kato, Takashi; Mizuno, Shinya; Ito, Akihiko

2014-01-01

The ICR-derived glomerulonephritis (ICGN) mouse is a unique model of nephrotic syndrome, and albuminuria becomes evident in a neonatal stage, due to a genetic mutation of tensin2. We previously provided evidence that an apparent decrease in nephrin, caused by tensin2-deficiencient states, leads to podocytopathy, albuminuria and eventually, chronic renal failure. In general, glomerular endothelial cells (ECs) function as a barrier through tight attachment of glomerular basement membrane to podocytes, while decreased ECs can worsen renal failure. Nevertheless, it is still unknown whether glomerular ECs are altered under the tensin-2-deficient states during the manifestation of chronic renal failure. Herein, we examined the changes of glomerular ECs, with focus on the expression of PECAM-1 and VE-cadherin (EC-specific markers), or of α-SMA (myofibroblast marker) in this mouse model by histological methods. Compared with the non-nephrotic (+/nep) mice, the nephrotic (nep/nep) mice exhibited the reduced expression of PECAM-1, or of VE-cadherin, in glomerular area. Notably, some glomerular ECs showed the positive stainings for both PECAM-1 and α-SMA, suggesting endothelial-to-mesenchymal transition (EndoMT) during progression of glomerular sclerosis. This is the first report showing that a decrease in glomerular ECs, at least in part, via EndoMT is involved in tensin2-deficient pathological conditions.

PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway.

PubMed

Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

2016-04-07

Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.

A Notch-dependent transcriptional hierarchy promotes mesenchymal transdifferentiation in the cardiac cushion.

PubMed

Chang, Alex C Y; Garside, Victoria C; Fournier, Michele; Smrz, Justin; Vrljicak, Pavle; Umlandt, Patricia; Fuller, Megan; Robertson, Gordon; Zhao, Yongjun; Tam, Angela; Jones, Steven J M; Marra, Marco A; Hoodless, Pamela A; Karsan, Aly

2014-07-01

Valvuloseptal defects are the most common congenital heart defects. Notch signaling-induced endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) cushions at murine embryonic day (E)9.5 is a required step during early valve development. Insights to the transcriptional network that is activated in endocardial cells (EC) during EMT and how these pathways direct valve maturation are lacking. We show that at E11.5, AVC-EC retain the ability to undergo Notch-dependent EMT when explanted on collagen. EC-Notch inhibition at E10.5 blocks expression of known mesenchymal genes in E11.5 AVC-EC. To understand the genetic network and AVC development downstream of Notch signaling beyond E9.5, we constructed Tag-Seq libraries corresponding to different cell types of the E11.5 AVC and atrium in wild-type mice and in EC-Notch inhibited mice. We identified 1,400 potential Notch targets in the AVC-EC, of which 124 are transcription factors (TF). From the 124 TFs, we constructed a transcriptional hierarchy and identify 10 upstream TFs within the network. We validated 4 of the upstream TFs as Notch targets that are enriched in AVC-EC. Functionally, we show these 4 TFs regulate EMT in AVC explant assays. These novel signaling pathways downstream of Notch are potentially relevant to valve development. © 2014 Wiley Periodicals, Inc.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis.

PubMed

Mittal, Rahul; Prasadarao, Nemani V

2011-11-22

Despite the fundamental function of neutrophils (polymorphonuclear leukocytes (PMNs)) in innate immunity, their role in Escherichia coli K1 (EC-K1) -induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1 infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by downregulating rac1, rac2 and gp91(phox) transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface-expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis

PubMed Central

Mittal, Rahul; Prasadarao, Nemani V.

2012-01-01

Despite the fundamental function of neutrophils (PMNs) in innate immunity, their role in Escherichia coli K1 (EC-K1) induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by down regulating rac1, rac2 and gp91phox transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis. PMID:22109526

Cancer cells cause vascular endothelial cell (vEC) retraction via 12(S)HETE secretion; the possible role of cancer cell derived microparticle.

PubMed

Uchide, Keiji; Sakon, Masato; Ariyoshi, Hideo; Nakamori, Syouji; Tokunaga, Masaru; Monden, Morito

2007-02-01

Cancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7. In order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM. These findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.

Cell-to-cell interactions in changed gravity: Ground-based and flight experiments

NASA Astrophysics Data System (ADS)

Buravkova, L.; Romanov, Yu.; Rykova, M.; Grigorieva, O.; Merzlikina, N.

2005-07-01

Cell-to-cell interactions play an important role in all physiological processes and are mediated by humoral and mechanical factors. Mechanosensitive cells (e.g., osteocytes, chondrocytes, and fibroblasts) can be studied ex vivo to understand the effects of an altered gravity environment. In particular, cultured endothelial cells (EC) are very sensitive to a broad spectrum of mechanical and biochemical stimuli. Earlier, we demonstrated that clinorotation leads to cytoskeletal remodeling in cultured ECs. Long-term gravity vector changes also modulate the expression of surface adhesion molecules (ICAM-1, E-selectin, VCAM-1) on cultured ECs. To study the interactions of geterological cells, we cocultured endothelial monolayers and human lymphocytes, immune cells and myeloleucemic (K-560) cells. It was found that, although clinorotation did not alter the basal adhesion level of non-activated immune cells on endothelial monolayers, the adhesion of PMA-activated lymphocytes was increased. During flight experiments onboard the Russian segment of the International Space Station, we measured the cytotoxic activity of natural killer (NK) cells incubated with labeled target cells. It was found that immune cells in microgravity retained their ability to contact, recognize, and destroy oncogenic cells in vitro. Together, our data concerning the effects of simulated and real microgravity suggest that, despite changes in the cytoskeleton, cell motility, and expression of adhesion molecules, cell-cell interactions are not compromised, thus preserving the critical physiological functions of immune and endothelial cells.

Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts.

PubMed

Chen, Joseph C; Johnson, Brittni A; Erikson, David W; Piltonen, Terhi T; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C; Greene, Warner C; Giudice, Linda C; Roan, Nadia R

2014-06-01

How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and pro-chemotactic cytokines, as well as pro-angiogenic and proliferative growth factors (P < 0.05) in both eEC and eSF. Finally, functional assays revealed that conditioned media from SP-treated eEC and eSF significantly increased (P < 0.05) chemotaxis of CD14+ monocytes and CD4+ T cells. This study is limited to in vitro analyses of the effects of SP on endometrial cells. In addition, the measured response to SP was conducted in the absence of the ovarian hormones estradiol and progesterone, as well as epithelial-stromal paracrine signaling. While this study focused on establishing the baseline cellular response of endometrial cells to SP, future work should assess how hormone signaling in the presence of appropriate paracrine interactions affects SP-induced genes in these cells. The results of this study support previous findings that SP and semen contain bioactive factors capable of eliciting chemotactic responses in the uterus, which can lead to recruitment of leukocytes to the endometrium. Future directions will explore if similar changes in gene expression do indeed occur after coitus in vivo, and how the signaling cascades initiated by SP in the endometrium can affect reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure. This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11-1-0562 (W.C.G.); NIH 5K12-DK083021-04, NIH 1K99AI104262-01A1, The UCSF Hellman Award (N.R.R.). The authors have nothing to disclose.

Phosphatidylserine-exposing blood and endothelial cells contribute to the hypercoagulable state in essential thrombocythemia patients.

PubMed

Tong, Dongxia; Yu, Muxin; Guo, Li; Li, Tao; Li, Jihe; Novakovic, Valerie A; Dong, Zengxiang; Tian, Ye; Kou, Junjie; Bi, Yayan; Wang, Jinghua; Zhou, Jin; Shi, Jialan

2018-04-01

The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P < 0.001)] than those in controls. Among ET patients, those with JAK2 mutations showed higher levels of PS-positive erythrocytes, platelets, neutrophils, and serum-cultured ECs than TN patients or those with CALR mutations, which show similar levels. Coagulation function assays showed that higher levels of PS-positive blood cells and serum-cultured ECs led to markedly shortened coagulation time and dramatically increased levels of FXa, thrombin, and fibrin production. This procoagulant activity could be largely blocked by addition of lactadherin (approx. 70% inhibition). Confocal microscopy showed that the FVa/FXa complex and fibrin fibrils colocalized with PS on ET serum-cultured ECs. Additionally, we found a relationship between D-dimer, prothrombin fragment F1 + 2, and PS exposure. Our study reveals a previously unrecognized link between hypercoagulability and exposed PS on cells, which might also be associated with distinct thrombotic risks among mutational subtypes in ET. Thus, blocking PS-binding sites may represent a new therapeutic target for preventing thrombosis in ET.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

PubMed

He, Jinlong; Bao, Qiankun; Zhang, Yan; Liu, Mingming; Lv, Huizhen; Liu, Yajin; Yao, Liu; Li, Bochuan; Zhang, Chenghu; He, Shuang; Zhai, Guijin; Zhu, Yan; Liu, Xin; Zhang, Kai; Wang, Xiu-Jie; Zou, Ming-Hui; Zhu, Yi; Ai, Ding

2018-02-16

Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2. © 2018 American Heart Association, Inc.

Five-year follow-up of the endothelial progenitor cell capturing stent versus the paxlitaxel-eluting stent in de novo coronary lesions with a high risk of coronary restenosis.

PubMed

Woudstra, Pier; Kalkman, Deborah N; Beijk, Marcel A; Klomp, Margo; Damman, Peter; Koch, Karel T; Henriques, Jose P S; Baan, Jan; Vis, Marije M; Piek, Jan J; Tijssen, Jan G P; de Winter, Robbert J

2018-06-01

To assess the long-term safety and clinical efficacy of the Genous endothelial progenitor cell capturing stent (ECS) compared with the TAXUS Liberté paclitaxel-eluting stent (PES) in lesions with a high risk of restenosis. Instead of the use of cytotoxic or cytostatic drugs in drug-eluting stents, a "pro-healing" approach in ECS may overcome impeded healing response due to delayed functional endothelialization of the stent struts. In the prospective, randomized TRIAS pilot study 193 patients with coronary artery lesions carrying a high risk of restenosis were included (ECS: n = 98, PES: n = 95). The primary focus of this analysis was target vessel failure (TVF) at 5 years. Dual antiplatelet therapy was prescribed for ≥1 month after ECS and for ≥6 months after PES. At 5 years follow-up, no significant differences were found in TVF (ECS 24% vs. PES 29%, risk difference 95% confidence interval (RDCI) -17.3% to 7.4%). Between 2 and 5 years after the index procedure, low numbers of TVF were observed in ECS compared with PES (ECS 4% vs. PES 16%, RDCI -20.8% to -2.3%). There was no definite stent thrombosis in ECS compared with four patients in the PES group. This is the first randomized study providing very long-term clinical efficacy and safety of the ECS in lesions carrying a high risk of restenosis. At 5 years follow-up, TVF rates in ECS group are numerically lower compared with PES due to an increase of events between 2 and 5 years after the index procedure. © 2017 Wiley Periodicals, Inc.

Minimizing antibody surface density on liposomes while sustaining cytokine-activated EC targeting.

PubMed

Almeda, Dariela; Wang, Biran; Auguste, Debra T

2015-02-01

Liposomes may be engineered to target inflamed endothelium by mimicking ligand-receptor interactions between leukocytes and cytokine-activated endothelial cells (ECs). The upregulation and assembly of vascular cell adhesion molecule-1 (VCAM1) and E-selectin on the cell membrane upon exposure to cytokines have shown potential for drug delivery vehicles to target sites of chronic endothelial inflammation, such as atherosclerosis and cancer. Herein, we characterized EC surfaces by measuring the E-selectin and VCAM1 surface densities and adhesion forces of aVCAM1 and aE-selectin to ECs. We quantified the antibody density, ratio, and diffusivity of liposomes to achieve significant binding and internalization. At 1 h, the 1:1 ratio of VCAM1:E-selectin antibodies was significantly higher than 1:0 and 0:1. Significant binding and uptake was achieved at aE-selectin densities as low as 400 molecules/μm(2). The highest levels of binding and uptake were achieved when using a 1:1 ratio of VCAM1:E-selectin antibodies at a density of 1000 molecules/μm(2); this density is 85% lower than previous reports. The binding and uptake of functionalized liposomes were reduced to levels comparable to IgG functionalized liposomes upon a 10-fold reduction in liposome membrane diffusivity. We conclude with a liposomal design that discriminates between healthy and inflamed endothelium while reducing antibody surface presentation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative gene expression analysis between coronary arteries and internal mammary arteries identifies a role for the TES gene in endothelial cell functions relevant to coronary artery disease.

PubMed

Archacki, Stephen R; Angheloiu, George; Moravec, Christine S; Liu, Hui; Topol, Eric J; Wang, Qing Kenneth

2012-03-15

Coronary artery disease (CAD) is the leading cause of death worldwide. It has been established that internal mammary arteries (IMA) are resistant to the development of atherosclerosis, whereas left anterior descending (LAD) coronary arteries are athero-prone. The contrasting properties of these two arteries provide an innovative strategy to identify the genes that play important roles in the development of atherosclerosis. We carried out microarray analysis to identify genes differentially expressed between IMA and LAD. Twenty-nine genes showed significant differences in their expression levels between IMA and LAD, which included the TES gene encoding Testin. The role of TES in the cardiovascular system is unknown. Here we show that TES is involved in endothelial cell (EC) functions relevant to atherosclerosis. Western blot analysis showed higher TES expression in IMA than in LAD. Reverse transcription polymerase chain reaction and western blot analyses showed that TES was consistently and markedly down-regulated by more than 6-fold at both mRNA and protein levels in patients with CAD compared with controls without CAD (P= 0.000049). The data suggest that reduced TES expression is associated with the development of CAD. Knockdown of TES expression by small-interfering RNA promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while the over-expression of TES in ECs blunted these processes. These results demonstrate association between reduced TES expression and CAD, establish a novel role for TES in EC functions and raise the possibility that reduced TES expression increases susceptibility to the development of CAD.

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

PubMed

Testa, Jacqueline E; Chrastina, Adrian; Oh, Phil; Li, Yan; Witkiewicz, Halina; Czarny, Malgorzata; Buss, Tim; Schnitzer, Jan E

2009-08-01

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

The ectodomain of cadherin-11 binds to erbB2 and stimulates Akt phosphorylation to promote cranial neural crest cell migration

PubMed Central

Mathavan, Ketan; Khedgikar, Vikram; Bartolo, Vanessa

2017-01-01

During development, a multi-potent group of cells known as the cranial neural crest (CNC) migrate to form craniofacial structures. Proper migration of these cells requires proteolysis of cell adhesion molecules, such as cadherins. In Xenopus laevis, preventing extracellular cleavage of cadherin-11 impairs CNC migration. However, overexpression of the soluble cleavage product (EC1-3) is capable of rescuing this phenotype. The mechanism by which EC1-3 promotes CNC migration has not been investigated until now. Here we show that EC1-3 stimulates phosphorylation of Akt, a target of PI3K, in X.laevis CNC. Through immunoprecipitation experiments, we determined that EC1-3 interacts with all ErbB receptors, PDGFRα, and FGFR1. Of these receptors, only ErbB2 was able to produce an increase in Akt phosphorylation upon treatment with a recombinant EC1-3. This increase was abrogated by mubritinib, an inhibitor of ErbB2. We were able to recapitulate this decrease in Akt phosphorylation in vivo by knocking down ErbB2 in CNC cells. Knockdown of the receptor also significantly reduced CNC migration in vivo. We confirmed the importance of ErbB2 and ErbB receptor signaling in CNC migration using mubritinib and canertinib, respectively. Mubritinib and the PI3K inhibitor LY294002 significantly decreased cell migration while canertinib nearly prevented it altogether. These data show that ErbB2 and Akt are important for CNC migration and implicate other ErbB receptors and Akt-independent signaling pathways. Our findings provide the first example of a functional interaction between the extracellular domain of a type II classical cadherin and growth factor receptors. PMID:29190819

Can vitamin d suppress endothelial cells apoptosis in multiple sclerosis patients?

PubMed

Dehghani, Leila; Meamar, Rokhsareh; Etemadifar, Masoud; Sheshde, Zahra Dehghani; Shaygannejad, Vahid; Sharifkhah, Mostafa; Tahani, Soheil

2013-05-01

Multiple sclerosis (MS) is an autoimmune disease of central nerves system, in which neurological disabilities occur in young adults. Despite increasing number of studies on MS, some aspects of this disorder are still unclear. In the previous studies, it has been proven that there is direct relation between MS incidence and vitamin D deficiency. Thereby, strong evidence in MS pathogenesis suggests that endothelial cells (EC) could be harmed in MS. In addition, functional changes in EC and macrovascular injuries lead blood-brain barrier disruption in MS. Current study is the first investigation to elucidate positive influences of vitamin D against EC apoptosis in MS. Human umbilical vein endothelial cells (HUVECs) were cultured and then treated with sera from patients with active MS (in relapse) and sera from healthy volunteer participants as control group (each group n=15). 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay for cell surveillance and cell-death detection kit for evaluating apoptosis were used in this study. There was a significant decrease in apoptosis rate by the serum of patients, just when 1,25(OH)2D3 applied before treating HUVECs with sera from active MS (in relapse). Furthermore, the cells surveillance increased markedly with the presence of 1,25(OH)(2)D(3) in culture, too. Withregard to increment in EC apoptosis rate, which treated by the sera from MS patients and decrement in apoptosis rate by the presence of vitamin D in culture media, it could be proposed that vitamin D pre-treatment can be used for MS patients, due to its beneficial effects on protecting EC apoptosis.

MiR-328 suppresses the survival of esophageal cancer cells by targeting PLCE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Na; Zhao, Wenchao; Zhang, Zhongmian

2016-01-29

Esophageal cancer (EC) is the sixth leading cause of death worldwide. Recent studies have highlighted the vital role of microRNAs (miRNAs) in EC development and diagnosis. In our study, qPCR analysis showed that miRNA-328 was expressed at significantly low levels in EC109 and EC9706 cells. The results also showed that overexpression of miR-328 by lentivirus-mediated gene transfer markedly inhibited cell proliferation and invasion, and enhanced apoptosis; whereas, inhibition of miR-328 significantly promoted cell proliferation and invasion, and suppressed apoptosis in EC109 and EC9706 cells. Dual-luciferase reporter assay confirmed that miR-328 directly targeted phospholipase C epsilon 1 (PLCE1) by binding to target sequencesmore » in the 3′-UTR. qPCR and Western blot analysis showed that the PLCE1 was overexpressed in EC109 and EC9706 cells. Additionally, we found that miR-328 overexpression decreased PLCE1 mRNA and protein levels, while miR-328 inhibition enhanced the PLCE1 expression. Further analysis showed that PLCE1 overexpression rescued the inhibitory effect of miR-328 on cell proliferation and invasion, and repressed the promotive effect of miR-328 on cell apoptosis. In conclusion, our results suggest that miR-328 suppresses the survival of EC cells by regulating PLCE1 expression, which might be a potential therapeutic method for EC. - Highlights: • PLCE1 was a target gene of miR-328. • MiR-328 overexpression decreased PLCE1 expression. • PLCE1 overexpression rescued the inhibitory effect of miR-328 on the survival of EC cells.« less

Electrical and Network Neuronal Properties Are Preferentially Disrupted in Dorsal, But Not Ventral, Medial Entorhinal Cortex in a Mouse Model of Tauopathy

PubMed Central

Booth, Clair A.; Ridler, Thomas; Murray, Tracey K.; Ward, Mark A.; de Groot, Emily; Goodfellow, Marc; Phillips, Keith G.; Randall, Andrew D.

2016-01-01

The entorhinal cortex (EC) is one of the first areas to be disrupted in neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia. The responsiveness of individual neurons to electrical and environmental stimuli varies along the dorsal–ventral axis of the medial EC (mEC) in a manner that suggests this topographical organization plays a key role in neural encoding of geometric space. We examined the cellular properties of layer II mEC stellate neurons (mEC-SCs) in rTg4510 mice, a rodent model of neurodegeneration. Dorsoventral gradients in certain intrinsic membrane properties, such as membrane capacitance and afterhyperpolarizations, were flattened in rTg4510 mEC-SCs, while other cellular gradients [e.g., input resistance (Ri), action potential properties] remained intact. Specifically, the intrinsic properties of rTg4510 mEC-SCs in dorsal aspects of the mEC were preferentially affected, such that action potential firing patterns in dorsal mEC-SCs were altered, while those in ventral mEC-SCs were unaffected. We also found that neuronal oscillations in the gamma frequency band (30–80 Hz) were preferentially disrupted in the dorsal mEC of rTg4510 slices, while those in ventral regions were comparatively preserved. These alterations corresponded to a flattened dorsoventral gradient in theta-gamma cross-frequency coupling of local field potentials recorded from the mEC of freely moving rTg4510 mice. These differences were not paralleled by changes to the dorsoventral gradient in parvalbumin staining or neurodegeneration. We propose that the selective disruption to dorsal mECs, and the resultant flattening of certain dorsoventral gradients, may contribute to disturbances in spatial information processing observed in this model of dementia. SIGNIFICANCE STATEMENT The medial entorhinal cortex (mEC) plays a key role in spatial memory and is one of the first areas to express the pathological features of dementia. Neurons of the mEC are anatomically arranged to express functional dorsoventral gradients in a variety of neuronal properties, including grid cell firing field spacing, which is thought to encode geometric scale. We have investigated the effects of tau pathology on functional dorsoventral gradients in the mEC. Using electrophysiological approaches, we have shown that, in a transgenic mouse model of dementia, the functional properties of the dorsal mEC are preferentially disrupted, resulting in a flattening of some dorsoventral gradients. Our data suggest that neural signals arising in the mEC will have a reduced spatial content in dementia. PMID:26758825

Gene therapy of murine teratocarcinoma: separate functions for insulin-like growth factors I and II in immunogenicity and differentiation.

PubMed Central

Trojan, J; Johnson, T R; Rudin, S D; Blossey, B K; Kelley, K M; Shevelev, A; Abdul-Karim, F W; Anthony, D D; Tykocinski, M L; Ilan, J

1994-01-01

Teratocarcinoma is a germ-line carcinoma giving rise to an embryoid tumor with structures derived from the three embryonic layers: mesoderm, endoderm, and ectoderm. Teratocarcinoma is widely used as an in vitro model system to study regulation of cell determination and differentiation during mammalian embryogenesis. Murine embryonic carcinoma (EC) PCC3 cells express insulin-like growth factor I(IGF-I) and its receptor, while all derivative tumor structures express IGF-I and IGF-II and their receptors. Therefore the system lends itself to dissect the role of these two growth factors during EC differentiation. With an episomal antisense strategy, we define a role for IGF-I in tumorigenicity and evasion of immune surveillance. Antisense IGF-I EC transfectants are shown to elicit a curative anti-tumor immune response with tumor regression at distal sites. In contrast, IGF-II is shown to drive determination and differentiation in EC cells. Since IGF-I and IGF-II bind to type I receptor and antisense sequence used for IGF-II cannot form duplex with endogenous IGF-I transcripts, it follows that this receptor is not involved in determination and differentiation. Images PMID:8016120

Procedure for the Isolation of Endothelial Cells from Human Cerebral Arteriovenous Malformation (cAVM) Tissues.

PubMed

Hao, Qiang; Chen, Xiao-Lin; Ma, Li; Wang, Tong-Tong; Hu, Yue; Zhao, Yuan-Li

2018-01-01

In this study, we successfully established a stable method for the isolation of endothelial cells (ECs) from human cerebral arteriovenous malformation (cAVM) tissues. Despite human cAVM tissues having a minor population of ECs, they play an important role in the manifestation and development of cAVM as well as in hemorrhagic stroke and thrombogenesis. To characterize and understand the biology of ECs in human cAVM (cAVM-ECs), methods for the isolation and purification of these cells are necessary. We have developed this method to reliably obtain pure populations of ECs from cAVMs. To obtain pure cell populations, cAVM tissues were mechanically and enzymatically digested and the resulting single cAVM-ECs suspensions were then labeled with antibodies of specific cell antigens and selected by flow cytometry. Purified ECs were detected using specific makers of ECs by immunostaining and used to study different cellular mechanisms. Compared to the different methods of isolating ECs from tissues, we could isolate ECs from cAVMs confidently, and the numbers of cAVM-ECs harvested were almost similar to the amounts present in vessel components. In addition to optimizing the protocol for isolation of ECs from human cAVM tissues, the protocol could also be applied to isolate ECs from other human neurovascular-diseased tissues. Depending on the tissues, the whole procedure could be completed in about 20 days.

Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical wnt signaling.

PubMed

Zhou, Yulian; Nathans, Jeremy

2014-10-27

Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp coreceptor and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. These experiments identify Gpr124 as a ligand-specific coactivator of canonical Wnt signaling.

Interactions of Human Endothelial and Multipotent Mesenchymal Stem Cells in Cocultures

PubMed Central

Ern, Christina; Krump-Konvalinkova, Vera; Docheva, Denitsa; Schindler, Stefanie; Rossmann, Oliver; Böcker, Wolfgang; Mutschler, Wolf; Schieker, Matthias

2010-01-01

Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC). The main goal of this study was to establish a coculture system of hMSC and EC for the purposes of bone tissue engineering. Therefore, the cell behaviour, proliferation and differentiation capacity in various cell culture media as well as cell interactions in the cocultures were evaluated. The differentiation capacity of hMSC along osteogenic, chondrogenic, and adipogenic lineage was impaired in EC medium while in a mixed EC and hMSC media, hMSC maintained osteogenic differentiation. In order to identify and trace EC in the cocultures, EC were transduced with eGFP. Using time-lapse imaging, we observed that hMSC and EC actively migrated towards cells of their own type and formed separate clusters in long term cocultures. The scarcity of hMSC and EC contacts in the cocultures suggest the influence of growth factor-mediated cell interactions and points to the necessity of further optimization of the coculture conditions. PMID:21625373

Cancer-associated fibroblasts promote endometrial cancer growth via activation of interleukin-6/STAT-3/c-Myc pathway.

PubMed

Subramaniam, Kavita S; Omar, Intan Sofia; Kwong, Soke Chee; Mohamed, Zahurin; Woo, Yin Ling; Mat Adenan, Noor Azmi; Chung, Ivy

2016-01-01

Cancer-associated fibroblasts (CAFs) secrete various pro-tumorigenic cytokines, yet the role of these cytokines in the progression of endometrial cancer remains unclear. We found that CAFs isolated from human endometrial cancer (EC) tissues secreted high levels of interleukin-6 (IL-6), which promotes EC cell proliferation in vitro. Neutralizing IL-6 in CAF-conditioned media reduced (47% inhibition) while IL-6 recombinant protein increased cell proliferation (~2.4 fold) of both EC cell lines and primary cultures. IL-6 receptors (IL-6R and gp130) were expressed only in EC epithelial cells but not in CAF, indicating a one-way paracrine signaling. In the presence of CAF-conditioned media, Janus kinase/signal transducers and activators of transcription (JAK/STAT3) pathway was activated in EC cells. Treatment with JAK and STAT3 specific inhibitors, AD412 and STATTIC, respectively, significantly abrogated CAF-mediated cell proliferation, indicating the role of IL-6 activation in EC cell proliferation. We further showed that one of STAT-3 target genes, c-Myc, was highly induced in EC cells after exposure to CAF-conditioned medium at both mRNA (>105-fold vs. control) and protein level (>2-fold vs. control). EC cell proliferation was dependent on c-Myc expression, as RNAi-mediated c-Myc down-regulation led to a significant 46% reduction in cell viability when compared with scrambled control. Interestingly, CAF-conditioned media failed to promote proliferation in EC cells with reduced c-Myc expression, suggesting that CAF-mediated cell proliferation was also dependent on c-Myc expression. Subcutaneous tumor xenograft model showed that EC cells grew at least 1.4 times larger when co-injected with CAF, when compared to those injected with EC cells alone. Mice injected with EC cells with down-regulated c-Myc expression, however, showed at least 2.5 times smaller tumor compared to those in control group. Notably, there was no increase of tumor size when co-injected with CAFs. Further immunohistochemical staining on human tissues showed positive expression of IL-6 receptors, phosphorylated-STAT3 and c-Myc in human EC tissues with less signals in benign endometrium. Taken together, our data suggests that IL-6 secreted by CAF induces c-Myc expression to promote EC proliferation in vitro and in vivo. IL-6 pathway can be a potential target to disrupt tumor-stroma interaction in endometrial cancer progression.

Heme oxygenase-1-derived bilirubin counteracts HIV protease inhibitor-mediated endothelial cell dysfunction

PubMed Central

Liu, Xiao-Ming; Durante, Zane E.; Peyton, Kelly J.; Durante, William

2016-01-01

The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-L-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS–Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy. PMID:26968795

Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice.

PubMed

Pan, Minglin; Han, Ying; Basu, Aninda; Dai, Anzhi; Si, Rui; Willson, Conor; Balistrieri, Angela; Scott, Brian T; Makino, Ayako

2018-03-07

Coronary microvascular rarefaction due to endothelial cell (EC) dysfunction is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial functions. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca 2+ concentration ([Ca 2+ ] mito ) compared to the control. Among several regulatory proteins for [Ca 2+ ] mito , HK1 and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from non-diabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in the MCEC. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca 2+ ] mito , but also reduced mitochondrial ROS production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.

Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function.

PubMed

Biwer, Lauren A; Good, Miranda E; Hong, Kwangseok; Patel, Rahul K; Agrawal, Neha; Looft-Wilson, Robin; Sonkusare, Swapnil K; Isakson, Brant E

2018-01-01

In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure. © 2017 American Heart Association, Inc.

P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation

PubMed Central

Simion, Viorel; Constantinescu, Cristina Ana; Stan, Daniela; Deleanu, Mariana; Tucureanu, Monica Madalina; Butoi, Elena; Manduteanu, Ileana; Simionescu, Maya

2016-01-01

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs' inflammation, in a mouse model of acute inflammation. PMID:27703301

Elabela-apelin receptor signaling pathway is functional in mammalian systems.

PubMed

Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

2015-02-02

Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

Effect and mechanism of PAR-2 on the proliferation of esophageal cancer cells.

PubMed

Quanjun, D; Qingyu, Z; Qiliang, Z; Liqun, X; Jinmei, C; Ziquan, L; Shike, H

2016-11-01

Esophageal Cancer (EC) is a common malignant tumor occurred in the digestive tract. In this study, we investigated the mechanism of Protease Activated Receptor 2 (PAR-2) on the proliferation of esophageal cancer cell. Transfected esophageal cancer (EC) cell (PAR-2shRNA EC109) was established with low stable PAR-2 expression. EC109 cell was treated with PAR-2 agonist, PAR-2 anti-agonist and MAPK inhibitor respectively; Untreated EC109 cell (blank control) and PAR-2shRNA EC109 cell were used for analysis also. The mRNA expressions of PAR-2, ERK1, Cyclin D1, and c-fos in each group were detected by reverse transcript and polymerase chain reaction. Western blot was used to detect the protein expressions in each group. The cell growth curves were drawn to compare the cell growth. Compared with the blank control, the mRNA and protein expressions of PAR-2, Cyclin D1, and c-fos in PAR-2 agonist group increased significantly (p < 0.05), while decreased significantly in PAR-2shRNA EC109 cell and MAPK inhibitor group (p < 0.05). The mRNA expression of ERK1 and protein expression of p-ERK1 increased in PAR-2 agonist group, decreased in PAR-2shRNA EC109 cell and MAPK inhibitor group when compared with blank control (p < 0.05). The growth of cells was upward in PAR-2 agonist group at cell growth phase when compared with blank control, while decreased in PAR-2 shRNA EC109 cell and MAPK inhibitor group with statistical difference (p < 0.05). PAR-2 regulate cell proliferation through the MAPK pathway in esophageal carcinoma cell, and Cyclin D1, c-fos are involved in this process.

Stromal deletion of the APC tumor suppressor in mice triggers development of endometrial cancer

PubMed Central

Tanwar, Pradeep S.; Zhang, LiHua; Roberts, Drucilla J.; Teixeira, Jose M.

