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Sample records for brain endothelial cells

  1. Hypertension alters phosphorylation of VASP in brain endothelial cells.

    Science.gov (United States)

    Arlier, Zulfikar; Basar, Murat; Kocamaz, Erdogan; Kiraz, Kemal; Tanriover, Gamze; Kocer, Gunnur; Arlier, Sefa; Giray, Semih; Nasırcılar, Seher; Gunduz, Filiz; Senturk, Umit K; Demir, Necdet

    2015-04-01

    Hypertension impairs cerebral vascular function. Vasodilator-stimulated phosphoprotein (VASP) mediates active reorganization of the cytoskeleton via membrane ruffling, aggregation and tethering of actin filaments. VASP regulation of endothelial barrier function has been demonstrated by studies using VASP(-/-) animals under conditions associated with tissue hypoxia. We hypothesize that hypertension regulates VASP expression and/or phosphorylation in endothelial cells, thereby contributing to dysfunction in the cerebral vasculature. Because exercise has direct and indirect salutary effects on vascular systems that have been damaged by hypertension, we also investigated the effect of exercise on maintenance of VASP expression and/or phosphorylation. We used immunohistochemistry, Western blotting and immunocytochemistry to examine the effect of hypertension on VASP expression and phosphorylation in brain endothelial cells in normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rats under normal and exercise conditions. In addition, we analyzed VASP regulation in normoxia- and hypoxia-induced endothelial cells. Brain endothelial cells exhibited significantly lower VASP immunoreactivity and phosphorylation at the Ser157 residue in SHR versus WKY rats. Exercise reversed hypertension-induced alterations in VASP phosphorylation. Western blotting and immunocytochemistry indicated reduction in VASP phosphorylation in hypoxic versus normoxic endothelial cells. These results suggest that diminished VASP expression and/or Ser157 phosphorylation mediates endothelial changes associated with hypertension and exercise may normalize these changes, at least in part, by restoring VASP phosphorylation. PMID:24894047

  2. Nanoparticle accumulation and transcytosis in brain endothelial cell layers

    NARCIS (Netherlands)

    Ye, Dong; Raghnaill, Michelle Nic; Bramini, Mattia; Mahon, Eugene; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A

    2013-01-01

    The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight juncti

  3. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

    Science.gov (United States)

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. PMID:22771710

  4. Neutral amino acid transport across brain microvessel endothelial cell monolayers

    International Nuclear Information System (INIS)

    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with [3H]-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, α-methyldopa, L-DOPA, α-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB

  5. Neutral amino acid transport across brain microvessel endothelial cell monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Audus, K.L.; Borchardt, R.T.

    1986-03-01

    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.

  6. Shear Stress Inhibits Apoptosis of Ischemic Brain Microvascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Xiafeng Shen

    2013-01-01

    Full Text Available As a therapeutic strategy for ischemic stroke, to restore or increase cerebral blood flow (CBF is the most fundamental option. Laminar shear stress (LS, as an important force generated by CBF, mainly acts on brain microvascular endothelial cells (BMECs. In order to study whether LS was a protective factor in stroke, we investigated LS-intervented ischemic apoptosis of rat BMECs (rBMECs through PE Annexin V/7-AAD, JC-1 and Hoechst 33258 staining to observe the membranous, mitochondrial and nuclear dysfunction. Real-time PCR and western blot were also used to test the gene and protein expressions of Tie-2, Bcl-2 and Akt, which were respectively related to maintain membranous, mitochondrial and nuclear norm. The results showed that LS could be a helpful stimulus for ischemic rBMECs survival. Simultaneously, membranous, mitochondrial and nuclear regulation played an important role in this process.

  7. Polylactic Acid Nanoparticles Targeted to Brain Microvascular Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Huafang; HU Yu; SUN Wangqiang; XIE Changsheng

    2005-01-01

    In this work, blank polylactic acid (PLA) nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells (BMECs) targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can be loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 (T-80) can facilitate BMECs targeting of PLA nanoparticles.

  8. The Secretome of Endothelial Progenitor Cells Promotes Brain Endothelial Cell Activity through PI3-Kinase and MAP-Kinase

    Science.gov (United States)

    Di Santo, Stefano; Seiler, Stefanie; Fuchs, Anna-Lena; Staudigl, Jennifer; Widmer, Hans Rudolf

    2014-01-01

    Background Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. Methods Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. Results Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. Conclusion The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects. PMID:24755675

  9. Isolation and expansion of human and mouse brain microvascular endothelial cells.

    Science.gov (United States)

    Navone, Stefania E; Marfia, Giovanni; Invernici, Gloria; Cristini, Silvia; Nava, Sara; Balbi, Sergio; Sangiorgi, Simone; Ciusani, Emilio; Bosutti, Alessandra; Alessandri, Giulio; Slevin, Mark; Parati, Eugenio A

    2013-09-01

    Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d. PMID:23928501

  10. Changes in the permeability of blood brain barrier and endothelial cell damage after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Ke Liu; Jiansheng Li

    2006-01-01

    OBJECTIVE: To investigate the effect of endothelial cells on the permeability of blood brain barrier (BBB) after brain injury and its effect mechanism.DATA SOURCES: We searched for the articles of permeability of BBB and endothelial cell injury after brain ischemia, which were published between January 1982 and December 2005, with the key words of "cerebral ischemia damage,blood brain barrier ( BBB),permeability,effect of endothelial cell (EC) and its variation mechanism"in English.STUDY SELECTION: The materials were primarily selected. The articles related to the changes in the permeability of BBB and the effect of endothelial cells as well as the change mechanism after cerebral ischemia damage were chosen. Repetitive studies or review articles were excluded.DATA EXTRACTION: Totally 55 related articles were collected, and 35 were excluded due to repetitive or review articles, finally 20 articles were involved.DATA SYNTHESIS: The content or viewpoints of involved literatures were analyzed. Cerebral ischemia had damage for endothelial cells, such as the inflow of a lot of Ca2+, the production of nitrogen monoxide and oxygen free radical, and aggravated destruction of BBB. After acceptors of inflammatory mediators on cerebrovascular endothelial cell membrane, such as histamine, bradykinin , 5-hydroxytryptamine and so on are activated, endothelial cells shrink and the permeability of BBB increases. Its mechanism involves in the inflow of extracellular Ca2+and the release of intracellular Ca2+ in the cells. Glycocalyx molecule on the surface of endothelial cell, having structural polytropy, is the determinative factor of the permeability of BBB. VEGF, intensively increasing the vasopermeability and mainly effecting on postcapillary vein and veinlet, is the strongest known blood vessel permeation reagent. Its chronic overexpression in the brain can lead the destruction of BBB.CONCLUSION: The injury of endothelial cell participants in the pathological mechanism of BBB

  11. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    OpenAIRE

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment.

  12. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

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    Haqqani Arsalan S

    2013-01-01

    Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

  13. Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier.

    Science.gov (United States)

    Demeuse, Ph; Kerkhofs, A; Struys-Ponsar, C; Knoops, B; Remacle, C; van den Bosch de Aguilar, Ph

    2002-11-15

    The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function. PMID:12393158

  14. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-brain...... barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  15. Transport and regulation mechanism of the colloidal gold liposomes in the brain microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    WANG Lipeng; CHANG Yanzhong

    2015-01-01

    Objective:Blood-brain barrier is the key barrier of brain in the innate immune. It can prevent the harmful substances from the blood into the brain. In order to keep the brain in a relatively stable environment and maintain the normal function of the nervous system, it can also pump harmful substances or excess substances outside the brain selectively. Among them, brain microvascular endothelial cell tissue is a key part in the blood-brain barrier's function. The number of the patients with central nervous system ( CNS) diseases increased year by year. The therapeutic drug is usually inhibited by the blood-brain barrier and is difficult to work. Therefore, how to modify the drug and to make it easier to cross the blood brain barrier is the key point to cure CNS. At present, more than 95% research focus only on how nano drugs can enter the cell, the way and efficiency to enter the cell and the research of effect of nano drug etc. For the process of drug carrier in endocytosis, intracellular transport and release and regulation of research are rarely reported. Clathrin and P-glycoprotein are related protein in endo-cytosis and exocytosis with nano drug. Clathrin is located on the plasma membrane. It participates in endocytosis of some nutrients, and maybe the entry into the cell of some drugs. P-glycoprotein is located in the membrane of cer-ebral capillary endothelial cells. It can efflux drugs relying on ATP. Although there is a certain understanding of the cell in the inner swallow and efflux. But the process of the liposome drug is not clear. To solve the above prob-lems, using colloidal gold liposome nano materials to trace liposome's transport and regulation mechanism in brain microvascular endothelial cells, and study endocytosis, release, distribution and regulation mechanism of nano lipo-somes in brain microvascular. The solution of this problem can guide to construct reasonable drug carrier, and look forward to clarifing the molecular basis and mechanism of

  16. Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

    Science.gov (United States)

    Veszelka, Szilvia; Pásztói, Mária; Farkas, Attila E; Krizbai, István; Ngo, Thi Khue Dung; Niwa, Masami; Abrahám, Csongor S; Deli, Mária A

    2007-01-01

    Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

  17. Hyaluronan oligosaccharides perturb lymphocyte slow rolling on brain vascular endothelial cells: Implications for inflammatory demyelinating disease

    OpenAIRE

    Winkler, Clayton W.; Foster, Scott C.; Itakura, Asako; Matsumoto, Steven G.; Asari, Akira; McCarty, Owen J. T.; Sherman, Larry S.

    2013-01-01

    Inflammatory demyelinating diseases like multiple sclerosis are characterized by mononuclear cell infiltration into the central nervous system. The glycosaminoglycan hyaluronan and its receptor, CD44, are implicated in the initiation and progression of a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Digestion of hyaluronan tethered to brain vascular endothelial cells by a hyaluronidase blocks the slow rolling of lymphocytes along activated brain vascular ...

  18. Notch4 is activated in endothelial and smooth muscle cells in human brain arteriovenous malformations

    OpenAIRE

    ZhuGe, Qichuan; Wu, Zhebao; Huang, Lijie; Zhao, Bei; Zhong, Ming; Zheng, Weiming; GouRong, Chen; Mao, XiaoOu; XIE, Lin; Wang, Xiangdong; Jin, Kunlin

    2013-01-01

    Up-regulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically resected brain AVMs and found that Notch4 was up-regulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expre...

  19. Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells

    Science.gov (United States)

    Gräfe, C.; Slabu, I.; Wiekhorst, F.; Bergemann, C.; von Eggeling, F.; Hochhaus, A.; Trahms, L.; Clement, J. H.

    2016-06-01

    Crossing the blood–brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood–brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles’ shape, material, size, and coating.

  20. Ethanol suppression of peripheral blood mononuclear cell trafficking across brain endothelial cells in immunodeficiency virus infection

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    Lola C Hudson

    2010-01-01

    Full Text Available Lola C Hudson1, Brenda A Colby1, Rick B Meeker21Department of Molecular Biosciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; 2Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAAbstract: Earlier studies suggested that the combination of alcohol use and immunodeficiency virus infection resulted in more severe neurologic disease than either condition individually. These deleterious interactions could be due to increased immune cell and virus trafficking or may result from interactions between ethanol and human immunodeficiency virus (HIV-associated toxicity within the brain. To determine the extent to which increased trafficking played a role, we examined the effect of ethanol on the migration of different peripheral blood mononuclear cell (PBMCs subsets across a brain endothelial cell monolayer. We utilized combinations of feline brain endothelial cells with astrocytes, and/or microglia with either acute exposure to 0.08 g/dL ethanol, a combination of ethanol and feline immunodeficiency virus (FIV, or FIV alone. Adherence of PBMCs to endothelium was increased in all combinations of cells with the addition of ethanol. Despite increased PBMC adhesion with ethanol treatment, transmigration of B cells, monocytes, CD4 T cells and CD8 T cells was not increased and was actually decreased in the presence of astrocytes. Expression of three common adhesion molecules, intercellular adhesion molecule-1 (ICAM1, ICAM2, and vascular cell adhesion molecule, was unchanged or slightly decreased by ethanol. This indicated that although adherence is increased by ethanol it is not due to an increased expression of adhesion molecules. RANTES, MIP1α, MIP1β, and MCP-1 mRNA expression was also studied in brain endothelial cells, astrocytes and microglia by reverse transcriptase-polymerase chain reaction. Ethanol treatment of astrocytes resulted in modest changes of

  1. Interactions of Haemophilus parasuis and its LOS with porcine brain microvascular endothelial cells

    OpenAIRE

    Bouchet, Bénédicte; Vanier, Ghyslaine; Jacques, Mario; Gottschalk, Marcelo

    2008-01-01

    International audience Haemophilus parasuis is a swine pathogen that causes Glässer's disease, which is characterized by polyserositis and meningitis. The pathogenesis of the H. parasuis infection is poorly understood. To cause meningitis, H. parasuis has to cross the blood-brain barrier (BBB) to gain access to the central nervous system (CNS). We recently showed that H. parasuis adheres to and invades porcine brain microvascular endothelial cells (PBMEC). The aim of this study was to eval...

  2. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    Science.gov (United States)

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

  3. IL-1alpha induces angiogenesis in brain endothelial cells in vitro: implications for brain angiogenesis after acute injury.

    Science.gov (United States)

    Salmeron, Kathleen; Aihara, Takuma; Redondo-Castro, Elena; Pinteaux, Emmanuel; Bix, Gregory

    2016-02-01

    Inflammation is a major contributor to neuronal injury and is associated with poor outcome after acute brain injury such as stroke. The pro-inflammatory cytokine interleukin (IL)-1 is a critical regulator of cerebrovascular inflammation after ischemic injury, mainly through action of both of its isoforms, IL-1α and IL-1β, at the brain endothelium. In contrast, the differential action of these ligands on endothelial activation and post-stroke angiogenesis is largely unknown. Here, we demonstrate that IL-1α is chronically elevated in the brain after experimental stroke suggesting that it is present during post-stroke angiogenic periods. Furthermore, we demonstrate that IL-1α is a potent mediator of endothelial activation and inducer of angiogenic markers in endothelial cells in vitro. Using brain endothelial cell lines, we found that IL-1α was significantly more potent than IL-1β at inducing endothelial cell activation, as measured by expression of the pro-angiogenic chemokine CXCL-1. IL-1α also induced strong expression of the angiogenic mediator IL-6 in a concentration-dependent manner. Furthermore, IL-1α induced significant proliferation and migration of endothelial cells, and promoted formation of tube-like structures that are established key hallmarks of angiogenesis in vitro. Finally, all of those responses were blocked by the IL-1 receptor antagonist (IL-1RA). In conclusion, our data highlights a potential new role for IL-1 in brain repair mechanisms and identifies IL-1α as a potential new therapy to promote post-stroke angiogenesis. Inflammation is a major contributor to neuronal injury and is associated with poor outcome after neurotrauma. We demonstrate that cytokine IL-1α is chronically elevated in the brain after experimental stroke suggesting that it is present chronically post-stroke. We demonstrate that IL-1α is a potent mediator of endothelial activation and inducer of angiogenic markers in endothelial cells. Our data highlights a new

  4. Brain microvascular endothelial cell association and distribution of a 5 nm ceria engineered nanomaterial

    Directory of Open Access Journals (Sweden)

    Dan M

    2012-07-01

    with the capillary fraction. Electron microscopy showed the ceria ENM located on the endothelial cell luminal surface.Conclusion: Ceria ENM association with brain capillary endothelial cells saturated between 20 and 60 seconds and ceria ENM brain uptake was not diffusion-mediated. During the 120-second ceria ENM perfusion, ceria ENM predominately associated with the surface of the brain capillary cells, providing the opportunity for its cell uptake or redistribution back into circulating blood.Keywords: ceria engineered nanomaterial, brain microvascular endothelial cell association, in situ brain perfusion, capillary depletion

  5. Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models.

    Directory of Open Access Journals (Sweden)

    Eduard Urich

    Full Text Available Brain microvascular endothelial cells (BEC constitute the blood-brain barrier (BBB which forms a dynamic interface between the blood and the central nervous system (CNS. This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local

  6. Mining a Yeast Library for Brain Endothelial Cell-Binding Antibodies

    OpenAIRE

    Wang, Xin Xiang; Cho, Yong Ku; Shusta, Eric V.

    2007-01-01

    We describe the use of yeast surface display for the identification of antibodies that bind the plasma membranes of living cells. Yeast panning with a nonimmune human single-chain antibody library identified 34 unique lead antibodies that bind (Kd = 82 ± 15 nM) and in some cases internalize into rat brain endothelial cells. In addition, a novel yeast display immunoprecipitation procedure was employed for initial characterization of the cognate antigens.

  7. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors

    OpenAIRE

    Andreas Noack; Sandra Noack; Manuela Buettner; Naim, Hassan Y.; Wolfgang Löscher

    2016-01-01

    The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not kno...

  8. Circulating endothelial progenitor cells in traumatic brain injury: an emerging therapeutic target?

    Institute of Scientific and Technical Information of China (English)

    WEI Hui-jie; JIANG Rong-cai; LIU Li; ZHANG Jian-ning

    2010-01-01

    Traumatic brain injury (TBI) is a major cause ofmortality and morbidity in the world. Recent clinical investigations and basic researches suggest that strategies to improve angiogenesis following TBI may provide promising opportunities to improve clinical outcomes and brain functional recovery. More and more evidences show that circulating endothelial progenitor cells (EPCs), which have been identified in the peripheral blood, may play an important role in the pathologic and physiological angiogenesis in adults. Moreover, impressive data demonstrate that EPCs are mobilized from bone marrow to blood circulation in response to traumatic or inflammatory stimulations.In this review, we discussed the role of EPCs in the repair of brain injury and the possible therapeutic implication for functional recovery of TBl in the future.

  9. Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

    Science.gov (United States)

    Faille, Dorothée; El-Assaad, Fatima; Mitchell, Andrew J; Alessi, Marie-Christine; Chimini, Giovanna; Fusai, Thierry; Grau, Georges E; Combes, Valéry

    2012-08-01

    Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

  10. Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood–brain barrier models for the study of T cell extravasation

    OpenAIRE

    Steiner, Oliver; Coisne, Caroline; Engelhardt, Britta; Lyck, Ruth

    2010-01-01

    Important insights into the molecular mechanism of T cell extravasation across the blood–brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally sui...

  11. PECAM-1 is involved in neutrophil transmigration across Histophilus somni treated bovine brain endothelial cells.

    Science.gov (United States)

    Tiwari, Raksha; Sullivan, J; Czuprynski, C J

    2009-09-01

    Histophilus somni (H. somni) is a gram-negative bacterial pathogen that causes respiratory, reproductive, and central nervous system disease in cattle. The hallmark of systemic H. somni infection is diffused vasculitis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because platelet endothelial cell adhesion molecule-1 (PECAM-1) and endothelial nitric oxide synthase (eNOS) play fundamental roles in maintaining homeostasis in blood vessels, we sought to determine if PECAM-1 and eNOS expression play a role in events related to the pathogenesis of TME. Our findings demonstrate that neutrophil transmigration across H. somni-treated TBBEC (SV-40 transformed bovine brain endothelial cell line) was reduced by treatment with anti-PECAM-1 antibodies. Confocal microscopy indicated that H. somni treatment leads to redistribution of PECAM-1 and eNOS on the surface of TBBEC. These findings suggest that PECAM-1 and eNOS may play a role in the early pathogenesis of TME. PMID:19524660

  12. Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation.

    Science.gov (United States)

    Gama Sosa, Miguel A; De Gasperi, Rita; Hof, Patrick R; Elder, Gregory A

    2016-01-01

    Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1-/- embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1-/- cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1-/- cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1-/- cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1-/- cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1-/- cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway. PMID:27443835

  13. Adhesive properties of Enterobacter sakazakii to human epithelial and brain microvascular endothelial cells

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    Pospischil Andreas

    2006-06-01

    Full Text Available Abstract Background Enterobacter sakazakii is an opportunistic pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. However, up to now little is known about the mechanisms of pathogenicity in E. sakazakii. A necessary state in the successful colonization, establishment and ultimately production of disease by microbial pathogens is the ability to adhere to host surfaces such as mucous membranes, gastric and intestinal epithelial or endothelial tissue. This study examined for the first time the adherence ability of 50 E. sakazakii strains to the two epithelial cell lines HEp-2 and Caco-2, as well as the brain microvascular endothelial cell line HBMEC. Furthermore, the effects of bacterial culture conditions on the adherence behaviour were investigated. An attempt was made to characterize the factors involved in adherence. Results Two distinctive adherence patterns, a diffuse adhesion and the formation of localized clusters of bacteria on the cell surface could be distinguished on all three cell lines. In some strains, a mixture of both patterns was observed. Adherence was maximal during late exponential phase, and increased with higher MOI. The adhesion capacity of E. sakazakii to HBMEC cells was affected by the addition of blood to the bacteria growth medium. Mannose, hemagglutination, trypsin digestion experiments and transmission electron microscopy suggested that the adhesion of E. sakazakii to the epithelial and endothelial cells is mainly non-fimbrial based. Conclusion Adherence experiments show heterogeneity within different E. sakazakii strains. In agreement with studies on E. cloacae, we found no relationship between the adhesive capacities in E. sakazakii and the eventual production of specific fimbriae. Further studies will have to be carried out in order to determine the adhesin(s involved in the interaction of E. sakazakii with cells and to

  14. Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis

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    Pittet Camille L

    2011-11-01

    Full Text Available Abstract Background Multiple sclerosis (MS, an inflammatory disease of the central nervous system (CNS, is characterized by blood-brain barrier (BBB disruption and massive infiltration of activated immune cells. Engagement of programmed cell death-1 (PD-1 expressed on activated T cells with its ligands (PD-L1 and PD-L2 suppresses T cell responses. We recently demonstrated in MS lesions elevated PD-L1 expression by glial cells and absence of PD-1 on many infiltrating CD8 T cells. We have now investigated whether human brain endothelial cells (HBECs, which maintain the BBB, can express PD-L1 or PD-L2 and thereby modulate T cells. Methods We used primary cultures of HBECs isolated from non-tumoral CNS tissue either under basal or inflamed conditions. We assessed the expression of PD-L1 and PD-L2 using qPCR and flow cytometry. Human CD8 T cells were isolated from peripheral blood of healthy donors and co-cultured with HBECs. Following co-culture with HBECs, proliferation and cytokine production by human CD8 T cells were measured by flow cytometry whereas transmigration was determined using a well established in vitro model of the BBB. The functional impact of PD-L1 and PD-L2 provided by HBECs was determined using blocking antibodies. We performed immunohistochemistry for the detection of PD-L1 or PD-L2 concurrently with caveolin-1 (a cell specific marker for endothelial cells on post-mortem human brain tissues obtained from MS patients and normal controls. Results Under basal culture conditions, PD-L2 is expressed on HBECs, whilst PD-L1 is not detected. Both ligands are up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 leads to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increases CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions does not express detectable PD-L1; in contrast, all blood vessels in normal

  15. Melatonin promotes blood-brain barrier integrity in methamphetamine-induced inflammation in primary rat brain microvascular endothelial cells.