2011-01-01

The contribution of the stromal microenvironment to the progression of endometrial cancer (EC) has not been well explored. We have conditionally expressed a mutant allele of adenomatous polyposis coli (APCcKO) in murine uterine stroma cells to study its effect on uterine development and function. In addition to metrorrhagia, the mice develop complex atypical endometrial gland hyperplasia that progresses to endometrial carcinoma in situ and endometrial adenocarcinoma as evidenced by myometrial invasion. Stromal cells subjacent to the carcinoma cells express αSMA with fewer cells expressing PDGFR-α compared to normal stromal cells suggesting that the mutant stromal cells have acquired a more myofibroblastic phenotype, which have been described as cancer-associated fibroblasts and have been shown to induce carcinogenesis in other organ systems. Analyses of human EC specimens showed substantial αSMA expression in the stroma compared with normal endometrial stroma cells. We also show that APCcKO mutant uteri and human EC have decreased stromal levels of TGFβ and BMP activities and that the mutant uteri failed to respond to exogenous estradiol stimulation. The mutant stroma cells also had higher levels of VEGF and SDF signaling components and diminished expression of ERα and PR which is common in advanced stages of human EC and is an indicator of poor prognosis. Our results indicate that de novo mutation or loss of heterozygosity in stromal APC is sufficient to induce endometrial hyperplasia and endometrial carcinogenesis by mechanisms that are consistent with unopposed estrogen signaling in the endometrial epithelium. PMID:21363919

Enhanced Therapeutic and Long-Term Dynamic Vascularization Effects of Human Pluripotent Stem Cell-Derived Endothelial Cells Encapsulated in a Nanomatrix Gel.

PubMed

Lee, Shin-Jeong; Sohn, Young-Doug; Andukuri, Adinarayana; Kim, Sangsung; Byun, Jaemin; Han, Ji Woong; Park, In-Hyun; Jun, Ho-Wook; Yoon, Young-Sup

2017-11-14

Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5 + cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis. The resultant hPSC-derived CDH5 + cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs. © 2017 American Heart Association, Inc.

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394

Vascular Endothelial Growth Factor Secretion by Nonmyocytes Modulates Connexin-43 Levels in Cardiac Organoids

PubMed Central

Iyer, Rohin K.; Odedra, Devang; Chiu, Loraine L.Y.; Vunjak-Novakovic, Gordana

2012-01-01

We previously showed that the sequential, but not simultaneous, culture of endothelial cells (ECs), fibroblasts (FBs), and cardiomyocytes (CMs) resulted in elongated, beating cardiac organoids. We hypothesized that the expression of Cx43 and contractile function are mediated by vascular endothelial growth factor (VEGF) released by nonmyocytes during the preculture period. Cardiac organoids (∼200 μm diameter) were cultivated in microchannels to enable rapid screening. Three experimental groups were formed: (i) Simultaneous Preculture (ECs+FBs for 48 h, followed by CMs), (ii) Sequential Preculture (ECs for 24 h, FBs for 24 h, followed by CMs), and (iii) Simultaneous Triculture (ECs+FBs+CMs). Controls included CMs only, FBs only, and ECs only groups, and preculture with ECs only or FBs only. The highest VEGF levels were found in the Preculture groups [Simultaneous Preculture, 8.9±2.7 ng/(mL·h−1); Sequential Preculture, 16.6±3.4 ng/(mL·h−1)], as compared with Simultaneous Triculture where VEGF was not detectable, as shown by enzyme-linked immunosorbent assay. Analytical flow cytometry showed that VEGFR2 was expressed by ECs (86%±2 VEGFR2+), FBs (44%±1 VEGFR2+), and CMs (49%±2 VEGFR2+), showing that all three cell types were capable of responding to changes in VEGF. Addition of anti-VEGF neutralizing IgG (0.4 μg/mL) to Simultaneous Preculture resulted in 3-fold decrease in Cx43 mRNA and 1.5-fold decrease in Cx43 protein, while Simultaneous Triculture supplemented with VEGF ligand (30 ng/mL) had a threefold increase in Cx43 mRNA and a twofold increase in Cx43 protein. Addition of a small molecule inhibitor of the VEGFR2 receptor (19.4 nM) to Sequential Preculture caused a 1.4-fold decrease in Cx43 mRNA and a 4.1-fold decrease in Cx43 protein. Cx43 was localized within CMs, and not within FBs or ECs. Enriched CM organoids and Sequential Preculture organoids grown in the presence of VEGFR2 inhibitor displayed low levels of Cx43 and poor functional properties. Taken together, these results suggest that endogenous VEGF-VEGFR2 signaling enhanced Cx43 expression and cardiac function in engineered cardiac organoids. PMID:22519405

Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: atomic force microscopy force mapping of pulmonary endothelial monolayer.

PubMed

Birukova, Anna A; Arce, Fernando T; Moldobaeva, Nurgul; Dudek, Steven M; Garcia, Joe G N; Lal, Ratnesh; Birukov, Konstantin G

2009-03-01

Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.

Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation.

PubMed

Pang, Xue-Li; He, Gang; Liu, Yang-Bo; Wang, Yan; Zhang, Bo

2013-03-21

To investigate the role of endoplasmic reticulum (ER) stress in cancer radiotherapy and its molecular mechanism. Tunicamycin (TM) was applied to induce ER stress in human esophageal cancer cell line EC109, and the radiosensitization effects were detected by acute cell death and clonogenic survival assay. Cell cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium iodide. Apoptosis of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose polymerase. Autophagic response was determined by acridine orange (AO) staining and Western blotting of microtubule-associated protein-1 light chain-3 (LC3) and autophagy related gene 5 (ATG5). In order to test the biological function of autophagy, specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy, and its effect on cell apoptosis was thus detected. Additionally, involvement of the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway was also detected by Western blotting. Finally, male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model observations. Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation (IR), which suggested an obvious radiosensitization effect of TM. Moreover, TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with IR alone. We also observed an increase of AO positive cells, and the protein level of LC3-II and ATG5 was induced by TM treatment, which suggested an autophagic response in EC109 cells. However, inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability, which suggested a cytoprotective role of autophagy in stressed EC109 cells. Furthermore, TM treatment also activated mTORC1, and in turn reduced Akt phosphorylation, which suggested the PI3K/Akt/mTOR signal pathway was involved in the TM-induced autophagic response in EC109 cells. Tumor xenograft results also showed synergistic retarded tumor growth by TM treatment and IR, as well as the involvement of the PI3K/Akt/mTOR pathway. Our data showed that TM treatment sensitized human esophageal cancer cells to radiation via apoptosis and autophagy both in vitro and in vivo.

Tissue Inhibitor of Metalloproteinase-3 (TIMP3) Promotes Endothelial Apoptosis via a Caspase-Independent Mechanism

PubMed Central

Qi, Jian Hua; Anand-Apte, Bela

2015-01-01

Tissue Inhibitor of Metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in endothelial cells (ECs) in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in endothelial cells expressing KDR (PAE/KDR), but not in endothelial cells expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway. PMID:25558000

Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be; Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be; Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com

Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment ofmore » venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.« less

Random-Walk Model of Diffusion in Three Dimensions in Brain Extracellular Space: Comparison with Microfiberoptic Photobleaching Measurements

PubMed Central

Jin, Songwan; Zador, Zsolt; Verkman, A. S.

2008-01-01

Diffusion through the extracellular space (ECS) in brain is important in drug delivery, intercellular communication, and extracellular ionic buffering. The ECS comprises ∼20% of brain parenchymal volume and contains cell-cell gaps ∼50 nm. We developed a random-walk model to simulate macromolecule diffusion in brain ECS in three dimensions using realistic ECS dimensions. Model inputs included ECS volume fraction (α), cell size, cell-cell gap geometry, intercellular lake (expanded regions of brain ECS) dimensions, and molecular size of the diffusing solute. Model output was relative solute diffusion in water versus brain ECS (Do/D). Experimental Do/D for comparison with model predictions was measured using a microfiberoptic fluorescence photobleaching method involving stereotaxic insertion of a micron-size optical fiber into mouse brain. Do/D for the small solute calcein in different regions of brain was in the range 3.0–4.1, and increased with brain cell swelling after water intoxication. Do/D also increased with increasing size of the diffusing solute, particularly in deep brain nuclei. Simulations of measured Do/D using realistic α, cell size and cell-cell gap required the presence of intercellular lakes at multicell contact points, and the contact length of cell-cell gaps to be least 50-fold smaller than cell size. The model accurately predicted Do/D for different solute sizes. Also, the modeling showed unanticipated effects on Do/D of changing ECS and cell dimensions that implicated solute trapping by lakes. Our model establishes the geometric constraints to account quantitatively for the relatively modest slowing of solute and macromolecule diffusion in brain ECS. PMID:18469079

Random-walk model of diffusion in three dimensions in brain extracellular space: comparison with microfiberoptic photobleaching measurements.

PubMed

Jin, Songwan; Zador, Zsolt; Verkman, A S

2008-08-01

Diffusion through the extracellular space (ECS) in brain is important in drug delivery, intercellular communication, and extracellular ionic buffering. The ECS comprises approximately 20% of brain parenchymal volume and contains cell-cell gaps approximately 50 nm. We developed a random-walk model to simulate macromolecule diffusion in brain ECS in three dimensions using realistic ECS dimensions. Model inputs included ECS volume fraction (alpha), cell size, cell-cell gap geometry, intercellular lake (expanded regions of brain ECS) dimensions, and molecular size of the diffusing solute. Model output was relative solute diffusion in water versus brain ECS (D(o)/D). Experimental D(o)/D for comparison with model predictions was measured using a microfiberoptic fluorescence photobleaching method involving stereotaxic insertion of a micron-size optical fiber into mouse brain. D(o)/D for the small solute calcein in different regions of brain was in the range 3.0-4.1, and increased with brain cell swelling after water intoxication. D(o)/D also increased with increasing size of the diffusing solute, particularly in deep brain nuclei. Simulations of measured D(o)/D using realistic alpha, cell size and cell-cell gap required the presence of intercellular lakes at multicell contact points, and the contact length of cell-cell gaps to be least 50-fold smaller than cell size. The model accurately predicted D(o)/D for different solute sizes. Also, the modeling showed unanticipated effects on D(o)/D of changing ECS and cell dimensions that implicated solute trapping by lakes. Our model establishes the geometric constraints to account quantitatively for the relatively modest slowing of solute and macromolecule diffusion in brain ECS.

RhoC and ROCKs regulate cancer cell interactions with endothelial cells.

PubMed

Reymond, Nicolas; Im, Jae Hong; Garg, Ritu; Cox, Susan; Soyer, Magali; Riou, Philippe; Colomba, Audrey; Muschel, Ruth J; Ridley, Anne J

2015-06-01

RhoC is a member of the Rho GTPase family that is implicated in cancer progression by stimulating cancer cell invasiveness. Here we report that RhoC regulates the interaction of cancer cells with vascular endothelial cells (ECs), a crucial step in the metastatic process. RhoC depletion by RNAi reduces PC3 prostate cancer cell adhesion to ECs, intercalation between ECs as well as transendothelial migration in vitro. Depletion of the kinases ROCK1 and ROCK2, two known RhoC downstream effectors, similarly decreases cancer interaction with ECs. RhoC also regulates the extension of protrusions made by cancer cells on vascular ECs in vivo. Transient RhoC depletion is sufficient to reduce both early PC3 cell retention in the lungs and experimental metastasis formation in vivo. Our results indicate RhoC plays a central role in cancer cell interaction with vascular ECs, which is a critical event for cancer progression. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Fibroblast growth factor 10 upregulates the expression of mucins in rat conjunctival epithelial cells

PubMed Central

Ma, Mingming; Zhang, Zhengwei; Niu, Weiran; Zheng, Wenjing; Kelimu, Jiang

2011-01-01

Purpose This in vitro study aimed to gain insight into the function of fibroblast growth factor 10 (FGF10) on the ocular surface, especially its effect on mRNA expression of the mucins Muc1, Muc4, and Muc5ac, and mucin protein synthesis. Methods We isolated primary cultured rat conjunctival epithelial cells (Cj-ECs) and treated them with FGF10 (1 ng/ml, 10 ng/ml, 100 ng/ml, and 200 ng/ml) and basic fibroblast growth factor 2 (FGF2; 10 ng/ml) for 24 h or 48 h. The proliferation of Cj-ECs was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). mRNA levels of Muc1, Muc4, and Muc5ac were determined by real-time PCR. Synthesis levels of MUC1 and MUC4 were measured by western blot. Flow cytometry and Annexin V/PI double staining revealed degrees of apoptosis. Results In primary culture, the epithelial cells were compact and cobblestone pavement in shape. Most of the cells were positive for cytokeratin (CK). FGF10 and FGF2 significantly stimulated Muc1, Muc4, and Muc5ac mRNA expression, cell proliferation, and synthesis of MUC1 and MUC4 proteins. FGF10 was more potent than FGF2 in these regards. FGF10 did not restrain the apoptosis of Cj-ECs. Conclusions The results of this study demonstrated that FGF10 is associated with the promotion of Cj-EC proliferation and mucin production. The effects of FGF10 on Cj-ECs support a rationale to investigate its therapeutic potential for ocular surface diseases. PMID:22065934

Endothelial FoxM1 Mediates Bone Marrow Progenitor Cell-Induced Vascular Repair and Resolution of Inflammation following Inflammatory Lung Injury

PubMed Central

Zhao, Yidan D.; Huang, Xiaojia; Yi, Fan; Dai, Zhiyu; Qian, Zhijian; Tiruppathi, Chinnaswamy; Tran, Khiem; Zhao, You-Yang

2015-01-01

Adult stem cell treatment is a potential novel therapeutic approach for acute respiratory distress syndrome. Given the extremely low rate of cell engraftment, it is believed that these cells exert their beneficial effects via paracrine mechanisms. However, the endogenous mediator(s) in the pulmonary vasculature remains unclear. Employing the mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO), here we show that endothelial expression of the reparative transcriptional factor FoxM1 is required for the protective effects of bone marrow progenitor cells (BMPC) against LPS-induced inflammatory lung injury and mortality. BMPC treatment resulted in rapid induction of FoxM1 expression in WT but not FoxM1 CKO lungs. BMPC-induced inhibition of lung vascular injury, resolution of lung inflammation, and survival, as seen in WT mice, were abrogated in FoxM1 CKO mice following LPS challenge. Mechanistically, BMPC treatment failed to induce lung EC proliferation in FoxM1 CKO mice, which was associated with impaired expression of FoxM1 target genes essential for cell cycle progression. We also observed that BMPC treatment enhanced endothelial barrier function in WT, but not in FoxM1-deficient EC monolayers. Restoration of β-catenin expression in FoxM1-deficient ECs normalized endothelial barrier enhancement in response to BMPC treatment. These data demonstrate the requisite role of endothelial FoxM1 in the mechanism of BMPC-induced vascular repair to restore vascular integrity and accelerate resolution of inflammation, thereby promoting survival following inflammatory lung injury. PMID:24578354

N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures.

PubMed

Tinh, Nguyen Thi Ngoc; Asanka Gunasekara, R A Y S; Boon, Nico; Dierckens, Kristof; Sorgeloos, Patrick; Bossier, Peter

2007-10-01

Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.

Inhibition of extracellular matrix mediated TGF-β signalling suppresses endometrial cancer metastasis

PubMed Central

Sahoo, Subhransu S.; Quah, Min Yuan; Nielsen, Sarah; Atkins, Joshua; Au, Gough G.; Cairns, Murray J.; Nahar, Pravin; Lombard, Janine M.; Tanwar, Pradeep S.

2017-01-01

Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-β signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-β pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-β signalling in non-glandular colonies. Importantly, alteration of TGF-β pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-β pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-β signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells. PMID:29069715

Three-dimensional growth of human endothelial cells in an automated cell culture experiment container during the SpaceX CRS-8 ISS space mission - The SPHEROIDS project.

PubMed

Pietsch, Jessica; Gass, Samuel; Nebuloni, Stefano; Echegoyen, David; Riwaldt, Stefan; Baake, Christin; Bauer, Johann; Corydon, Thomas J; Egli, Marcel; Infanger, Manfred; Grimm, Daniela

2017-04-01

Human endothelial cells (ECs) were sent to the International Space Station (ISS) to determine the impact of microgravity on the formation of three-dimensional structures. For this project, an automatic experiment unit (EU) was designed allowing cell culture in space. In order to enable a safe cell culture, cell nourishment and fixation after a pre-programmed timeframe, the materials used for construction of the EUs were tested in regard to their biocompatibility. These tests revealed a high biocompatibility for all parts of the EUs, which were in contact with the cells or the medium used. Most importantly, we found polyether ether ketones for surrounding the incubation chamber, which kept cellular viability above 80% and allowed the cells to adhere as long as they were exposed to normal gravity. After assembling the EU the ECs were cultured therein, where they showed good cell viability at least for 14 days. In addition, the functionality of the automatic medium exchange, and fixation procedures were confirmed. Two days before launch, the ECs were cultured in the EUs, which were afterwards mounted on the SpaceX CRS-8 rocket. 5 and 12 days after launch the cells were fixed. Subsequent analyses revealed a scaffold-free formation of spheroids in space. Copyright © 2017 Elsevier Ltd. All rights reserved.

Convergent evidence from systematic analysis of GWAS revealed genetic basis of esophageal cancer.

PubMed

Gao, Xue-Xin; Gao, Lei; Wang, Jiu-Qiang; Qu, Su-Su; Qu, Yue; Sun, Hong-Lei; Liu, Si-Dang; Shang, Ying-Li

2016-07-12

Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with risk of esophageal cancer (EC). However, investigation of genetic basis from the perspective of systematic biology and integrative genomics remains scarce.In this study, we explored genetic basis of EC based on GWAS data and implemented a series of bioinformatics methods including functional annotation, expression quantitative trait loci (eQTL) analysis, pathway enrichment analysis and pathway grouped network analysis.Two hundred and thirteen risk SNPs were identified, in which 44 SNPs were found to have significantly differential gene expression in esophageal tissues by eQTL analysis. By pathway enrichment analysis, 170 risk genes mapped by risk SNPs were enriched into 38 significant GO terms and 17 significant KEGG pathways, which were significantly grouped into 9 sub-networks by pathway grouped network analysis. The 9 groups of interconnected pathways were mainly involved with muscle cell proliferation, cellular response to interleukin-6, cell adhesion molecules, and ethanol oxidation, which might participate in the development of EC.Our findings provide genetic evidence and new insight for exploring the molecular mechanisms of EC.

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

PubMed Central

Dudek, Steven M.; Chiang, Eddie T.; Camp, Sara M.; Guo, Yurong; Zhao, Jing; Brown, Mary E.; Singleton, Patrick A.; Wang, Lichun; Desai, Anjali; Arce, Fernando T.; Lal, Ratnesh; Van Eyk, Jennifer E.; Imam, Syed Z.

2010-01-01

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement. PMID:20861316

Optimization of immunolocalization of cell cycle proteins in human corneal endothelial cells.

PubMed

He, Zhiguo; Campolmi, Nelly; Ha Thi, Binh-Minh; Dumollard, Jean-Marc; Peoc'h, Michel; Garraud, Olivier; Piselli, Simone; Gain, Philippe; Thuret, Gilles

2011-01-01

En face observation of corneal endothelial cells (ECs) using flat-mounted whole corneas is theoretically much more informative than observation of cross-sections that show only a few cells. Nevertheless, it is not widespread for immunolocalization (IL) of proteins, probably because the endothelium, a superficial monolayer, behaves neither like a tissue in immunohistochemistry (IHC) nor like a cell culture in immunocytochemistry (ICC). In our study we optimized IL for ECs of flat-mounted human corneas to study the expression of cell cycle-related proteins. We systematically screened 15 fixation and five antigen retrieval (AR) methods on 118 human fresh or stored corneas (organ culture at 31 °C), followed by conventional immunofluorescence labeling. First, in an attempt to define a universal protocol, we selected combinations able to correctly localize four proteins that are perfectly defined in ECs (zonula occludens-1 [ZO-1] and actin) or ubiquitous (heterogeneous nuclear ribonucleoprotein L [hnRNP L] and histone H3). Second, we screened protocols adapted to the revelation of 9 cell cycle proteins: Ki67, proliferating cell nuclear antigen (PCNA), minichromosome maintenance protein 2 (MCM2), cyclin D1, cyclin E, cyclin A, p16(Ink4a), p21(Cip1) and p27(Kip1). Primary antibody controls (positive controls) were performed on both epithelial cells of the same, simultaneously-stained whole corneas, and by ICC on human ECs in in vitro non-confluent cultures. Both controls are known to contain proliferating cells. IL efficiency was evaluated by two observers in a masked fashion. Correct localization at optical microscopy level in ECs was define as clear labeling with no background, homogeneous staining, agreement with previous works on ECs and/or protein functions, as well as a meaningful IL in proliferating cells of both controls. The common fixation with 4% formaldehyde (gold standard for IHC) failed to reveal 12 of the 13 proteins. In contrast, they were all revealed using either 0.5% formaldehyde at room temperature (RT) during 30 min alone or followed by AR with sodium dodecyl sulfate or trypsin, or pure methanol for 30 min at RT. Individual optimization was nevertheless often required to optimize the labeling. Ki67 was absent in both fresh and stored corneas, whereas PCNA was found in the nucleus, and MCM2 in the cytoplasm, of all ECs. Cyclin D1 was found in the cytoplasm in a paranuclear pattern much more visible after corneal storage. Cyclin E and cyclin A were respectively nuclear and cytoplasmic, unmodified by storage. P21 was not found in ECs with three different antibodies. P16 and p27 were exclusively nuclear, unmodified by storage. IL in ECs of flat-mounted whole human corneas requires a specific sample preparation, especially to avoid overfixation with aldehydes that probably easily masks epitopes. En face observation allows easy analysis of labeling pattern within the endothelial layer and clear subcellular localization, neither of which had previously been described for PCNA, MCM2, or cyclin D1.

Annonacin Exerts Antitumor Activity through Induction of Apoptosis and Extracellular Signal-regulated Kinase Inhibition

PubMed Central

Yap, Chee Voon; Subramaniam, Kavita S.; Khor, Sik Wey; Chung, Ivy

2017-01-01

Background: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Annonacin, a natural pure compound extracted from the seeds of Annona muricata, is a potential alternative therapeutic agent to treat EC. Objective: To study the antitumor activity of annonacin and its mechanism of action in EC cells (ECCs). Materials and Methods: Viability of ECCs treated with annonacin for 72 h was determined using methyl thiazolyl tetrazolium assay. The induction of cell cycle arrest and apoptotic cell death was evaluated using propidium iodide and annexin V-PE/7-AAD assay, respectively. DNA strand breaks were visualized using transferase dUTP nick end labeling assay, and the effects of annonacin on survival signaling were determined using western blotting. Results: Annonacin exhibited antiproliferative effects on EC cell lines (ECC-1 and HEC-1A) and primary cells (EC6-ept and EC14-ept) with EC50values ranging from 4.62 to 4.92 μg/ml. EC cells were shown arrested at G2/M phase after treated with 4 μg/ml of annonacin for 72 h. This led to a significant increase in apoptotic cell death (65.7%) in these cells when compared to vehicle-treated cells (P < 0.005). We further showed that annonacin-mediated apoptotic cell death was associated with an increase in caspase-3 cleavage and DNA fragmentation. Cell apoptosis was accompanied with downregulation of extracellular signal-regulated kinase survival protein expression and induction of G2/M cell cycle arrest. Conclusion: Annonacin may be a potential novel therapeutic agent for EC patients. SUMMARY We aimed to study the antitumor activity of annonacin and its mechanism of action in endometrial cancer cells. Annonacin exerted antiproliferation effects on both endometrial cancer cell lines and primary cells via induction of apoptosis and inhibition of extracellular signal-regulated kinase. Our data represented that annonacin could be an alternative therapeutic treatment to combat endometrial cancer. Abbreviations Used: 7-AAD: 7-Amino-Actinomycin, ATP: Adenosine diphosphate, BSA: Bovine serum albumin, DNA: Deoxyribonucleic acid, EC: Endometrial cancer, ECC-1: Endometrial cancer cell-1, EC50: Half maximal effective concentration, Ept: Epithelial, FBS: Fetal bovine serum, HEC-1A: Human endometrial carcinoma-1A, MTT: Methyl thiazolyl tetrazolium, NaCl: Sodium chloride, NADH: Nicotinamide adenine dinucleotide, RPMI 1640: Roswell Park Memorial Institute Medium, SDS: Sodium dodecyl sulfate PMID:29263632

Endothelial Antioxidant-1: A key mediator of Copper-dependent wound healing in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Archita; Sudhahar, Varadarajan; Chen, Gin -Fu

Here, Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remains elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX) while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1 -/ - mice. Experiments using endothelial cell (EC)-specific Atox1 -/ - mice and gene transfer of nuclear-targetmore » Atox1 in Atox1 -/ - mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1 -/ - mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O 2 - production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an essential role to sense Cu to accelerate wound angiogenesis and healing.« less

EZH2 Modulates Angiogenesis In Vitro and in a Mouse Model of Limb Ischemia

PubMed Central

Mitić, Tijana; Caporali, Andrea; Floris, Ilaria; Meloni, Marco; Marchetti, Micol; Urrutia, Raul; Angelini, Gianni D; Emanueli, Costanza

2015-01-01

Epigenetic mechanisms may regulate the expression of pro-angiogenic genes, thus affecting reparative angiogenesis in ischemic limbs. The enhancer of zest homolog-2 (EZH2) induces thtrimethylation of lysine 27 on histone H3 (H3K27me3), which represses gene transcription. We explored (i) if EZH2 expression is regulated by hypoxia and ischemia; (ii) the impact of EZH2 on the expression of two pro-angiogenic genes: eNOS and BDNF; (iii) the functional effect of EZH2 inhibition on cultured endothelial cells (ECs); (iv) the therapeutic potential of EZH2 inhibition in a mouse model of limb ischemia (LI). EZH2 expression was increased in cultured ECs exposed to hypoxia (control: normoxia) and in ECs extracted from mouse ischemic limb muscles (control: absence of ischemia). EZH2 increased the H3K27me3 abundance onto regulatory regions of eNOS and BDNF promoters. In vitro RNA silencing or pharmacological inhibition by 3-deazaneplanocin (DZNep) of EZH2 increased eNOS and BDNF mRNA and protein levels and enhanced functional capacities (migration, angiogenesis) of ECs under either normoxia or hypoxia. In mice with experimentally induced LI, DZNep increased angiogenesis in ischaemic muscles, the circulating levels of pro-angiogenic hematopoietic cells and blood flow recovery. Targeting EZH2 for inhibition may open new therapeutic avenues for patients with limb ischemia. PMID:25189741

Endothelial Antioxidant-1: A key mediator of Copper-dependent wound healing in vivo

DOE PAGES

Das, Archita; Sudhahar, Varadarajan; Chen, Gin -Fu; ...

2016-09-26

Here, Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remains elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX) while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1 -/ - mice. Experiments using endothelial cell (EC)-specific Atox1 -/ - mice and gene transfer of nuclear-targetmore » Atox1 in Atox1 -/ - mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1 -/ - mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O 2 - production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an essential role to sense Cu to accelerate wound angiogenesis and healing.« less

Microarray pathway analysis indicated that mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin growth factor 1 signaling pathways were inhibited by small interfering RNA against AT-rich interactive domain 1A in endometrial cancer

PubMed Central

Yang, Ye; Bao, Wei; Sang, Zhengyu; Yang, Yongbing; Lu, Meng; Xi, Xiaowei

2018-01-01

Mutations in the gene encoding AT-rich interactive domain 1A (ARID1A) are frequently observed in endometrial cancer (EC) but the molecular mechanisms linking the genetic changes remain to be fully understood. The present study aimed to elucidate the influence of ARID1A mutations on signaling pathways. Missense, synonymous and nonsense heterozygous ARID1A mutations in the EC HEC-1-A cell line were verified by Sanger sequencing. Mutated ARID1A small interfering RNA was transfected into HEC-1-A cells. Biochemical microarray analysis revealed 13 upregulated pathways, 17 downregulated pathways, 14 significantly affected disease states and functions, 662 upstream and 512 downstream genes in mutated ARID1A-depleted HEC-1-A cells, among which the mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin-like growth factor-1 (IGF1) signaling pathways were the 2 most downregulated pathways. Furthermore, the forkhead box protein O1 pathway was upregulated, while the IGF1 receptor, insulin receptor substrate 1 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit b pathways were downregulated. Carcinoma tumorigenesis, tumor cell mitosis and tumor cell death were significantly upregulated disease states and functions, while cell proliferation and tumor growth were significantly downregulated. The results of the present study suggested that ARID1A may be a potential prognostic and therapeutic molecular drug target for the prevention of EC progression. PMID:29399196

Water filtration rate and infiltration/accumulation of low density lipoproteins in 3 different modes of endothelial/smooth muscle cell co-cultures.

PubMed

Ding, ZuFeng; Fan, YuBo; Deng, XiaoYan

2009-11-01

Using different endothelial/smooth muscle cell co-culture modes to simulate the intimal structure of blood vessels, the water filtration rate and the infiltration/accumulation of LDL of the cultured cell layers were studied. The three cell culture modes of the study were: (i) The endothelial cell monolayer (EC/Phi); (ii) endothelial cells directly co-cultured on the smooth muscle cell monolayer (EC-SMC); (iii) endothelial cells and smooth muscle cells cultured on different sides of a Millicell-CM membrane (EC/SMC). It was found that under the same condition, the water filtration rate was the lowest for the EC/SMC mode and the highest for the EC/Phi mode, while the infiltration/accumulation of DiI-LDLs was the lowest in the EC/Phi mode and the highest in the EC-SMC mode. It was also found that DiI-LDL infiltration/accumulation in the cultured cell layers increased with the increasing water filtration rate. The results from the in vitro model study therefore suggest that the infiltration/accumulation of the lipids within the arterial wall is positively correlated with concentration polarization of atherogenic lipids, and the integrity of the endothelium plays an important role in the penetration and accumulation of atherogenic lipids in blood vessel walls.

Soluble Factors Secreted by Endothelial Cells Allow for Productive and Latent HIV-1 Infection in Resting CD4+ T Cells.

PubMed

Morris, John Henry; Nguyen, Tran; Nwadike, Abuoma; Geels, Mackenzie L; Kamp, Derrick L; Kim, Bo Ram; Boyer, Jean D; Shen, Anding

2017-02-01

In vitro, it is difficult to infect resting CD4 + T cells with human immunodeficiency virus type 1 (HIV), but infections readily occur in vivo. Endothelial cells (ECs) interact with resting CD4 + T cells in vivo, and we found previously that EC stimulation leads to productive and latent HIV infection of resting CD4 + T cells. In this study, we further characterize the interactions between EC and resting T cells. We found that resting CD4 + T cells did not require direct contact with EC for productive and/or latent infection to occur, indicating the involvement of soluble factors. Among 30 cytokines tested in a multiplex enzyme-linked immunosorbent assay (ELISA), we found that expressions for IL-6, IL-8, and CCL2 were much higher in EC-stimulated resting T cells than resting T cells cultured alone. IL-6 was found to be the soluble factor responsible for inducing productive infection of resting T cells, although direct contact with EC had an added effect. However, none of the cytokines tested, IL-6, IL-8, or CCL2, induced additional latent infection in resting T cells, suggesting that unidentified cytokines were involved. Intracellular molecules MURR1, c-Jun N-terminal kinase (JNK), and glucose transporter-1 (GLUT1) were previously shown in blocking HIV infection of resting CD4 + T cells. We found that the concentrations of these proteins were not significantly different in resting T cells before and after stimulation by EC; therefore, they are not likely involved in EC stimulation of resting CD4 + T cells, and a new mechanism is yet to be identified.

Soluble Factors Secreted by Endothelial Cells Allow for Productive and Latent HIV-1 Infection in Resting CD4+ T Cells

PubMed Central

Morris, John Henry; Nguyen, Tran; Nwadike, Abuoma; Geels, Mackenzie L.; Kamp, Derrick L.; Kim, Bo Ram; Boyer, Jean D.

2017-01-01

Abstract In vitro, it is difficult to infect resting CD4+ T cells with human immunodeficiency virus type 1 (HIV), but infections readily occur in vivo. Endothelial cells (ECs) interact with resting CD4+ T cells in vivo, and we found previously that EC stimulation leads to productive and latent HIV infection of resting CD4+ T cells. In this study, we further characterize the interactions between EC and resting T cells. We found that resting CD4+ T cells did not require direct contact with EC for productive and/or latent infection to occur, indicating the involvement of soluble factors. Among 30 cytokines tested in a multiplex enzyme-linked immunosorbent assay (ELISA), we found that expressions for IL-6, IL-8, and CCL2 were much higher in EC-stimulated resting T cells than resting T cells cultured alone. IL-6 was found to be the soluble factor responsible for inducing productive infection of resting T cells, although direct contact with EC had an added effect. However, none of the cytokines tested, IL-6, IL-8, or CCL2, induced additional latent infection in resting T cells, suggesting that unidentified cytokines were involved. Intracellular molecules MURR1, c-Jun N-terminal kinase (JNK), and glucose transporter-1 (GLUT1) were previously shown in blocking HIV infection of resting CD4+ T cells. We found that the concentrations of these proteins were not significantly different in resting T cells before and after stimulation by EC; therefore, they are not likely involved in EC stimulation of resting CD4+ T cells, and a new mechanism is yet to be identified. PMID:27599784

Human embryonic stem cell-derived endothelial cells as cellular delivery vehicles for treatment of metastatic breast cancer.