    Science.gov (United States)

    Jumnongprakhon, Pichaya; Govitrapong, Piyarat; Tocharus, Chainarong; Tocharus, Jiraporn

    2016-09-01

    Melatonin is a neurohormone and has high potent of antioxidant that is widely reported to be active against methamphetamine (METH)-induced toxicity to neuron, glial cells, and brain endothelial cells. However, the role of melatonin on the inflammatory responses which are mostly caused by blood-brain barrier (BBB) impairment by METH administration has not been investigated. This study used the primary rat brain microvascular endothelial cells (BMVECs) to determine the protective mechanism of melatonin on METH-induced inflammatory responses in the BBB via nuclear factor-ĸB (NF-κB) and nuclear factor erythroid 2-related factor-2 (Nrf2) signaling. Herein, we demonstrated that melatonin reduced the level of the inflammatory mediators, including intercellular adhesion molecules (ICAM)-1, vascular cell adhesion molecules (VCAM)-1, matrix metallopeptidase (MMP)-9, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) caused by METH. These responses were related to the decrease of the expression and translocation of the NF-κB p65 subunit and the activity of NADPH oxidase (NOX)-2. In addition, melatonin promoted the antioxidant processes, modulated the expression and translocation of Nrf2, and also increased the level of heme oxygenase (HO)-1, NAD (P) H: quinone oxidoreductase (NQO)-1, γ-glutamylcysteine synthase (γ-GCLC), and the activity of superoxide dismutase (SOD) through NOX2 mechanism. In addition, we found that the protective role of melatonin in METH-induced inflammatory responses in the BBB was mediated through melatonin receptors (MT1/2). We concluded that the interaction of melatonin with its receptor prevented METH-induced inflammatory responses by suppressing the NF-κB signaling and promoting the Nrf2 signaling before BBB impairment. PMID:27268413

  16. Differential activation of acid sphingomyelinase and ceramide release determines invasiveness of Neisseria meningitidis into brain endothelial cells.

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    Alexander Simonis

    2014-06-01

    Full Text Available The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains.

  17. Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells

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    Kanmogne GD

    2012-05-01

    Full Text Available Georgette D Kanmogne1, Sangya Singh1, Upal Roy1, Xinming Liu1, JoEllyn McMillan1, Santhi Gorantla1, Shantanu Balkundi1, Nathan Smith1, Yazen Alnouti2, Nagsen Gautam2, You Zhou3, Larisa Poluektova1, Alexander Kabanov2, Tatiana Bronich2, Howard E Gendelman11Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, 2Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE; 3Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE, USAAbstract: Despite the successes of antiretroviral therapy (ART, HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART (atazanavir, ritonavir, indinavir, and efavirenz. We demonstrate that nanoART transfer from mononuclear phagocytes (MP to human brain microvascular endothelial cells (HBMEC can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γcnull mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and

  18. In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs)

    OpenAIRE

    Gallagher, Erin; Minn, IL; Chambers, Janice E.; Searson, Peter C.

    2016-01-01

    Background Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. Methods To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-...

  19. Uptake of codeine into intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells.

    Science.gov (United States)

    Fischer, Wiebke; Bernhagen, Jennifer; Neubert, Reinhard H H; Brandsch, Matthias

    2010-09-11

    Orally administered codeine has to permeate both the intestinal and the blood-brain barrier in order to act as analgesic and cough suppressant. In this study we characterized the uptake of codeine at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells. At both cell types, uptake of [(3)H]codeine was independent of an inwardly directed Na(+) gradient. Uptake was, however, strongly stimulated by an outwardly directed H(+) gradient and inhibited by the protonophore FCCP. [(3)H]Codeine uptake into Caco-2 cells was strongly temperature dependent. In the presence of excess amounts of unlabeled codeine, the uptake was inhibited by up to 87% (Caco-2) or 94% (RBE4), respectively. Synthetic opioids and some non-opioid organic cations like propranolol, pyrilamine and quinidine potently inhibited [(3)H]codeine uptake. Several prototype substrates of known transporters for amino acids, neurotransmitters and organic cations were ineffective. Our data are consistent with a hypothetic saturable, H(+)-dependent (antiport) mechanism not yet identified on a molecular level. The pH dependence of codeine uptake and its intracellular accumulation can partially also be explained by a model comprising diffusional membrane permeation of unionized species of codeine followed by codeine sequestration into acidic vesicles and distribution into cellular lipids. PMID:20510359

  20. Acute Modulation of Sugar Transport in Brain Capillary Endothelial Cell Cultures during Activation of the Metabolic Stress Pathway*

    OpenAIRE

    Cura, Anthony J.; Carruthers, Anthony

    2010-01-01

    GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete i...

  1. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors.

    Science.gov (United States)

    Noack, Andreas; Noack, Sandra; Buettner, Manuela; Naim, Hassan Y; Löscher, Wolfgang

    2016-01-01

    The blood-brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity. PMID:27375084

  2. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells.

    Science.gov (United States)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-08-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. PMID:27235552

  3. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    International Nuclear Information System (INIS)

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

  4. P. falciparum isolate-specific distinct patterns of induced apoptosis in pulmonary and brain endothelial cells.

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    Nadine N'Dilimabaka

    Full Text Available The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF, PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria.

  5. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    Science.gov (United States)

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy.

  6. Quercetin protects human brain microvascular endothelial cells from fibrillar β-amyloid1–40-induced toxicity

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    Yongjie Li

    2015-01-01

    Full Text Available Amyloid beta-peptides (Aβ are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature in the majority of Alzheimer׳s disease. Quercetin is a natural flavonoid molecule and has been demonstrated to have potent neuroprotective effects, but its protective effect on endothelial cells under Aβ-damaged condition is unclear. In the present study, the protective effects of quercetin on brain microvascular endothelial cells injured by fibrillar Aβ1–40 (fAβ1–40 were observed. The results show that fAβ1–40-induced cytotoxicity in human brain microvascular endothelial cells (hBMECs can be relieved by quercetin treatment. Quercetin increases cell viability, reduces the release of lactate dehydrogenase, and relieves nuclear condensation. Quercetin also alleviates intracellular reactive oxygen species generation and increases superoxide dismutase activity. Moreover, it strengthens the barrier integrity through the preservation of the transendothelial electrical resistance value, the relief of aggravated permeability, and the increase of characteristic enzyme levels after being exposed to fAβ1–40. In conclusion, quercetin protects hBMECs from fAβ1–40-induced toxicity.

  7. Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures.

    Science.gov (United States)

    Mao, Xiaoou; Xie, Lin; Greenberg, David A

    2015-12-01

    Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.

  8. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

    Science.gov (United States)

    Brailoiu, G Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2016-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor. PMID:26467159

  9. Hepatitis C virus infection induces elevation of CXCL10 in human brain microvascular endothelial cells.

    Science.gov (United States)

    Liu, Yuan; Chen, Li; Zou, Ziying; Zhu, Bing; Hu, Zonghai; Zeng, Ping; Wu, Lijuan; Xiong, Jie

    2016-09-01

    Hepatitis C virus (HCV) primarily infects liver tissues, while pathogenesis of extrahepatic tissues has been reported. About 50% of patients with HCV infection suffer from neurological disease. The underlying molecular mechanisms remain unclear. In the present study, we aimed to investigate the induction of CXC chemokine ligand 10 (CXCL10) in human brain microvascular endothelial cells (HBMECs) by HCV infection. CXCL10 and its receptor CXCR3 were constitutively expressed in HBMECs. HCV infection induced CXCL10 elevation in HBMECs. The elevation of CXCL10 in HBMECs was eliminated when HCV infection was blocked by neutralizing antibodies. NF-κB is a positive regulator for CXCL10 transcription. HCV infection led to an increased phosphorylation of NF-κB (ser536) in HBMECs, and CXCL10 induced by HCV was slightly decreased when an inhibitor of NF-κB was added. IL1 beta and IFN gama were also upregulated in HCV infected HBMECs, and could be depressed by inhibitor of NF-κB. Thus, HCV infection leads to upregulated expression of CXCL10 in HBMECs, which is probably via the phosphorylation of NF-κB. The findings of this study provide potential mechanisms and novel targets for HCV induced neuroinflammation. J. Med. Virol. 88:1596-1603, 2016. © 2016 Wiley Periodicals, Inc. PMID:26895737

  10. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Dennis J Grab

    Full Text Available BACKGROUND: Using human brain microvascular endothelial cells (HBMECs as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain. In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs known as protease activated receptors (PARs that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi we found that in vitro PAR-2 gene (F2RL1 expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49% and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q with Pasteurella multocida toxin (PMT. PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

  11. Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Filipa L Cardoso

    Full Text Available BACKGROUND: Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS is known to alter the integrity of the blood-brain barrier (BBB, little is known on the effects of unconjugated bilirubin (UCB and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC. METHODOLOGY/PRINCIPAL FINDINGS: Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. CONCLUSIONS: LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

  12. Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.

    Science.gov (United States)

    Zhao, Zhenjun; Johnson, Michael S; Chen, Biyi; Grace, Michael; Ukath, Jaysree; Lee, Vivienne S; McRobb, Lucinda S; Sedger, Lisa M; Stoodley, Marcus A

    2016-06-01

    OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation

  13. Brain endothelial TAK1 and NEMO safeguard the neurovascular unit

    OpenAIRE

    Ridder, Dirk A.; Wenzel, Jan; Müller, Kristin; Töllner, Kathrin; Tong, Xin-Kang; Assmann, Julian C; Stroobants, Stijn; Weber, Tobias; Niturad, Cristina; Fischer, Lisanne; Lembrich, Beate; Wolburg, Hartwig; Grand'Maison, Marilyn; Papadopoulos, Panayiota; Korpos, Eva

    2015-01-01

    Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood–brain barrier (BB...

  14. Effect of curcumin on the adhesion of platelets to brain microvascular endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Li ZHANG; Zhen-lun GU; Zheng-hong QIN; Zhong-qin LIANG

    2008-01-01

    Aim: To determine whether curcumin prevents the adhesion of platelets to brain microvascular endothelial cells (BMECs) cultured in vitro. Methods: [3H]Ad-chine-labeled platelets were incubated with BMECs to investigate the role of curcumin in the adhesion of platelets to BMECs. The number of platelets adher-ing to the BMECs monolayer was determined by liquid scintillation spectroscopy. The thrombin-induced expression of platelets P-selectin, glycoprotein Ⅱb (GPⅡb), and glycoprotein Ⅲa (GPⅢa) on the cell surface, was measured by flow cytometry. P-selectin mRNA levels of BMECs were determined by RT-PCR. The TNF-α-induced expressions of P-selectin and E-selectin on the surface of BMECs were determined by Western blotting. Results: The adhesion between thrombin-acti-vated platelets and normal BMECs, and that of TNF-α-activated BMECs and normal platelets were significantly increased, and this increase could be inhibited by curcumin (30-240 μmol/L) in a concentration-dependant manner. The platelets activated with thrombin and BMECs stimulated by TNF-α demonstrated an upregulated expressions of P-selectin and E-selectin, and this increase, when pretreated with curcumin for 30 min, could be restrained dose dependently. Curcumin also inhibited the increase of the GPⅡb/GPⅢa expression of thrombin-activated platelets in a concentration-dependent manner. Conclusion: Curcumin can inhibit the platelets to BMECs. This effect may be related to the decreased expressions of P-selectin, E-selectin, and GPⅡb/GPⅢa on platelets and BMECs.

  15. Role of astrocytic leptin receptor subtypes on leptin permeation across hCMEC/D3 human brain endothelial cells

    OpenAIRE

    Hsuchou, Hung; Kastin, Abba J; Tu, Hong; Abbott, N Joan; Couraud, Pierre-Olivier; Pan, Weihong

    2010-01-01

    Astrocytic leptin receptors (ObR) can be upregulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb overex...

  16. Morphine induces expression of platelet-derived growth factor in human brain microvascular endothelial cells: implication for vascular permeability.

    Directory of Open Access Journals (Sweden)

    Hongxiu Wen

    Full Text Available Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling and loss-of-function (Egr-1 approaches demonstrated the role of mitogen-activated protein kinases (MAPKs, PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

  17. Activation of sonic hedgehog signaling attenuates oxidized low-density lipoprotein-stimulated brain microvascular endothelial cells dysfunction in vitro.

    Science.gov (United States)

    Jiang, Xiu-Long; Chen, Ting; Zhang, Xu

    2015-01-01

    The study was performed to investigate the role of sonic hedgehog (SHH) in the oxidized low-density lipoprotein (oxLDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to oxLDL. The results indicated that treatment of MBMECs with oxLDL decreased the cell viability, and oxidative stress was involved in oxLDL-induce MBMECs dysfunction with increasing intracellular ROS and MDA formation as well as decreasing NO release and eNOS mRNA expression. In addition, SHH signaling components, such as SHH, Smo and Gli1, mRNA and protein levels were significantly decreased after incubation with increasing concentrations of oxLDL. Treatment with oxLDL alone or SHH loss-of-function significantly increased the permeability of MBMECs, and overexpression of SHH attenuated oxLDL-induced elevation of permeability in MBMECs. Furthermore, SHH gain-of-function could reverse oxLDL-induced apoptosis through inhibition caspase3 and caspase8 levels in MBMECs. Taken together, these results demonstrated that the suppression of SHH in MBMECs might contribute to the oxLDL-induced disruption of endothelial barrier. However, the overexpression of SHH could reverse oxLDL-induced endothelial cells dysfunction in vitro. PMID:26722472

  18. B7-H1 shapes T-cell–mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity

    Science.gov (United States)

    Klotz, Luisa; Kuzmanov, Ivan; Hucke, Stephanie; Gross, Catharina C.; Posevitz, Vilmos; Dreykluft, Angela; Schulte-Mecklenbeck, Andreas; Janoschka, Claudia; Lindner, Maren; Herold, Martin; Schwab, Nicholas; Ludwig-Portugall, Isis; Kurts, Christian; Meuth, Sven G.; Kuhlmann, Tanja; Wiendl, Heinz

    2016-01-01

    Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood–brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4+ T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity. PMID:27671636

  19. T11TS inhibits Angiopoietin-1/Tie-2 signaling, EGFR activation and Raf/MEK/ERK pathway in brain endothelial cells restraining angiogenesis in glioma model.

    Science.gov (United States)

    Bhattacharya, Debanjan; Chaudhuri, Suhnrita; Singh, Manoj Kumar; Chaudhuri, Swapna

    2015-06-01

    Malignant gliomas represent one of the most aggressive and hypervascular primary brain tumors. Angiopoietin-1, the peptide growth factor activates endothelial Tie-2 receptor promoting vessel maturation and vascular stabilization steps of angiogenesis in glioma. Epidermal growth factor receptor (EGFR) and Tie-2 receptor on endothelial cells once activated transmits signals through downstream Raf/MEK/ERK pathway promoting endothelial cell proliferation and migration which are essential for angiogenesis induction. The in vivo effect of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) on angiopoietin-1/Tie-2 axis, EGFR signaling and Raf/MEK/ERK pathway in glioma associated endothelial cells has not been investigated previously. The present study performed with rodent glioma model aims to investigate the effect of T11TS treatment on angiopoietin-1/Tie-2 signaling, EGFR activity and Raf/MEK/ERK pathway in glioma associated endothelial cells within glioma milieu. T11TS administration in rodent glioma model inhibited angiopoietin-1 expression and attenuated Tie-2 expression and activation in glioma associated brain endothelial cells. T11TS treatment also downregulated total and phosphorylated EGFR expression in glioma associated endothelial cells. Additionally T11TS treatment inhibited Raf-1 expression, MEK-1 and ERK-1/2 expression and phosphorylation in glioma associated brain endothelial cells. Thus T11TS therapy remarkably inhibits endothelial angiopoietin-1/Tie-2 signaling associated with vessel maturation and simultaneously antagonizes endothelial cell proliferation signaling by blocking EGFR activation and components of Raf/MEK/ERK pathway. Collectively, the findings demonstrate a multi-targeted anti-angiogenic activity of T11TS which augments the potential for clinical translation of T11TS as an effective angiogenesis inhibitor for glioma treatment.

  20. Repressed Ca(2+) clearance in parthenolide-treated murine brain bEND.3 endothelial cells.

    Science.gov (United States)

    Tsai, Tien-Yao; Lou, Shyh-Liang; Cheng, Ka-Shun; Wong, Kar-Lok; Wang, Mei-Ling; Su, Tzu-Hui; Chan, Paul; Leung, Yuk-Man

    2015-12-15

    Parthenolide is a sesquiterpene lactone compound isolated from the leaves and flowerheads of the plant feverfew (Tanacetum parthenium). The anticancer effects of parthenolide have been well studied and this lactone compound is currently under clinical trials. Parthenolide is also a protective agent in cardiac reperfusion injury via its inhibition of nuclear factor-κB (NF-κB). Not much is known if this compound affects signal transduction in non-tumor cells. We investigated whether parthenolide affected Ca(2+) signaling in endothelial cells, key components in regulating the vascular tone. In this work using mouse cortical microvascular bEND.3 endothelial cells, we found that a 15-h treatment with parthenolide resulted in amplified ATP-triggered Ca(2+) signal; the latter had a very slow decay rate suggesting suppression of Ca(2+) clearance. Evidence suggests parthenolide suppressed Ca(2+) clearance by inhibiting the plasmalemmal Ca(2+) pump; such suppression did not result from decreased expression of the plasmalemmal Ca(2+) pump protein. Rather, such suppression was possibly a consequence of endoplasmic reticulum (ER) stress, since salubrinal (an ER stress protector) was able to alleviate parthenolide-induced Ca(2+) clearance suppression. Given the current deployment of parthenolide as an anti-cancer drug in clinical trials and the potential usage of this lactone as a cardioprotectant, it is important to examine in details the perturbing effects of parthenolide on Ca(2+) homeostasis in endothelial cells and neighboring vascular smooth muscle cells, activities of which exert profound effects on hemodynamics. PMID:26607466

  1. Opiates Upregulate Adhesion Molecule Expression in Brain MicroVascular Endothelial Cells (BMVEC: Implications for Altered Blood Brain Barrier (BBB Permeability

    Directory of Open Access Journals (Sweden)

    Madhavan P.N. Nair

    2006-01-01

    Full Text Available The blood-brain barrier (BBB is an intricate cellular system composed of vascular endothelial cells and perivascular astrocytes that restrict the passage of immunocompetent cells into the central nervous system (CNS. Expression of the adhesion molecules, intercellular adhesion molecule 1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 on brain microvascular endothelial cells (BMVEC and their interaction with human immunodeficiency virus (HIV-1 viral proteins may help enhance viral adhesion and virus-cell fusion resulting in increased infectivity. Additionally, transmigration through the BBB is facilitated by both endothelial and monocyte/macrophage-derived nitric oxide (NO. Dysregulated production of NO by BMVEC due to opiates and HIV-1 viral protein interactions play a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB. Opioids act as co-factors in the neuropathogenesis of HIV-1 by facilitating BBB dysfunction however, no studies have been done to investigate the role of opiates alone or in combination with HIV-1 viral proteins on adhesion molecule expression in BMVEC. We hypothesize that opiates such as heroin and morphine in conjunction with the HIV-1 viral protein gp120 increase the expression of adhesion molecules ICAM-1 and VCAM-1 and these effects are mediated via the modulation of NO. Results show that opiates alone and in synergy with gp120 increase both the genotypic and phenotypic expression of ICAM-1 and VCAM-1 by BMVEC, additionally, these opiate induced effects may be the result of increased NO production. These studies will provide a better understanding of how opiate abuse in conjunction with HIV-1 infection facilitates the breakdown of the BBB and exacerbates the neuropathogenesis of HIV-1. Elucidation of the mechanisms of BBB modulation will provide new therapeutic approaches to maintain BBB integrity

  2. Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier

    DEFF Research Database (Denmark)

    Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette;

    2016-01-01

    The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of the present study......-culture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane...... proteins was analysed using RT-qPCR, mass spectrometry, and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the mono-culture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major...

  3. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  4. Delivering minocycline into brain endothelial cells with liposome-based technology

    Science.gov (United States)

    Xing, Changhong; Levchenko, Tatyana; Guo, Shuzhen; Stins, Monique; Torchilin, Vladimir P; Lo, Eng H

    2012-01-01

    Minocycline has been proposed as a way to blunt neurovascular injury from matrix metalloproteinases (MMPs) during stroke. However, recent clinical trials suggest that high levels of minocycline may have deleterious side-effects. Here, we showed that very high minocycline concentrations damage endothelial cells via calpain/caspase pathways. To alleviate this potential cytotoxicity, we encapsulated minocycline in liposomes. Low concentrations of minocycline could not reduce tumor necrosis factor α (TNFα)-induced MMP-9 release from endothelial cells. But low concentrations of minocycline-loaded liposomes significantly reduced TNFα-induced MMP-9 release. This study provides proof-of-concept that liposomes may be used to deliver lower levels of minocycline for targeting MMPs in cerebral endothelium. PMID:22491155

  5. Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro.

    Directory of Open Access Journals (Sweden)

    Roberta Paolinelli

    Full Text Available Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

  6. Delivering minocycline into brain endothelial cells with liposome-based technology

    OpenAIRE

    Xing, Changhong; Levchenko, Tatyana; Guo, Shuzhen; Stins, Monique; Torchilin, Vladimir P.; Eng H Lo

    2012-01-01

    Minocycline has been proposed as a way to blunt neurovascular injury from matrix metalloproteinases (MMPs) during stroke. However, recent clinical trials suggest that high levels of minocycline may have deleterious side-effects. Here, we showed that very high minocycline concentrations damage endothelial cells via calpain/caspase pathways. To alleviate this potential cytotoxicity, we encapsulated minocycline in liposomes. Low concentrations of minocycline could not reduce tumor necrosis facto...

  7. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling

    Directory of Open Access Journals (Sweden)

    Chia-Wei Huang

    2015-01-01

    Full Text Available Neonatal hypoxic-ischemic (HI brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs from adipose-derived stem cells (ASCs and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1 and vascular endothelial growth factor receptor 2 (VEGFR2 was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.

  8. The multifaceted responses of primary human astrocytes and brain microvascular endothelial cells to the Lyme disease spirochete, Borrelia burgdorferi

    Directory of Open Access Journals (Sweden)

    Catherine A. Brissette

    2013-08-01

    Full Text Available The vector-borne pathogen, Borrelia burgdorferi, causes a multi-system disorder including neurological complications. These neurological disorders, collectively termed neuroborreliosis, can occur in up to 15% of untreated patients. The neurological symptoms are probably a result of a glial-driven, host inflammatory response to the bacterium. However, the specific contributions of individual glial and other support cell types to the pathogenesis of neuroborreliosis are relatively unexplored. The goal of this project was to characterize specific astrocyte and endothelial cell responses to B. burgdorferi. Primary human astrocytes and primary HBMEC (human brain microvascular endothelial cells were incubated with B. burgdorferi over a 72-h period and the transcriptional responses to the bacterium were analyzed by real-time PCR arrays. There was a robust increase in several surveyed chemokine and related genes, including IL (interleukin-8, for both primary astrocytes and HBMEC. Array results were confirmed with individual sets of PCR primers. The production of specific chemokines by both astrocytes and HBMEC in response to B. burgdorferi, including IL-8, CXCL-1, and CXCL-10, were confirmed by ELISA. These results demonstrate that primary astrocytes and HBMEC respond to virulent B. burgdorferi by producing a number of chemokines. These data suggest that infiltrating phagocytic cells, particularly neutrophils, attracted by chemokines expressed at the BBB (blood–brain barrier may be important contributors to the early inflammatory events associated with neuroborreliosis.