PubMed

Su, Weijun; Wang, Lina; Zhou, Manqian; Liu, Ze; Hu, Shijun; Tong, Lingling; Liu, Yanhua; Fan, Yan; Kong, Deling; Zheng, Yizhou; Han, Zhongchao; Wu, Joseph C; Xiang, Rong; Li, Zongjin

2013-01-01

Endothelial progenitor cells (EPCs) have shown tropism towards primary tumors or metastases and are thus potential vehicles for targeting tumor therapy. However, the source of adult EPCs is limited, which highlights the need for a consistent and renewable source of endothelial cells for clinical applications. Here, we investigated the potential of human embryonic stem cell-derived endothelial cells (hESC-ECs) as cellular delivery vehicles for therapy of metastatic breast cancer. In order to provide an initial assessment of the therapeutic potency of hESC-ECs, we treated human breast cancer MDA-MB-231 cells with hESC-EC conditioned medium (EC-CM) in vitro. The results showed that hESC-ECs could suppress the Wnt/β-catenin signaling pathway and thereby inhibit the proliferation and migration of MDA-MB-231 cells. To track and evaluate the possibility of hESC-EC-employed therapy, we employed the bioluminescence imaging (BLI) technology. To study the therapeutic potential of hESC-ECs, we established lung metastasis models by intravenous injection of MDA-MB-231 cells labeled with firefly luciferase (Fluc) and green fluorescent protein (GFP) to NOD/SCID mice. In mice with lung metastases, we injected hESC-ECs armed with herpes simplex virus truncated thymidine kinase (HSV-ttk) intravenously on days 11, 16, 21, and 26 after MDA-MB-231 cell injection. The NOD/SCID mice were subsequently treated with ganciclovir (GCV), and the growth status of tumor was monitored by Fluc imaging. We found that MDA-MB-231 tumors were significantly inhibited by intravenously injected hESC-ECs. The tumor-suppressive effects of the hESC-ECs, by inhibiting Wnt/β-catenin signaling pathway and inducing tumor cell death through bystander effect in human metastatic breast cancer model, provide previously unexplored therapeutic modalities for cancer treatment.

Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clarkin, Claire E.; Garonna, Elena; Pitsillides, Andrew A.

In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE{sub 2} on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure ofmore » ECs to PGE{sub 2} increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF{sub 1{alpha}} release and EC proliferation. In contrast, PGE{sub 2} attenuated VEGF{sub 165}-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE{sub 2} restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH{sub 2} (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.« less

Targeting the lactate transporter MCT1 in endothelial cells inhibits lactate-induced HIF-1 activation and tumor angiogenesis.

PubMed

Sonveaux, Pierre; Copetti, Tamara; De Saedeleer, Christophe J; Végran, Frédérique; Verrax, Julien; Kennedy, Kelly M; Moon, Eui Jung; Dhup, Suveera; Danhier, Pierre; Frérart, Françoise; Gallez, Bernard; Ribeiro, Anthony; Michiels, Carine; Dewhirst, Mark W; Feron, Olivier

2012-01-01

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities.

Increased endothelial apoptotic cell density in human diabetic erectile tissue--comparison with clinical data.

PubMed

Costa, Carla; Soares, Raquel; Castela, Angela; Adães, Sara; Hastert, Véronique; Vendeira, Pedro; Virag, Ronald

2009-03-01

Erectile dysfunction (ED) is a common complication of diabetes. Endothelial cell (EC) dysfunction is one of the main mechanisms of diabetic ED. However, loss of EC integrity has never been assessed in human diabetic corpus cavernosum. To identify and quantify apoptotic cells in human diabetic and normal erectile tissue and to compare these results with each patient's clinical data and erection status. Eighteen cavernosal samples were collected, 13 from diabetics with ED and 5 from nondiabetic individuals. Cavernosal structure and cell proliferation status were evaluated by immunohistochemistry. Tissue integrity was assessed by terminal transferase dUTP nick end labeling assay, an index of apoptotic cell density (ACD) established and compared with each patient age, type of diabetes, arterial risk factors number, arterial/veno-occlusive disease, response to intracavernous vasoactive injections (ICI), and penile nitric oxide release test (PNORT). Establish an index of ACD and correlate those results with patient clinical data. Nondiabetic samples presented few scattered cells in apoptosis and an ACD of 7.15 +/- 0.44 (mean apoptotic cells/tissue area mm(2) +/- standard error). The diabetic group showed an increased ACD of 23.82 +/- 1.53, and apoptotic cells were located specifically at vascular sites. Rehabilitation of these endothelial lesions seemed impaired, as no evidence of EC proliferation was observed. Furthermore, higher ACD in diabetic individuals correlated to poor response to PNORT and to ICI. We provided evidence for the first time that loss of cavernosal EC integrity is a crucial event involved in diabetic ED. Furthermore, we were able to establish a threshold between ACD values and cavernosal tissue functionality, as assessed by PNORT and vasoactive ICI.

Targeting the Lactate Transporter MCT1 in Endothelial Cells Inhibits Lactate-Induced HIF-1 Activation and Tumor Angiogenesis

PubMed Central

Sonveaux, Pierre; Copetti, Tamara; De Saedeleer, Christophe J.; Végran, Frédérique; Verrax, Julien; Kennedy, Kelly M.; Moon, Eui Jung; Dhup, Suveera; Danhier, Pierre; Frérart, Françoise; Gallez, Bernard; Ribeiro, Anthony; Michiels, Carine

2012-01-01

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities. PMID:22428047

Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling.

PubMed

Sugihara, Kei; Nishiyama, Koichi; Fukuhara, Shigetomo; Uemura, Akiyoshi; Arima, Satoshi; Kobayashi, Ryo; Köhn-Luque, Alvaro; Mochizuki, Naoki; Suda, Toshio; Ogawa, Hisao; Kurihara, Hiroki

2015-12-01

Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC) behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

PubMed

Kahaleh, M B; Fan, P S; Otsuka, T

1999-05-01

In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc. Copyright 1999 Academic Press.

Concise Review: Therapeutic Potential of Adipose Tissue-Derived Angiogenic Cells

PubMed Central

Brinchmann, Jan E.

2012-01-01

Inadequate blood supply to tissues is a leading cause of morbidity and mortality today. Ischemic symptoms caused by obstruction of arterioles and capillaries are currently not treatable by vessel replacement or dilatation procedures. Therapeutic angiogenesis, the treatment of tissue ischemia by promoting the proliferation of new blood vessels, has recently emerged as one of the most promising therapies. Neovascularization is most often attempted by introduction of angiogenic cells from different sources. Emerging evidence suggests that adipose tissue (AT) is an excellent reservoir of autologous cells with angiogenic potential. AT yields two cell populations of importance for neovascularization: AT-derived mesenchymal stromal cells, which likely act predominantly as pericytes, and AT-derived endothelial cells (ECs). In this concise review we discuss different physiological aspects of neovascularization, briefly present cells isolated from the blood and bone marrow with EC properties, and then discuss isolation and cell culture strategies, phenotype, functional capabilities, and possible therapeutic applications of angiogenic cells obtained from AT. PMID:23197872

FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer

PubMed Central

2014-01-01

Background Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear. Methods FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor–formation assays. Results We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn’t influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth. Conclusions These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway. It indicated that FOXA1 and AR may serve as potential gene therapy in EC. PMID:24512546

Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature

NASA Astrophysics Data System (ADS)

Dai, Guohao; Kaazempur-Mofrad, Mohammad R.; Natarajan, Sripriya; Zhang, Yuzhi; Vaughn, Saran; Blackman, Brett R.; Kamm, Roger D.; García-Cardeña, Guillermo; Gimbrone, Michael A., Jr.

2004-10-01

Atherosclerotic lesion localization to regions of disturbed flow within certain arterial geometries, in humans and experimental animals, suggests an important role for local hemodynamic forces in atherogenesis. To explore how endothelial cells (EC) acquire functional/dysfunctional phenotypes in response to vascular region-specific flow patterns, we have used an in vitro dynamic flow system to accurately reproduce arterial shear stress waveforms on cultured human EC and have examined the effects on EC gene expression by using a high-throughput transcriptional profiling approach. The flow patterns in the carotid artery bifurcations of several normal human subjects were characterized by using 3D flow analysis based on actual vascular geometries and blood flow profiles. Two prototypic arterial waveforms, "athero-prone" and "athero-protective," were defined as representative of the wall shear stresses in two distinct regions of the carotid artery (carotid sinus and distal internal carotid artery) that are typically "susceptible" or "resistant," respectively, to atherosclerotic lesion development. These two waveforms were applied to cultured EC, and cDNA microarrays were used to analyze the differential patterns of EC gene expression. In addition, the differential effects of athero-prone vs. athero-protective waveforms were further characterized on several parameters of EC structure and function, including actin cytoskeletal organization, expression and localization of junctional proteins, activation of the NF-B transcriptional pathway, and expression of proinflammatory cytokines and adhesion molecules. These global gene expression patterns and functional data reveal a distinct phenotypic modulation in response to the wall shear stresses present in atherosclerosis-susceptible vs. atherosclerosis-resistant human arterial geometries.

Coculture with endothelial cells enhances osteogenic differentiation of periodontal ligament stem cells via cyclooxygenase-2/prostaglandin E2/vascular endothelial growth factor signaling under hypoxia.

PubMed

Zhao, Lixing; Wu, Yeke; Tan, Lijun; Xu, Zhenrui; Wang, Jun; Zhao, Zhihe; Li, Xiaoyu; Li, Yu; Yang, Pu; Tang, Tian

2013-12-01

During periodontitis and orthodontic tooth movement, periodontal vasculature is severely impaired, leading to a hypoxic microenvironment of periodontal cells. However, the impact of hypoxia on periodontal cells is poorly defined. The present study investigates responses of cocultured endothelial cells (ECs) and periodontal ligament stem cells (PDLSCs) to hypoxia. Osteogenic differentiation, molecular characterization, and various behaviors of PDLSCs and human umbilical venous ECs under hypoxia were assessed by quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Moreover, the effect of ECs on PDLSC osteogenic differentiation was tested using NS398 (cyclooxygenase 2 blocker), SU5416 (vascular endothelial growth factor [VEGF] receptor inhibitor), AH6809, L-798106, and L-161982 (EP1/2/3/4 antagonists). First, hypoxia promoted osteogenic differentiation in PDLSCs and enhanced EC migration, whereas PD98059 (extracellular signal-regulated protein kinase [ERK] inhibitor) blocked, and cocultured ECs further enhanced, hypoxia-induced osteogenic differentiation. Second, NS398 impaired EC migration and prostaglandin E2 (PGE2)/VEGF release, whereas cocultured PDLSCs and exogenous PGE2 partially reversed it. Third, NS398 (pretreated ECs) decreased PGE2/VEGF concentrations. NS398-treated ECs and AH6809/SU5416-treated PDLSCs impaired cocultured EC-induced enhancement of PDLSC osteogenic differentiation. Hypoxia enhances ERK-mediated osteogenic differentiation in PDLSCs. Coculture with EC further augments PDLSC osteogenic differentiation via cyclooxygenase-2/PGE2/VEGF signaling.

Perfluoroalkyl-substituted ethylene carbonates: Novel electrolyte additives for high-voltage lithium-ion batteries

NASA Astrophysics Data System (ADS)

Zhu, Ye; Casselman, Matthew D.; Li, Yan; Wei, Alexander; Abraham, Daniel P.

2014-01-01

A new family of polyfluoroalkyl-substituted ethylene carbonates is synthesized and tested as additives in lithium-ion cells containing EC:EMC + LiPF6-based electrolyte. The influence of these compounds is investigated in Li1.2Ni0.15Mn0.55Co0.1O2//graphite cells via a combination of galvanostatic cycling and electrochemical impedance spectroscopy (EIS) tests. Among the four additives studied in this work (4-(trifluoromethyl)-1,3-dioxolan-2-one (TFM-EC), 4-(perfluorobutyl)-1,3-dioxolan-2-one (PFB-EC), 4-(perfluorohexyl)-1,3-dioxolan-2-one (PFH-EC), and 4-(perfluorooctyl)-1,3-dioxolan-2-one (PFO-EC)), small amounts (0.5 wt%) of PFO-EC is found to be most effective in lessening cell performance degradation during extended cycling. Linear sweep voltammetry (LSV), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy are used to further characterize the effects of PFO-EC on the positive and negative electrodes. LSV data from the electrolyte, and XPS analyses of electrodes harvested after cycling, suggest that PFO-EC is oxidized on the cathode forming surface films that slow electrode/cell impedance rise. Differential capacity (dQ/dV) plots from graphite//Li cells suggest that PFO-EC is involved in solid electrolyte interphase (SEI) formation. Raman data from anodes after cycling suggest that structural disordering of graphite is reduced by the addition of PFO-EC, which may explain the improved cell capacity retention.

Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo

CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulatingmore » the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis. -- Highlights: •Knockdown of CD82 decreases EC migration, proliferation and angiogenesis. •Anti-CD82 mAb 4F9 inhibits EC migration, proliferation and angiogenesis. •4F9 inhibits VEGFR2 phosphorylation via control of CD82 distribution in lipid rafts.« less

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

PubMed

Wang, Xiangmin; Pan, Bin; Honda, Goichi; Wang, Xintao; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-06-14

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Here, we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from GPR15 knock-out mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal ECs numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. Use of TME5C may be a promising treatment strategy to prevent or treat lethal complications such as sinusoidal obstruction syndrome whose pathogenesis is based on endothelial insults. Copyright © 2018, Ferrata Storti Foundation.

Elabela-Apelin Receptor Signaling Pathway is Functional in Mammalian Systems

PubMed Central

Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

2015-01-01

Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development. PMID:25639753

let-7 Contributes to Diabetic Retinopathy but Represses Pathological Ocular Angiogenesis

PubMed Central

Zhou, Qinbo; Frost, Robert J. A.; Anderson, Chastain; Zhao, Fangkun; Ma, Jing; Yu, Bo

2017-01-01

ABSTRACT The in vivo function of microRNAs (miRs) in diabetic retinopathy (DR) and age-related macular degeneration (AMD) remains unclear. We report here that let-7 family members are expressed in retinal and choroidal endothelial cells (ECs). In ECs, overexpression of let-7 by adenovirus represses EC proliferation, migration, and networking in vitro, whereas inhibition of the let-7 family with a locked nucleic acid (LNA)–anti-miR has the opposite effect. Mechanistically, silencing of the let-7 target HMGA2 gene mimics the phenotype of let-7 overexpression in ECs. let-7 transgenic (let-7-Tg) mice show features of nonproliferative DR, including tortuous retinal vessels and defective pericyte coverage. However, these mice develop significantly less choroidal neovascularization (CNV) compared to wild-type controls after laser injury. Consistently, silencing of let-7 in the eye increased laser-induced CNV in wild-type mice. Together, our data establish a causative role of let-7 in nonproliferative diabetic retinopathy and a repressive function of let-7 in pathological angiogenesis, suggesting distinct implications of let-7 in the pathogenesis of DR and AMD. PMID:28584193

Removal of metals in leachate from sewage sludge using electrochemical technology.

PubMed

Meunier, N; Drogui, P; Gourvenec, C; Mercier, G; Hausler, R; Blais, J F

2004-02-01

Heavy metals in acidic leachates from sewage sludge are usually removed by chemical precipitation, which often requires high concentration of chemicals and induces high metallic sludge production. Electrochemical technique has been explored as an alternative method in a laboratory pilot scale reactor for heavy metals (Cu and Zn) removal from sludge leachate. Three electrolytic cell arrangements using different electrodes materials were tested: mild steel or aluminium bipolar electrode (EC cell), Graphite/stainless steel monopolar electrodes (ER cell) and iron-monopolar electrodes (EC-ER cell). Results showed that the best performances of metal removal were obtained with EC and EC-ER cells using mild steel electrodes operated respectively at current intensities of 0.8 and 2.0 A through 30 and 60 min of treatment. The yields of Cu and Zn removal from leachate varied respectively from 92.4 to 98.9% and from 69.8 to 76.6%. The amounts of 55 and 44 kg tds(-1) of metallic sludge were respectively produced using EC and EC-ER cells. EC and EC-ER systems involved respectively a total cost of 21.2 and 13.1 CAN dollars per ton of dry sludge treated including only energy consumption and metallic sludge disposal. The treatment using EC-ER system was found to be effective and more economical than the traditional metal precipitation using either Ca(OH)2 and/or NaOH.

Discovering the Deregulated Molecular Functions Involved in Malignant Transformation of Endometriosis to Endometriosis-Associated Ovarian Carcinoma Using a Data-Driven, Function-Based Analysis

PubMed Central

Chang, Chia-Ming; Yang, Yi-Ping; Chuang, Jen-Hua; Chuang, Chi-Mu; Lin, Tzu-Wei; Wang, Peng-Hui; Yu, Mu-Hsien

2017-01-01

The clinical characteristics of clear cell carcinoma (CCC) and endometrioid carcinoma EC) are concomitant with endometriosis (ES), which leads to the postulation of malignant transformation of ES to endometriosis-associated ovarian carcinoma (EAOC). Different deregulated functional areas were proposed accounting for the pathogenesis of EAOC transformation, and there is still a lack of a data-driven analysis with the accumulated experimental data in publicly-available databases to incorporate the deregulated functions involved in the malignant transformation of EOAC. We used the microarray gene expression datasets of ES, CCC and EC downloaded from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database. Then, we investigated the pathogenesis of EAOC by a data-driven, function-based analytic model with the quantified molecular functions defined by 1454 Gene Ontology (GO) term gene sets. This model converts the gene expression profiles to the functionome consisting of 1454 quantified GO functions, and then, the key functions involving the malignant transformation of EOAC can be extracted by a series of filters. Our results demonstrate that the deregulated oxidoreductase activity, metabolism, hormone activity, inflammatory response, innate immune response and cell-cell signaling play the key roles in the malignant transformation of EAOC. These results provide the evidence supporting the specific molecular pathways involved in the malignant transformation of EAOC. PMID:29113136

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

PubMed Central

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-01-01

AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators. PMID:12632483

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray.

PubMed

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-03-01

To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

PubMed Central

Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

2014-01-01

STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. STUDY DESIGN, SIZE, DURATION This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. PARTICIPANTS/MATERIALS, SETTING, METHODS eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. MAIN RESULTS AND THE ROLE OF CHANCE Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and pro-chemotactic cytokines, as well as pro-angiogenic and proliferative growth factors (P < 0.05) in both eEC and eSF. Finally, functional assays revealed that conditioned media from SP-treated eEC and eSF significantly increased (P < 0.05) chemotaxis of CD14+ monocytes and CD4+ T cells. LIMITATIONS, REASONS FOR CAUTION This study is limited to in vitro analyses of the effects of SP on endometrial cells. In addition, the measured response to SP was conducted in the absence of the ovarian hormones estradiol and progesterone, as well as epithelial-stromal paracrine signaling. While this study focused on establishing the baseline cellular response of endometrial cells to SP, future work should assess how hormone signaling in the presence of appropriate paracrine interactions affects SP-induced genes in these cells. WIDER IMPLICATIONS OF THE FINDINGS The results of this study support previous findings that SP and semen contain bioactive factors capable of eliciting chemotactic responses in the uterus, which can lead to recruitment of leukocytes to the endometrium. Future directions will explore if similar changes in gene expression do indeed occur after coitus in vivo, and how the signaling cascades initiated by SP in the endometrium can affect reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure. STUDY FUNDING/COMPETING INTEREST(S) This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11-1-0562 (W.C.G.); NIH 5K12-DK083021-04, NIH 1K99AI104262-01A1, The UCSF Hellman Award (N.R.R.). The authors have nothing to disclose. PMID:24626806

Characterization of cathepsin B gene from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

PubMed

Wei, Shina; Huang, Youhua; Huang, Xiaohong; Cai, Jia; Yan, Yang; Guo, Chuanyu; Qin, Qiwei

2014-01-01

The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ethyl carbamate induces cell death through its effects on multiple metabolic pathways.

PubMed

Liu, Huichang; Cui, Bo; Xu, Yi; Hu, Chaoyang; Liu, Ying; Qu, Guorun; Li, Dawei; Wu, Yongning; Zhang, Dabing; Quan, Sheng; Shi, Jianxin

2017-11-01

Ethyl carbamate (EC), a multisite carcinogenic chemical causing tumors in various animal species, is probably carcinogenic to humans. However, information about the possible carcinogenic and toxicological effects of EC in humans is quite limited. Because EC is found in many dietary foods (such as fermented foods) and tobacco and its products, and exposure of humans to EC often occurs inevitably, its toxicological effects in humans need to be studied. This study was conducted to understand the metabolomic and transcriptomic changes in human hepatocellular carcinoma cells (HepG2) exposed to 100 mM EC for short term (4 h) and long term (12 h) period, respectively. The results revealed multiple influences of EC on the metabolome and transcriptome of HepG2 cells, which was exposure time-dependent and well correlated with the kinetic changes of cell viability and mortality. EC treatment affected multiple metabolic pathways, inducing oxidative stress, reducing detoxification capacity, depleting energy, decreasing reducing power, disrupting membrane integrity, and damaging DNA and protein. These metabolomic and transcriptomic biomarkers of EC on human cell metabolism identified in this study would facilitate further studies on the risk assessment and the mitigation of dietary EC. Copyright © 2017 Elsevier B.V. All rights reserved.

A distinct entorhinal cortex to hippocampal CA1 direct circuit for olfactory associative learning.

PubMed

Li, Yiding; Xu, Jiamin; Liu, Yafeng; Zhu, Jia; Liu, Nan; Zeng, Wenbo; Huang, Ning; Rasch, Malte J; Jiang, Haifei; Gu, Xiang; Li, Xiang; Luo, Minhua; Li, Chengyu; Teng, Junlin; Chen, Jianguo; Zeng, Shaoqun; Lin, Longnian; Zhang, Xiaohui

2017-04-01

Lateral and medial parts of entorhinal cortex (EC) convey nonspatial 'what' and spatial 'where' information, respectively, into hippocampal CA1, via both the indirect EC layer 2→ hippocampal dentate gyrus→CA3→CA1 and the direct EC layer 3→CA1 paths. However, it remains elusive how the direct path transfers distinct information and contributes to hippocampal learning functions. Here we report that lateral EC projection neurons selectively form direct excitatory synapses onto a subpopulation of morphologically complex, calbindin-expressing pyramidal cells (PCs) in the dorsal CA1 (dCA1), while medial EC neurons uniformly innervate all dCA1 PCs. Optogenetically inactivating the distinct lateral EC-dCA1 connections or the postsynaptic dCA1 calbindin-expressing PC activity slows olfactory associative learning. Moreover, optetrode recordings reveal that dCA1 calbindin-expressing PCs develop more selective spiking responses to odor cues during learning. Thus, our results identify a direct lateral EC→dCA1 circuit that is required for olfactory associative learning.

Requirement of a Relatively High Threshold Level of Mg2+ for Cell Growth of a Rhizoplane Bacterium, Sphingomonas yanoikuyae EC-S001

PubMed Central

Hoo, Henny; Hashidoko, Yasuyuki; Islam, Md. Tofazzal; Tahara, Satoshi

2004-01-01

Mg2+ is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg2+) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg2+ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg2+ or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg2+ requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg2+-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg2+ for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg2+ to Mg2+-free HSG medium. Our studies concluded that Mg2+ is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg2+ or another specific essential element for their growth. PMID:15345402

A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment

PubMed Central

Lauridsen, Holly M.; Pober, Jordan S.; Gonzalez, Anjelica L.

2014-01-01

Neutrophil extravasation occurs across postcapillary venules, structures composed of endothelial cells (ECs), pericytes (PCs), and basement membrane (BM). We constructed composite models of the human postcapillary venule, combining ECs with PCs or PC-deposited BM, to better study this process. Quiescent and tumor necrosis factor α (TNF-α)-activated composites demonstrated in situ-like expression of cadherins, E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet-endothelial cell adhesion molecule 1 (PECAM-1), CD99, and interleukin 8 (IL-8). After TNF-α activation, the ECs supported greater neutrophil adhesion (66.1 vs. 23.7% of input cells) and transmigration (35.1 vs. 7.20% of input cells) than did the PCs, but the composites behaved comparably (no significant difference) to ECs in both assays. TNF-α-activated EC-conditioned medium (CM) increased transmigration across the PCs, whereas TNF-α-activated PC-CM decreased transmigration across the ECs, and culturing on PC-derived BM decreased both adhesion to and transmigration across the ECs. Anti-very late antigen 4 (VLA-4; on neutrophils) inhibited adhesion to TNF-α-activated composites, but not to ECs alone. Anti-CD99 (expressed on all 3 cell types) inhibited transmigration across the composites (14.5% of control) more than across the ECs (39.0% of control), and venular shear stress reduced transmigration across the ECs (17.3% of static) more than across the composites (36.7% of static). These results provide proof of concept that our composite human EC/PC/BM venular construct can reveal new interactions in the inflammatory cascade.—Lauridsen, H. M., Pober, J. S., Gonzalez, A. L. A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment. PMID:24297702

Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue

PubMed Central

Luo, Wei; Hu, Qiang; Wang, Dan; Deeb, Kristin K.; Ma, Yingyu; Morrison, Carl D.; Liu, Song; Johnson, Candace S.; Trump, Donald L.

2013-01-01

Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissues obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed genes were overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer. PMID:23978847

Grouper translationally controlled tumor protein prevents cell death and inhibits the replication of Singapore grouper iridovirus (SGIV).

PubMed

Wei, Jingguang; Guo, Minglan; Ji, Huasong; Yan, Yang; Ouyang, Zhengliang; Huang, Xiaohong; Hang, Youhua; Qin, Qiwei

2012-10-01

Translationally controlled tumor protein (TCTP) is an important molecule involved in multiple biological processes, such as cell growth, cell cycle progression, malignant transformation, and enhancement of the anti-apoptotic activity. In this study, the TCTP from orange-spotted grouper Epinephelus coioides (Ec-TCTP) was cloned and characterized. The full-length cDNA of Ec-TCTP was comprised of 1057 bp with a 510 bp open reading frame that encodes a putative protein of 170 amino acids. Recombinant Ec-TCTP (rEc-TCTP) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Ec-TCTP serum preparation. The rEc-TCTP fusion protein was demonstrated to possess antioxidant activity, which conferred resistance to H(2)O(2) damage. Quantitative real-time PCR analysis revealed that Ec-TCTP mRNA is predominately expressed in the liver, and the expression was up-regulated in the liver of grouper after viral challenge with Singapore grouper iridovirus (SGIV). Intracellular localization revealed that Ec-TCTP expression was distributed predominantly in the cytoplasm. Although human TCTP has a role in apoptosis regulation, it is not known if grouper TCTP has any role in apoptosis regulation. Strikingly, grouper TCTP, when overexpressed in fathead minnow (FHM) cells, protected them from cell death induced by cycloheximide (CHX). In addition, overexpressed Ec-TCTP in grouper spleen (GS) cells inhibited the replication of SGIV. These results suggest that Ec-TCTP may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans. Copyright © 2012 Elsevier Ltd. All rights reserved.

Herbal remedy magnolol suppresses IL-6-induced STAT3 activation and gene expression in endothelial cells

PubMed Central

Chen, Shih-Chung; Chang, Ying-Ling; Wang, Danny Ling; Cheng, Jing-Jy

2006-01-01

Magnolol (Mag), an active constituent isolated from the Chinese herb Hou p'u (Magnolia officinalis) has long been used to suppress inflammatory processes. Chronic inflammation is well known to be involved in vascular injuries such as atherosclerosis in which interleukin (IL)-6 may participate. Signal transducer and activator of transcription protein 3 (STAT3), a transcription factor involved in inflammation and the cell cycle, is activated by IL-6. In this study, we evaluated whether Mag can serve as an anti-inflammatory agent during endothelial injuries. The effects of Mag on IL-6-induced STAT3 activation and downstream target gene induction in endothelial cells (ECs) were examined. Pretreatment of ECs with Mag dose dependently inhibited IL-6-induced Tyr705 and Ser727 phosphorylation in STAT3 without affecting the phosphorylation of JAK1, JAK2, and ERK1/2. Mag pretreatment of these ECs dose dependently suppressed IL-6-induced promoter activity of intracellular cell adhesion molecule (ICAM)-1 that contains functional IL-6 response elements (IREs). An electrophoretic mobility shift assay (EMSA) revealed that Mag treatment significantly reduced STAT3 binding to the IRE region. Consistently, Mag treatment markedly inhibited ICAM-1 expression on the endothelial surface. As a result, reduced monocyte adhesion to IL-6-activated ECs was observed. Furthermore, Mag suppressed IL-6-induced promoter activity of cyclin D1 and monocyte chemotactic protein (MCP)-1 for which STAT3 activation plays a role. In conclusion, our results indicate that Mag inhibits IL-6-induced STAT3 activation and subsequently results in the suppression of downstream target gene expression in ECs. These results provide a therapeutic basis for the development of Mag as an anti-inflammatory agent for vascular disorders including atherosclerosis. PMID:16520748

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.

PubMed

Zheng, Chunyu; Azcutia, Veronica; Aikawa, Elena; Figueiredo, Jose-Luiz; Croce, Kevin; Sonoki, Hiroyuki; Sacks, Frank M; Luscinskas, Francis W; Aikawa, Masanori

2013-02-01

Activation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo. Physiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor κB (NF-κB) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-κB activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr(-/-) mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect. These findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.

Importins α and β signaling mediates endothelial cell inflammation and barrier disruption.

PubMed

Leonard, Antony; Rahman, Arshad; Fazal, Fabeha

2018-04-01

Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and β importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser 536 , showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin β1, but not β2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or β1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or β1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and β1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation. Copyright © 2018 Elsevier Inc. All rights reserved.

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC).

PubMed

Arosa, F A; Irwin, C; Mayer, L; de Sousa, M; Posnett, D N

1998-05-01

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28- phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8+CD28- T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8+ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8+CD28+ and CD8+CD28- T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8+CD28- cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8+CD28- phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vbeta subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8+CD28- T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8+CD28- T cells in the iIEL compartment.

Differential expression of survival proteins during decreased intracellular oxygen tension in brain endothelial cells of grey mullets.

PubMed

Ekambaram, Padmini; Narayanan, Meenakshi; Parasuraman, Parimala

2017-02-15

The brain requires constant oxygen supply to perform its biological functions essential for survival. Because of low oxygen capacity and poor oxygen diffusibility of water, many fish species have evolved various adaptive mechanisms to cope with depleted oxygen. Endothelial cells (EC) are the primary components responsible for controlled environment of brain. Brain homeostasis largely depends on integrity of the EC. To elucidate their adaptive strategy, EC were isolated from the fish brain of Kovalam-control site and Ennore estuary-test/field hypoxic site and were subjected to low oxygen tension in laboratory. Cell viability, 4-hydroxynonenal (4HNE) and total antioxidant capacity (TAC) were analyzed to ascertain stress. Hypoxic insult, cytoprotective role of HSPs and apoptotic effect were analyzed by assessing hypoxia-inducible-factor-α (HIF1α), heat-shock-protein-70 (HSP70), heme-oxygenase 1 (HO-1), and apoptosis signal regulating kinase-1 (ASK1). This study evidenced that HSP70 and HO-1 are the key stress proteins, confer high tolerance to decreased oxygen tension mediated stress. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transplantation of Endothelial Cells to Mitigate Acute and Chronic Radiation Injury to Vital Organs.