  9. Blockade of Apoptosis Signal-Regulating Kinase 1 Attenuates Matrix Metalloproteinase 9 Activity in Brain Endothelial Cells and the Subsequent Apoptosis in Neurons after Ischemic Injury.

    Science.gov (United States)

    Cheon, So Y; Cho, Kyoung J; Kim, So Y; Kam, Eun H; Lee, Jong E; Koo, Bon-Nyeo

    2016-01-01

    Conditions of increased oxidative stress including cerebral ischemia can lead to blood-brain barrier dysfunction via matrix metalloproteinase (MMP). It is known that MMP-9 in particular is released from brain endothelial cells is involved in the neuronal cell death that occurs after cerebral ischemia. In the intracellular signaling network, apoptosis signal-regulating kinase 1 (ASK1) is the main activator of the oxidative stress that is part of the pathogenesis of cerebral ischemia. ASK1 also promotes apoptotic cell death and brain infarction after ischemia and is associated with vascular permeability and the formation of brain edema. However, the relationship between ASK1 and MMP-9 after cerebral ischemia remains unknown. Therefore, the aim of the present study was to determine whether blocking ASK1 would affect MMP-9 activity in the ischemic brain and cultured brain endothelial cells. Our results showed that ASK1 inhibition efficiently reduced MMP-9 activity in vivo and in vitro. In endothelial cell cultures, ASK1 inhibition upregulated phosphatidylinositol 3-kinase/Akt/nuclear factor erythroid 2 [NF-E2]-related factor 2/heme oxygenase-1 signals and downregulated cyclooxygenase-2 signals after hypoxia/reperfusion. Additionally, in neuronal cell cultures, cell death occurred when neurons were incubated with endothelial cell-conditioned medium (EC-CM) obtained from the hypoxia/reperfusion group. However, after incubation with EC-CM and following treatment with the ASK1 inhibitor NQDI-1, neuronal cell death was efficiently decreased. We conclude that suppressing ASK1 decreases MMP-9 activity in brain endothelial cells, and leads to decreased neuronal cell death after ischemic injury. PMID:27642277

  10. Platelets alter gene expression profile in human brain endothelial cells in an in vitro model of cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Mathieu Barbier

    Full Text Available Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC and human brain microvascular endothelial cells (HBEC and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

  11. Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2015-04-10

    Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.

  12. Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

    Institute of Scientific and Technical Information of China (English)

    Weihong Yang; Ling Li; Ruxun Huang; Zhong Pei; Songjie Liao; Jinsheng Zeng

    2012-01-01

    Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation -induced hypoxia inducible factor-1α expression. In this study, we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression. MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner. Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α. Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression; however, DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA. These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

  13. The effect of beta-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells

    DEFF Research Database (Denmark)

    Sorensen, M; Steenberg, B; Knipp, G T;

    1997-01-01

    PURPOSE: To investigate the effects of the beta-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). METHODS: The effective...... permeability coefficients (Peff) of the model peptides were determined across BBMEC monolayers. The dimensions of the aqueous pores in the tight junctions (TJs) of the BBMEC monolayers were determined using a series of hydrophilic permeants. This value and the molecular radius of each peptide were used...... to calculate the theoretical paracellular (PP*) and transcellular (PT*) permeability coefficients for each peptide. RESULTS: A comparison of the theoretical PP* values with the observed Peff values was made for a series of model peptides. For the most hydrophobic peptides (Ac-PheProXaaIle-NH2 and Ac...

  14. Brain endothelial TAK1 and NEMO safeguard the neurovascular unit

    Science.gov (United States)

    Ridder, Dirk A.; Wenzel, Jan; Müller, Kristin; Töllner, Kathrin; Tong, Xin-Kang; Assmann, Julian C.; Stroobants, Stijn; Weber, Tobias; Niturad, Cristina; Fischer, Lisanne; Lembrich, Beate; Wolburg, Hartwig; Grand’Maison, Marilyn; Papadopoulos, Panayiota; Korpos, Eva; Truchetet, Francois; Rades, Dirk; Sorokin, Lydia M.; Schmidt-Supprian, Marc; Bedell, Barry J.; Pasparakis, Manolis; Balschun, Detlef; D’Hooge, Rudi; Löscher, Wolfgang; Hamel, Edith

    2015-01-01

    Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood–brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB–independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1–NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP. PMID:26347470

  15. Interactions of primary neuroepithelial progenitor and brain endothelial cells: distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

    Institute of Scientific and Technical Information of China (English)

    Miguel A Gama Sosa; Rita De Gasperi; Anne B Rocher; Gissel M Perez; Keila Simons; Daniel E Cruz; Patrick R Hof; Gregory A Elder

    2007-01-01

    Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.

  16. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

    Science.gov (United States)

    Toyoda, Keisuke; Tanaka, Kunihiko; Nakagawa, Shinsuke; Thuy, Dinh Ha Duy; Ujifuku, Kenta; Kamada, Kensaku; Hayashi, Kentaro; Matsuo, Takayuki; Nagata, Izumi; Niwa, Masami

    2013-05-01

    Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

  17. Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2.

    Science.gov (United States)

    Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra

    2016-01-01

    Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells.

  18. Effect of low-dose methylprednisolone on peripheral blood endothelial progenitor cells and its significance in rats after brain injury

    Directory of Open Access Journals (Sweden)

    Bin ZHANG

    2011-05-01

    Full Text Available Objective To explore the effects of low-dose methylprednisolone(MP treatment after traumatic brain injury(TBI in rats on the number of peripheral blood endothelial progenitor cells(EPCs and injury area of the brain.Methods One hundred and fifty-four adult male Wistar rats were involved in the present study,and they were randomly divided into normal control group(n=18,TBI control group(n=38,MP control group(n=30,MP+TBI group(n=30 and TBI+MP group(n=38.The TBI model was reproduced by fluid percussion injury(FPI.MP(5mg/kg was intraperitoneally administered once a day for 4 days.Peripheral venous blood samples were taken on day 1,3,7 and 14,and the counts of EPCs were determined by flow cytometry.The rats were sacrificed on day 1 and 3,brain edema was estimated by dry-wet weight method,and the blood-brain barrier(BBB permeability was determined by Evans-blue extravasation.Results The counts of peripheral blood EPCs were significantly higher in MP control group,MP+TBI group and TBI+MP group on day 1,3 and 7 than that in normal control and TBI control group,and it returned to the level of normal control group on day 14.The BBB permeability was improved and brain edema alleviated in MP+TBI and TBI+MP group on day 3.Conclusion The administration of low-dose MP may increase the count of peripheral blood EPCs in rats,decrease BBB damage,and alleviate brain edema.

  19. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Yamazaki, Daiju [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu; Yamamura, Hisao [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2011-07-29

    Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  20. Design and physicochemical characterization of poly(amidoamine) nanoparticles and the toxicological evaluation in human endothelial cells: applications to peptide delivery to the brain

    NARCIS (Netherlands)

    Coue, G.M.J.P.C.; Freese, C.; Unger, R.E.; Kirkpatrick, C.J.; Pickl, K.E.; Sinner, F.M.; Engbersen, J.F.J.

    2013-01-01

    In this study, we investigated nanoparticles formulated by self-assembly of a biodegradable poly(amidoamine) (PAA) and a fluorescently labeled peptide, in their capacity to internalize in endothelial cells and deliver the peptide, with possible applications for brain drug delivery. The nanoparticles

  1. Lack of adrenomedullin in mouse endothelial cells results in defective angiogenesis, enhanced vascular permeability, less metastasis, and more brain damage.

    Science.gov (United States)

    Ochoa-Callejero, Laura; Pozo-Rodrigálvarez, Andrea; Martínez-Murillo, Ricardo; Martínez, Alfredo

    2016-01-01

    Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. AM plays critical roles in blood vessels, including regulation of vascular stability and permeability. To elucidate the autocrine/paracrine function of AM in endothelial cells (EC) in vivo, a conditional knockout of AM in EC (AM(EC-KO)) was used. The amount of vascularization of the matrigel implants was lower in AM(EC-KO) mice indicating a defective angiogenesis. Moreover, ablation of AM in EC revealed increased vascular permeability in comparison with wild type (WT) littermates. In addition, AM(EC-KO) lungs exhibited significantly less tumor growth than littermate WT mice using a syngeneic model of metastasis. Furthermore, following middle cerebral artery permanent occlusion, there was a significant infarct size decrease in animals lacking endothelial AM when compared to their WT counterparts. AM is an important regulator of EC function, angiogenesis, tumorigenesis, and brain response to ischemia. Studies of AM should bring novel approaches to the treatment of vascular diseases. PMID:27640364

  2. Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-κB pathway in mouse brain microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Lin Chih-Chung

    2013-01-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 is a proinflammatory mediator and elevated in the regions of several brain injury and inflammatory diseases. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the regulation of cyclooxygenase-2 (COX-2/prostaglandin E2 (PGE2 system in various cell types. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. Herein we investigated the effects of ET-1 in COX-2 regulation in mouse brain microvascular endothelial (bEnd.3 cells. Results The data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that ET-1-induced COX-2 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi- and Gq-protein-coupled ETB receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP and promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its corresponding binding sites in COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, up-regulation of COX-2 by ET-1 promoted PGE2 release in these cells. Conclusions These results suggested that in mouse bEnd.3 cells, activation of NF-κB by ETB-dependent MAPK cascades is essential for ET-1-induced up-regulation of COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rationally therapeutic interventions for brain injury or inflammatory diseases.

  3. Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Long Min

    2012-02-01

    Full Text Available Abstract Background Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC, is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown. Methods In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1 of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated. Results We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network. Conclusion These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.

  4. Solubilization of flurbiprofen into aptamer-modified PEG-PLA micelles for targeted delivery to brain-derived endothelial cells in vitro.

    Science.gov (United States)

    Mu, Chaofeng; Dave, Nimita; Hu, Jing; Desai, Pankaj; Pauletti, Giovanni; Bai, Shuhua; Hao, Jiukuan

    2013-01-01

    Novel aptamer-functionalized polyethylene glycol-polylactic acid (PEG-PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer's disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.

  5. Role of astrocytic leptin receptor subtypes on leptin permeation across hCMEC/D3 human brain endothelial cells.

    Science.gov (United States)

    Hsuchou, Hung; Kastin, Abba J; Tu, Hong; Joan Abbott, N; Couraud, Pierre-Olivier; Pan, Weihong

    2010-12-01

    Astrocytic leptin receptors (ObR) can be up-regulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb over-expression in C6 cells increased leptin permeation whereas ObRa over-expression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to the sodium fluorescein control was unchanged by over-expression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the over-expression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the evidence that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability. PMID:20977476

  6. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  7. Effect of baicalin and berberine on transport of nimodipine on primary-cultured, rat brain microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Dong-mei ZHANG; Hai-yan LIU; Lin XIE; Xiao-dong LIU

    2007-01-01

    Aim: To investigate whether baicalin and berberine affects the transport of nimodipine (NMD) across the blood-brain barrier (BBB). Methods: Primary-cultured, rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the BBB. When cells became confluent, the steady-state uptake of NMD by rBMEC with or without baicalin and berberine was measured. The ef-fects of baicalin and berberine on the efflux of NMD from rBMEC were also studied.Results: Baicalin (2-5 μg/mL) increased the uptake of NMD, and baicalin (10-20 μg/mL) decreased the uptake. The steady-state uptake of NMD was higher than that of control group in the presence of 0.01-1 μg/mL berberine, but was lower in the presence of 2-10 μg/mL berberine. Conclusion: The bidirectional effect of baicalin and berberine on the uptake of NMD by rBMEC was found. Higher concentration showed an inhibitory effect, and lower concentration demonstrated an increasing effect.

  8. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

    Science.gov (United States)

    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways.

  9. Prevention of Escherichia coli K1 Penetration of the Blood-Brain Barrier by Counteracting the Host Cell Receptor and Signaling Molecule Involved in E. coli Invasion of Human Brain Microvascular Endothelial Cells▿

    OpenAIRE

    Zhu, Longkun; Pearce, Donna; Kim, Kwang Sik

    2010-01-01

    Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interacti...

  10. Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection

    Directory of Open Access Journals (Sweden)

    Wenying Luo

    2015-01-01

    Full Text Available 2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC, to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

  11. HIV-1 Tat Regulates Occludin and Aβ Transfer Receptor Expression in Brain Endothelial Cells via Rho/ROCK Signaling Pathway

    Science.gov (United States)

    Chen, Yanlan; Jiang, Wenlin; Wu, Xianghong; Ye, Biao; Zhou, Xiaoting

    2016-01-01

    HIV-1 transactivator protein (Tat) has been shown to play an important role in HIV-associated neurocognitive disorders. The aim of the present study was to evaluate the relationship between occludin and amyloid-beta (Aβ) transfer receptors in human cerebral microvascular endothelial cells (hCMEC/D3) in the context of HIV-1-related pathology. The protein expressions of occludin, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in hCMEC/D3 cells were examined using western blotting and immunofluorescent staining. The mRNA levels of occludin, RAGE, and LRP1 were measured using quantitative real-time polymerase chain reaction. HIV-1 Tat at 1 µg/mL and the Rho inhibitor hydroxyfasudil (HF) at 30 µmol/L, with 24 h exposure, had no significant effect on hCMEC/D3 cell viability. Treatment with HIV-1 Tat protein decreased mRNA and protein levels of occludin and LRP1 and upregulated the expression of RAGE; however, these effects were attenuated by HF. These data suggest that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-mediated changes in occludin, RAGE, and LRP1 in hCMEC/D3 cells. HF may have a beneficial influence by protecting the integrity of the blood-brain barrier and the expression of Aβ transfer receptors.

  12. Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

    OpenAIRE

    Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

    2014-01-01

    P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. ...

  13. Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

    Directory of Open Access Journals (Sweden)

    Simona F Spampinato

    Full Text Available The ability of the Blood Brain Barrier (BBB to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS: fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

  14. Targeting Cells With MR Imaging Probes: Cellular Interaction And Intracellular Magnetic Iron Oxide Nanoparticles Uptake In Brain Capillary Endothelial and Choroidal Plexus Epithelial Cells

    Science.gov (United States)

    Cambianica, I.; Bossi, M.; Gasco, P.; Gonzalez, W.; Idee, J. M.; Miserocchi, G.; Rigolio, R.; Chanana, M.; Morjan, I.; Wang, D.; Sancini, G.

    2010-10-01

    Magnetic iron oxide nanoparticles (NPs) are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging. In delivery application, the critical step is the transport across cell layers and the internalization of NPs into specific cells, a process often limited by poor targeting specificity and low internalization efficiency. The development of the models of brain endothelial cells and choroidal plexus epithelial cells in culture has allowed us to investigate into these mechanisms. Our strategy is aimed at exploring different routes to the entrapment of iron oxide NPs in these brain related cells. Here we demonstrated that not only cells endowed with a good phagocytic activity like activated macrophages but also endothelial brain capillary and choroidal plexus epithelial cells do internalize iron oxide NPs. Our study of the intracellular trafficking of NPs by TEM, and confocal microscopy revealed that NPs are mainly internalized by the endocytic pathway. Iron oxide NPs were dispersed in water and coated with 3,4-dihydroxyl-L-phenylalanine (L-DOPA) using standard procedures. Magnetic lipid NPs were prepared by NANOVECTOR: water in oil in water (W/O/W) microemulsion process has been applied to directly coat different iron based NPs by lipid layer or to encapsulate them into Solid Lipid Nanoparticles (SLNs). By these coating/loading the colloidal stability was improved without strong alteration of the particle size distribution. Magnetic lipid NPs could be reconstituted after freeze drying without appreciable changes in stability. L-DOPA coated NPs are stable in PBS and in MEM (Modified Eagle Medium) medium. The magnetic properties of these NPs were not altered by the coating processes. We investigated the cellular uptake, cytotoxicity, and interaction of these NPs with rat brain capillary endothelial (REB4) and choroidal plexus epithelial (Z310) cells. By means of widefield, confocal

  15. Brain pericytes increase the lipopolysaccharide-enhanced transcytosis of HIV-1 free virus across the in vitro blood–brain barrier: evidence for cytokine-mediated pericyte-endothelial cell crosstalk

    Science.gov (United States)

    2013-01-01

    Background Human immunodeficiency virus-1 (HIV-1) enters the brain by crossing the blood–brain barrier (BBB) as both free virus and within infected immune cells. Previous work showed that activation of the innate immune system with lipopolysaccharide (LPS) enhances free virus transport both in vivo and across monolayer monocultures of brain microvascular endothelial cells (BMECs) in vitro. Methods Here, we used monocultures and co-cultures of brain pericytes and brain endothelial cells to examine the crosstalk between these cell types in mediating the LPS-enhanced permeation of radioactively-labeled HIV-1 (I-HIV) across BMEC monolayers. Results We found that brain pericytes when co-cultured with BMEC monolayers magnified the LPS-enhanced transport of I-HIV without altering transendothelial electrical resistance, indicating that pericytes affected the transcytotic component of HIV-1 permeation. As LPS crosses the BBB poorly if at all, and since pericytes are on the abluminal side of the BBB, we postulated that luminal LPS acts indirectly on pericytes through abluminal secretions from BMECs. Consistent with this, we found that the pattern of secretion of cytokines by pericytes directly exposed to LPS was different than when the pericytes were exposed to the abluminal fluid from LPS-treated BMEC monolayers. Conclusion These results are evidence for a cellular crosstalk in which LPS acts at the luminal surface of the brain endothelial cell, inducing abluminal secretions that stimulate pericytes to release substances that enhance the permeability of the BMEC monolayer to HIV. PMID:23816186

  16. Induction of heme oxygenase-1 attenuates lipopolysaccharide-induced cyclooxygenase-2 expression in mouse brain endothelial cells

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    Yang Chuen-Mao

    2010-11-01

    Full Text Available Abstract Background Prostaglandin E2 (PGE2, an arachidonic acid metabolite converted by cyclooxygenase-2 (COX-2, plays important roles in the regulation of endothelial functions in response to bacterial infection. The enzymatic activity of COX-2 can be down-regulated by heme oxygenase-1 (HO-1 induction. However, the mechanisms underlying HO-1 modulating COX-2 protein expression are not known. Objective The aim of the present study was to investigate whether the up-regulation of HO-1 regulates COX-2 expression induced by lipopolysaccharide (LPS, an endotoxin produced by Gram negative bacteria, in mouse brain endothelial cells (bEnd.3 Methods Cultured bEnd.3 cells were used to investigate LPS-induced COX-2 expression and PGE2 production. Cobalt protoporphyrin IX (CoPP, an HO-1 inducer, infection with a recombinant adenovirus carried with HO-1 gene (Adv-HO-1, or zinc protoporphyrin (ZnPP, an HO-1 inhibitor was used to stimulate HO-1 induction or inhibit HO-1 activity. The expressions of COX-2 and HO-1 were evaluated by western blotting. PGE2 levels were detected by an enzyme-linked immunoassay. Hemoglobin (a chelator of carbon monoxide, CO, one of metabolites of HO-1 and CO-RM2 (a CO releasing molecule were used to investigate the mechanisms of HO-1 regulating COX-2 expression. Results We found that LPS-induced COX-2 expression and PGE2 production were mediated through NF-κB (p65 via activation of Toll-like receptor 4 (TLR4. LPS-induced COX-2 expression was inhibited by HO-1 induction by pretreatment with CoPP or infection with Adv-HO-1. This inhibitory effect of HO-1 was reversed by pretreatment with either ZnPP or hemoglobin. Pretreatment with CO-RM2 also inhibited TLR4/MyD88 complex formation, NF-κB (p65 activation, COX-2 expression, and PGE2 production induced by LPS. Conclusions We show here a novel inhibition of HO-1 on LPS-induced COX-2/PGE2 production in bEnd.3. Our results reinforce the emerging role of cerebral endothelium-derived HO-1

  17. The Physiochemistry of Capped Nanosilver Predicts Its Biological Activity in Rat Brain Endothelial Cells (REBEC4)

    Science.gov (United States)

    The “capping” or coating of nanosilver (nanoAg) extends its potency by limiting its oxidation and aggregation and stabilizing its size and shape. The ability of such coated nanoAg to alter the permeability and activate oxidative stress pathways in rat brain endothelia...

  18. HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

    Science.gov (United States)

    Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

    2015-01-01

    Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury.

  19. Endotoxin-activated microglia injure brain derived endothelial cells via NF-κB, JAK-STAT and JNK stress kinase pathways

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    Yenari Midori A

    2011-03-01

    Full Text Available Abstract Background We previously showed that microglia damage blood brain barrier (BBB components following ischemic brain insults, but the underlying mechanism(s is/are not well known. Recent work has established the contribution of toll-like receptor 4 (TLR4 activation to several brain pathologies including ischemia, neurodegeneration and sepsis. The present study established the requirement of microglia for lipopolysaccharide (LPS mediated endothelial cell death, and explored pathways involved in this toxicity. LPS is a classic TLR4 agonist, and is used here to model aspects of brain conditions where TLR4 stimulation occurs. Methods/Results In monocultures, LPS induced death in microglia, but not brain derived endothelial cells (EC. However, LPS increased EC death when cocultured with microglia. LPS led to nitric oxide (NO and inducible NO synthase (iNOS induction in microglia, but not in EC. Inhibiting microglial activation by blocking iNOS and other generators of NO or blocking reactive oxygen species (ROS also prevented injury in these cocultures. To assess the signaling pathway(s involved, inhibitors of several downstream TLR-4 activated pathways were studied. Inhibitors of NF-κB, JAK-STAT and JNK/SAPK decreased microglial activation and prevented cell death, although the effect of blocking JNK/SAPK was rather modest. Inhibitors of PI3K, ERK, and p38 MAPK had no effect. Conclusions We show that LPS-activated microglia promote BBB disruption through injury to endothelial cells, and the specific blockade of JAK-STAT, NF-κB may prove to be especially useful anti-inflammatory strategies to confer cerebrovascular protection.

  20. West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

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    Kelsey Roe

    Full Text Available Characterizing the mechanisms by which West Nile virus (WNV causes blood-brain barrier (BBB disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE. Infection with WNV (NY99 strain significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1 did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101 strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

  1. 1α,25-Dihydroxyvitamin D3-Liganded Vitamin D Receptor Increases Expression and Transport Activity of P-glycoprotein in Isolated Rat Brain Capillaries and Human and Rat Brain Microvessel Endothelial Cells

    OpenAIRE

    Durk, Matthew R.; Chan, Gary N.Y.; Campos, Christopher R.; Peart, John C.; Chow, Edwin C.Y.; Lee, Eason; Cannon, Ronald E.; Bendayan, Reina; Miller, David S.; Pang, K. Sandy

    2012-01-01

    MDR1/P-gp induction by the vitamin D receptor (VDR) was investigated in isolated rat brain capillaries and rat (RBE4) and human (hCMEC/D3) brain microvessel endothelial cell lines. Incubation of isolated rat brain capillaries with 10 nM of the VDR ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] for 4 h increased P-gp protein expression (4-fold). Incubation with 1,25(OH)2D3 for 4 or 24 h increased P-gp transport activity (specific luminal accumulation of NBD-CSA, the fluorescent P-gp substrate...