PubMed

Rafii, Shahin; Ginsberg, Michael; Scandura, Joseph; Butler, Jason M; Ding, Bi-Sen

2016-08-01

Current therapeutic approaches for treatment of exposure to radiation involve the use of antioxidants, chelating agents, recombinant growth factors and transplantation of stem cells (e.g., hematopoietic stem cell transplantation). However, exposure to high-dose radiation is associated with severe damage to the vasculature of vital organs, often leading to impaired healing, tissue necrosis, thrombosis and defective regeneration caused by aberrant fibrosis. It is very unlikely that infusion of protective chemicals will reverse severe damage to the vascular endothelial cells (ECs). The role of irradiated vasculature in mediating acute and chronic radiation syndromes has not been fully appreciated or well studied. New approaches are necessary to replace and reconstitute ECs in organs that are irreversibly damaged by radiation. We have set forth the novel concept that ECs provide paracrine signals, also known as angiocrine signals, which not only promote healing of irradiated tissue but also direct organ regeneration without provoking fibrosis. We have developed innovative technologies that enable manufacturing and banking of human GMP-grade ECs. These ECs can be transplanted intravenously to home to and engraft to injured tissues where they augment organ repair, while preventing maladaptive fibrosis. In the past, therapeutic transplantation of ECs was not possible due to a shortage of availability of suitable donor cell sources and preclinical models, a lack of understanding of the immune privilege of ECs, and inadequate methodologies for expansion and banking of engraftable ECs. Recent advances made by our group as well as other laboratories have breached the most significant of these obstacles with the development of technologies to manufacture clinical-scale quantities of GMP-grade and human ECs in culture, including genetically diverse reprogrammed human amniotic cells into vascular ECs (rAC-VECs) or human pluripotent stem cells into vascular ECs (iVECs). This approach provides a path to therapeutic EC transplantation that can be infused concomitantly or sequentially with hematopoietic stem cell transplantation more than 24 h after irradiation to support multi-organ regeneration, thereby improving immediate and long-term survival, while limiting long-term morbidity resulting from nonregenerative damage repair pathways.

Orchestration of Angiogenesis by Immune Cells

PubMed Central

Bruno, Antonino; Pagani, Arianna; Pulze, Laura; Albini, Adriana; Dallaglio, Katiuscia; Noonan, Douglas M.; Mortara, Lorenzo

2014-01-01

It is widely accepted that the tumor microenvironment (TUMIC) plays a major role in cancer and is indispensable for tumor progression. The TUMIC involves many “players” going well beyond the malignant-transformed cells, including stromal, immune, and endothelial cells (ECs). The non-malignant cells can acquire tumor-promoting functions during carcinogenesis. In particular, these cells can “orchestrate” the “symphony” of the angiogenic switch, permitting the creation of new blood vessels that allows rapid expansion and progression toward malignancy. Considerable attention within the context of tumor angiogenesis should focus not only on the ECs, representing a fundamental unit, but also on immune cells and on the inflammatory tumor infiltrate. Immune cells infiltrating tumors typically show a tumor-induced polarization associated with attenuation of anti-tumor functions and generation of pro-tumor activities, among these angiogenesis. Here, we propose a scenario suggesting that the angiogenic switch is an immune switch arising from the pro-angiogenic polarization of immune cells. This view links immunity, inflammation, and angiogenesis to tumor progression. Here, we review the data in the literature and seek to identify the “conductors” of this “orchestra.” We also suggest that interrupting the immune → inflammation → angiogenesis → tumor progression process can delay or prevent tumor insurgence and malignant disease. PMID:25072019

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

PubMed

Dehler, Carola E; Boudinot, Pierre; Martin, Samuel A M; Collet, Bertrand

2016-08-01

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

Comparative study of P19 EC stem cell differentiation in between conventional hanging drop and the zebrafish chorion as a bio-derived material.

PubMed

Dae Seok Na; Lee, Hwang; Sun Uk Kim; Chang Nam Hwang; Sang Ho Lee; Ji Yoon Kang; Jai Kyeong Kim; James Jungho Pak

2008-07-01

Various materials including glass and polymers have been widely used for stem cell culture due to their biocompatibility. However, the roles of these materials are fundamentally limited because they cannot realize or imitate the complex biological functions of living tissues, except in very simple cases. Here, the development of a bio-derived material suitable for stem cell culture and improvement of differentiation efficiency to specific cell lineages with no stimulating agents by using a chorion obtained from a fertilized zebrafish egg through the removal of the yolk and embryonic cell mass from the egg is reported. Mouse P19 EC stem cells introduced into the empty chorion form a uniform embryoid body (EB) without addition of any inducing agent. It is demonstrated that the zebrafish chorion with nanopores improves efficiencies greatly in the EB formation, cell proliferation, and lineage-specific differentiations compared to those of the conventional hanging drop culture method.

Smooth muscle cells improve endothelial cell retention on polytetrafluoroethylene grafts in vivo.

PubMed

Yu, Hong; Dai, Wangde; Yang, Zhe; Kirkman, Paul; Weaver, Fred A; Eton, Darwin; Rowe, Vincent L

2003-09-01

We investigated the influence of smooth muscle cells (SMC) on endothelial cell (EC) retention on polytetrafluoroethylene (PTFE) grafts and the effect of SMC seeding on intimal hyperplasia in vivo in a rabbit model. Fibronectin-coated PTFE grafts (4 mm diameter) were seeded with either EC alone, SMC alone, or SMC followed 24 hours later by EC. The grafts were connected to an extracorporal aortic shunt for 1 hour or were individually implanted for 1, 30, and 100 days into the infrarenal aorta as an end-to-side bypass graft. The number of retained cells was compared at 1 hour and at 1 day after implantation. Neointimal thickness was measured 30 and 100 days after implantation. After 1-hour exposure to blood flow, EC retention rate was greater (P <.005) if seeded on top of SMC (98% +/- 2%; n = 8) versus being seeded alone (65 +/- 11%; n = 8). SMC retention rate was 95 +/- 5% (n = 8) when seeded alone. Similar cell retention was obtained 1 day after implantation. After 30-day implantation the neointima was thicker in grafts seeded with EC and SMC (282 +/- 136 microm; n = 3) than with EC only (52 +/- 45 microm; n = 3; P <.001). However, the neointimal thickness for dual-cell-seeded grafts (126 +/- 60 microm; n = 3) was not significantly different (P =.09) from EC-seeded grafts (79 +/- 48 microm; n = 3) after 100-day implantation. EC retention on PTFE grafts in vivo is improved if seeded over a layer of SMC. Further studies are needed to determine whether overlying EC modulate proliferation of underlying SMC.

A Microfluidic System for the Investigation of Tumor Cell Extravasation.

PubMed

Kühlbach, Claudia; da Luz, Sabrina; Baganz, Frank; Hass, Volker C; Mueller, Margareta M

2018-05-23

Metastatic dissemination of cancer cells is a very complex process. It includes the intravasation of cells into the metastatic pathways, their passive distribution within the blood or lymph flow, and their extravasation into the surrounding tissue. Crucial steps during extravasation are the adhesion of the tumor cells to the endothelium and their transendothelial migration. However, the molecular mechanisms that are underlying this process are still not fully understood. Novel three dimensional (3D) models for research on the metastatic cascade include the use of microfluidic devices. Different from two dimensional (2D) models, these devices take cell⁻cell, structural, and mechanical interactions into account. Here we introduce a new microfluidic device in order to study tumor extravasation. The device consists of three different parts, containing two microfluidic channels and a porous membrane sandwiched in between them. A smaller channel together with the membrane represents the vessel equivalent and is seeded separately with primary endothelial cells (EC) that are isolated from the lung artery. The second channel acts as reservoir to collect the migrated tumor cells. In contrast to many other systems, this device does not need an additional coating to allow EC growth, as the primary EC that is used produces their own basement membrane. VE-Cadherin, an endothelial adherence junction protein, was expressed in regular localization, which indicates a tight barrier function and cell⁻cell connections of the endothelium. The EC in the device showed in vivo-like behavior under flow conditions. The GFP-transfected tumor cells that were introduced were of epithelial or mesenchymal origin and could be observed by live cell imaging, which indicates tightly adherent tumor cells to the endothelial lining under different flow conditions. These results suggest that the new device can be used for research on molecular requirements, conditions, and mechanism of extravasation and its inhibition.

Expression of HES and HEY genes in infantile hemangiomas.

PubMed

Adepoju, Omotinuwe; Wong, Alvin; Kitajewski, Alex; Tong, Karen; Boscolo, Elisa; Bischoff, Joyce; Kitajewski, Jan; Wu, June K

2011-08-11

Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs. HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR. HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes. HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent

PubMed Central

Carter, Emma J.; Barnes, David; Hoppe, Hans-Jürgen; Hughes, Jennifer; Cobbold, Stephen; Harper, James; Morreau, Hans; Surakhy, Mirvat; Hassan, A. Bassim

2016-01-01

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity. PMID:27566565

Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood–brain barrier disruption after ischemic brain injury

PubMed Central

Jiang, Xiaoyan; Zhang, Lili; Pu, Hongjian; Hu, Xiaoming; Zhang, Wenting; Cai, Wei; Gao, Yanqin; Leak, Rehana K.; Keep, Richard F.; Bennett, Michael V. L.; Chen, Jun

2017-01-01

The damage borne by the endothelial cells (ECs) forming the blood–brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs—namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30–60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs—but not within neurons—ameliorated BBB impairment 1–24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown. PMID:28137866

Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle Abnormalities, Cachexia and Exercise Intolerance in Mice with Congestive Heart Failure

PubMed Central

Okutsu, Mitsuharu; Call, Jarrod A.; Lira, Vitor A.; Zhang, Mei; Donet, Jean A.; French, Brent A.; Martin, Kyle S.; Peirce-Cottler, Shayn M.; Rembold, Christopher M.; Annex, Brian H.; Yan, Zhen

2014-01-01

Background Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide (NO)-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of the NO-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. Methods and Results We demonstrated that systemic administration of endogenous nitric oxide donor S-Nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis [muscle creatine kinase (MCK)-EcSOD] in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF [α-myosin heavy chain (MHC)-calsequestrin] MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced heart failure. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria and vascular rarefaction in skeletal muscle. Conclusions EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF. PMID:24523418

Magnetic beads (Dynabead) toxicity to endothelial cells at high bead concentration: implication for tissue engineering of vascular prosthesis.

PubMed

Tiwari, A; Punshon, G; Kidane, A; Hamilton, G; Seifalian, A M

2003-10-01

Magnetic beads (Dynabeads) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads per cell may need to be used. The effect of large numbers of CD31 Dynabeads on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads-coated cells in culture compared to controls (%) by day 6 were 30.7 +/- 2.56, 41.3 +/- 9.8 and 59.2 +/- 7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7 +/- 1.2, 61.8 +/- 1.4, and 72.1 +/- 4.3. The seeded grafts showed reduced metabolism with the Dynabeads-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads will need to be used with resultant reduction in the number of available EC.

Mechano-sensing and transduction by endothelial surface glycocalyx: composition, structure, and function

PubMed Central

Fu, Bingmei M.; Tarbell, John M.

2014-01-01

The endothelial cells (ECs) lining every blood vessel wall are constantly exposed to the mechanical forces generated by blood flow. The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system. To ensure proper EC mechano-sensing and transduction, there are a variety of mechano-sensors and transducers that have been identified on the EC surface, intra- and trans-EC membrane and within the EC cytoskeleton. Among them, the most recent candidate is the endothelial surface glycocalyx (ESG), which is a matrix-like thin layer covering the luminal surface of the EC. It consists of various proteoglycans, glycosaminoglycans, and plasma proteins, and is close to other prominent EC mechano-sensors and transducers. The ESG thickness was found to be in the order of 0.1–1 μm by different visualization techniques and in different types of vessels. Detailed analysis on the electron microscopy (EM) images of the microvascular ESG revealed a quasi-periodic substructure with the ESG fiber diameter of 10–12 and 20 nm spacing between adjacent fibers. Atomic force microscopy and optical tweezers were applied to investigate the mechanical properties of the ESG on the cultured EC monolayers and in solutions. Enzymatic degradation of specific ESG glycosaminoglycan components was used to directly elucidate the role of the ESG in EC mechano-sensing and transduction by measuring the shear-induced productions of nitric oxide and prostacyclin, two characteristic responses of the ECs to the flow. The unique location, composition, and structure of the ESG determine its role in EC mechano-sensing and transduction. PMID:23401243

Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch

The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-IImore » binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.« less

Involvement of Mst1 in tumor necrosis factor-{alpha}-induced apoptosis of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohtsubo, Hideki; Ichiki, Toshihiro; Imayama, Ikuyo

2008-03-07

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-{alpha}-induced apoptosis of ECs. Western blot analysis revealed that TNF-{alpha} induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-{alpha}-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting ofmore » propidium iodide-stained cells showed that TNF-{alpha} induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-{alpha}-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-{alpha}-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-{alpha}-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.« less

Hyperglycemia-induced PATZ1 negatively modulates endothelial vasculogenesis via repression of FABP4 signaling.

PubMed

Chen, Ren-An; Sun, Xiao-Mian; Yan, Chang-You; Liu, Li; Hao, Miao-Wang; Liu, Qiang; Jiao, Xi-Ying; Liang, Ying-Min

2016-09-02

Vascular endothelial dysfunction, a central hallmark of diabetes, predisposes diabetic patients to numerous cardiovascular complications. The POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1), is an important transcriptional regulatory factor and regulates divergent pathways depending on the cellular context, but its role in endothelial cells remains poorly understood. Herein, we report for the first time that endothelial PATZ1 expression was abnormally upregulated in diabetic endothelial cells (ECs) regardless of diabetes classification. This stimulatory effect was further confirmed in the high glucose-treated human umbilical vein endothelial cells (HUVECs). From a functional standpoint, transgenic overexpression of PATZ1 in endothelial colony forming cells (ECFCs) blunted angiogenesis in vivo and rendered endothelial cells unresponsive to established angiogenic factors. Mechanistically, PATZ1 acted as a potent transcriptional corepressor of fatty acid-binding protein 4 (FABP4), an essential convergence point for angiogenic and metabolic signaling pathways in ECs. Taken together, endothelial PATZ1 thus potently inhibits endothelial function and angiogenesis via inhibition of FABP4 expression, and abnormal induction of endothelial PATZ1 may contribute to multiple aspects of vascular dysfunction in diabetes. Copyright © 2016. Published by Elsevier Inc.

Functional toxicity and tolerance patterns of bioavailable Pd(II), Pt(II), and Rh(III) on suspended Saccharomyces cerevisiae cells assayed in tandem by a respirometric biosensor.

PubMed

Frazzoli, Chiara; Dragone, Roberto; Mantovani, Alberto; Massimi, Cristiana; Campanella, Luigi

2007-12-01

Toxicological implications of exposure to bioavailable platinum group metals, here Pd, Pt, and Rh, are still to be clarified. This study obtained by a biosensor-based method preliminary information on potential effects on cellular metabolism as well as on possible tolerance mechanisms. Aerobic respiration was taken as the toxicological end point to perform tandem tests, namely functional toxicity test and tolerance test. Cells were suspended in the absence of essential constituents for growth. The dose-response curves obtained by exposure (2 h) to the metals (nanogram per gram range) suggested the same mechanisms of action, with Rh showing the greatest curve steepness and the lowest EC50 value. Conservative (95% lower confidence interval) EC10 values were 187, 85 and 51 ng g(-1) for Pt, Pd, and Rh respectively. Tolerance patterns were tested during the same runs. The full tolerance obtained after 12 h of exposure to each metal suggested mitochondrial inhibition of aerobic respiration as a target effect. The hazard rating of the metals in the tolerance test changed in the Rh EC50 range, where Rh showed the lowest toxicity. The observed tolerance might suggest a protective mechanism such as metallothionein induction at concentrations around the EC50 values. The performance of the bioassay was satisfactory, in terms of the limit of detection, repeatability, reproducibility, roboustness, sensibility, and stability; the method's critical uncertainty sources were identified for improvements.

Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

PubMed Central

Garonna, Elena; Botham, Kathleen M.; Birdsey, Graeme M.; Randi, Anna M.; Gonzalez-Perez, Ruben R.; Wheeler-Jones, Caroline P. D.

2011-01-01

Background The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs. Methodology/Principal Findings Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr1175. Phosphorylation of VEGFR2, p38MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo. Conclusions/Significance We conclude that a functional endothelial p38MAPK/Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals. PMID:21533119

Barrier Epithelial Cells and the Control of Type 2 Immunity.

PubMed

Hammad, Hamida; Lambrecht, Bart N

2015-07-21

Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor.

PubMed

Sivarapatna, Amogh; Ghaedi, Mahboobe; Le, Andrew V; Mendez, Julio J; Qyang, Yibing; Niklason, Laura E

2015-01-01

Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Specific cellular accumulation of photofrin-II in EC cells promotes photodynamic treatment efficacy in esophageal cancer.

PubMed

Gao, Shegan; Liang, Shuo; Ding, Kaili; Qu, Zhifeng; Wang, Ying; Feng, Xiaoshan

2016-06-01

Photodynamic therapy (PDT), which uses a light-sensitive compound and laser irradiation, is a light-based oncological treatment modality. PDT offers an alternative, less invasive treatment for various malignant tumors, such as esophageal cancer (EC), through a photochemical reaction induced by photofrin-II or other oncotropic photosensitizers without severe complications. Previous studies has shown that cancerous tissues accumulated more photosensitizers than paired normal tissues, however, whether it is cellular or vascular mechanisms remains unknown. Herein, in vivo and in vitro examinations were performed to study the mechanisms by which photofrin-II effectively and specifically killed EC cells. In this study, EC tissue of patients treated with photofrin-II, human ESCC cellline SHEEC and parental normal cellline SHEE, primary culture cells of EC tissue were used. The concentration of photofrin-II in cells were evaluated by high-performance liquid chromatography (HPLC). The results exhibited that accumulation of photofrin-II in cancerous cells were significantly higher than that in non-cancerous cells (p<0.05) under certain dose and time period of incubation of photofrin-II. In summary, our study showed that, photofrin-II specifically accumulated in EC cells in vivo and in vitro after controlling for vascular factors, which provided strong evidence that maybe the cellular factor is the main mechanism by which photofrin-II-mediated PDT selectively caused EC cells death. Copyright © 2016 Elsevier B.V. All rights reserved.

B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers.

PubMed

Fukazawa, Yoshinori; Lum, Richard; Okoye, Afam A; Park, Haesun; Matsuda, Kenta; Bae, Jin Young; Hagen, Shoko I; Shoemaker, Rebecca; Deleage, Claire; Lucero, Carissa; Morcock, David; Swanson, Tonya; Legasse, Alfred W; Axthelm, Michael K; Hesselgesser, Joseph; Geleziunas, Romas; Hirsch, Vanessa M; Edlefsen, Paul T; Piatak, Michael; Estes, Jacob D; Lifson, Jeffrey D; Picker, Louis J

2015-02-01

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

Functional subregions of the human entorhinal cortex

PubMed Central

Maass, Anne; Berron, David; Libby, Laura A; Ranganath, Charan; Düzel, Emrah

2015-01-01

The entorhinal cortex (EC) is the primary site of interactions between the neocortex and hippocampus. Studies in rodents and nonhuman primates suggest that EC can be divided into subregions that connect differentially with perirhinal cortex (PRC) vs parahippocampal cortex (PHC) and with hippocampal subfields along the proximo-distal axis. Here, we used high-resolution functional magnetic resonance imaging at 7 Tesla to identify functional subdivisions of the human EC. In two independent datasets, PRC showed preferential intrinsic functional connectivity with anterior-lateral EC and PHC with posterior-medial EC. These EC subregions, in turn, exhibited differential connectivity with proximal and distal subiculum. In contrast, connectivity of PRC and PHC with subiculum followed not only a proximal-distal but also an anterior-posterior gradient. Our data provide the first evidence that the human EC can be divided into functional subdivisions whose functional connectivity closely parallels the known anatomical connectivity patterns of the rodent and nonhuman primate EC. DOI: http://dx.doi.org/10.7554/eLife.06426.001 PMID:26052749

Erythro-myeloid progenitors can differentiate from endothelial cells and modulate embryonic vascular remodeling

PubMed Central

Kasaai, Bahar; Caolo, Vincenza; Peacock, Hanna M.; Lehoux, Stephanie; Gomez-Perdiguero, Elisa; Luttun, Aernout; Jones, Elizabeth A. V.

2017-01-01

Erythro-myeloid progenitors (EMPs) were recently described to arise from the yolk sac endothelium, just prior to vascular remodeling, and are the source of adult/post-natal tissue resident macrophages. Questions remain, however, concerning whether EMPs differentiate directly from the endothelium or merely pass through. We provide the first evidence in vivo that EMPs can emerge directly from endothelial cells (ECs) and demonstrate a role for these cells in vascular development. We find that EMPs express most EC markers but late EMPs and EMP-derived cells do not take up acetylated low-density lipoprotein (AcLDL), as ECs do. When the endothelium is labelled with AcLDL before EMPs differentiate, EMPs and EMP-derived cells arise that are AcLDL+. If AcLDL is injected after the onset of EMP differentiation, however, the majority of EMP-derived cells are not double labelled. We find that cell division precedes entry of EMPs into circulation, and that blood flow facilitates the transition of EMPs from the endothelium into circulation in a nitric oxide-dependent manner. In gain-of-function studies, we inject the CSF1-Fc ligand in embryos and found that this increases the number of CSF1R+ cells, which localize to the venous plexus and significantly disrupt venous remodeling. This is the first study to definitively establish that EMPs arise from the endothelium in vivo and show a role for early myeloid cells in vascular development. PMID:28272478

Preparation-free method for detecting Escherichia coli O157:H7 in the presence of spinach, spring lettuce mix, and ground beef particulates.

PubMed

Maraldo, David; Mutharasan, Raj

2007-11-01

We show the detection of 100 cells per ml of Escherichia coli O157:H7 in the presence of spinach, spring lettuce mix, and ground beef washes and particulate matter with piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. The PEMC sensors (sensing area, 2 mm2) were immobilized with polyclonal antibody specific to E. coli O157:H7 (EC) and were exposed to 10 aqueous washes of locally purchased spinach, spring lettuce mix, and ground beef for testing if EC was present. Absence of resonance frequency shift indicated that EC was not present in the 30 samples tested. Following the last sample in each food matrix, 1,000 cells per ml of EC were spiked into the sample container, and resonance frequency change was monitored. The total resonance frequency change was 880 +/- 5, 1,875 +/- 8, and 1,417 +/- 4 Hz for spinach, spring lettuce mix, and ground beef, respectively. A mixture of the three food matrices spiked with 100 cells per ml of EC gave a sensor response of 260 +/- 15 Hz. The resonance frequency changes are approximately 40% lower than our previously reported study on ground beef. It is suggested that the reduction in sensitivity is due to differences in pathogen adherence to food matrices, which affects target binding to the sensor surface. We conclude that detection selectivity is conserved in the three food matrices examined and that the magnitude of sensor response is a function of the food matrix.

Epigenetic and Epitranscriptomic Factors Make a Mark on Hematopoietic Stem Cell Development.

PubMed

Kasper, Dionna M; Nicoli, Stefania

2018-03-01

Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity, however the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between 'epi'-machineries. Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

p38 Signaling and Receptor Recycling Events in a Microfluidic Endothelial Cell Adhesion Assay

PubMed Central

Vickers, Dwayne A. L.; Chory, Emma J.; Harless, Megan C.; Murthy, Shashi K.

2013-01-01

Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs) is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV) and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome. PMID:23762436

Role of nesprin-1 in nuclear deformation in endothelial cells under static and uniaxial stretching conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anno, Toshiro; Sakamoto, Naoya, E-mail: sakan@me.kawasaki-m.ac.jp; Sato, Masaaki

Highlights: Black-Right-Pointing-Pointer Nesprin-1 knockdown decreases widths of nuclei in ECs under static condition. Black-Right-Pointing-Pointer Nuclear strain caused by stretching is increased by nesprin-1 knockdown in ECs. Black-Right-Pointing-Pointer We model mechanical interactions of F-actin with the nucleus in stretched cells. Black-Right-Pointing-Pointer F-actin bound to nesprin-1 may cause sustainable force transmission to the nucleus. -- Abstract: The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filamentsmore » (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.« less

CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism.

PubMed

Guo, Lu; Tian, Shuang; Chen, Yuguo; Mao, Yun; Cui, Sumei; Hu, Aihua; Zhang, Jianliang; Xia, Shen-Ling; Su, Yunchao; Du, Jie; Block, Edward R; Wang, Xing Li; Cui, Zhaoqiang

2015-10-01

Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with β-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hydraulic Conductivity of Endothelial Cell-Initiated Arterial Cocultures

PubMed Central

Mathura, Rishi A.; Russell-Puleri, Sparkle; Cancel, Limary; Tarbell, John M.

2014-01-01

This study describes cocultures of arterial smooth muscle cells (SMC) and endothelial cells (EC) and the influences of their heterotypic interactions on hydraulic conductivity (Lp), an important transport property. A unique feature of these cocultures is that ECs were first grown to confluence and then SMCs were inoculated. Bovine aortic smooth muscle cells (BASMCs) and bovine aortic endothelial cells (BAECs) were cocultured on Transwell Permeable Supports, and then exposed to a pressure-driven transmural flow. Lp across each culture was measured using a bubble tracking apparatus that determined water flux (Jv). Our results indicate that arterial Lp is significantly modulated by EC-SMC proximity, and serum content in culture. The Lp of cocultures was also compared to the predictions of a resistances-in-series model to distinguish the contributions of heterotypic interactions between SMCs and ECs. Conditions that lead to significantly reduced coculture Lp, compared to BAEC monoculture controls, have been uncovered and the lowest Lp in the literature for an in-vitro system are reported. In addition, VE-cadherin immunostaining of intact BAEC monolayers in each culture configuration reveals that EC-SMC proximity on a porous membrane has a dramatic influence on EC morphology patterns. The cocultures with the lowest Lp have ECs with significantly elongated morphology. Confocal imaging indicates that there are no direct EC-SMC contacts in coculture. PMID:24264601

p53 Hypersensitivity Is the Predominant Mechanism of the Unique Responsiveness of Testicular Germ Cell Tumor (TGCT) Cells to Cisplatin

PubMed Central

Gutekunst, Matthias; Oren, Moshe; Weilbacher, Andrea; Dengler, Michael A.; Markwardt, Christiane; Thomale, Jürgen; Aulitzky, Walter E.; van der Kuip, Heiko

2011-01-01

Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis plays a central role in this hypersensitive phenotype. The role of the tumor suppressor p53 in this response, however, remains controversial. Here we show that siRNA-mediated silencing of p53 is sufficient to completely abrogate hypersensitivity not only to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. The close relationship between p53 protein levels and induction of apoptosis is lost upon short-term differentiation, indicating that this predominant pro-apoptotic function of p53 is unique in pluripotent embryonal carcinoma (EC) cells. RNA interference experiments as well as microarray analysis demonstrated a central role of the pro-apoptotic p53 target gene NOXA in the p53-dependent apoptotic response of these cells. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to p53 activation. Furthermore, in the very specific cellular context of germ cell-derived pluripotent EC cells, p53 function appears to be limited to induction of apoptosis. PMID:21532991

Axon guidance molecules in vascular patterning.

PubMed

Adams, Ralf H; Eichmann, Anne

2010-05-01

Endothelial cells (ECs) form extensive, highly branched and hierarchically organized tubular networks in vertebrates to ensure the proper distribution of molecular and cellular cargo in the vertebrate body. The growth of this vascular system during development, tissue repair or in disease conditions involves the sprouting, migration and proliferation of endothelial cells in a process termed angiogenesis. Surprisingly, specialized ECs, so-called tip cells, which lead and guide endothelial sprouts, share many feature with another guidance structure, the axonal growth cone. Tip cells are motile, invasive and extend numerous filopodial protrusions sensing growth factors, extracellular matrix and other attractive or repulsive cues in their tissue environment. Axonal growth cones and endothelial tip cells also respond to signals belonging to the same molecular families, such as Slits and Roundabouts, Netrins and UNC5 receptors, Semaphorins, Plexins and Neuropilins, and Eph receptors and ephrin ligands. Here we summarize fundamental principles of angiogenic growth, the selection and function of tip cells and the underlying regulation by guidance cues, the Notch pathway and vascular endothelial growth factor signaling.

In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures

PubMed Central

Ni, Chao; Chen, Yuhui; Zeng, Musheng; Pei, Rongjuan; Du, Yong; Tang, Linquan; Wang, Mengyi; Hu, Yazhuo; Zhu, Hanyu; He, Meifang; Wei, Xiawei; Wang, Shan; Ning, Xiangkai; Wang, Manna; Wang, Jufang; Ma, Li; Chen, Xinwen; Sun, Qiang; Tang, Hong; Wang, Ying; Wang, Xiaoning

2015-01-01

Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel “in-cell infection” mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified “in-cell infection” as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that “in-cell infection” is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs. PMID:25916549

Derivation of vascular endothelial cells from human embryonic stem cells under GMP-compliant conditions: towards clinical studies in ischaemic disease.

PubMed

Kaupisch, A; Kennedy, L; Stelmanis, V; Tye, B; Kane, N M; Mountford, J C; Courtney, A; Baker, A H

2012-10-01

Revascularisation of ischaemic tissue remains an area of substantial unmet clinical need in cardiovascular disease. Strategies to induce therapeutic angiogenesis are therefore attractive. Our recent focus has been on human embryonic stem cell (hESC) strategies since hESC can be maintained in a pluripotent state or differentiated into any desired cell type, including endothelial cells (EC), under defined differentiation culture conditions. We recently published a protocol for non-good manufacturing practice (GMP) feeder- and serum-free hESC-EC-directed monolayer differentiation to vascular EC demonstrating the potential to generate hESC-derived EC in a GMP-compliant manner suitable for use in clinical trials. In this study we modified that laboratory protocol to GMP compliance. EC production was confirmed by flow cytometry, qRT-PCR and production of vascular structures in Matrigel®, yielding approximately 30 % mature VE-cadherin(+)/PECAM-1(+) cells using the GMP-compliant hESC line RC13. In conclusion, we have successfully demonstrated the production of vascular EC under GMP-compliant conditions suitable for clinical evaluation.

Endothelial cell metabolism: A novel player in atherosclerosis? Basic principles and therapeutic opportunities.

PubMed

Pircher, Andreas; Treps, Lucas; Bodrug, Natalia; Carmeliet, Peter

2016-10-01

Atherosclerosis is a leading cause of morbidity and mortality in Western society. Despite improved insight into disease pathogenesis and therapeutic options, additional treatment strategies are required. Emerging evidence highlights the relevance of endothelial cell (EC) metabolism for angiogenesis, and indicates that EC metabolism is perturbed when ECs become dysfunctional to promote atherogenesis. In this review, we overview the latest insights on EC metabolism and discuss current knowledge on how atherosclerosis deregulates EC metabolism, and how maladaptation of deregulated EC metabolism can contribute to atherosclerosis progression. We will also highlight possible therapeutic avenues, based on targeting EC metabolism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

In vivo microscopic imaging of the bronchial mucosa using an endo-cytoscopy system.

PubMed

Shibuya, Kiyoshi; Fujiwara, Taiki; Yasufuku, Kazuhiro; Alaa, Mohamed; Chiyo, Masako; Nakajima, Takahiro; Hoshino, Hidehisa; Hiroshima, Kenzo; Nakatani, Yukio; Yoshino, Ichiro

2011-05-01

We investigated the capabilities of an endo-cytoscopy system (ECS) that enables microscopic imaging of the tracheobronchial tree during bronchoscopy, including normal bronchial epithelium, dysplastic mucosa and squamous cell carcinoma. The newly developed ECS has a 3.2 mm diameter that can be passed through the 4.2 mm working channel of a mother endoscope for insertion of the ECS. It has a high magnification of 570× on a 17 in. video monitor. Twenty-two patients (7 squamous cell carcinoma, 11 squamous dysplasia and 4 after PDT therapies) were underwent white light, NBI light and AFI bronchoscopy. Both abnormal areas of interest and normal bronchial mucosa were stained with 0.5% methylene blue and examined with ECS at high magnification (570×). Histological examinations using haematoxylin and eosin staining were made of biopsied specimens. Analyzed ECS images were compared with the corresponding histological examinations. In normal bronchial mucosa, ciliated columnar epithelial cells were visible. In bronchial squamous dysplasia, superficial cells with abundant cytoplasm were arranged regularly. In squamous cell carcinoma, large, polymorphic tumor cells showed increased cellular densities with irregular stratified patterns. These ECS images corresponded well with the light-microscopic examination of conventional histology. ECS was useful for the discrimination between normal bronchial epithelial cells and dysplastic cells or malignant cells during bronchoscopy in real time. This novel technology has an excellent potential to provide in vivo diagnosis during bronchoscopic examinations. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

AIP1-mediated stress signaling in atherosclerosis and arteriosclerosis

PubMed Central

Zhang, Jiqin; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

2016-01-01

AIP1 (encoded by the DAB2IP gene), a signaling scaffolding protein, is abundantly expressed in vascular endothelial cells (EC). While it was initially discovered as an ASK1-interacting protein, AIP1 broadly suppresses inflammatory responses triggered by cytokines and stresses such as TNF, LPS, VEGF and ER stress in EC (therefore AIP1 is an Anti-Inflammatory Protein). Human genome-wide association study (GWAS) has identified DAB2IP gene variants conferring susceptibility to cardiovascular diseases. Consistently, a global or vascular EC-specific deletion of DAB2IP in mice strongly enhances inflammatory responses and exacerbates atherosclerosis and graft arteriosclerosis progression in mouse models. Mechanisms for AIP1 function and regulation associated with human cardiovascular diseases need further investigations. PMID:25732743

MCP-1-Induced Protein Promotes Endothelial-Like and Angiogenic Properties in Human Bone Marrow Monocytic Cells

PubMed Central

Wang, Kangkai; Zhelyabovska, Olga; Saad, Yasser; Kolattukudy, Pappachan E.

2013-01-01

Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3′-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases. PMID:24008336

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

PubMed

Nakasaki, Tae; Tanaka, Toshiyuki; Okudaira, Shinichi; Hirosawa, Michi; Umemoto, Eiji; Otani, Kazuhiro; Jin, Soojung; Bai, Zhongbin; Hayasaka, Haruko; Fukui, Yoshinori; Aozasa, Katsuyuki; Fujita, Naoya; Tsuruo, Takashi; Ozono, Keiichi; Aoki, Junken; Miyasaka, Masayuki

2008-11-01

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

The endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) and synthetic cannabinoid WIN55,212-2 abate the inflammatory activation of human endothelial cells.

PubMed

Wilhelmsen, Kevin; Khakpour, Samira; Tran, Alphonso; Sheehan, Kayla; Schumacher, Mark; Xu, Fengyun; Hellman, Judith

2014-05-09

Although cannabinoids, such as Δ(9)-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB2R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB1R/CB2R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.

Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

PubMed Central

Kim, Jeffrey; Carlson, Morgan E.; Watkins, Bruce A.

2014-01-01

Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS) in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA) are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25 μM of arachidonate (AA) or docosahexaenoate (DHA), 25 μM of EC [anandamide (AEA), 2-arachidonoylglycerol (2-AG), docosahexaenoylethanolamide (DHEA)], 1 μM of CB1 antagonist NESS0327, and CB2 inverse agonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts. PMID:24711795

Antithrombotic Effects of Nur77 and Nor1 Are Mediated Through Upregulating Thrombomodulin Expression in Endothelial Cells.

PubMed

Yang, Ping; Wei, Xin; Zhang, Jian; Yi, Bing; Zhang, Guan-Xin; Yin, Litian; Yang, Xiao-Feng; Sun, Jianxin

2016-02-01

Thrombomodulin is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, antifibrinolytic, and anti-inflammatory activities in the vessel wall. However, the regulation of thrombomodulin expression in ECs remains largely unknown. In this study, we characterized nuclear receptor 4A family as a novel regulator of thrombomodulin expression in vascular ECs. We demonstrated that both nuclear receptors 4A, Nur77 and Nor1, robustly increase thrombomodulin mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus-mediated transduction of Nur77 and Nor1 cDNAs. Moreover, Nur77 deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and vascular endothelial growth factor165-stimulated thrombomodulin expression. Mechanistically, we found that Nur77 and Nor1 increase thrombomodulin expression by acting through 2 different mechanisms. We showed that Nur77 barely affects thrombomodulin promoter activity, but significantly increases thrombomodulin mRNA stability, whereas Nor1 enhances thrombomodulin expression mainly through induction of Kruppel-like factors 2 and 4 in vascular ECs. Furthermore, we demonstrated that both Nur77 and Nor1 significantly increase protein C activity and inhibit tumor necrosis factor α-induced prothrombotic effects in human ECs. Deficiency of Nur77 increases susceptibility to arterial thrombosis, whereas enhanced expression of Nur77 and Nor1 protects mice from arterial thrombus formation. Our results identified nuclear receptors 4A as novel regulators of thrombomodulin expression and function in vascular ECs and provided a proof-of-concept demonstration that targeted increasing expression of Nur77 and Nor1 in the vascular endothelium might represent a novel therapeutic approach for the treatment of thrombotic disorders. © 2015 American Heart Association, Inc.

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells*

PubMed Central

Wilhelmsen, Kevin; Khakpour, Samira; Tran, Alphonso; Sheehan, Kayla; Schumacher, Mark; Xu, Fengyun; Hellman, Judith

2014-01-01

Although cannabinoids, such as Δ9-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB2R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB1R/CB2R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation. PMID:24644287

Enhancement of CCL15 expression and monocyte adhesion to endothelial cells (ECs) after hypoxia/reoxygenation and induction of ICAM-1 expression by CCL15 via the JAK2/STAT3 pathway in ECs.

PubMed

Park, Keun Hyung; Lee, Tae Hoon; Kim, Chan Woo; Kim, Jiyoung

2013-06-15

CCL15, a member of the CC chemokine family, is a potent chemoattractant for leukocytes and endothelial cells (ECs). Given that chemokines play key roles in vascular inflammation, we investigated the effects of hypoxia/reoxygenation (H/R) on expression of human CCL15 and a role of CCL15 in upregulating ICAM-1 in ECs. We found that exposure of ECs to H/R increased expression of CCL15 and ICAM-1, which resulted in an increase in monocyte adhesivity to the ECs. Further studies revealed that knockdown of CCL15 or CCR1 attenuated expression of ICAM-1 in ECs after H/R, suggesting that expression of ICAM-1 is upregulated by CCL15. Stimulation of ECs with CCL15 significantly increased expression of ICAM-1 predominantly via the CCR1 receptor. We observed that phosphorylation of JAK2 and STAT3 was stimulated by CCL15 treatment of ECs. Results from reporter and chromatin immunoprecipitation assays revealed that CCL15 activates transcription from the IFN-γ activation site promoter and stimulates binding of STAT3 to the ICAM-1 promoter. Our data also showed that CCL15 increased cell adhesion of human monocytes to ECs under static and shear-stress conditions. Pretreatment of these cells with inhibitors for JAK, PI3K, and AKT prevented the CCL15-induced expression of ICAM-1 and monocyte adhesion to ECs, suggesting the involvement of those signaling molecules in ICAM-1 gene activation by CCL15. The results suggest that CCR1 and its ligands may be a potential target for treating inflammatory diseases involving upregulation of cell adhesion molecules.

Defining the extent of cables loss in endometrial cancer subtypes and its effectiveness as an inhibitor of cell proliferation in malignant endometrial cells in vitro and in vivo.

PubMed

DeBernardo, Robert L; Littell, Ramey D; Luo, Hongwei; Duska, Linda R; Oliva, Esther; Kirley, Sandra D; Lynch, Maureen P; Zukerberg, Lawrence R; Rueda, Bo R

2005-01-01

Loss of Cables expression is associated with a high incidence of endometrial hyperplasia and endometrial adenocarcinoma in humans. The Cables mutant mouse develops endometrial hyperplasia and following exposure to chronic estrogen develops early endometrial adenocarcinoma. The objectives of the current study were to determine if: (1) loss of Cables expression occurred in high grade endometrioid adenocarcinoma, uterine serous and clear cell carcinoma as observed in endometrial hyperplasia and low grade endometrial adenocarcinoma; (2) overexpression of Cables inhibited cell proliferation in endometrial cancer (EC) cells in vitro and in vivo; and (3) progesterone could regulate the expression of Cables mRNA. Hyperplastic endometrium and low and high grade endometrioid adenocarcinoma showed loss of Cables expression when compared to benign control secretory endometrium. Loss of Cables expression in serous and clear cell tumors was similar to that observed in endometrioid adenocarcinomas with greater than 80% showing loss of protein expression. Treatment of EC lines with progesterone increased cables expression in low-grade EC whereas it had no effect on cables expression in cells derived from high-grade EC. The progesterone-induced increase in cables was abrogated in the presence of a progesterone receptor (PR) antagonist, suggesting the PR mediates the increase. Cables overexpression inhibited cell proliferation of well differentiated EC cells and had no effect on the poorly differentiated EC cells. The capacity to form tumors was dramatically reduced in the Cables overexpressing cell lines compared to those cells containing the control vector. Collectively these results suggest that Cables is an important regulator of cell proliferation and loss of Cables expression contributes to the development of all types of EC.

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

PubMed

Graham, Mindy Kim; Principessa, Lorenzo; Antony, Lizamma; Meeker, Alan K; Isaacs, John T

2017-03-01

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16 INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

PubMed Central

Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

2014-01-01

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells. PMID:24830447

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

PubMed

Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

2014-05-16

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

Maintenance of HIV-Specific Memory B-Cell Responses in Elite Controllers Despite Low Viral Burdens.

PubMed

Buckner, Clarisa M; Kardava, Lela; Zhang, Xiaozhen; Gittens, Kathleen; Justement, J Shawn; Kovacs, Colin; McDermott, Adrian B; Li, Yuxing; Sajadi, Mohammad M; Chun, Tae-Wook; Fauci, Anthony S; Moir, Susan

2016-08-01

Human immunodeficiency virus (HIV)-specific B-cell responses in infected individuals are maintained by active HIV replication. Suppression of viremia by antiretroviral therapy (ART) leads to quantitative and qualitative changes that remain unclear. Accordingly, B-cell responses were investigated in elite controllers (ECs), who maintain undetectable HIV levels without ART, and in individuals whose viremia was suppressed by ART. Despite a higher HIV burden in the ART group, compared with the EC group, frequencies of HIV-specific B cells were higher in the EC group, compared with those in the ART group. However, the initiation of ART in several ECs was associated with reduced frequencies of HIV-specific B cells, suggesting that responses are at least in part sustained by HIV replication. Furthermore, B-cell responses to tetanus toxin but not influenza hemagglutinin in the ART group were lower than those in the EC group. Thus, the superior HIV-specific humoral response in ECs versus ART-treated individuals is likely due to a more intact humoral immune response in ECs and/or distinct responses to residual HIV replication. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

PubMed Central

Jambou, Ronan; Combes, Valery; Jambou, Marie-Jose; Weksler, Babeth B.; Couraud, Pierre-Olivier; Grau, Georges E.

2010-01-01

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria. PMID:20686652

Cell Selective Cardiovascular Biology of Microsomal Prostaglandin E Synthase-1

PubMed Central

Chen, Lihong; Yang, Guangrui; Xu, Xiufeng; Grant, Gregory; Lawson, John A.; Bohlooly-Y, Mohammad; FitzGerald, Garret A.

2013-01-01

Background Global deletion of microsomal prostaglandin E synthase (mPGES) -1 in mice attenuates the response to vascular injury without a predisposition to thrombogenesis or hypertension. However, enzyme deletion results in cell specific differential utilization by prostaglandin (PG) synthases of the accumulated PGH2 substrate. Here, we generated mice deficient in mPGES-1 in vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and myeloid cells further to elucidate the cardiovascular function of this enzyme. Methods and Results VSMC and EC mPGES-1 deletion did not alter blood pressure at baseline or in response to a high salt diet. The propensity to evoked macrovascular and microvascular thrombogenesis was also unaltered. However, both VSMC and EC mPGES-1 deficient mice exhibited a markedly exaggerated neointimal hyperplastic response to wire injury of the femoral artery compared to their littermate controls. The hyperplasia was associated with increased proliferating cell nuclear antigen (PCNA) and tenascin-C (TN-C) expression. In contrast, the response to injury was markedly suppressed by myeloid cell depletion of mPGES-1 with decreased hyperplasia, leukocyte infiltration and expression of PCNA and TN-C. Conditioned medium derived from mPGES-1 deficient macrophages less potently induced VSMC proliferation and migration than that from wild type macrophages. Conclusion Deletion of mPGES-1 in the vasculature and myeloid cells differentially modulates the response to vascular injury, implicating macrophage mPGES-1 as a cardiovascular drug target. PMID:23204105

Molecular and Cellular Characterization of Space Flight Effects on Microvascular Endothelial Cell Function - PreparatoryWork for the SFEF Project

NASA Astrophysics Data System (ADS)

Balsamo, Michele; Barravecchia, Ivana; Mariotti, Sara; Merenda, Alessandra; De Cesari, Chiara; Vukich, Marco; Angeloni, Debora

2014-12-01

Exposure to microgravity during space flight (SF) of variable length induces suffering of the endothelium (the cells lining all blood vessels), mostly responsible for health problems found in astronauts and animals returning from space. Of interest to pre-nosological medicine, the effects of microgravity on astronauts are strikingly similar to the consequences of sedentary life, senescence and degenerative diseases on Earth, although SF effects are accelerated and reversible. Thus, microgravity is a significant novel model for better understanding of common pathologies. A comprehensive cell and molecular biology study is needed in order to explain pathophysiological findings after SFs. This project will study the effects of microgravity and cosmic radiation on endothelial cells (ECs) cultured on the International Space Station through analysis of 1) cell transcriptome, 2) DNA methylome, 3) DNA damage and cell senescence, 4) variations in cell cycle and cell morphology. This project has been selected by the European Space Agency and the Italian Space Agency and is presently in preparation. The ground study presented here was performed to determine the biological and engineering requirements that will allow us to retrieve suitable samples after culturing, fixing and storing ECs in space. We expect to identify molecular pathways activated by space microgravity in microvascular ECs, which may shed light on pathogenic molecular mechanisms responsible for endothelial suffering shared by astronauts and individuals affected with aging, degenerative and sedentary life-associated pathologies on Earth.

Increased numbers of circulating ECs are associated with systemic GVHD.

PubMed

Yan, Z; Zeng, L; Jia, L; Xu, S; Ding, S

2011-10-01

Circulating endothelial cells (ECs) are known to reflect endothelial injury, and endothelial injury is associated with graft-versus-host disease (GVHD). We hypothesised that circulating ECs might be associated with systemic acute graft-versus-host disease (aGVHD). BALB/c (H-2k(d) ) mice were treated with total body irradiation and then infused with C57B/6-derived T-cell-depleted bone marrow (TCD-BM) cells or TCD-BM cells and splenocytes. Cyclosporine was used to prevent aGVHD. Circulating ECs and allogeneic lymphocytes were analysed by flow cytometry at multiple time points. The morphology and ultrastructure of the endothelium were examined by light microscopy or transmission electron microscopy. The results indicated that the number of circulating ECs peaked at day 5 after lethal irradiation in all mice; allogenic transplanted mice (TCD-BM cells and splenocytes) developed typical aGVHD beginning at day 7, exhibiting both histological and clinical symptoms of disease. Circulating ECs peaked a second time at day 9 with aGVHD progression. However, following the administration of CSA, an absence of or a reduction in the amount of subsequent endothelial injury was observed. Circulating ECs might be associated with systemic aGVHD. © 2011 Blackwell Publishing Ltd.

Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

PubMed

Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Zhang, Wei; Lanza, Robert; Lu, Shi-Jiang; Jonas, Jost B; Xu, Liang

2017-09-01

To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 μl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 μl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. By integrating into damaged retinal vessels and differentiating into ECs, intravitreally administered hESC-HBs may have partially repaired a retinal vascular injury caused by OIR model and DR. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Cerebroprotection of Flavanol (−)-Epicatechin after Traumatic Brain Injury via Nrf2-dependent and –independent Pathways

PubMed Central

Cheng, Tian; Wang, Wenzhu; Li, Qian; Han, Xiaoning; Xing, Jing; Qi, Cunfang; Lan, Xi; Wan, Jieru; Potts, Alexa; Guan, Fangxia; Wang, Jian

2016-01-01

Traumatic brain injury (TBI), which leads to disability, dysfunction, and even death, is a prominent health problem worldwide with no effective treatment. A brain-permeable flavonoid named (−)-epicatechin (EC) modulates redox/oxidative stress and has been shown to be beneficial for vascular and cognitive function in humans and for ischemic and hemorrhagic stroke in rodents. Here we examined whether EC is able to protect the brain against TBI-induced brain injury in mice and if so, whether it exerts neuroprotection by modulating the NF-E2-related factor (Nrf2) pathway. We used the controlled cortical impact model to mimic TBI. EC was administered orally at 3 h after TBI and then every 24 h for either 3 or 7 days. We evaluated lesion volume, brain edema, white matter injury, neurologic deficits, cognitive performance and emotion-like behaviors, neutrophil infiltration, reactive oxygen species (ROS), and a variety of injury-related protein markers. Nrf2 knockout mice were used to determine the role of the Nrf2 signaling pathway after EC treatment. In wild-type mice, EC significantly reduced lesion volume, edema, and cell death and improved neurologic function on days 3 and 28; cognitive performance and depression-like behaviors were also improved with EC administration. In addition, EC reduced white matter injury, heme oxygenase-1 expression, and ferric iron deposition after TBI. These changes were accompanied by attenuation of neutrophil infiltration and oxidative insults, reduced activity of matrix metalloproteinase 9, decreased Keap 1 expression, increased Nrf2 nuclear accumulation, and increased expression of superoxide dismutase 1 and quinone 1. However, EC did not significantly reduce lesion volume or improve neurologic deficits in Nrf2 knockout mice after TBI. Our results show that EC protects the TBI brain by activating the Nrf2 pathway, inhibiting heme oxygenase-1 protein expression, and reducing iron deposition. The latter two effects could represent an Nrf2-independent mechanism in this model of TBI. PMID:26724590

Ester-Based Electrolytes for Low-Temperature Li-Ion Cells

NASA Technical Reports Server (NTRS)

Smart, Marshall; Bugga, Ratnakumar

2005-01-01

Electrolytes comprising LiPF6 dissolved at a concentration of 1.0 M in five different solvent mixtures of alkyl carbonates have been found to afford improved performance in rechargeable lithium-ion electrochemical cells at temperatures as low as -70 C. These and other electrolytes have been investigated in continuing research directed toward extending the lower limit of practical operating temperatures of Li-ion cells. This research at earlier stages, and the underlying physical and chemical principles, were reported in numerous previous NASA Tech Briefs articles, the most recent being Low-EC-Content Electrolytes for Low-Temperature Li-Ion Cells (NPO-30226), NASA Tech Briefs, Vol. 27, No. 1 (January 2003), page 46. The ingredients of the present solvent mixtures are ethylene carbonate (EC), ethyl methyl carbonate (EMC), methyl butyrate (MB), methyl propionate (MP), ethyl propionate (EP), ethyl butyrate (EB), and ethyl valerate (EV). In terms of volume proportions of these ingredients, the present solvent mixtures are 1EC + 1EMC + 8MB, 1EC + 1EMC + 8EB, 1EC + 1EMC + 8MP, 1EC + 1EMC + 8EV, and 1EC + 9EMC. These electrolytes were placed in Liion cells containing carbon anodes and LiNi0.8Co0.2O2 cathodes, and the low-temperature electrical performances of the cells were measured. The cells containing the MB and MP mixtures performed best.

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC)

PubMed Central

Arosa, F A; Irwin, C; Mayer, L; De Sousa, M; Posnett, D N

1998-01-01

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28− phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8+ CD28− T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8+ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8+ CD28+ and CD8+ CD28− T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8+ CD28− cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8+ CD28− phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vβ subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8+ CD28− T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8+ CD28− T cells in the iIEL compartment. PMID:9649184

Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers.

PubMed

Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu; Hickman, Taylor; Cronin, Jacqueline; Pimenova, Dina; Walker, Bruce D; Lichterfeld, Mathias; Yu, Xu G

2015-06-01

The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes.

Differential activation of human T cells to allogeneic endothelial cells, epithelial cells and fibroblasts in vitro

PubMed Central

2012-01-01

Background In the direct pathway, T cells recognize intact donor major histocompatability complexes and allogeneic peptide on the surface of donor antigen presenting cells (APCs). Indirect allorecognition results from the recognition of processed alloantigen by self MHC complexes on self APCs. In this study, we wished to evaluate the relative contribution of different intragraft cells to the alloactivation of nave and memory T cells though the direct and the indirect pathway of allorecognition. Methods The processing of membrane fragments from IFN-treated single donor endothelial cells (EC), fibroblasts or renal epithelial cells (RPTEC) was evaluated by DiOC labeling of each cell type and flow cytometry following interaction with PBMC. Direct pathway activation of nave CD45RA+ or memory CD45RO+ CD4+ T cells was evaluated following coculture with IFN-treated and MHC class II-expressing EC, fibroblasts or RPTEC. Indirect pathway activation was assessed using CD45RA+ or CD45RO+ CD4+ T cells cocultured with autologous irradiated APCs in the absence or presence of sonicates derived from IFN-treated allogeneic EC, fibroblasts or RPTEC. Activation of T cells was assessed by [3H]thymidine incorporation and by ELISpot assays. Results We find that CD14+ APCs readily acquire membrane fragments from fibroblasts and RPTEC, but fail to acquire membrane fragments from intact EC. However, APCs process membranes from EC undergoing apoptosis.There was a notable direct pathway alloproliferative response of CD45RO+ CD4+ T cells to IFN-treated EC, but not to fibroblasts or RPTEC. Also, there was a minimal direct pathway response of CD45RA+ CD4+ T cells to all cell types. In contrast, we found that both CD45RA+ and CD45RO+ CD4+ T cells proliferated following coculture with autologous APCs in the presence of sonicates derived from IFN-treated EC, fibroblasts or RPTEC. By ELISpot, we found that these T cells stimulated via the indirect pathway also produced the cytokines IFN, IL-2, IL-4 and IL-5. Conclusions Recipient APCs may readily process membrane fragments from allogeneic intragraft cells, but not from EC unless they are undergoing apoptosis. This processing is sufficient for indirect pathway alloactivation of both CD45RA+ and CD45RO+ CD4+ T cells. Only graft vascular EC mediate direct pathway reactivation of CD4+ T cells. PMID:23369287

Initial investigation of SU-8 photopolymer as a material for noninvasive endothelial cell research platforms

NASA Astrophysics Data System (ADS)

Westwood, S.; Gojova, A.; Kuo, B.; Barakat, A. I.; Gray, B. L.

2007-01-01

This paper presents a preliminary investigation in the usage of the micromachining polymer material SU-8 for the noninvasive shape control and functional study of vascular endothelial cells (ECs). We previously demonstrated a silicon and glass modular microinstrument platform that allowed for a wide range of EC functional response studies. However, we expect SU-8 to provide a more versatile fabrication technology and material for microchannel fabrication and instrumentation, since it is capable of achieving high aspect ratio sensor-compatible structures through simple photopatterning. In this paper, SU-8 microchannels were fabricated on glass slides for straightforward optical observation and biological sampling. Channel widths ranged from 50 to 210 µm, length varied from 100 to 2100 µm, with depth fixed at 100 µm. We plated bovine aortic endothelial cells (BAECs) in the microchannels and used image analysis to determine cellular elongation and orientation. Similar to silicon-on-glass microchannels, the cells become more elongated and oriented along the microchannel axis as the width of the microchannel decreases. Initial results indicate cells plate in the microchannels and on the SU-8 surfaces, whereas in a previous silicon microchannel study, cells plated exclusively on the glass bottom surfaces. This finding has implications for SU-8 as a structural material for microchannel instrumentation.

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

PubMed Central

Mir, Enrique; Rovira, Montse; Escolar, Ginés; Carreras, Enric; Diaz-Ricart, Maribel

2016-01-01

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. PMID:26755708

MicroRNA-372 inhibits endometrial carcinoma development by targeting the expression of the Ras homolog gene family member C (RhoC)

PubMed Central

Liu, Bo-Liang; Sun, Kai-Xuan; Zong, Zhi-Hong; Chen, Shuo; Zhao, Yang

2016-01-01

Here we explore the role of microRNA-372 (miR-372) in tumorigenesis and development of endometrial adenocarcinoma (EC) and analyze the underlying mechanism. We found that miR-372 expression is much lower in EC than normal endometrial specimens. Cell function experiments demonstrated that miR-372 overexpression suppressed cell proliferation, migration, and invasion, and led to a G1 phase arrest and promoted the apoptosis of endometrial carcinoma cells in vitro. The nude mouse xenograft assay demonstrated that miR-372 overexpression suppressed tumor growth. RT-PCR and Western blot assays detected the expression of known targets of miR-372 in other malignant tumors and found Cyclin A1 and Cyclin-dependent Kinase 2 (CDK2) was downregulated by miR-372. Bioinformatic predictions and dual-luciferase reporter assays found that RhoC was a possible target of miR-372. RT-PCR and Western blot assays demonstrated that miR-372 transfection reduced the expression of RhoC, matrix metalloproteinase 2 (MMP2) and MMP9, while it increased the expression of cleaved poly (ADP ribose) polymerase (PARP) and bcl-2-associated X protein (Bax). The cell function experiments that transfected siRNA with RhoC showed the same trend as those which were transfected with miR-372. Taken together, our results demonstrated for the first time that miR-372 suppresses tumorigenesis and the development of EC; RhoC is a new and potentially important therapeutic target. PMID:26673619

Adhesion behavior of endothelial progenitor cells to endothelial cells in simple shear flow

NASA Astrophysics Data System (ADS)

Gong, Xiao-Bo; Li, Yu-Qing; Gao, Quan-Chao; Cheng, Bin-Bin; Shen, Bao-Rong; Yan, Zhi-Qiang; Jiang, Zong-Lai

2011-12-01

The adhesion of endothelial progenitor cells (EPCs) on endothelial cells (ECs) is one of the critical physiological processes for the regenesis of vascular vessels and the prevention of serious cardiovascular diseases. Here, the rolling and adhesion behavior of EPCs on ECs was studied numerically. A two-dimensional numerical model was developed based on the immersed boundary method for simulating the rolling and adhesion of cells in a channel flow. The binding force arising from the catch bond of a receptor and ligand pair was modeled with stochastic Monte Carlo method and Hookean spring model. The effect of tumor necrosis factor alpha (TNF- α) on the expression of the number of adhesion molecules in ECs was analyzed experimentally. A flow chamber system with CCD camera was set up to observe the top view of the rolling of EPCs on the substrate cultivated with ECs. Numerical results prove that the adhesion of EPC on ECs is closely related to membrane stiffness of the cell and shear rate of the flow. It also suggests that the adhesion force between EPC and EC by P-selectin glycoprotein ligand-1 only is not strong enough to bond the cell onto vessel walls unless contributions of other catch bond are considered. Experimental results demonstrate that TNF- α enhanced the expressions of VCAM, ICAM, P-selectin and E-selectin in ECs, which supports the numerical results that the rolling velocity of EPC on TNF- α treated EC substrate decreases obviously compared with its velocity on the untreated one. It is found that because the adhesion is affected by both the rolling velocity and the deformability of the cell, an optimal stiffness of EPC may exist at a given shear rate of flow for achieving maximum adhesion rates.

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2–mediated heparin-binding EGF signaling in endothelial cells

PubMed Central

Svensson, Katrin J.; Kucharzewska, Paulina; Christianson, Helena C.; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C.; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-01-01

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein–coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2–mediated activation of hypoxic ECs. We show that PAR-2–dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2–dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa–dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2–mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors. PMID:21788507

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.

PubMed

Svensson, Katrin J; Kucharzewska, Paulina; Christianson, Helena C; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-08-09

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

PubMed

Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

2017-11-01

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

Evaluation of ex vivo produced endothelial progenitor cells for autologous transplantation in primates.

PubMed

Qin, Meng; Guan, Xin; Zhang, Yu; Shen, Bin; Liu, Fang; Zhang, Qingyu; Ma, Yupo; Jiang, Yongping

2018-01-22

Autologous transplantation of endothelial progenitor cells (EPCs) is a promising therapeutic approach in the treatment of various vascular diseases. We previously reported a two-step culture system for scalable generation of human EPCs derived from cord blood CD34 + cells ex vivo. Here, we now apply this culture system to expand and differentiate human and nonhuman primate EPCs from mobilized peripheral blood (PB) CD34 + cells for the therapeutic potential of autologous transplantation. The human and nonhuman primate EPCs from mobilized PB CD34 + cells were cultured according to our previously reported system. The generated adherent cells were then characterized by the morphology, surface markers, nitric oxide (NO)/endothelial NO synthase (eNOS) levels and Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake/fluorescein isothiocyanate (FITC)-lectin binding actives. Furthermore, the efficacy and safety studies were performed by autologous transplantation via hepatic portal vein injection in a nonhuman primate model with acute liver sinusoidal endothelial cell injury. The mobilized PB CD34 + cells from both human and nonhuman primate were efficiently expanded and differentiated. Over 2 × 10 8 adherent cells were generated from 20 mL mobilized primate PB (1.51 × 10 6  ± 3.39 × 10 5 CD34 + cells) by 36-day culture and more than 80% of the produced cells were identified as EPCs/endothelial cells (ECs). In the autologous transplant model, the injected EPC/ECs from nonhuman primate PB were scattered in the intercellular spaces of hepatocytes at the hepatic tissues 14 days post-transplantation, indicating successful migration and reconstitution in the liver structure as the functional EPCs/ECs. We successfully applied our previous two-step culture system for the generation of primate EPCs from mobilized PB CD34 + cells, evaluated the phenotypes ex vivo, and transplanted autologous EPCs/ECs in a nonhuman primate model. Our study indicates that it may be possible for these ex-vivo high-efficient expanded EPCs to be used in clinical cell therapy.

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Hydraulic conductivity of endothelial cell-initiated arterial cocultures.

PubMed

Mathura, Rishi A; Russell-Puleri, Sparkle; Cancel, Limary M; Tarbell, John M

2014-04-01

This study describes cocultures of arterial smooth muscle cells (SMCs) and endothelial cells (ECs) and the influences of their heterotypic interactions on hydraulic conductivity (L p ), an important transport property. A unique feature of these cocultures is that ECs were first grown to confluence and then SMCs were inoculated. Bovine aortic smooth muscle cells and bovine aortic endothelial cells (BAECs) were cocultured on Transwell Permeable Supports, and then exposed to a pressure-driven transmural flow. L p across each culture was measured using a bubble tracking apparatus that determined water flux (J v ). Our results indicate that arterial L p is significantly modulated by EC-SMC proximity, and serum content in culture. The L p of cocultures was also compared to the predictions of a resistances-in-series model to distinguish the contributions of heterotypic interactions between SMCs and ECs. Conditions that lead to significantly reduced coculture L p , compared to BAEC monoculture controls, have been uncovered and the lowest L p in the literature for an in vitro system are reported. In addition, VE-cadherin immunostaining of intact BAEC monolayers in each culture configuration reveals that EC-SMC proximity on a porous membrane has a dramatic influence on EC morphology patterns. The cocultures with the lowest L p have ECs with significantly elongated morphology. Confocal imaging indicates that there are no direct EC-SMC contacts in coculture.

Effects of Disturbed Flow on Vascular Endothelium: Pathophysiological Basis and Clinical Perspectives

PubMed Central

Chiu, Jeng-Jiann; Chien, Shu

2013-01-01

Vascular endothelial cells (ECs) are exposed to hemodynamic forces, which modulate EC functions and vascular biology/pathobiology in health and disease. The flow patterns and hemodynamic forces are not uniform in the vascular system. In straight parts of the arterial tree, blood flow is generally laminar and wall shear stress is high and directed; in branches and curvatures, blood flow is disturbed with nonuniform and irregular distribution of low wall shear stress. Sustained laminar flow with high shear stress upregulates expressions of EC genes and proteins that are protective against atherosclerosis, whereas disturbed flow with associated reciprocating, low shear stress generally upregulates the EC genes and proteins that promote atherogenesis. These findings have led to the concept that the disturbed flow pattern in branch points and curvatures causes the preferential localization of atherosclerotic lesions. Disturbed flow also results in postsurgical neointimal hyperplasia and contributes to pathophysiology of clinical conditions such as in-stent restenosis, vein bypass graft failure, and transplant vasculopathy, as well as aortic valve calcification. In the venous system, disturbed flow resulting from reflux, outflow obstruction, and/or stasis leads to venous inflammation and thrombosis, and hence the development of chronic venous diseases. Understanding of the effects of disturbed flow on ECs can provide mechanistic insights into the role of complex flow patterns in pathogenesis of vascular diseases and can help to elucidate the phenotypic and functional differences between quiescent (nonatherogenic/nonthrombogenic) and activated (atherogenic/thrombogenic) ECs. This review summarizes the current knowledge on the role of disturbed flow in EC physiology and pathophysiology, as well as its clinical implications. Such information can contribute to our understanding of the etiology of lesion development in vascular niches with disturbed flow and help to generate new approaches for therapeutic interventions. PMID:21248169

Fluid and mass transport modelling to drive the design of cell-packed hollow fibre bioreactors for tissue engineering applications.

PubMed

Shipley, Rebecca J; Waters, Sarah L

2012-12-01

A model for fluid and mass transport in a single module of a tissue engineering hollow fibre bioreactor (HFB) is developed. Cells are seeded in alginate throughout the extra-capillary space (ECS), and fluid is pumped through a central lumen to feed the cells and remove waste products. Fluid transport is described using Navier-Stokes or Darcy equations as appropriate; this is overlaid with models of mass transport in the form of advection-diffusion-reaction equations that describe the distribution and uptake/production of nutrients/waste products. The small aspect ratio of a module is exploited and the option of opening an ECS port is explored. By proceeding analytically, operating equations are determined that enable a tissue engineer to prescribe the geometry and operation of the HFB by ensuring the nutrient and waste product concentrations are consistent with a functional cell population. Finally, results for chondrocyte and cardiomyocyte cell populations are presented, typifying two extremes of oxygen uptake rates.