  2. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

    OpenAIRE

    Haqqani Arsalan S; Delaney Christie E; Tremblay Tammy-Lynn; Sodja Caroline; Sandhu Jagdeep K; Stanimirovic Danica B

    2013-01-01

    Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain ...

  3. Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloid-β peptide in mouse brain capillary endothelial cells.

    Science.gov (United States)

    Ito, Shingo; Ohtsuki, Sumio; Murata, Sho; Katsukura, Yuki; Suzuki, Hiroya; Funaki, Miho; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-01-01

    Cerebral clearance of amyloid-β peptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

  4. Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats.

    Science.gov (United States)

    Fang, Lili; Li, Xue; Zhong, Yinbo; Yu, Jing; Yu, Lina; Dai, Haibin; Yan, Min

    2015-10-01

    Cerebral microvascular endothelial cells (ECs) are crucial for brain vascular repair and maintenance, but their physiological function may be impaired during ischemic stroke and diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, could exacerbate ischemia-induced EC injury and dysfunction. We investigated the protective effect of autophagy on cultured human brain microvascular endothelial cells (HBMEC) that underwent MGO treatment. A further study was conducted to explore the underlying mechanisms of the protective effect. Autophagic activity was assessed by evaluating protein levels, using western blot. 3-methyladenine (3-MA), bafilomycin A1, ammonium chloride (AC), Beclin 1 siRNA, and chloroquine (CQ) were used to cause autophagy inhibition. Alarmar blue assay and lactate dehydrogenase release assay were used to evaluate cell viability. Streptozotocin was administered to induce type I diabetes in rats and post-permanent middle cerebral artery occlusion was performed to elicit cerebral ischemia. Blood-brain barrier permeability was also assessed. Our study found that MGO reduced HBMEC cell viability in a concentration- and time-dependent manner, and triggered the responsive autophagy activation. Autophagy inhibitors bafilomycin A1, AC, 3-MA, and BECN1 siRNA exacerbated MGO-induced HBMEC injury. FAK phosphorylation inhibitor PF573228 inhibited MGO-triggered autophagy and enhanced lactate dehydrogenase release. Meanwhile, similar autophagy activation in brain vascular ECs was observed during permanent middle cerebral artery occlusion-induced cerebral ischemia in diabetic rats, while chloroquine-induced autophagy inhibition enhanced blood-brain barrier permeability. Taken together, our study indicates that autophagy triggered by MGO defends HBMEC against injuries. PMID:26251121

  5. Impact of polymer-modified gold nanoparticles on brain endothelial cells: exclusion of endoplasmic reticulum stress as a potential risk factor.

    Science.gov (United States)

    Anspach, Laura; Unger, Ronald E; Brochhausen, Christoph; Gibson, Matthew I; Klok, Harm-Anton; Kirkpatrick, C James; Freese, Christian

    2016-11-01

    A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood-brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modifications) for various times and evaluated their effects on cytotoxicity, proinflammatory mediators, barrier functions and factors involved in ER stress. We demonstrated a time-dependent uptake of all AuNPs and no cytotoxicity for up to 72 h of exposure. Exposure to certain AuNPs resulted in a time-dependent increase in the proinflammatory markers IL-8, MCP-1, sVCAM, sICAM. However, none of the AuNPs induced an increase in expression of the chaperones and stress sensor proteins BiP and GRP94, respectively, or the transcription factors ATF4 and ATF6. Furthermore, no upregulation of the UPR stress sensor receptor PERK, no active splicing product of the transcription factor XBP1 and no upregulation of the transcription factor CHOP were detectable. In conclusion, the results of the present study indicate that effects of different-sized gold nanoparticles modified with various polymers were not related to the induction of ER stress in brain microvascular endothelial cells or led to apoptosis. PMID:27492761

  6. Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

    Science.gov (United States)

    Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M; Rice, Kenner C; Gannon, Brenda M; Fantegrossi, William E; Gonzalez, Carmen; Paule, Merle G; Ali, Syed F

    2016-08-26

    Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic. PMID:27320055

  7. Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Tahanian E

    2011-05-01

    Full Text Available Elizabeth Tahanian¹, Luis Arguello Sanchez¹, Tze Chieh Shiao², René Roy², Borhane Annabi¹¹Centre de Recherche BioMED, ²Centre de Recherche PharmaQAM, Département de chimie, Université du Québec à Montréal, QC, CanadaAbstract: Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB. Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9 and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA, a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors.Keywords: blood–brain barrier, flavonoids, neuroinflammation, NF-κB signal transduction inhibitors

  8. Rhizoma Chuanxiong regulates vascular endothelial growth factor production in hypoxic human umbilical vein endothelial cells in vitro and in peri-infarct rat brain tissue

    Institute of Scientific and Technical Information of China (English)

    Muke Zhou; Mi Yang; Ning Chen; Yucai Wang; Jian Guo; Xue Yang; Zhijian Zhang; Dong Zhou; Li He

    2009-01-01

    BACKGROUND: Vascular endothelial growth factor (VEGF) acts as "molecular bridge" following ischemic stroke to improve and restore blood supply and reduce infarction volume. Clinical studies have demonstrated the efficacy of Rhizoma Chuanxiong (Chuanxiong) in the treatment of ischemic cerebrovascular diseases. However, whether it promotes endogenous VEGF expression in ischemic stroke remains unknown.OBJECTIVE: To investigate the influence of Rhizoma Chuanxiong on VEGF production in vitro cultured human umbilical vein endothelial cells and on VEGF expression in ischemic cerebral tissues to explore its role in angiogenesis.DESIGN, TIME AND SE'B'ING: In vitro basic comparison of traditional Chinese drug-containing serum pharmacology; in vivo randomized, controlled, animal experiment. This study was performed at the Medical Laboratory of West China Hospital, Sichuan University between December 2002 and April 2004.MATERIALS: Two Chinese rabbits were selected. One was intragastrically perfused with 5.8 g/kg Rhizoma Chuanxiong extract twice per day for three consecutive days to prepare Rhizoma Chuanxiong extract-containing serum. The remaining rabbit was intragastdcally perfused with the same volume of normal saline twice per day for three consecutive days. Rhizoma Chuanxiong extract was provided by Beijing Traditional Chinese Medicine Research Institute, predominantly composed of ligustrazine, ligustilide, and ferulic acid. ChemiKineTM human VEGF Kit was purchased from Chemicon, USA; mouse anti-VEGF monoclonal antibody and biotin-goat anti-mouse IgG were purchased from Santa Cruz Biotechnology. Inc., USA.METHODS: (1) In vitro experiment: in vitro cultured human umbilical vein endothelial cells were separately incubated in rabbit serum with 10% Rhizoma Chuanxiong extract, normal medium without rabbit serum, and rabbit serum without Rhizoma Chuanxiong extract (blank control). In addition, cells from the three groups were incubated under normoxia (5% CO2, 95% air) and

  9. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Science.gov (United States)

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

  10. Interactions of Neuropathogenic Escherichia coli K1 (RS218 and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Farzana Abubakar Yousuf

    2014-01-01

    Full Text Available Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin, adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (IbeA, CNF1, metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (CNF1, metabolism (D-serine catabolism abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

  11. The protective role of isorhamnetin on human brain microvascular endothelial cells from cytotoxicity induced by methylglyoxal and oxygen-glucose deprivation.

    Science.gov (United States)

    Li, Wenlu; Chen, Zhigang; Yan, Min; He, Ping; Chen, Zhong; Dai, Haibin

    2016-02-01

    As the first target of stroke, cerebral endothelial cells play a key role in brain vascular repair and maintenance, and their function is impeded in diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, accumulates in diabetic patients. MGO and MGO-induced advanced glycation end-products (AGEs) could ameliorate stroke-induced brain vascular damage, closely related with ECs dysfunction. Using MGO plus oxygen-glucose deprivation (OGD) to mimic diabetic stroke, we reported the protective effect of isorhamnetin on OGD-induced cytotoxicity after MGO treatment on primary human brain microvascular endothelial cells (HBMEC) and explored the underlying mechanisms. Treatment of MGO for 24 h significantly enhanced 3-h OGD-induced HBMEC toxic effect, which was inhibited by pretreatment of isorhamnetin (100 μmol/L). Moreover, the protective effect of isorhamnetin is multiple function dependent, which includes anti-inflammation, anti-oxidative stress and anti-apoptosis effects. Besides its well-known inhibition on the mitochondria-dependent or intrinsic apoptotic pathway, isorhamnetin also reduced activation of the extrinsic apoptotic pathway, as characterized by the decreased expression and activity of caspase 3 and caspase 8. Furthermore, pretreatment with isorhamnetin specifically inhibited FAS/FASL expression and suppressed nuclear factor-kappa B nuclear translocation. Taken together, our results indicated that isorhamnetin protected against OGD-induced cytotoxicity after MGO treatment in cultured HBMEC due to its multiple protective effects and could inhibit Fas-mediated extrinsic apoptosis. Therefore, isorhamnetin is a promising reagent for the treatment of hyperglycemia and ischemia-induced cerebral vascular degeneration. A proposed model of the potential protective mechanism of isorhamnetin, a metabolite of quercetin, on methylglyoxal (MGO) treatment plus oxygen-glucose deprivation (OGD) exposure-induced cytotoxicity in cultured human

  12. Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

    Science.gov (United States)

    2014-01-01

    Background Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood–brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer’s disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model. Results Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, “reduced-exposure” brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1. Conclusions In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content. PMID:24755281

  13. Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

    Science.gov (United States)

    Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

    2014-01-01

    P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

  14. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells

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    Axel Haarmann

    2015-08-01

    Full Text Available Dimethyl fumarate (DMF is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  15. Endothelial potential of human embryonic stem cells

    OpenAIRE

    Levenberg, Shulamit; Zoldan, Janet; Basevitch, Yaara; Langer, Robert

    2007-01-01

    Growing interest in using endothelial cells for therapeutic purposes has led to exploring human embryonic stem cells as a potential source for endothelial progenitor cells. Embryonic stem cells are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. However, there are many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells in the clinic can be realize...

  16. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

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    Ken Kono

    Full Text Available 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.

  17. Retinal Endothelial Cell Apoptosis Stimulates Recruitment of Endothelial Progenitor Cells

    Science.gov (United States)

    Bhatwadekar, Ashay D.; Glenn, Josephine V.; Curtis, Tim M.; Grant, Maria B.; Stitt, Alan W.; Gardiner, Tom A.