Lyophilized plasma attenuates vascular permeability, inflammation and lung injury in hemorrhagic shock.

PubMed

Pati, Shibani; Peng, Zhanglong; Wataha, Katherine; Miyazawa, Byron; Potter, Daniel R; Kozar, Rosemary A

2018-01-01

In severe trauma and hemorrhage the early and empiric use of fresh frozen plasma (FFP) is associated with decreased morbidity and mortality. However, utilization of FFP comes with the significant burden of shipping and storage of frozen blood products. Dried or lyophilized plasma (LP) can be stored at room temperature, transported easily, reconstituted rapidly with ready availability in remote and austere environments. We have previously demonstrated that FFP mitigates the endothelial injury that ensues after hemorrhagic shock (HS). In the current study, we sought to determine whether LP has similar properties to FFP in its ability to modulate endothelial dysfunction in vitro and in vivo. Single donor LP was compared to single donor FFP using the following measures of endothelial cell (EC) function in vitro: permeability and transendothelial monolayer resistance; adherens junction preservation; and leukocyte-EC adhesion. In vivo, using a model of murine HS, LP and FFP were compared in measures of HS- induced pulmonary vascular inflammation and edema. Both in vitro and in vivo in all measures of EC function, LP demonstrated similar effects to FFP. Both FFP and LP similarly reduced EC permeability, increased transendothelial resistance, decreased leukocyte-EC binding and persevered adherens junctions. In vivo, LP and FFP both comparably reduced pulmonary injury, inflammation and vascular leak. Both FFP and LP have similar potent protective effects on the vascular endothelium in vitro and in lung function in vivo following hemorrhagic shock. These data support the further development of LP as an effective plasma product for human use after trauma and hemorrhagic shock.

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus

PubMed Central

Lu, Yingjuan; Parker, Nikki; Kleindl, Paul J; Cross, Vicky A; Wollak, Kristin; Westrick, Elaine; Stinnette, Torian W; Gehrke, Mark A; Wang, Kevin; Santhapuram, Hari Krishna R; You, Fei; Hahn, Spencer J; Vaughn, Jeremy F; Klein, Patrick J; Vlahov, Iontcho R; Low, Philip S; Leamon, Christopher P

2015-01-01

Folate receptor (FR)-β has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid–derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a “macrophage-rich” model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted. PMID:26181632

Targeting of tumor endothelial cells combining 2 Gy/day of X-ray with Everolimus is the effective modality for overcoming clinically relevant radioresistant tumors

PubMed Central

Kuwahara, Yoshikazu; Mori, Miyuki; Kitahara, Shuji; Fukumoto, Motoi; Ezaki, Taichi; Mori, Shiro; Echigo, Seishi; Ohkubo, Yasuhito; Fukumoto, Manabu

2014-01-01

Radiotherapy is widely used to treat cancer because it has the advantage of physically and functionally conserving the affected organ. To improve radiotherapy and investigate the molecular mechanisms of cellular radioresistance, we established a clinically relevant radioresistant (CRR) cell line, SAS-R, from SAS cells. SAS-R cells continue to proliferate when exposed to fractionated radiation (FR) of 2 Gy/day for more than 30 days in vitro. A xenograft tumor model of SAS-R was also resistant to 2 Gy/day of X-rays for 30 days. The density of blood vessels in SAS-R tumors was higher than in SAS tumors. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, sensitized microvascular endothelial cells to radiation, but failed to radiosensitize SAS and SAS-R cells in vitro. Everolimus with FR markedly reduced SAS and SAS-R tumor volumes. Additionally, the apoptosis of endothelial cells (ECs) increased in SAS-R tumor tissues when both Everolimus and radiation were administered. Both CD34-positive and tomato lectin-positive blood vessel densities in SAS-R tumor tissues decreased remarkably after the Everolimus and radiation treatment. Everolimus-induced apoptosis of vascular ECs in response to radiation was also followed by thrombus formation that leads to tumor necrosis. We conclude that FR combined with Everolimus may be an effective modality to overcome radioresistant tumors via targeting tumor ECs. PMID:24464839

Endothelial ERK signaling controls lymphatic fate specification

PubMed Central

Deng, Yong; Atri, Deepak; Eichmann, Anne; Simons, Michael

2013-01-01

Lymphatic vessels are thought to arise from PROX1-positive endothelial cells (ECs) in the cardinal vein in response to induction of SOX18 expression; however, the molecular event responsible for increased SOX18 expression has not been established. We generated mice with endothelial-specific, inducible expression of an RAF1 gene with a gain-of-function mutation (RAF1S259A) that is associated with Noonan syndrome. Expression of mutant RAF1S259A in ECs activated ERK and induced SOX18 and PROX1 expression, leading to increased commitment of venous ECs to the lymphatic fate. Excessive production of lymphatic ECs resulted in lymphangiectasia that was highly reminiscent of abnormal lymphatics seen in Noonan syndrome and similar “RASopathies.” Inhibition of ERK signaling during development abrogated the lymphatic differentiation program and rescued the lymphatic phenotypes induced by expression of RAF1S259A. These data suggest that ERK activation plays a key role in lymphatic EC fate specification and that excessive ERK activation is the basis of lymphatic abnormalities seen in Noonan syndrome and related diseases. PMID:23391722

Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

PubMed Central

Ohkawara, Hiroshi; Ishibashi, Toshiyuki; Sugimoto, Koichi; Ikeda, Kazuhiko; Ogawa, Kazuei; Takeishi, Yasuchika

2014-01-01

Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs. PMID:25162582

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayama, Hironao; Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115; Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295

Class 3 semaphorins were discovered as a family of axon guidance molecules, but are now known to be involved in diverse biologic processes. In this study, we investigated the anti-angiogenic potential of SEMA3E and SEMA3F (SEMA3E&F) in infantile hemangioma (IH). IH is a common vascular tumor that involves both vasculogenesis and angiogenesis. Our lab has identified and isolated hemangioma stem cells (HemSC), glucose transporter 1 positive (GLUT1{sup +}) endothelial cells (designated as GLUT1{sup sel} cells) based on anti-GLUT1 magnetic beads selection and GLUT1-negative endothelial cells (named HemEC). We have shown that these types of cells play important roles in hemangiogenesis.more » We report here that SEMA3E inhibited HemEC migration and proliferation while SEMA3F was able to suppress the migration and proliferation in all three types of cells. Confocal microscopy showed that stress fibers in HemEC were reduced by SEMA3E&F and that stress fibers in HemSC were decreased by SEMA3F, which led to cytoskeletal collapse and loss of cell motility in both cell types. Additionally, SEMA3E&F were able to inhibit vascular endothelial growth factor (VEGF)-induced sprouts in all three types of cells. Further, SEMA3E&F reduced the level of p-VEGFR2 and its downstream p-ERK in HemEC. These results demonstrate that SEMA3E&F inhibit IH cell proliferation and suppress the angiogenic activities of migration and sprout formation. SEMA3E&F may have therapeutic potential to treat or prevent growth of highly proliferative IH. - Highlights: • SEMA3E&F reduce actin stress fibers and induce cytoskeletal collapse in HemEC. • SEMA3E&F inhibit angiogenic activities of HemEC. • SEMA3E&F can interrupt the VEGF-A-VEGFR2-ERK signaling pathway in HemEC. • Plexin D1 and NRP2 are induced during HemSC/GLUT1{sup sel}-to-EC differentiation.« less

Cytokine-mediated induction of endothelial adhesion molecule and histocompatibility leukocyte antigen expression by cytomegalovirus-activated T cells.

PubMed Central

Waldman, W. J.; Knight, D. A.

1996-01-01

Cytomegalovirus (CMV) has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system; however, mechanisms by which such interaction might exacerbate the rejection process remain unresolved. Here we test the hypothesis that host immune activity, triggered by CMV-infected graft endothelial cells (ECs), can result in the production of cytokines capable of enhancing the alloimmunogenicity of nearby uninfected endothelia. To model these phenomena in vitro, confluent monolayers of ECs derived from human umbilical vein or adult gonadal vein were incubated 5 days beneath trans-well culture inserts containing CMV-seropositive or CMV-seronegative donor-derived CD3+ or CD4+ T cells alone or in combination with CMV-infected or uninfected allogeneic ECs. The extent of T cell proliferation was determined by [3H]thymidine labeling of trans-well contents after transfer to microtiter plates. Endothelial responses to soluble factors elaborated by CMV-activated T cells were determined by immunohistochemical staining and immunofluorescence flow cytometric analysis of underlying EC monolayers. Results of experiments with CMV-seropositive donor-derived CD4+ T cells demonstrated enhancement of ICAM-1 and histocompatibility leukocyte antigen class I, as well as induction of histocompatibility leukocyte antigen DR on ECs incubated beneath T cell/EC/CMV trans-well co-cultures. Total (CD3+) T cells co-cultured with EC/CMV induced VCAM-1 as well. Furthermore, [3H]thymidine incorporation by these T cells indicated a strong proliferative response. Endothelial responses to T cells alone or in combination with uninfected ECs were minimal, and T cells cultured under these conditions showed little proliferative activity. Similarly, little or no endothelial responses were apparent in monolayers beneath trans-wells containing T cells isolated from CMV-seronegative individuals regardless of the CMV status of stimulator ECs. Finally, experiments employing blocking antibodies identified interferon-gamma and tumor necrosis factor-alpha as inducing agents in this co-culture system. These findings suggest that allograft endothelium harboring CMV has the potential to activate host T cells and that the consequent release of cytokines shows potential to raise surrounding endothelia to a fully activated, highly immunogenic state. Results of these studies thus provide insight into mechanisms that help elucidate the association between CMV and transplantation-associated arteriosclerosis and/or allograft rejection. Images Figure 1 Figure 5 PMID:8546198

P2Y(2)R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production.

PubMed

Eun, So Young; Park, Sang Won; Lee, Jae Heun; Chang, Ki Churl; Kim, Hye Jung

2014-04-01

Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y2 receptor (P2Y2R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y2R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y2R agonists ATP/UTP for 1h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y2R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y2R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y2R-mediated ROS production. Treatment with oxLDL for 24h induced P2Y2R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y2R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y2R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y2R or RAGE, suggesting that P2Y2R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y2R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer.

PubMed

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-10-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer

PubMed Central

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-01-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer. PMID:28943918

Establishment and characterization of a novel uterine carcinosarcoma cell line, TU-ECS-1, with mutations of TP53 and KRAS.

PubMed

Chiba, Yohei; Sato, Seiya; Itamochi, Hiroaki; Suga, Yasuko; Fukagawa, Tomoyuki; Oumi, Nao; Oishi, Tetsuro; Harada, Tasuku; Sugai, Tamotsu; Sugiyama, Toru

2017-04-01

A new human uterine carcinosarcoma (UCS) cell line, TU-ECS-1, was established and characterized. The morphological appearance of the cultured cells was an insular of epithelial-like cells arranged in the form of a jigsaw puzzle and mesenchymal-like cells with a spindle-shaped or fibroblast-like morphology. A relatively high proliferation rate was observed with a doubling time of 18.2 h. The chromosome number ranged from 44 to 49 and had an extra chromosome 12 (trisomy 12). The respective half-maximal inhibitory concentrations of cisplatin, paclitaxel, and doxorubicin were 2.9 µM, 154 nM, and 219 ng/mL, respectively. Mutational analysis revealed that TU-ECS-1 cells have mutations of TP53 in exons 4, 6, and 8 and of KRAS at codon 12 (G12D) in exon 2, which is a mutation hot spot on this gene. Western blot analysis showed that p53 protein was overexpressed in TU-ECS-1 cells. Immunostaining of the cultured cells and in vivo tumors showed that the TU-ECS-1 cells and xenografts were positive for epithelial marker cytokeratin AE1/3 and mesenchymal marker vimentin. These results suggested that TU-ECS-1 cells might have both epithelial and mesenchymal characteristics. This cell line may be useful to study the carcinogenesis of UCS and contribute to the development of novel treatment strategies.

Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor.

PubMed

Blenau, W; Balfanz, S; Baumann, A

2000-03-01

Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin-induced cyclic AMP production in a dose-dependent manner with an EC50 of approximately 130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of approximately 3 microM) and less efficacious than tyramine. Receptor-encoding mRNA has a wide-spread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher-order brain functions.

The Role of Endothelial Cells in Myofiber Differentiation and the Vascularization and Innervation of Bioengineered Muscle Tissue in vivo

DTIC Science & Technology

2012-10-08

differentiation of co- cultured cells in vivo and in vitro. We first utilized a co- culture of fluorescently labeled endothelial cells (ECs) and muscle...of 10T1/2 cells as pericytes (PCs) to the culture of MPCs and ECs can result in the stabilization of bioengineered vessels [10]. In the current study...Burlingame, CA). 2.2. Cell culture 2.2.1. MPC, EC, PC isolation and culture Green fluorescent protein (GFP)-labeled muscle progenitor cells (GFPþ MPCs

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

PubMed

Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

2017-03-01

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

PubMed Central

Teoh-Fitzgerald, ML; Fitzgerald, MP; Zhong, W; Askeland, RW; Domann, FE

2013-01-01

Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment. PMID:23318435

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

PubMed Central

Cai, Qing; Brissova, Marcela; Reinert, Rachel B.; Pan, Fong Cheng; Brahmachary, Priyanka; Jeansson, Marie; Shostak, Alena; Radhika, Aramandla; Poffenberger, Greg; Quaggin, Susan E.; Jerome, W. Gray; Dumont, Daniel J.; Powers, Alvin C.

2012-01-01

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a “tet-on” inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that 1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; 2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; 3) Angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization. PMID:22546694

Both internalization and AIP1 association are required for tumor necrosis factor receptor 2-mediated JNK signaling.

PubMed

Ji, Weidong; Li, Yonghao; Wan, Ting; Wang, Jing; Zhang, Haifeng; Chen, Hong; Min, Wang

2012-09-01

The proinflammtory cytokine tumor necrosis factor (TNF), primarily via TNF receptor 1 (TNFR1), induces nuclear factor-κB (NF-κB)-dependent cell survival, and c-Jun N-terminal kinase (JNK) and caspase-dependent cell death, regulating vascular endothelial cell (EC) activation and apoptosis. However, signaling by the second receptor, TNFR2, is poorly understood. The goal of this study was to dissect how TNFR2 mediates NF-κB and JNK signaling in vascular EC, and its relevance to in vivo EC function. We show that TNFR2 contributes to TNF-induced NF-κB and JNK signaling in EC as TNFR2 deletion or knockdown reduces the TNF responses. To dissect the critical domains of TNFR2 that mediate the TNF responses, we examine the activity of TNFR2 mutant with a specific deletion of the TNFR2 intracellular region, which contains conserved domain I, domain II, domain III, and 2 TNFR-associated factor-2-binding sites. Deletion analyses indicate that different sequences on TNFR2 have distinct roles in NF-κB and JNK activation. Specifically, deletion of the TNFR-associated factor-2-binding sites (TNFR2-59) diminishes the TNFR2-mediated NF-κB, but not JNK activation; whereas, deletion of domain II or domain III blunts TNFR2-mediated JNK but not NF-κB activation. Interestingly, we find that the TNFR-associated factor-2-binding sites ensure TNFR2 on the plasma membrane, but the di-leucine LL motif within the domain II and aa338-355 within the domain III are required for TNFR2 internalization as well as TNFR2-dependent JNK signaling. Moreover, domain III of TNFR2 is responsible for association with ASK1-interacting protein-1, a signaling adaptor critical for TNF-induced JNK signaling. While TNFR2 containing the TNFR-associated factor-2-binding sites prevents EC cell death, a specific activation of JNK without NF-κB activation by TNFR2-59 strongly induces caspase activation and EC apoptosis. Our data reveal that both internalization and ASK1-interacting protein-1 association are required for TNFR2-dependent JNK and apoptotic signaling. Controlling TNFR2-mediated JNK and apoptotic signaling in EC may provide a novel strategy for the treatment of vascular diseases.

Chemoprevention of Cigarette Smoke–Induced Alterations of MicroRNA Expression in Rat Lungs

PubMed Central

Izzotti, Alberto; Calin, George A.; Steele, Vernon E.; Cartiglia, Cristina; Longobardi, Mariagrazia; Croce, Carlo M.; De Flora, Silvio

2015-01-01

We previously showed that exposure to environmental cigarette smoke (ECS) for 28 days causes extensive downregulation of microRNA expression in the lungs of rats, resulting in the overexpression of multiple genes and proteins. In the present study, we evaluated by microarray the expression of 484 microRNAs in the lungs of either ECS-free or ECS-exposed rats treated with the orally administered chemopreventive agents N-acetylcysteine, oltipraz, indole-3-carbinol, 5,6-benzoflavone, and phenethyl isothiocyanate (as single agents or in combinations). This is the first study of microRNA modulation by chemopreventive agents in nonmalignant tissues. Scatterplot, hierarchical cluster, and principal component analyses of microarray and quantitative PCR data showed that none of the above chemopreventive regimens appreciably affected the baseline microRNA expression, indicating potential safety. On the other hand, all of them attenuated ECS-induced alterations but to a variable extent and with different patterns, indicating potential preventive efficacy. The main ECS-altered functions that were modulated by chemopreventive agents included cell proliferation, apoptosis, differentiation, Ras activation, P53 functions, NF-κB pathway, transforming growth factor–related stress response, and angiogenesis. Some micro-RNAs known to be polymorphic in humans were downregulated by ECS and were protected by chemopreventive agents. This study provides proof-of-concept and validation of technology that we are further refining to screen and prioritize potential agents for continued development and to help elucidate their biological effects and mechanisms. Therefore, microRNA analysis may provide a new tool for predicting at early carcinogenesis stages both the potential safety and efficacy of cancer chemopreventive agents. PMID:20051373

Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine.

PubMed

Albany, Costantine; Hever-Jardine, Mary P; von Herrmann, Katherine M; Yim, Christina Y; Tam, Janice; Warzecha, Joshua M; Shin, Leah; Bock, Sarah E; Curran, Brian S; Chaudhry, Aneeq S; Kim, Fred; Sandusky, George E; Taverna, Pietro; Freemantle, Sarah J; Christensen, Brock C; Einhorn, Lawrence H; Spinella, Michael J

2017-01-10

Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A substantial portion of TGCT patients are refractory to cisplatin. There are no effective therapies for these patients, many of whom die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. In prior in vitro studies we found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). Here, as an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. These preclinical findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients.

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance.

PubMed

Palomo, Marta; Mir, Enrique; Rovira, Montse; Escolar, Ginés; Carreras, Enric; Diaz-Ricart, Maribel

2016-03-31

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. © 2016 by The American Society of Hematology.

Androgen actions on endothelium functions and cardiovascular diseases

PubMed Central

Cai, Jing-Jing; Wen, Juan; Jiang, Wei-Hong; Lin, Jian; Hong, Yuan; Zhu, Yuan-Shan

2016-01-01

The roles of androgens on cardiovascular physiology and pathophysiology are controversial as both beneficial and detrimental effects have been reported. Although the reasons for this discrepancy are unclear, multiple factors such as genetic and epigenetic variation, sex-specificity, hormone interactions, drug preparation and route of administration may contribute. Recently, growing evidence suggests that androgens exhibit beneficial effects on cardiovascular function though the mechanism remains to be elucidated. Endothelial cells (ECs) which line the interior surface of blood vessels are distributed throughout the circulatory system, and play a crucial role in cardiovascular function. Endothelial progenitor cells (EPCs) are considered an indispensable element for the reconstitution and maintenance of an intact endothelial layer. Endothelial dysfunction is regarded as an initiating step in development of atherosclerosis and cardiovascular diseases. The modulation of endothelial functions by androgens through either genomic or nongenomic signal pathways is one possible mechanism by which androgens act on the cardiovascular system. Obtaining insight into the mechanisms by which androgens affect EC and EPC functions will allow us to determine whether androgens possess beneficial effects on the cardiovascular system. This in turn may be critical in the prevention and therapy of cardiovascular diseases. This article seeks to review recent progress in androgen regulation of endothelial function, the sex-specificity of androgen actions, and its clinical applications in the cardiovascular system. PMID:27168746

Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila.

PubMed

Park, Joung-Sun; Jeon, Ho-Jun; Pyo, Jung-Hoon; Kim, Young-Shin; Yoo, Mi-Ae

2018-03-07

Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11 , Rad50 , Nbs1 , ATM , ATR , Chk1 , and Chk2 , which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

Electroconvection in one-dimensional liquid crystal cells

NASA Astrophysics Data System (ADS)

Huh, Jong-Hoon

2018-04-01

We investigate the alternating current (ac) -driven electroconvection (EC) in one-dimensional cells (1DCs) under the in-plane switching mode. In 1DCs, defect-free EC can be realized. In the presence and absence of external multiplicative noise, the features of traveling waves (TWs), such as their Hopf frequency fH and velocity, are examined in comparison with those of conventional two-dimensional cells (2DCs) accompanying defects of EC rolls. In particular, we show that the defects significantly contribute to the features of the TWs. Additionally, owing to the defect-free EC in the 1DCs, the effects of the ac and noise fields on the TW are clarified. The ac field linearly increases fH, independent of the ac frequency f . The noise increases fH monotonically, but fH does not vary below a characteristic noise intensity VN*. In addition, soliton-like waves and unfamiliar oscillation of EC vortices in 1DCs are observed, in contrast to the localized EC (called worms) and the oscillation of EC rolls in 2DCs.

Organotypic vasculature: From descriptive heterogeneity to functional pathophysiology.

PubMed

Augustin, Hellmut G; Koh, Gou Young

2017-08-25

Blood vessels form one of the body's largest surfaces, serving as a critical interface between the circulation and the different organ environments. They thereby exert gatekeeper functions on tissue homeostasis and adaptation to pathologic challenge. Vascular control of the tissue microenvironment is indispensable in development, hemostasis, inflammation, and metabolism, as well as in cancer and metastasis. This multitude of vascular functions is mediated by organ-specifically differentiated endothelial cells (ECs), whose cellular and molecular heterogeneity has long been recognized. Yet distinct organotypic functional attributes and the molecular mechanisms controlling EC differentiation and vascular bed-specific functions have only become known in recent years. Considering the involvement of vascular dysfunction in numerous chronic and life-threatening diseases, a better molecular understanding of organotypic vasculatures may pave the way toward novel angiotargeted treatments to cure hitherto intractable diseases. This Review summarizes recent progress in the understanding of organotypic vascular differentiation and function. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system.

PubMed

Ohtani-Kaneko, Rsituko; Sato, Kenjiro; Tsutiya, Atsuhiro; Nakagawa, Yuka; Hashizume, Kazutoshi; Tazawa, Hidekatsu

2017-10-09

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.

Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

PubMed

Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

2018-02-01

The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Low-EC-Content Electrolytes for Low-Temperature Li-Ion Cells

NASA Technical Reports Server (NTRS)

Smart, Marshall; Bugga, Ratnakumar; Surampudi, Subbarao

2003-01-01

Electrolytes comprising LiPF6 dissolved at a concentration of 1.0 M in three different mixtures of alkyl carbonates have been found well suited for use in rechargeable lithium-ion electrochemical cells at low temperatures. These and other electrolytes have been investigated in continuing research directed toward extending the lower limit of practical operating temperatures of Li-ion cells down to -60 C. This research at earlier stages was reported in numerous previous NASA Tech Briefs articles, the three most recent being "Ethyl Methyl Carbonate as a Cosolvent for Lithium-Ion Cells" (NPO-20605), Vol. 25, Low-EC-Content Electrolytes for Low-Temperature Li-Ion Cells No. 6 (June 2001), page 53; "Alkyl Pyrocarbonate Electrolyte Additives for Li-Ion Cells" (NPO-20775), Vol. 26, No. 5 (May 2002), page 37; and "Fluorinated Alkyl Carbonates as Cosolvents in Li-Ion Cells (NPO-21076), Vol. 26, No. 5 (May 2002), page 38. The present solvent mixtures, in terms of volume proportions of their ingredients, are 1 ethylene carbonate (EC) + 1 diethyl carbonate (DEC) + 1 dimethyl carbonate (DMC) + 3 ethyl methyl carbonate (EMC); 3EC + 3DMC + 14EMC; and 1EC + 1DEC + 1DMC + 4EMC. Relative to similar mixtures reported previously, the present mixtures, which contain smaller proportions of EC, have been found to afford better performance in experimental Li-ion cells at temperatures < -20 C.

Hepatoprotective effects of litchi (Litchi chinensis) procyanidin A2 on carbon tetrachloride-induced liver injury in ICR mice

PubMed Central

Chen, Lih-Geeng; Chang, Cheng-Wei; Tsay, Jwu-Guh; Weng, Brian Bor-Chun

2017-01-01

Drug tolerance, lacking liver regenerative activity and inconclusive inhibition of steatosis and cirrhosis by silymarin treatment during chronic liver injury have increased the demand for novel alternative or synergistic treatments for liver damage. Litchi fruit is abundant in polyphenolic compounds and is used in traditional Chinese medicine for treatments that include the strengthening of hepatic and pancreatic functions. Unique polyphenolic compounds obtained from litchi pericarp extract (LPE) were studied in vitro and in vivo for hepatoprotection. Epicatechin (EC) and procyanidin A2 (PA2) of LPE were obtained by fractionated-extraction from pulverized litchi pericarps. All fractions, including LPE, were screened against silymarin in carbon tetrachloride (CCl4)-treated murine embryonic liver cell line (BNL). The effects of daily gavage-feeding of LPE, silymarin (200 mg/kg body weight) or H2O in CCl4-intoxicated male ICR mice were evaluated by studying serum chemicals, liver pathology and glutathione antioxidative enzymes. The effects of EC and PA2 on liver cell regenerative activity were investigated using a scratch wound healing assay and flow cytometric cell cycle analysis; the results of which demonstrated that LPE protected BNL from CCl4-intoxication. Gavage-feeding of LPE decreased serum glutamic oxaloacetate transaminase and glutamic pyruvic transaminase levels, and exhibited superior retention of the hexagonal structure of hepatocytes and reduced necrotic cells following liver histopathological examinations in CCl4-intoxicated ICR mice. Glutathione peroxidise and glutathione reductase activities were preserved as the normal control level in LPE groups. EC and PA2 were principle components of LPE. PA2 demonstrated liver cell regenerative activity in scratch wound healing assays and alcohol-induced liver cell injury in vitro. The present findings suggest that litchi pericarp polyphenolic extracts, including EC and PA2, may be a synergistic alternative to silymarin in hepatoprotection and liver cell regeneration. PMID:28587348

Hepatoprotective effects of litchi (Litchi chinensis) procyanidin A2 on carbon tetrachloride-induced liver injury in ICR mice.

PubMed

Chen, Lih-Geeng; Chang, Cheng-Wei; Tsay, Jwu-Guh; Weng, Brian Bor-Chun

2017-06-01

Drug tolerance, lacking liver regenerative activity and inconclusive inhibition of steatosis and cirrhosis by silymarin treatment during chronic liver injury have increased the demand for novel alternative or synergistic treatments for liver damage. Litchi fruit is abundant in polyphenolic compounds and is used in traditional Chinese medicine for treatments that include the strengthening of hepatic and pancreatic functions. Unique polyphenolic compounds obtained from litchi pericarp extract (LPE) were studied in vitro and in vivo for hepatoprotection. Epicatechin (EC) and procyanidin A2 (PA2) of LPE were obtained by fractionated-extraction from pulverized litchi pericarps. All fractions, including LPE, were screened against silymarin in carbon tetrachloride (CCl 4 )-treated murine embryonic liver cell line (BNL). The effects of daily gavage-feeding of LPE, silymarin (200 mg/kg body weight) or H 2 O in CCl 4 -intoxicated male ICR mice were evaluated by studying serum chemicals, liver pathology and glutathione antioxidative enzymes. The effects of EC and PA2 on liver cell regenerative activity were investigated using a scratch wound healing assay and flow cytometric cell cycle analysis; the results of which demonstrated that LPE protected BNL from CCl 4 -intoxication. Gavage-feeding of LPE decreased serum glutamic oxaloacetate transaminase and glutamic pyruvic transaminase levels, and exhibited superior retention of the hexagonal structure of hepatocytes and reduced necrotic cells following liver histopathological examinations in CCl 4- intoxicated ICR mice. Glutathione peroxidise and glutathione reductase activities were preserved as the normal control level in LPE groups. EC and PA2 were principle components of LPE. PA2 demonstrated liver cell regenerative activity in scratch wound healing assays and alcohol-induced liver cell injury in vitro . The present findings suggest that litchi pericarp polyphenolic extracts, including EC and PA2, may be a synergistic alternative to silymarin in hepatoprotection and liver cell regeneration.

Inward rectifier potassium (Kir2.1) channels as end‐stage boosters of endothelium‐dependent vasodilators

PubMed Central

Dalsgaard, Thomas; Bonev, Adrian D.; Nelson, Mark T.

2016-01-01

Key points Increase in endothelial cell (EC) calcium activates calcium‐sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium‐dependent vasodilatation.Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium‐dependent vasodilatation is not clear.In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform.Endothelium‐dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown.These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. Abstract Endothelium‐dependent vasodilators, such as acetylcholine, increase intracellular Ca2+ through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca2+‐sensitive intermediate and small conductance K+ (IK and SK, respectively) channels. Although strong inward rectifier K+ (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC‐dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC‐dependent vasodilatation of resistance‐sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC‐specific Kir2.1 knockdown (EC‐Kir2.1 −/−) mice. Elevation of extracellular K+ to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba2+, ML‐133) or in the arteries from EC‐Kir2.1 −/− mice. Potassium‐induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba2+, did not affect currents through TRPV4, IK or SK channels. Endothelial cell‐dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC‐Kir2.1 −/−. In angiotensin II‐induced hypertension, the Kir channel function was not altered, although the endothelium‐dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca2+‐dependent activation of IK and SK channels. PMID:26840527

SOX15 regulates proliferation and migration of endometrial cancer cells.

PubMed

Rui, Xiaohui; Xu, Yun; Jiang, Xiping; Guo, Caixia; Jiang, Jingting

2017-10-31

The study aimed to investigate the effects of Sry-like high mobility group box 15 ( SOX15 ) on proliferation and migration of endometrial cancer (EC) cells. Immunohistochemistry (IHC) was applied to determine the expression of SOX15 in EC tissues and adjacent tissues. We used cell transfection method to construct the HEC-1-A and Ishikawa cell lines with stable overexpression and low expression SOX15 Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot were performed to examine expression of SOX15 mRNA and SOX15 protein, respectively. By conducting a series of cell proliferation assay and migration assay, we analyzed the influence of SOX15 overexpression or low expression on EC cell proliferation and migration. The expression of SOX15 mRNA and protein in EC tissues was significantly lower than that in adjacent tissues. After lentivirus-transfecting SOX15 , the expression level of SOX15 mRNA and protein was significantly increased in cells of SOX15 group, and decreased in sh- SOX15 group. Overexpression of SOX15 could suppress cell proliferation, while down-regulation of SOX15 increased cell proliferation. Flow cytometry results indicated that overexpression of SOX15 induced the ratio of cell-cycle arrest in G 1 stage. In addition, Transwell migration assay results showed that SOX15 overexpression significantly inhibited cell migration, and also down-regulation of SOX15 promoted the migration. As a whole, SOX15 could regulate the proliferation and migration of EC cells and up- regulation of SOX15 could be valuable for EC treatment. © 2017 The Author(s).

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

2015-01-01

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

Impact of pH on the structure and function of neural cadherin.

PubMed

Jungles, Jared M; Dukes, Matthew P; Vunnam, Nagamani; Pedigo, Susan

2014-12-02

Neural (N-) cadherin is a transmembrane protein within adherens junctions that mediates cell-cell adhesion. It has 5 modular extracellular domains (EC1-EC5) that bind 3 calcium ions between each of the modules. Calcium binding is required for dimerization. N-Cadherin is involved in diverse processes including tissue morphogenesis, excitatory synapse formation and dynamics, and metastasis of cancer. During neurotransmission and tumorigenesis, fluctuations in extracellular pH occur, causing tissue acidosis with associated physiological consequences. Studies reported here aim to determine the effect of pH on the dimerization properties of a truncated construct of N-cadherin containing EC1-EC2. Since N-cadherin is an anionic protein, we hypothesized that acidification of solution would cause an increase in stability of the apo protein, a decrease in the calcium-binding affinity, and a concomitant decrease in the formation of adhesive dimer. The stability of the apo monomer was increased and the calcium-binding affinity was decreased at reduced pH, consistent with our hypothesis. Surprisingly, analytical SEC studies showed an increase in calcium-induced dimerization as solution pH decreased from 7.4 to 5.0. Salt-dependent dimerization studies indicated that electrostatic repulsion attenuates dimerization affinity. These results point to a possible electrostatic mechanism for moderating dimerization affinity of the Type I cadherin family. Extrapolating these results to cell adhesion in vivo leads to the assertion that decreased pH promotes adhesion by N-cadherin, thereby stabilizing synaptic junctions.

Voltage clamp methods for the study of membrane currents and SR Ca2+ release in adult skeletal muscle fibres

PubMed Central

Hernández-Ochoa, Erick O.; Schneider, Martin F.

2012-01-01

Skeletal muscle excitation-contraction (E-C)1 coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)2 Ca2+ release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca2+, due to depolarization-initiated SR Ca2+ release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or ‘high resistance gap’ techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca2+ signalling properties of mouse skeletal muscle membranes are being investigated. PMID:22306655

Examination of tetrachlorosalicylanilide (TCSA) photoallergy using in vitro photohapten-modified Langerhans cell-enriched epidermal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerberick, G.F.; Ryan, C.A.; Von Bargen, E.C.

Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis responsemore » compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent.« less

Induced Pluripotent Stem Cell‐Derived Endothelial Cells Overexpressing Interleukin‐8 Receptors A/B and/or C‐C Chemokine Receptors 2/5 Inhibit Vascular Injury Response

PubMed Central

Giordano, Samantha; Zhao, Xiangmin; Chen, Yiu‐Fai; Litovsky, Silvio H.; Hage, Fadi G.; Townes, Tim M.; Sun, Chiao‐Wang; Wu, Li‐Chen; Oparil, Suzanne

2017-01-01

Abstract Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C‐C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat‐induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS‐ECs overexpressing IL8RA/B (RiPS‐IL8RA/B‐ECs), CCR2/5 (RiPS‐CCR2/5‐ECs), or both receptors (RiPS‐IL8RA/B+CCR2/5‐ECs) will inhibit inflammatory responses and neointima formation in balloon‐injured rat carotid artery. Twelve‐week‐old male Sprague‐Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS‐Null‐ECs (ECs transduced with empty virus), (c) RiPS‐IL8RA/B‐ECs, (d) RiPS‐CCR2/5‐ECs, or (e) RiPS‐IL8RA/B+CCR2/5‐ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS‐ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury‐induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS‐ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS‐ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168–1177 PMID:28233474

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells

PubMed Central

Dong, Lixue; Krewson, Elizabeth A.; Yang, Li V.

2017-01-01

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the “Warburg effect”), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4-induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment. PMID:28134810

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells.

PubMed

Dong, Lixue; Krewson, Elizabeth A; Yang, Li V

2017-01-27

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

Rebound spiking in layer II medial entorhinal cortex stellate cells: Possible mechanism of grid cell function

PubMed Central

Shay, Christopher F.; Ferrante, Michele; Chapman, G. William; Hasselmo, Michael E.

2015-01-01

Rebound spiking properties of medial entorhinal cortex (mEC) stellate cells induced by inhibition may underlie their functional properties in awake behaving rats, including the temporal phase separation of distinct grid cells and differences in grid cell firing properties. We investigated rebound spiking properties using whole cell patch recording in entorhinal slices, holding cells near spiking threshold and delivering sinusoidal inputs, superimposed with realistic inhibitory synaptic inputs to test the capacity of cells to selectively respond to specific phases of inhibitory input. Stellate cells showed a specific phase range of hyperpolarizing inputs that elicited spiking, but non-stellate cells did not show phase specificity. In both cell types, the phase range of spiking output occurred between the peak and subsequent descending zero crossing of the sinusoid. The phases of inhibitory inputs that induced spikes shifted earlier as the baseline sinusoid frequency increased, while spiking output shifted to later phases. Increases in magnitude of the inhibitory inputs shifted the spiking output to earlier phases. Pharmacological blockade of h-current abolished the phase selectivity of hyperpolarizing inputs eliciting spikes. A network computational model using cells possessing similar rebound properties as found in vitro produces spatially periodic firing properties resembling grid cell firing when a simulated animal moves along a linear track. These results suggest that the ability of mEC stellate cells to fire rebound spikes in response to a specific range of phases of inhibition could support complex attractor dynamics that provide completion and separation to maintain spiking activity of specific grid cell populations. PMID:26385258

Comparison of the triple-point temperatures of {sup 20}Ne, {sup 22}Ne and normal Ne

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakano, T.; Tamura, O.; Nagao, K.

2013-09-11

At the National Metrology Institute of Japan (NMIJ), the triple points of {sup 20}Ne and {sup 22}Ne were realized using modular sealed cells, Ec3Ne20 and Ec8Ne22, made by the Istituto Nazionale di Ricerca Metrologica (INRiM) in Italy. The difference of the triple-point temperatures of {sup 20}Ne and {sup 22}Ne was estimated by using the sub-range of standard platinum resistance thermometers (SPRTs) calibrated by NMIJ on the International Temperature Scale of 1990 (ITS-90). The melting curves obtained with the Ec3Ne20 and Ec8Ne22 cells show narrow widths (0.1 mK) over a wide range of the inverse of the melted fraction (1/F) frommore » 1/F=1 to 1/F=10. The liquidus point T{sub tp} estimated by the melting curves from F∼0.5 to F∼0.85 using the Ec8Ne22 is 0.146 29 (4) K higher than that using the Ec3Ne20 cell, which is in good agreement with that observed by INRiM using the same cells. After correction of the effect of impurities and other isotopes for Ec3Ne20 and Ec8Ne22 cells, the difference of T{sub tp} between pure {sup 20}Ne and pure {sup 22}Ne is estimated to be 0.146 61 (4) K, which is consistent with the recent results reported elsewhere. The sub-ranges of SPRTs computed by using the triple point of {sup 20}Ne or {sup 22}Ne realized by the Ec3Ne20 cell or the Ec8Ne22 cell in place of the triple point of Ne for the defining fixed point of the ITS-90 are in good agreement with those realized on the basis of the ITS-90 at NMIJ within 0.03 mK, which is much smaller than the non-uniqueness and the sub-range inconsistency of SPRTs.« less

Molecules mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells.

PubMed Central

Prieto, J; Beatty, P G; Clark, E A; Patarroyo, M

1988-01-01

Leucocytes interact with vascular endothelial cells (EC), and adhesion between these two cell types in vitro is modulated by phorbol ester. Monocytes were found to display the highest basal adhesion to EC, followed by Epstein-Barr virus-immortalized normal B cells (EBV-B), T cells and granulocytes. Phorbol ester treatment increased the adhesion of all types of leucocytes, except monocytes. In the presence of this compound, monoclonal antibody 60.3 to GP90 (CD18, a leucocyte-adhesion protein which is non-covalently associated to either GP160, GP155, or GP130) was found to inhibit the adhesion of the four types of leucocytes to a considerable extent, while anti-lymphocyte function-associated antigen-1 (LFA-1) antibody to GP160 (CD11a) inhibited the adhesion of T and B cells only. Antibody 60.1 to GP155 (CD11b) had a major inhibitory activity exclusively on granulocytes, while antibody LB-2, which recognizes a distinct adhesion molecule (GP84) and, in contrast to the previous antibodies, reacts with EC, mainly inhibited adhesion of EBV-B and did not increase the inhibition obtained with antibody 60.3 alone. Fab fragments of antibody 60.3 inhibited leucocyte adhesion more efficiently, in either the absence or presence of phorbol ester, than the intact antibody molecule. It is concluded the GP90, either alone or associated to the larger glycoproteins, mediates the adhesion in all types of leucocytes, while GP84 mediates the adhesion of the activated B cells. Images Figure 2 PMID:3259203

Biochemical and Genetic Analysis of the Chlamydia GroEL Chaperonins

PubMed Central

Illingworth, Melissa; Hooppaw, Anna J.; Ruan, Lu

2017-01-01

ABSTRACT Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm Escherichia coli chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of groEL and groES homologs, groEL paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. Chlamydia spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three groEL genes, Chlamydia groEL (ChgroEL), ChgroEL2, and ChgroEL3. We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel Chlamydia-specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the in vivo essentiality of the three ChgroEL genes through targeted mutagenesis in Chlamydia trachomatis. ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not ChgroEL, leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL. IMPORTANCE Chlamydia is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL from E. coli and Helicobacter pylori, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle. PMID:28396349

MiR-218 inhibits HMGB1-mediated autophagy in endometrial carcinoma cells during chemotherapy.

PubMed

Ran, Xiaomin; Yang, Juan; Liu, Chaoxia; Zhou, Ping; Xiao, Linzhi; Zhang, Keqiang

2015-01-01

Endometrial carcinoma is the most common gynecological malignancy among women worldwide. Although treatment for EC has improved with the introduction of Paclitaxel (Tax) chemotherapy, the majority of patients will develop resistance to the treatment, leading to poor prognosis. One of the causes of chemoresistance is the increased ability to undergo autophagy. In this study, we identified that miR-218 was significantly down-regulated in Tax-resistant EC cells compared to the non-drug resistant cell lines, and overexpression of miR-218 sensitized paclitaxel resistant EC cells to paclitaxel. Moreover, we demonstrated that miR-218 directly binds to the 3'-UTR of HMGB1 gene. HMGB1 was upregulated in paclitaxel resistant EC cells, it mediated autophagy and contributed to chemotherapy resistance in endometrial carcinoma in vitro. HMGB1-mediated autophagy could be suppressed by miR-218 overexpression in Tax resistant EC cells. In summary, we determined the targeting role of miR-218 to HMGB1 and the regulation of miR-218 on the HMGB1-mediated cell autophagy during chemotherapy resistance in endometrial carcinoma cells. These results reveal novel potential role of miR-218 against chemotherapy resistance during the treatment of endometrial carcinoma.

Excess centrosomes perturb dynamic endothelial cell repolarization during blood vessel formation

PubMed Central

Kushner, Erich J.; Ferro, Luke S.; Yu, Zhixian; Bautch, Victoria L.

2016-01-01

Blood vessel formation requires dynamic movements of endothelial cells (ECs) within sprouts. The cytoskeleton regulates migratory polarity, and centrosomes organize the microtubule cytoskeleton. However, it is not well understood how excess centrosomes, commonly found in tumor stromal cells, affect microtubule dynamics and interphase cell polarity. Here we find that ECs dynamically repolarize during sprouting angiogenesis, and excess centrosomes block repolarization and reduce migration and sprouting. ECs with excess centrosomes initially had more centrosome-derived microtubules but, paradoxically, fewer steady-state microtubules. ECs with excess centrosomes had elevated Rac1 activity, and repolarization was rescued by blockade of Rac1 or actomyosin blockers, consistent with Rac1 activity promoting cortical retrograde actin flow and actomyosin contractility, which precludes cortical microtubule engagement necessary for dynamic repolarization. Thus normal centrosome numbers are required for dynamic repolarization and migration of sprouting ECs that contribute to blood vessel formation. PMID:27099371

Using Optical Tweezers to Study Cell Mechanics during Airway Reopening

NASA Astrophysics Data System (ADS)

Yalcin, Huseyin; Wang, Jing; Ghadiali, Samir; Ou-Yang, H. Daniel

2006-03-01

Patients suffering from the acute respiratory distress syndrome (ARDS) must be mechanically ventilated in order to survive. However, these ventilation protocols may generate injurious hydrodynamic stresses especially during low tidal volume (VT) ventilation when the flow of micron-sized air bubbles displace the surrounding liquid. In-vitro studies in our lab revealed that microbubble flows can severally damage lung epithelial cells (EC). The degree of injury was elevated for sub-confluent monolayers in small channel heights. Under these conditions, the micromechanics of individual EC may influence the degree of cellular injury. To investigate the role of cell mechanics, we used an oscillating Optical Tweezers (OT) technique to measure the intrinsic mechanical properties of EC before and after the flow of microbubbles. Knowledge of how the EC's micromechanical properties influence cell viability may lead to the development of novel treatment therapies that enhance the EC's ability to withstand injurious hydrodynamic stresses during ventilation treatment.

Design of a novel bioreactor and application in vascular tissue engineering

NASA Astrophysics Data System (ADS)

Zhang, Zhi-Xiong; Xi, Ting-Fei; Wang, Ying-Jun; Chen, Xiao-Song; Zhang, Jian; Wang, Chun-Ren; Gu, Yong-Quan; Chen, Liang; Li, Jian-Xin; Chen, Bing

2008-11-01

Endothelial cells (ECs) detachment under high shear stress at the early period of transplantation resulted in thrombosis and occlusion. To solve this problem, we developed a novel bioreactor. The bioreactor mimicked the formation of pulsatile flow in physiological conditions. Human umbilical vein ECs were seeded onto the lumen of living tissue conduits grown within dog peritoneal cavity. The shear stress generated by the bioreactor was increased step by step from 1.5 ± 0.8 dyn/cm 2 to 5.3 ± 2.4 dyn/cm 2, and was applied to ECs after static culture for 2 days. The results showed that completely confluent monolayer ECs were elongated, and were oriented parallel to the flow direction. The bioreactor could provide good environment for formation of endothelium. Stepwise increase shear stress could strengthen cell-cell and cell-extracellular matrix. The flow conditions of the bioreactor play a key role to determine the quality of the ECs lining.

Emerging contaminants at a closed and an operating landfill in Oklahoma

USGS Publications Warehouse

Andrews, William J.; Masoner, Jason R.; Cozzarelli, Isabelle M.

2012-01-01

Landfills are the final depositories for a wide range of solid waste from both residential and commercial sources, and therefore have the potential to produce leachate containing many organic compounds found in consumer products such as pharmaceuticals, plasticizers, disinfectants, cleaning agents, fire retardants, flavorings, and preservatives, known as emerging contaminants (ECs). Landfill leachate was sampled from landfill cells of three different age ranges from two landfills in Central Oklahoma. Samples were collected from an old cell containing solid waste greater than 25 years old, an intermediate age cell with solid waste between 16 and 3 years old, and operating cell with solid waste less than 5 years old to investigate the chemical variability and persistence of selected ECs in landfill leachate of differing age sources. Twenty-eight of 69 analyzed ECs were detected in one or more samples from the three leachate sources. Detected ECs ranged in concentration from 0.11 to 114 μg/L and included 4 fecal and plant sterols, 13 household\\industrial, 7 hydrocarbon, and 4 pesticide compounds. Four ECs were solely detected in the oldest leachate sample, two ECs were solely detected in the intermediate leachate sample, and no ECs were solely detected in the youngest leachate sample. Eleven ECs were commonly detected in all three leachate samples and are an indication of the contents of solid waste deposited over several decades and the relative resistance of some ECs to natural attenuation processes in and near landfills.

TET1-GPER-PI3K/AKT pathway is involved in insulin-driven endometrial cancer cell proliferation.

PubMed

Xie, Bing-Ying; Lv, Qiao-Ying; Ning, Cheng-Cheng; Yang, Bing-Yi; Shan, Wei-Wei; Cheng, Ya-Li; Gu, Chao; Luo, Xue-Zhen; Zhang, Zhen-Bo; Chen, Xiao-Jun; Xi, Xiao-Wei; Feng, You-Ji

2017-01-22

Large amount of clinical evidence has demonstrated that insulin resistance is closely related to oncogenesis of endometrial cancer (EC). Despite recent studies showed the up-regulatory role of insulin in G protein-coupled estrogen receptor (GPER/GPR30) expression, GPER expression was not decreased compared to control when insulin receptor was blocked even in insulin treatment. The purpose of this study was to explore the possible mechanism by which insulin up-regulates GPER that drives EC cell proliferation. For this purpose, we first investigated the GPER expression in tissues of endometrial lesions, further explored the effect of GPER on EC cell proliferation in insulin resistance context. Then we analyzed the role of Ten-Eleven Translocation 1 (TET1) in insulin-induced GEPR expression and EC cell proliferation. The results showed that GPER was highly expressed in endometrial atypical hyperplasia and EC tissues. Mechanistically, insulin up-regulated TET1 expression and the latter played an important role in up-regulating GPER expression and activating PI3K/AKT signaling pathway. TET1 mediated GPER up-regulation was another mechanism that insulin promotes EC cell proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation of cell-mediated immunity by Morinda citrifolia fruit extract and its constituents.

PubMed

Murata, Kazuya; Abe, Yumi; Futamura-Masudaa, Megumi; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

2014-04-01

Morinda citrifolia, commonly known as noni, is a traditional natural medicine in French Polynesia and Hawaii. Functional foods derived from M. citrifolia fruit have been marketed to help prevent diseases and promote good health. The objective of this study was to assess the effects of M. citrifolia fruit on cell-mediated immunity. In the picryl chloride-induced contact dermatitis test, M. citrifolia fruit extract (Noni-ext) inhibited the suppression of cell-mediated immunity by immunosuppressive substances isolated from freeze-dried ascites of Ehrlich carcinoma-bearing mice (EC-sup). In addition, Noni-ext inhibited reduction of IL-2 production in EC-sup-treated mice and activated natural killer cells in normal mice. These results suggest that Noni-ext has multiple effects on the recovery of cell-mediated immunity. Furthermore, we investigated the active principles of Noni-ext and identified an iridoid glycoside, deacetylasperulosidic acid. Oral administration of deacetylasperulosidic acid inhibited the reduction of ear swelling, and also cancelled the suppression of IL-2 production along with the activation of natural killer cells in the same manner as that of Noni-ext.

Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells' migration and the associated FAK-Rho GTPases signalling pathways

PubMed Central

Shen, Yang; Wang, Guixue; Huang, Xianliang; Zhang, Qin; Wu, Jiang; Tang, Chaojun; Yu, Qingsong; Liu, Xiaoheng

2012-01-01

Vascular endothelial cell (EC) adhesion and migration are essential processes in re-endothelialization of implanted biomaterials. There is no clear relationship and mechanism between EC adhesion and migration behaviour on surfaces with varying wettabilities. As model substrates, plasma SiOx:H nanocoatings with well-controlled surface wettability (with water contact angles in the range of 98.5 ± 2.3° to 26.3 ± 4.0°) were used in this study to investigate the effects of surface wettability on cell adhesion/migration and associated protein expressions in FAK-Rho GTPases signalling pathways. It was found that EC adhesion/migration showed opposite behaviour on the hydrophilic and hydrophobic surfaces (i.e. hydrophobic surfaces promoted EC migration but were anti-adhesions). The number of adherent ECs showed a maximum on hydrophilic surfaces, while cells adhered to hydrophobic surfaces exhibited a tendency for cell migration. The focal adhesion kinase (FAK) inhibitor targeting the Y-397 site of FAK could significantly inhibit cell adhesion/migration, suggesting that EC adhesion and migration on surfaces with different wettabilities involve (p)FAK and its downstream signalling pathways. Western blot results suggested that the FAK-Rho GTPases signalling pathways were correlative to EC migration on hydrophobic plasma SiOx:H surfaces, but uncertain to hydrophilic surfaces. This work demonstrated that surface wettability could induce cellular behaviours that were associated with different cellular signalling events. PMID:21715399

Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells.

PubMed

Wanjare, Maureen; Hou, Luqia; Nakayama, Karina H; Kim, Joseph J; Mezak, Nicholas P; Abilez, Oscar J; Tzatzalos, Evangeline; Wu, Joseph C; Huang, Ngan F

2017-07-25

Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 μm fiber diameter) or parallel-aligned (7 μm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

PubMed

Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

2017-06-01

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

A20 Regulates Atherogenic Interferon (IFN)-γ Signaling in Vascular Cells by Modulating Basal IFNβ Levels*

PubMed Central

Moll, Herwig P.; Lee, Andy; Minussi, Darlan C.; da Silva, Cleide G.; Csizmadia, Eva; Bhasin, Manoj; Ferran, Christiane

2014-01-01

IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment. PMID:25217635

Nitro-oleic acid inhibits vascular endothelial inflammatory responses and the endothelial-mesenchymal transition.

PubMed

Ambrozova, Gabriela; Fidlerova, Tana; Verescakova, Hana; Koudelka, Adolf; Rudolph, Tanja K; Woodcock, Steven R; Freeman, Bruce A; Kubala, Lukas; Pekarova, Michaela

2016-11-01

Inflammatory-mediated pathological processes in the endothelium arise as a consequence of the dysregulation of vascular homeostasis. Of particular importance are mediators produced by stimulated monocytes/macrophages inducing activation of endothelial cells (ECs). This is manifested by excessive soluble pro-inflammatory mediator production and cell surface adhesion molecule expression. Nitro-fatty acids are endogenous products of metabolic and inflammatory reactions that display immuno-regulatory potential and may represent a novel therapeutic strategy to treat inflammatory diseases. The purpose of our study was to characterize the effects of nitro-oleic acid (OA-NO2) on inflammatory responses and the endothelial-mesenchymal transition (EndMT) in ECs that is a consequence of the altered healing phase of the immune response. The effect of OA-NO2 on inflammatory responses and EndMT was determined in murine macrophages and murine and human ECs using Western blotting, ELISA, immunostaining, and functional assays. OA-NO2 limited the activation of macrophages and ECs by reducing pro-inflammatory cytokine production and adhesion molecule expression through its modulation of STAT, MAPK and NF-κB-regulated signaling. OA-NO2 also decreased transforming growth factor-β-stimulated EndMT and pro-fibrotic phenotype of ECs. These effects are related to the downregulation of Smad2/3. The study shows the pleiotropic effect of OA-NO2 on regulating EC-macrophage interactions during the immune response and suggests a role for OA-NO2 in the regulation of vascular endothelial immune and fibrotic responses arising during chronic inflammation. These findings propose the OA-NO2 may be useful as a novel therapeutic agent for treatment of cardiovascular disorders associated with dysregulation of the endothelial immune response. Copyright © 2016 Elsevier B.V. All rights reserved.

Myoepithelial cells are the main component in pleomorphic adenomas?

PubMed

Ponce Bravo, Santa; Ledesma Montes, Constantino; López Becerril, Uriel; Morales Sánchez, Israel

2007-03-01

The aim of this study was to quantify by immunohistochemistry the number of myoepithelial cells (MyECs) in pleomorphic adenomas (PAs). We retrieved the paraffin cubes of 27 PAs, new slides were done and they were stained with anti-S100 protein antibody. The amount of S-100 protein positive cells was quantified, their morphology was recorded and comparison among MyEC number with age, gender and involved gland were also done. With S-100 protein, MyECs in normal salivary gland tissue were seen surrounding the ductual structures only. In the analysed PAs a mean of 27.4% of the neoplastic cells were positive to the antibody. With the exception of one PA, in all the analysed cases the plasmacytoid cells were the most commonly identified cells (48,6%). Results of this study suggest that MyECs do not constitute the main cellular component of the neoplastic compartment in PAs and corroborate the previously reported evidence by different authors, who studying the PAs suggested that MyECs does not comprise the main cellular neoplastic component of these entities.

Enhanced endothelial cell senescence by lithium-induced matrix metalloproteinase-1 expression.

PubMed

Struewing, Ian T; Durham, Samuel N; Barnett, Corey D; Mao, Catherine D

2009-06-26

Endothelial cell (EC) senescence and dysfunction occurring after chronic injury and inflammation are highly associated with the development and progression of cardiovascular diseases. However, the factors involved in the establishment of EC senescence remain poorly understood. We have previously shown that lithium, an inhibitor of glycogen synthase kinase (GSK)-3beta and activator of the Wnt/beta-catenin signaling pathway, induces an EC senescent-like phenotype. Herein, we show that lithium induces a rapid and pronounced up-regulation of the matrix metalloproteinase (MMP)-1, an inflammation and senescent cell marker, at the mRNA and protein levels, whereas the induction of two other senescent cell markers is either weak (interleukin-8) or delayed (plasminogen activator inhibitor-1). Lithium effect on MMP-1 expression is also specific among other MMPs and not mediated by GSK3beta inhibition. Lithium affects MMP-1 expression mainly at the transcriptional level but neither the AP1/Ets regulatory sites nor the redox sensitive (-1607/2G) site in MMP-1 promoter are involved in lithium-dependent MMP-1 regulation. However, down-regulation of p53, a target of lithium in EC, dampens both basal and lithium-induced MMP-1 expression, which further links MMP-1 up-regulation with the establishment of cell senescence. Although increased MMP-1 levels are usually associated with angiogenesis in enabled proliferative EC, the exogenous addition of activated MMP-1 on lithium- arrested EC increases the number of EC positive for the senescent-associated-beta-galactosidase marker. Conversely, down-regulation of MMP-1 expression by small interfering RNAs blunts the lithium-dependent increase in senescent-associated-beta-galactosidase positive cells. Altogether our data indicate that lithium-induced MMP-1 may participate in the reinforcement of EC senescence and reveal a novel mechanism for lithium-induced tissue remodeling.

Mulberry Fruit Extract Affords Protection against Ethyl Carbamate-Induced Cytotoxicity and Oxidative Stress.

PubMed

Chen, Wei; Li, Yuting; Bao, Tao; Gowd, Vemana

2017-01-01

Ethyl carbamate (EC) is a food and environmental toxicant and is a cause of concern for human exposure. Several studies indicated that EC-induced toxicity was associated with oxidative stress. Mulberry fruits are reported to have a wide range of bioactive compounds and pharmacological activities. The present study was therefore aimed to investigate the protective property of mulberry fruit extract (MFE) on EC-induced cytotoxicity and oxidative stress. Chemical composition analysis showed that total phenolic content and total flavonoid content in MFE were 502.43 ± 5.10 and 219.12 ± 4.45 mg QE/100 g FW. Cyanidin -3-O- glucoside and cyanidin -3-O- rutinoside were the major anthocyanins in MFE. In vitro antioxidant studies (DPPH, ABTS, and FRAP assays) jointly exhibited the potent antioxidant capacity of MFE. Further study indicated that MFE protected human liver HepG2 cells from EC-induced cytotoxicity by scavenging overproduced cellular ROS. EC treatment promoted intracellular glutathione (GSH) depletion and caused mitochondrial membrane potential (MMP) collapse, as well as mitochondrial membrane lipid peroxidation, whereas MFE pretreatment significantly inhibited GSH depletion and restored the mitochondrial membrane function. Overall, our study suggested that polyphenolic-rich MFE could afford a potent protection against EC-induced cytotoxicity and oxidative stress.

Mulberry Fruit Extract Affords Protection against Ethyl Carbamate-Induced Cytotoxicity and Oxidative Stress

PubMed Central

Li, Yuting; Bao, Tao; Gowd, Vemana

2017-01-01

Ethyl carbamate (EC) is a food and environmental toxicant and is a cause of concern for human exposure. Several studies indicated that EC-induced toxicity was associated with oxidative stress. Mulberry fruits are reported to have a wide range of bioactive compounds and pharmacological activities. The present study was therefore aimed to investigate the protective property of mulberry fruit extract (MFE) on EC-induced cytotoxicity and oxidative stress. Chemical composition analysis showed that total phenolic content and total flavonoid content in MFE were 502.43 ± 5.10 and 219.12 ± 4.45 mg QE/100 g FW. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were the major anthocyanins in MFE. In vitro antioxidant studies (DPPH, ABTS, and FRAP assays) jointly exhibited the potent antioxidant capacity of MFE. Further study indicated that MFE protected human liver HepG2 cells from EC-induced cytotoxicity by scavenging overproduced cellular ROS. EC treatment promoted intracellular glutathione (GSH) depletion and caused mitochondrial membrane potential (MMP) collapse, as well as mitochondrial membrane lipid peroxidation, whereas MFE pretreatment significantly inhibited GSH depletion and restored the mitochondrial membrane function. Overall, our study suggested that polyphenolic-rich MFE could afford a potent protection against EC-induced cytotoxicity and oxidative stress. PMID:28819542

Genetically Engineered Escherichia coli Nissle 1917 Synbiotics Reduce Metabolic Effects Induced by Chronic Consumption of Dietary Fructose

PubMed Central

Somabhai, Chaudhari Archana; Raghuvanshi, Ruma; Nareshkumar, G.

2016-01-01

Aims To assess protective efficacy of genetically modified Escherichia coli Nissle 1917 (EcN) on metabolic effects induced by chronic consumption of dietary fructose. Materials and Methods EcN was genetically modified with fructose dehydrogenase (fdh) gene for conversion of fructose to 5-keto-D-fructose and mannitol-2-dehydrogenase (mtlK) gene for conversion to mannitol, a prebiotic. Charles foster rats weighing 150–200 g were fed with 20% fructose in drinking water for two months. Probiotic treatment of EcN (pqq), EcN (pqq-glf-mtlK), EcN (pqq-fdh) was given once per week 109 cells for two months. Furthermore, blood and liver parameters for oxidative stress, dyslipidemia and hyperglycemia were estimated. Fecal samples were collected to determine the production of short chain fatty acids and pyrroloquinoline quinone (PQQ) production. Results EcN (pqq-glf-mtlK), EcN (pqq-fdh) transformants were confirmed by restriction digestion and functionality was checked by PQQ estimation and HPLC analysis. There was significant increase in body weight, serum glucose, liver injury markers, lipid profile in serum and liver, and decrease in antioxidant enzyme activity in high-fructose-fed rats. However the rats treated with EcN (pqq-glf-mtlK) and EcN (pqq-fdh) showed significant reduction in lipid peroxidation along with increase in serum and hepatic antioxidant enzyme activities. Restoration of liver injury marker enzymes was also seen. Increase in short chain fatty acids (SCFA) demonstrated the prebiotic effects of mannitol and gluconic acid. Conclusions Our study demonstrated the effectiveness of probiotic EcN producing PQQ and fructose metabolizing enzymes against the fructose induced hepatic steatosis suggesting that its potential for use in treating fructose induced metabolic syndrome. PMID:27760187

Transgenic mice overexpressing the extracellular domain of NCAM are impaired in working memory and cortical plasticity

PubMed Central

Brennaman, Leann H.; Kochlamazashvili, Gaga; Stoenica, Luminita; Nonneman, Randall J.; Moy, Sheryl S.; Schachner, Melitta; Dityatev, Alexander; Maness, Patricia F.

2011-01-01

The neural cell adhesion molecule, NCAM, is a pivotal regulator of neural development, with key roles in axonal and dendritic growth and synaptic plasticity. Alterations in NCAM expression or proteolytic cleavage have been linked to human neuropsychiatric disorders such as schizophrenia, bipolar disorder and Alzheimer’s disease, and may contribute to cognitive dysfunction. We have generated mice overexpressing the NCAM extracellular (EC) proteolytic cleavage fragment which has been reported to be increased in schizophrenic versus normal brains. These mice show impaired GABAergic innervation and reduced number of apical dendritic spines on pyramidal neurons in the prefrontal cortex (PFC). Here, these NCAM-EC transgenic mice were subjected to behavioral tasks and electrophysiological measurements to determine the impact of structural abnormalities in the PFC on synaptic and cognitive functions. NCAM-EC mice exhibited impaired working memory in a delayed non-match-to-sample task, which requires PFC function, but showed no differences in anxiety, olfactory abilities, or sociability. Transgenic mice displayed impaired long- and short-term potentiation in the PFC but normal synaptic plasticity in the hippocampus, suggesting that the abnormal synaptic innervation in NCAM-EC mice impairs PFC plasticity and alters working memory. These findings may have implications for cognitive dysfunctions observed in neuropsychiatric disorders. PMID:21515372

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat.

PubMed

Giordano, Samantha; Zhao, Xiangmin; Xing, Daisy; Hage, Fadi; Oparil, Suzanne; Cooke, John P; Lee, Jieun; Nakayama, Karina H; Huang, Ngan F; Chen, Yiu-Fai

2016-03-15

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat

PubMed Central

Giordano, Samantha; Zhao, Xiangmin; Xing, Daisy; Hage, Fadi; Oparil, Suzanne; Cooke, John P.; Lee, Jieun; Nakayama, Karina H.; Huang, Ngan F.

2016-01-01

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 106 HiPS-ECs, 1.5 × 106 HiPS-Null-ECs, or 1.5 × 106 HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury. PMID:26801304

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines

PubMed Central

Rabquer, Bradley J.; Ohara, Ray A.; Stinson, William A.; Campbell, Phillip L.; Amin, M. Asif; Balogh, Beatrix; Zakhem, George; Renauer, Paul A.; Lozier, Ann; Arasu, Eshwar; Haines, G. Kenneth; Kahaleh, Bashar; Schiopu, Elena; Khanna, Dinesh; Koch, Alisa E.

2016-01-01

Objectives. Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. Methods. Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. Results. Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. Conclusion. Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc. PMID:26705326

Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes

PubMed Central

Moorthy, Sakthi D.; Davidson, Scott; Shchuka, Virlana M.; Singh, Gurdeep; Malek-Gilani, Nakisa; Langroudi, Lida; Martchenko, Alexandre; So, Vincent; Macpherson, Neil N.; Mitchell, Jennifer A.

2017-01-01

Transcriptional enhancers are critical for maintaining cell-type–specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes. PMID:27895109

Electrocorticographic high gamma activity versus electrical cortical stimulation mapping of naming.

PubMed

Sinai, Alon; Bowers, Christopher W; Crainiceanu, Ciprian M; Boatman, Dana; Gordon, Barry; Lesser, Ronald P; Lenz, Frederick A; Crone, Nathan E

2005-07-01

Subdural electrocorticographic (ECoG) recordings in patients undergoing epilepsy surgery have shown that functional activation is associated with event-related broadband gamma activity in a higher frequency range (>70 Hz) than previously studied in human scalp EEG. To investigate the utility of this high gamma activity (HGA) for mapping language cortex, we compared its neuroanatomical distribution with functional maps derived from electrical cortical stimulation (ECS), which remains the gold standard for predicting functional impairment after surgery for epilepsy, tumours or vascular malformations. Thirteen patients had undergone subdural electrode implantation for the surgical management of intractable epilepsy. Subdural ECoG signals were recorded while each patient verbally named sequentially presented line drawings of objects, and estimates of event-related HGA (80-100 Hz) were made at each recording site. Routine clinical ECS mapping used a subset of the same naming stimuli at each cortical site. If ECS disrupted mouth-related motor function, i.e. if it affected the mouth, lips or tongue, naming could not be tested with ECS at the same cortical site. Because naming during ECoG involved these muscles of articulation, the sensitivity and specificity of ECoG HGA were estimated relative to both ECS-induced impairments of naming and ECS disruption of mouth-related motor function. When these estimates were made separately for 12 electrode sites per patient (the average number with significant HGA), the specificity of ECoG HGA with respect to ECS was 78% for naming and 81% for mouth-related motor function, and equivalent sensitivities were 38% and 46%, respectively. When ECS maps of naming and mouth-related motor function were combined, the specificity and sensitivity of ECoG HGA with respect to ECS were 84% and 43%, respectively. This study indicates that event-related ECoG HGA during confrontation naming predicts ECS interference with naming and mouth-related motor function with good specificity but relatively low sensitivity. Its favourable specificity suggests that ECoG HGA can be used to construct a preliminary functional map that may help identify cortical sites of lower priority for ECS mapping. Passive recordings of ECoG gamma activity may be done simultaneously at all electrode sites without the risk of after-discharges associated with ECS mapping, which must be done sequentially at pairs of electrodes. We discuss the relative merits of these two functional mapping techniques.

Biomaterials trigger endothelial cell activation when co-incubated with human whole blood.

PubMed

Herklotz, Manuela; Hanke, Jasmin; Hänsel, Stefanie; Drichel, Juliane; Marx, Monique; Maitz, Manfred F; Werner, Carsten

2016-10-01

Endothelial cell activation resulting from biomaterial contact or biomaterial-induced blood activation may in turn also affect hemostasis and inflammatory processes in the blood. Current in vitro hemocompatibility assays typically ignore these modulating effects of the endothelium. This study describes a co-incubation system of human whole blood, biomaterial and endothelial cells (ECs) that was developed to overcome this limitation. First, human endothelial cells were characterized in terms of their expression of coagulation- and inflammation-relevant markers in response to various activators. Subsequently, their capacity to regulate hemostasis as well as complement and granulocyte activation was monitored in a hemocompatibility assay. After blood contact, quiescent ECs exhibited anticoagulant and anti-inflammatory properties. When they were co-incubated with surfaces exhibiting pro-coagulant or pro-inflammatory characteristics, the ECs down-regulated coagulation but not complement or leukocyte activation. Analysis of intracellular levels of the endothelial activation markers E-selectin and tissue factor showed that co-incubation with model surfaces and blood significantly increased the activation state of ECs. Finally, the coagulation- and inflammation-modulating properties of the ECs were tested after blood/biomaterial exposure. Pre-activation of ECs by biomaterials in the blood induced a pro-coagulant and pro-inflammatory state of the ECs, wherein the pro-coagulant response was higher for biomaterial/blood pre-activated ECs than for TNF-α-pre-activated cells. This work provides evidence that biomaterials, even without directly contacting the endothelium, affect the endothelial activation state with and have consequences for plasmatic and cellular reactions in the blood. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of mTOR complex 2 restrains tumor angiogenesis in multiple myeloma

PubMed Central

Lamanuzzi, Aurelia; Saltarella, Ilaria; Desantis, Vanessa; Frassanito, Maria Antonia; Leone, Patrizia; Racanelli, Vito; Nico, Beatrice; Ribatti, Domenico; Ditonno, Paolo; Prete, Marcella; Solimando, Antonio Giovanni; Dammacco, Francesco; Vacca, Angelo; Ria, Roberto

2018-01-01

The mammalian Target of Rapamycin (mTOR) is an intracellular serine/threonine kinase that mediates intracellular metabolism, cell survival and actin rearrangement. mTOR is made of two independent complexes, mTORC1 and mTORC2, activated by the scaffold proteins RAPTOR and RICTOR, respectively. The activation of mTORC1 triggers protein synthesis and autophagy inhibition, while mTORC2 activation promotes progression, survival, actin reorganization, and drug resistance through AKT hyper-phosphorylation on Ser473. Due to the mTOR pivotal role in the survival of tumor cells, we evaluated its activation in endothelial cells (ECs) from 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 47 patients with multiple myeloma (MM), and its involvement in angiogenesis. MM-ECs showed a significantly higher expression of mTOR and RICTOR than MGUS-ECs. These data were supported by the higher activation of mTORC2 downstream effectors, suggesting a major role of mTORC2 in the angiogenic switch to MM. Specific inhibition of mTOR activity through siRNA targeting RICTOR and dual mTOR inhibitor PP242 reduced the MM-ECs angiogenic functions, including cell migration, chemotaxis, adhesion, invasion, in vitro angiogenesis on Matrigel®, and cytoskeleton reorganization. In addition, PP242 treatment showed anti-angiogenic effects in vivo in the Chick Chorioallantoic Membrane (CAM) and Matrigel® plug assays. PP242 exhibited a synergistic effect with lenalidomide and bortezomib, suggesting that mTOR inhibition can enhance the anti-angiogenic effect of these drugs. Data to be shown indicate that mTORC2 is involved in MM angiogenesis, and suggest that the dual mTOR inhibitor PP242 may be useful for the anti-angiogenic management of MM patients. PMID:29755672

Autologous human plasma in stem cell culture and cryopreservation in the creation of a tissue-engineered vascular graft.

PubMed

Zhang, Ping; Policha, Aleksandra; Tulenko, Thomas; DiMuzio, Paul

2016-03-01

Previous work demonstrated the effectiveness of autologous adipose-derived stem cells (ASCs) as endothelial cell (EC) substitutes in vascular tissue engineering. We further this work toward clinical translation by evaluating ASC function after (1) replacement of fetal bovine serum (FBS) with autologous human plasma (HP) in culture and (2) cryopreservation. Human ASCs and plasma, isolated from periumbilical fat and peripheral blood, respectively, were collected from the same donors. ASCs were differentiated in endothelial growth medium supplemented with FBS (2%) vs HP (2%). Proliferation was measured by growth curves and MTT assay. Endothelial differentiation was measured by quantitative polymerase chain reaction, assessment of acetylated low-density lipoprotein uptake, and cord formation after plating on Matrigel (BD Biosciences, San Jose, Calif). Similar studies were conducted before and after cryopreservation of ASCs and included assessment of cell retention on the luminal surface of a vascular graft. ASCs expanded in HP-supplemented medium showed (1) similar proliferation to FBS-cultured ASCs, (2) consistent differentiation toward an EC lineage (increases in CD31, von Willebrand factor, and CD144 message; acetylated low-density lipoprotein uptake; and cord formation on Matrigel), and (3) retention on the luminal surface after seeding and subsequent flow conditioning. Cryopreservation did not significantly alter ASC viability, proliferation, acquisition of endothelial characteristics, or retention after seeding onto a vascular graft. This study suggests that (1) replacement of FBS with autologous HP--a step necessary for the translation of this technology into human use--does not significantly impair proliferation or endothelial differentiation of ASCs used as EC substitutes and (2) ASCs are tolerant to cryopreservation in terms of maintaining EC characteristics and retention on a vascular graft. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Bottom-up fabrication of artery-mimicking tubular co-cultures in collagen-based microchannel scaffolds.

PubMed

Tan, A; Fujisawa, K; Yukawa, Y; Matsunaga, Y T

2016-10-20

We developed a robust bottom-up approach to construct open-ended, tubular co-culture constructs that simulate the human vascular morphology and microenvironment. By design, these three-dimensional artificial vessels mimic the basic architecture of an artery: a collagen-rich extracellular matrix (as the tunica externa), smooth muscle cells (SMCs) (as the tunica media), and an endothelial cell (EC) lining (as the tunica interna). A versatile needle-based fabrication technique was employed to achieve controllable arterial layouts within a PDMS-hosted collagen microchannel scaffold (330 ± 10 μm in diameter): (direct co-culture) a SMC/EC bilayer to follow the structure of an arteriole-like segment; and (encapsulated co-culture) a lateral SMC multilayer covered by an EC monolayer lining to simulate the architecture of a larger artery. Optical and fluorescence microscopy images clearly evidenced the progressive cell elongation and sprouting behavior of SMCs and ECs along the collagen gel contour and within the gel matrix under static co-culture conditions. The progressive cell growth patterns effectively led to the formation of a tubular co-culture with an internal endothelial lining expressing prominent CD31 (cluster of differentiation 31) intercellular junction markers. During a 4-day static maturation period, the artery constructs showed modest alteration in the luminal diameters (i.e. less than 10% changes from the initial measurements). This argues in favor of stable and predictable arterial architecture achieved via the proposed fabrication protocols. Both co-culture models showed a high glucose metabolic rate during the initial proliferation phase, followed by a temporary quiescent (and thus, mature) stage. These proof-of-concept models with a controllable architecture create an important foundation for advanced vessel manipulations such as the integration of relevant physiological functionality or remodeling into a vascular disease-mimicking tissue.

Insights into the regulation of tumor dormancy by angiogenesis in experimental tumors.

PubMed

Indraccolo, Stefano

2013-01-01

While it is well established that an angiogenic switch marks escape from tumor dormancy in xenograft models, the molecular pathways involved in the control of tumor cell proliferation or survival by angiogenesis remain substantially uncharted. We recently demonstrated that signals stemming from angiogenic endothelial cells (EC) regulate the behavior of dormant cancer cells. Specifically, we observed that the Notch ligand Dll4, induced by angiogenic factors in EC, triggers Notch3 activation in neighboring tumor cells and promotes a tumorigenic phenotype. Evidence that Notch signaling is involved in tumor dormancy was further strengthened by the observation that MKP-1 levels-a broadly expressed phosphatase-are controlled by Notch3 by regulation of protein ubiquitination and stability. Notch3 and MKP-1 levels are consistently low in dormant tumors, and this is accompanied by relatively high levels of phosphorylated p38, a canonical MKP-1 target previously associated with maintenance of tumor dormancy. These results elucidate a novel angiogenesis-driven mechanism involving the Notch and MAPK pathways that controls tumor dormancy. More in general, angiogenic EC could form part of the vascular niche, a specialized microenvironment which appears to regulate metastatic outgrowth and future studies are needed to clarify the contribution of EC in the regulation of cancer stem cell behavior in the niche.The notion that EC could communicate signals to tumor cells raises questions about the possibility of achieving tumor dormancy by counteracting angiogenesis. In experimental tumors, anti-VEGF drugs typically prune the newly formed vasculature, thus reducing microvessel density, blood flow, and perfusion. These drugs eventually increase hypoxia and cause tumor necrosis but dormancy is rarely observed. Our group recently reported that anti-VEGF therapy causes a dramatic depletion of glucose and an exhaustion of ATP levels in tumors. Moreover, we found that the central metabolic checkpoint LKB1/AMPK-a cellular sensor of ATP levels that supports cell viability in response to energy stress-is activated by anti-VEGF therapy in experimental tumors and it has a key role in induction of sustained tumor regression. These functional links between activation of the LKB1/AMPK by anti-angiogenic therapy and tumor dormancy suggest a role for metabolism in the regulation of this phenomenon.

Toll-Like Receptor Function in Acute Wounds

PubMed Central

Chen, Lin; DiPietro, Luisa A.

2017-01-01

Significance: Inflammation is an integral part of immune response and supports optimal wound healing in adults. Inflammatory cells such as neutrophils, macrophages, dendritic cells, lymphocytes, and mast cells produce important cytokines, chemokines, and growth factors. These immune cells interact with keratinocytes, fibroblasts, and endothelial cells (ECs), as well as the extracellular matrix within a complicated network that promotes and regulates wound healing. Aberrant and persistent inflammation may result in delayed wound healing, scar formation, or chronic wounds. Targeting the molecules involved in the inflammatory response may have great potential therapeutic value. Recent Advances and Critical Issues: Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen-associated molecular patterns from microbes or danger-associated molecular patterns from damaged cells. The discovery of TLRs sheds new light on the mechanism by which the inflammatory or innate immune response is initiated in wound healing. Convincing evidence now shows that multiple types of cells, including infiltrating or resident inflammatory cells, keratinocytes, fibroblasts, and ECs, express specific types of TLRs. Experimental reduction of certain TLRs or treatment of wounds with TLR ligands has been shown to affect wound healing. A better understanding of the involvement of TLRs in the innate immune response during skin wound healing may suggest novel strategies to improve the quality of tissue repair. Future Directions: Despite the indisputable role of TLRs in regulating the immune response in acute wound healing, the functions of TLRs that are relevant to human wound healing and chronic wounds are poorly understood. PMID:29062591

Enhanced MEA Performance for PEMFCs under Low Relative Humidity and Low Oxygen Content Conditions via Catalyst Functionalization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xin, Le; Yang, Fan; Xie, Jian

2017-01-01

This work demonstrates that functionalizing annealed-Pt/Ketjen black EC300j (a-Pt/KB) and dealloyed-PtNi/Ketjen black EC300j (d-PtNi/KB) catalysts using p-phenyl sulfonic acid can effectively enhance performance in the membrane electrode assemblies (MEAs) of proton exchange membrane fuel cells (PEMFCs). The functionalization increased the size of both Pt and PtNi catalyst particles and resulted in the further leaching of Ni from the PtNi catalyst while promoting the formation of nanoporous PtNi nanoparticles. The size of the SO3H-Pt/KB and SO3H-PtNi/KB carbon-based aggregates decreased dramatically, leading to the formation of catalyst layers with narrower pore size distributions.MEA tests highlighted the benefits of the surface functionalization, inmore » which the cells with SO3H-Pt/KB and SO3H-PtNi/KB cathode catalysts showed superior high current density performance under reduced RH conditions, in comparison with cells containing annealed Pt/KB (a-Pt/KB) and de-alloyed PtNi/KB (d-PtNi/KB) catalysts. The performance improvement was particularly evident when using reactant gases with low relative humidity, indicating that the hydrophilic functional groups on the carbon improved the water retention in the cathode catalyst layer. These results show a new avenue for enhancing catalyst performance for the next generation of catalytic materials for PEMFCs.« less

Small size of metastatic lymph nodes with extracapsular spread greatly impacts treatment outcomes in oral squamous cell carcinoma patients.

PubMed

Michikawa, C; Izumo, T; Sumino, J; Morita, T; Ohyama, Y; Michi, Y; Uzawa, N

2018-07-01

Extracapsular spread (ECS) of metastatic lymph nodes from oral carcinoma is the most significant prognostic predictor of a poor treatment outcome. However, only a few reports on prognostic factors in ECS-positive cases have been investigated. To address this problem, a detailed examination of ECS pathology was conducted to determine the prognostic factors of oral squamous cell carcinoma (OSCC) with ECS of metastatic lymph nodes. This study involved 63 OSCC patients with at least one pathologically metastatic node with ECS. Among the 229 metastatic lymph nodes, 149 exhibited ECS. Univariate analysis revealed that a poor outcome and recurrence were significantly associated with the number of ECS-positive nodes, density of ECS, and the minor axis of the smallest ECS-positive node. However, multivariate analysis identified only small size of ECS-positive nodes as a significant and independent factor predicting recurrence and a poor outcome. Thus, small size of ECS-positive nodes is the most important prognostic indicator for OSCC with ECS in metastatic lymph nodes. The classification of ECS status using the minor axis of ECS-positive nodes may be useful for further prediction of a poorer prognosis in OSCC cases. Standardization of ECS diagnosis and multicenter prospective studies will be required to confirm and refine these findings. Copyright © 2017 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Stretchable and Photocatalytically Renewable Electrochemical Sensor Based on Sandwich Nanonetworks for Real-Time Monitoring of Cells.

PubMed

Wang, Ya-Wen; Liu, Yan-Ling; Xu, Jia-Quan; Qin, Yu; Huang, Wei-Hua

2018-05-15

Stretchable electrochemical (EC) sensors have broad prospects in real-time monitoring of living cells and tissues owing to their excellent elasticity and deformability. However, the redox reaction products and cell secretions are easily adsorbed on the electrode, resulting in sensor fouling and passivation. Herein, we developed a stretchable and photocatalytically renewable EC sensor based on Au nanotubes (NTs) and TiO 2 nanowires (NWs) sandwich nanonetworks. The external Au NTs are used for EC sensing, and internal TiO 2 NWs provide photocatalytic performance to degrade contaminants, which endows the sensor with excellent EC performance, high photocatalytic activity, and favorable mechanical tensile property. This allows highly sensitive recycling monitoring of NO released from endothelial cells and 5-HT released from mast cells under their stretching states in real time, therefore providing a promising tool to unravel elastic and mechanically sensitive cells, tissues, and organs.

Mass spectrometric methods for monitoring redox processes in electrochemical cells.

PubMed

Oberacher, Herbert; Pitterl, Florian; Erb, Robert; Plattner, Sabine

2015-01-01

Electrochemistry (EC) is a mature scientific discipline aimed to study the movement of electrons in an oxidation-reduction reaction. EC covers techniques that use a measurement of potential, charge, or current to determine the concentration or the chemical reactivity of analytes. The electrical signal is directly converted into chemical information. For in-depth characterization of complex electrochemical reactions involving the formation of diverse intermediates, products and byproducts, EC is usually combined with other analytical techniques, and particularly the hyphenation of EC with mass spectrometry (MS) has found broad applicability. The analysis of gases and volatile intermediates and products formed at electrode surfaces is enabled by differential electrochemical mass spectrometry (DEMS). In DEMS an electrochemical cell is sampled with a membrane interface for electron ionization (EI)-MS. The chemical space amenable to EC/MS (i.e., bioorganic molecules including proteins, peptides, nucleic acids, and drugs) was significantly increased by employing electrospray ionization (ESI)-MS. In the simplest setup, the EC of the ESI process is used to analytical advantage. A limitation of this approach is, however, its inability to precisely control the electrochemical potential at the emitter electrode. Thus, particularly for studying mechanistic aspects of electrochemical processes, the hyphenation of discrete electrochemical cells with ESI-MS was found to be more appropriate. The analytical power of EC/ESI-MS can further be increased by integrating liquid chromatography (LC) as an additional dimension of separation. Chromatographic separation was found to be particularly useful to reduce the complexity of the sample submitted either to the EC cell or to ESI-MS. Thus, both EC/LC/ESI-MS and LC/EC/ESI-MS are common. © 2013 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc.

Endocrine cells producing peptide hormones in the intestine of Nile tilapia: distribution and effects of feeding and fasting on the cell density.

PubMed

Pereira, Raquel Tatiane; de Freitas, Thaiza Rodrigues; de Oliveira, Izabela Regina Cardoso; Costa, Leandro Santos; Vigliano, Fabricio Andrés; Rosa, Priscila Vieira

2017-10-01

Endocrine cells (ECs) act as a luminal surveillance system responding to either the presence or absence of food in the gut through the secretion of peptide hormones. The aim of this study was to analyze the effects of feeding and fasting on the EC peptide-specific distribution along the intestine of Nile tilapia. We assessed the density of ECs producing gastrin (GAS), cholecystokinin-8 (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in nine segments of the intestine using immunohistochemistry. Our results show that ECs immunoreactive to CCK-8, GAS, NPY, and CGRP can be found along all the intestinal segments sampled, from the midgut to hindgut, although differences in their distribution along the gut were observed. Regarding nutrient status, we found that the anterior segments of the midgut seem to be the main site responding to luminal changes in Nile tilapia. The NPY+ and CGRP+ EC densities increased in the fasted group, while the amount of CCK-8+ ECs were higher in the fed group. No effects of fasting or feeding were found in the GAS+ EC densities. Changes in ECs density were found only at the anterior segments of the intestine which may be due to the correlation between vagus nerve anatomy, EC location, and peptide turnover. Lastly, ECs may need to be considered an active cell subpopulation that may adapt and respond to different nutrient status as stimuli. Due to the complexity of the enteroendocrine system and its importance in fish nutrition, much remains to be elucidated and it deserves closer attention.

Mass spectrometric methods for monitoring redox processes in electrochemical cells

PubMed Central

Oberacher, Herbert; Pitterl, Florian; Erb, Robert; Plattner, Sabine

2015-01-01

Electrochemistry (EC) is a mature scientific discipline aimed to study the movement of electrons in an oxidation–reduction reaction. EC covers techniques that use a measurement of potential, charge, or current to determine the concentration or the chemical reactivity of analytes. The electrical signal is directly converted into chemical information. For in-depth characterization of complex electrochemical reactions involving the formation of diverse intermediates, products and byproducts, EC is usually combined with other analytical techniques, and particularly the hyphenation of EC with mass spectrometry (MS) has found broad applicability. The analysis of gases and volatile intermediates and products formed at electrode surfaces is enabled by differential electrochemical mass spectrometry (DEMS). In DEMS an electrochemical cell is sampled with a membrane interface for electron ionization (EI)-MS. The chemical space amenable to EC/MS (i.e., bioorganic molecules including proteins, peptides, nucleic acids, and drugs) was significantly increased by employing electrospray ionization (ESI)-MS. In the simplest setup, the EC of the ESI process is used to analytical advantage. A limitation of this approach is, however, its inability to precisely control the electrochemical potential at the emitter electrode. Thus, particularly for studying mechanistic aspects of electrochemical processes, the hyphenation of discrete electrochemical cells with ESI-MS was found to be more appropriate. The analytical power of EC/ESI-MS can further be increased by integrating liquid chromatography (LC) as an additional dimension of separation. Chromatographic separation was found to be particularly useful to reduce the complexity of the sample submitted either to the EC cell or to ESI-MS. Thus, both EC/LC/ESI-MS and LC/EC/ESI-MS are common. PMID:24338642

Engineering biomimetic periosteum with β-TCP scaffolds to promote bone formation in calvarial defects of rats.

PubMed

Zhang, Dan; Gao, Peng; Li, Qin; Li, Jinda; Li, Xiaojuan; Liu, Xiaoning; Kang, Yunqing; Ren, Liling

2017-06-05

There is a critical need for the management of large bone defects. The purpose of this study was to engineer a biomimetic periosteum and to combine this with a macroporous β-tricalcium phosphate (β-TCP) scaffold for bone tissue regeneration. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were harvested and cultured in different culture media to form undifferentiated rBMSC sheets (undifferentiated medium (UM)) and osteogenic cell sheets (osteogenic medium (OM)). Simultaneously, rBMSCs were differentiated to induced endothelial-like cells (iECs), and the iECs were further cultured on a UM to form a vascularized cell sheet. At the same time, flow cytometry was used to detect the conversion rates of rBMSCs to iECs. The pre-vascularized cell sheet (iECs/UM) and the osteogenic cell sheet (OM) were stacked together to form a biomimetic periosteum with two distinct layers, which mimicked the fibrous layer and cambium layer of native periosteum. The biomimetic periostea were wrapped onto porous β-TCP scaffolds (BP/β-TCP) and implanted in the calvarial bone defects of rats. As controls, autologous periostea with β-TCP (AP/β-TCP) and β-TCP alone were implanted in the calvarial defects of rats, with a no implantation group as another control. At 2, 4, and 8 weeks post-surgery, implants were retrieved and X-ray, microcomputed tomography (micro-CT), histology, and immunohistochemistry staining analyses were performed. Flow cytometry results showed that rBMSCs were partially differentiated into iECs with a 35.1% conversion rate in terms of CD31. There were still 20.97% rBMSCs expressing CD90. Scanning electron microscopy (SEM) results indicated that cells from the wrapped cell sheet on the β-TCP scaffold apparently migrated into the pores of the β-TCP scaffold. The histology and immunohistochemistry staining results from in vivo implantation indicated that the BP/β-TCP and AP/β-TCP groups promoted the formation of blood vessels and new bone tissues in the bone defects more than the other two control groups. In addition, micro-CT showed that more new bone tissue formed in the BP/β-TCP and AP/β-TCP groups than the other groups. Inducing rBMSCs to iECs could be a good strategy to obtain an endothelial cell source for prevascularization. Our findings indicate that the biomimetic periosteum with porous β-TCP scaffold has a similar ability to promote osteogenesis and angiogenesis in vivo compared to the autologous periosteum. This function could result from the double layers of biomimetic periosteum. The prevascularized cell sheet served a mimetic fibrous layer and the osteogenic cell sheet served a cambium layer of native periosteum. The biomimetic periosteum with a porous ceramic scaffold provides a new promising method for bone healing.

A B cell follicle sanctuary permits persistent productive SIV infection in elite controllers

PubMed Central

Fukazawa, Yoshinori; Lum, Richard; Okoye, Afam A.; Park, Haesun; Matsuda, Kenta; Bae, Jin Young; Hagen, Shoko I.; Shoemaker, Rebecca; Deleage, Claire; Lucero, Carissa; Morcock, David; Swanson, Tonya; Legasse, Alfred W.; Axthelm, Michael K.; Hesselgesser, Joseph; Geleziunas, Romas; Hirsch, Vanessa M.; Edlefsen, Paul T.; Piatak, Michael; Estes, Jacob D.; Lifson, Jeffrey D.; Picker, Louis J.

2014-01-01

Chronic phase HIV/SIV replication is reduced by as much as 10,000-fold in elite controllers (EC) compared to typical progressors, but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here, we show that productive SIV infection in rhesus monkey EC is strikingly restricted to follicular helper CD4+ T cells (TFH), suggesting that while the potent SIV-specific CD8+ T cells of these monkeys can effectively clear productive infection from extra-follicular sites, their relative exclusion from B cell follicles limits elimination of infected TFH. Indeed, CD8+ lymphocyte depletion of EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH, with TFH restriction resuming upon CD8+ T cell recovery. Thus, B cell follicles constitute sanctuaries for persistent SIV replication in the presence of potent anti-viral CD8+ T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy. PMID:25599132

Effects of the micro-nano surface topography of titanium alloy on the biological responses of osteoblast.

PubMed

Yin, Chengcheng; Zhang, Yanjing; Cai, Qing; Li, Baosheng; Yang, Hua; Wang, Heling; Qi, Hua; Zhou, Yanmin; Meng, Weiyan

2017-03-01

In clinical applications, osseointegration is essential for the long-term stability of dental implants. Inspired by the hierarchical structure of natural bone, we applied the electrochemical etching (EC) technique to form a micro-nano structure on a titanium alloy (Ti6Al4V) substrate, called EC surface. Sand blasting and acid etching (SLA) and machined (M) methods were employed to generate micro and smooth textures, respectively, as the control groups. The surface topographies of the three substrates were characterized using scanning electron microscopy (SEM). Then, human osteoblast-like cells (MG63) were cultured on substrates, and adhesion, proliferation, morphology, alkaline phosphatase activity (ALP), and gene expression levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and type I collagen (COLIA 1) were analyzed. MG63 cells cultured on the EC Ti alloy substrates displayed better cell adhesion, significant proliferation, and a higher production level of ALP, gene expressions of RUNX2, OCN, OPN and COLIA 1 (p < 0.01 or p < 0.05) compared with those of SLA and M substrates. These results indicate that the micro-nano structure fabricated by electrochemical etching method is beneficial for the biological functions of MG63 cells and may be a promising candidate in dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 757-769, 2017. © 2016 Wiley Periodicals, Inc.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

PubMed

Panigrahi, Rashmi; Lemieux, M Joanne

2015-01-01

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Radiation-induced senescence-like phenotype in proliferating and plateau-phase vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Igarashi, Kaori; Sakimoto, Ippei; Kataoka, Keiko

The effects of ionizing radiation (IR) on tumor angiogenesis still remain largely unknown. In this study, we found that IR (8 Gy) induces a high-frequency (80-90%) senescence-like phenotype in vascular endothelial cells (ECs) undergoing exponential growth. This finding allowed us to characterize the IR-induced senescence-like (IRSL) phenotype by examining the gene expression profiles and in vitro angiogenic activities of these ECs. The expression levels of genes associated with cell cycle progression and DNA replication were remarkably reduced in the IRSL ECs. Additionally, the in vitro invasion and migration activities of these cells through Matrigel were significantly suppressed. We also foundmore » that confluent ECs exhibited a high-frequency IRSL phenotype when they were replated immediately after irradiation, whereas incubation in plateau-phase conditions reduced the induction of this phenotype and enhanced colony formation. The kinetics of DNA double-strand break repair, which showed a faster time course in confluent ECs than in growing ECs, may contribute to the protective mechanism associated with the IRSL phenotype. These results imply that the IRSL phenotype may be important for determining the angiogenic activity of ECs following irradiation. The present study should contribute to the understanding of the effects of IR on tumor angiogenesis.« less

Assessing and correcting spatial representativeness of tower eddy-covariance flux measurements

NASA Astrophysics Data System (ADS)

Metzger, S.; Xu, K.; Desai, A. R.; Taylor, J. R.; Kljun, N.; Blanken, P.; Burns, S. P.; Scott, R. L.

2014-12-01

Estimating the landscape-scale exchange of ecologically relevant trace gas and energy fluxes from tower eddy-covariance (EC) measurements is often complicated by surface heterogeneity. For example, a tower EC measurement may represent less than 1% of a grid cell resolved by mechanistic models (order 100-1000 km2). In particular for data assimilation or comparison with large-scale observations, it is hence critical to assess and correct the spatial representativeness of tower EC measurements. We present a procedure that determines from a single EC tower the spatio-temporally explicit flux field of its surrounding. The underlying principle is to extract the relationship between biophysical drivers and ecological responses from measurements under varying environmental conditions. For this purpose, high-frequency EC flux processing and source area calculations (≈60 h-1) are combined with remote sensing retrievals of land surface properties and subsequent machine learning. Methodological details are provided in our companion presentation "Towards the spatial rectification of tower-based eddy-covariance flux observations". We apply the procedure to one year of data from each of four AmeriFlux sites under different climate and ecological environments: Lost Creek shrub fen wetland, Niwot Ridge subalpine conifer, Park Falls mixed forest, and Santa Rita mesquite savanna. We find that heat fluxes from the Park Falls 122-m-high EC measurement and from a surrounding 100 km2 target area differ up to 100 W m-2, or 65%. Moreover, 85% and 24% of the EC flux observations are adequate surrogates of the mean surface-atmosphere exchange and its spatial variability across a 900 km2 target area, respectively, at 5% significance and 80% representativeness levels. Alternatively, the resulting flux grids can be summarized as probability density functions, and used to inform mechanistic models directly with the mean flux value and its spatial variability across a model grid cell. Lastly, for each site we evaluate the applicability of the procedure based on a full bottom-up uncertainty budget.

Cooperative fuzzy games approach to setting target levels of ECs in quality function deployment.

PubMed

Yang, Zhihui; Chen, Yizeng; Yin, Yunqiang

2014-01-01

Quality function deployment (QFD) can provide a means of translating customer requirements (CRs) into engineering characteristics (ECs) for each stage of product development and production. The main objective of QFD-based product planning is to determine the target levels of ECs for a new product or service. QFD is a breakthrough tool which can effectively reduce the gap between CRs and a new product/service. Even though there are conflicts among some ECs, the objective of developing new product is to maximize the overall customer satisfaction. Therefore, there may be room for cooperation among ECs. A cooperative game framework combined with fuzzy set theory is developed to determine the target levels of the ECs in QFD. The key to develop the model is the formulation of the bargaining function. In the proposed methodology, the players are viewed as the membership functions of ECs to formulate the bargaining function. The solution for the proposed model is Pareto-optimal. An illustrated example is cited to demonstrate the application and performance of the proposed approach.