    2013-01-01

    Purpose Bone marrow–derived endothelial progenitor cells (EPCs) contribute to vascular repair although it is uncertain how local endothelial cell apoptosis influences their reparative function. This study was conducted to determine how the presence of apoptotic bodies at sites of endothelial damage may influence participation of EPCs in retinal microvascular repair. Methods Microlesions of apoptotic cell death were created in monolayers of retinal microvascular endothelial cells (RMECs) by using the photodynamic drug verteporfin. The adhesion of early-EPCs to these lesions was studied before detachment of the apoptotic cells or after their removal from the wound site. Apoptotic bodies were fed to normal RMECs and mRNA levels for adhesion molecules were analyzed. Results Endothelial lesions where apoptotic bodies were left attached at the wound site showed a fivefold enhancement in EPC recruitment (P < 0.05) compared with lesions where the apoptotic cells had been removed. In intact RMEC monolayers exposed to apoptotic bodies, expression of ICAM, VCAM, and E-selectin was upregulated by 5- to 15-fold (P < 0.05– 0.001). EPCs showed a characteristic chemotactic response (P < 0.05) to conditioned medium obtained from apoptotic bodies, whereas analysis of the medium showed significantly increased levels of VEGF, IL-8, IL-6, and TNF-α when compared to control medium; SDF-1 remained unchanged. Conclusions The data indicate that apoptotic bodies derived from retinal capillary endothelium mediate release of proangiogenic cytokines and chemokines and induce adhesion molecule expression in a manner that facilitates EPC recruitment. PMID:19474402

  18. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

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    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  19. Inflammation Modulates RLIP76/RALBP1 Electrophile-Glutathione Conjugate Transporter and Housekeeping Genes in Human Blood-Brain Barrier Endothelial Cells.

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    Barbara Bennani-Baiti

    Full Text Available Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-κB. RLIP76 (a.k.a. RALBP1, an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-α pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-α on RLIP76, we faced the problem of choosing reference genes impervious to TNF-α. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-α, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-α, and show for the first time that RLIP76 expression is induced by TNF-α and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1, another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-α is not a general property of glutathione transporters. The reference geneset

  20. Nipah virus infection and glycoprotein targeting in endothelial cells

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    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

  1. SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection.

    Science.gov (United States)

    Zhe, Ma; Jie, Peng; Hui, Zhang; Bin, Xu; Xiaomeng, Pei; Huixing, Lin; Chengping, Lu; Hongjie, Fan

    2016-08-01

    Streptococcus equi ssp. zooepidemicus (SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7 %, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis. PMID:27178179

  2. G-CSF Protects Human Brain Vascular Endothelial Cells Injury Induced by High Glucose, Free Fatty Acids and Hypoxia through MAPK and Akt Signaling

    Science.gov (United States)

    Tao, Yinghong; Guo, Jingchun; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Dong, Qiang; Hu, Renming

    2015-01-01

    Granulocyte-colony stimulating factor (G-CSF) has been shown to play a neuroprotective role in ischemic stroke by mobilizing bone marrow (BM)-derived endothelial progenitor cells (EPCs), promoting angiogenesis, and inhibiting apoptosis. Impairments in mobilization and function of the BM-derived EPCs have previously been reported in animal and human studies of diabetes where there is both reduction in the levels of the BM-derived EPCs and its ability to promote angiogenesis. This is hypothesized to account for the pathogenesis of diabetic vascular complications such as stroke. Here, we sought to investigate the effects of G-CSF on diabetes-associated cerebral vascular defect. We observed that pretreatment of the cultured human brain vascular endothelial cells (HBVECs) with G-CSF largely prevented cell death induced by the combination stimulus with high glucose, free fatty acids (FFA) and hypoxia by increasing cell viability, decreasing apoptosis and caspase-3 activity. Cell ultrastructure measured by transmission electron microscope (TEM) revealed that G-CSF treatment nicely reduced combination stimulus-induced cell apoptosis. The results from fluorescent probe Fluo-3/AM showed that G-CSF greatly suppressed the levels of intracellular calcium ions under combination stimulus. We also found that G-CSF enhanced the expression of cell cycle proteins such as human cell division cycle protein 14A (hCdc14A), cyclinB and cyclinE, inhibited p53 activity, and facilitated cell cycle progression following combination stimulus. In addition, activation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt, and deactivation of c-Jun N terminal kinase (JNK) and p38 were proved to be required for the pro-survival effects of G-CSF on HBVECs exposed to combination stimulus. Overall, G-CSF is capable of alleviating HBVECs injury triggered by the combination administration with high glucose, FFA and hypoxia involving the mitogen-activated protein kinases (MAPK) and Akt signaling

  3. Endothelial cell growth factor and ionophore A23187 stimulation of production of inositol phosphates in porcine aorta endothelial cells.

    OpenAIRE

    Moscat, J; Moreno, F.; Herrero, C.; C. López; García-Barreno, P.

    1988-01-01

    The existence of a bovine brain-derived endothelial cell growth factor has recently been reported, but its mode of action is unknown. We show that the endothelial cell growth factor is a potent stimulant of inositol monophosphate release in porcine aorta endothelial cells. Although the activation of phospholipase C by this factor does not appear to be dependent on Ca2+, the Ca2+ ionophore A23187 stimulates release of inositol phosphates. It is suggested that the inositol 1,4,5-trisphosphate 3...

  4. Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation.

    Science.gov (United States)

    Lopez-Ramirez, Miguel Alejandro; Reijerkerk, Arie; de Vries, Helga E; Romero, Ignacio Andres

    2016-08-01

    Brain endothelial cells constitute the major cellular element of the highly specialized blood-brain barrier (BBB) and thereby contribute to CNS homeostasis by restricting entry of circulating leukocytes and blood-borne molecules into the CNS. Therefore, compromised function of brain endothelial cells has serious consequences for BBB integrity. This has been associated with early events in the pathogenesis of several disorders that affect the CNS, such as multiple sclerosis, HIV-associated neurologic disorder, and stroke. Recent studies demonstrate that brain endothelial microRNAs play critical roles in the regulation of BBB function under normal and neuroinflammatory conditions. This review will focus on emerging evidence that indicates that brain endothelial microRNAs regulate barrier function and orchestrate various phases of the neuroinflammatory response, including endothelial activation in response to cytokines as well as restoration of inflamed endothelium into a quiescent state. In particular, we discuss novel microRNA regulatory mechanisms and their contribution to cellular interactions at the neurovascular unit that influence the overall function of the BBB in health and during neuroinflammation.-Lopez-Ramirez, M. A., Reijerkerk, A., de Vries, H. E., Romero, I. A. Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation. PMID:27118674

  5. Escherichia coli K1 RS218 Interacts with Human Brain Microvascular Endothelial Cells via Type 1 Fimbria Bacteria in the Fimbriated State

    Science.gov (United States)

    Teng, Ching-Hao; Cai, Mian; Shin, Sooan; Xie, Yi; Kim, Kee-Jun; Khan, Naveed Ahmed; Di Cello, Francescopaolo; Kim, Kwang Sik

    2005-01-01

    Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC. PMID:15845498

  6. [Transplantation of corneal endothelial cells].

    Science.gov (United States)

    Amano, Shiro

    2002-12-01

    Though conventional corneal transplantation has achieved great success, it still has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases. Reconstructing clinically usable corneas by applying the technology of regenerative medicine can offer a solution to these problems, as well as making corneal transplantation a non-emergency surgery and enabling the usage of banked corneal cells. In the present study, we focused on corneal endothelium that is critical for corneal transparency and investigated the reconstruction of cornea utilizing cultured human corneal endothelial cells (HCECs). We succeeded in steadily culturing HCECs by using culture dishes pre-coated with extracellular matrix produced by calf corneal endothelial cells and culture media that contained basic fibroblast growth factor and fetal bovine serum. We performed the following analysis utilizing these cultured HCECs. The older the donor was, the more frequently large senescent cells appeared in the passaged HCECs. The telomeres of HCECs were measured as terminal restriction fragments (TRF) by Southern blotting. HCECs, in vivo from donors in their seventies had a long TRFs of over 12 kilobases. Passaging shortened the TRFs but there was no difference in TRFs among donors of various ages. These results indicated that shortening of telomere length is not related to senescence of HCECs. We investigated the role of advanced glycation end products (AGEs) in the senescence of in vivo HCECs. The results indicated that AGE-protein in the aqueous humor is endocytosed into HCECs via AGE receptors expressed on the surface of HCECs and damages HCECs by producing reactive oxygen species and inducing apoptosis, suggesting that AGEs, at least partly, cause the senescence of HECEs. HCECs were cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prions. Primary and passage

  7. Vascular endothelial growth factor:an attractive target in the treatment of hypoxic/ischemic brain injury

    Institute of Scientific and Technical Information of China (English)

    Hui Guo; Hui Zhou; Jie Lu; Yi Qu; Dan Yu; Yu Tong

    2016-01-01

    Cerebral hypoxia or ischemia results in cell death and cerebral edema, as well as other cellular reactions such as angiogenesis and the reestablishment of functional microvasculature to promote recovery from brain injury. Vascular endothelial growth factor is expressed in the central nervous system after hypoxic/ischemic brain injury, and is involved in the process of brain repairvia the regulation of angiogenesis, neurogenesis, neurite outgrowth, and cerebral edema, which all require vascular endothelial growth factor signaling. In this review, we focus on the role of the vascular endothelial growth factor signaling pathway in the response to hypoxic/ischemic brain injury, and discuss potential therapeutic interventions.

  8. Citicoline induces angiogenesis improving survival of vascular/human brain microvessel endothelial cells through pathways involving ERK1/2 and insulin receptor substrate-1

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    Krupinski Jerzy

    2012-12-01

    Full Text Available Abstract Background Citicoline is one of the neuroprotective agents that have been used as a therapy in stroke patients. There is limited published data describing the mechanisms through which it acts. Methods We used in vitro angiogenesis assays: migration, proliferation, differentiation into tube-like structures in Matrigel™ and spheroid development assays in human brain microvessel endothelial cells (hCMEC/D3. Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. An analysis of citicoline signalling pathways was previously studied using a Kinexus phospho-protein screening array. A staurosporin/calcium ionophore-induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. In a pilot in vivo study, transient MCAO in rats was carried out with and without citicoline treatment (1000 mg/Kg applied at the time of occlusion and subsequently every 3 days until euthanasia (21 days. Vascularity of the stroke-affected regions was examined by immunohistochemistry. Results Citicoline presented no mitogenic and chemotactic effects on hCMEC/D3; however, it significantly increased wound recovery, the formation of tube-like structures in Matrigel™ and enhanced spheroid development and sprouting. Citicoline induced the expression of phospho-extracellular-signal regulated kinase (ERK-1/2. Kinexus assays showed an over-expression of insulin receptor substrate-1 (IRS-1. Knock-down of IRS-1 with targeted siRNA in our hCMEC/D3 inhibited the pro-angiogenic effects of citicoline. The percentage of surviving cells was higher in the presence of citicoline. Citicoline treatment significantly increased the numbers of new, active CD105-positive microvessels following MCAO. Conclusions The findings demonstrate both a pro-angiogenic and protective effect of citicoline on hCMEC/D3 in vitro and following middle cerebral artery occlusion (MCAO in vivo.

  9. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  10. Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

    OpenAIRE

    Eigenmann, Daniela E; Xue, Gongda; Kwang S Kim; Moses, Ashlee V.; Hamburger, Matthias; Oufir, Mouhssin

    2013-01-01

    Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellu...

  11. Astrocytes drive upregulation of the multidrug resistance transporter ABCB1 (P-Glycoprotein) in endothelial cells of the blood-brain barrier in mutant superoxide dismutase 1-linked amyotrophic lateral sclerosis.

    Science.gov (United States)

    Qosa, Hisham; Lichter, Jessica; Sarlo, Mark; Markandaiah, Shashirekha S; McAvoy, Kevin; Richard, Jean-Philippe; Jablonski, Michael R; Maragakis, Nicholas J; Pasinelli, Piera; Trotti, Davide

    2016-08-01

    The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313. PMID:27158936

  12. Estetrol modulates endothelial nitric oxide synthesis in human endothelial cells

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    Maria Magdalena eMontt-Guevara

    2015-07-01

    Full Text Available Estetrol (E4 is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO is a key player for vascular function and disease during pregnancy and throughout ageing in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS in cultured human umbilical vein endothelial cells (HUVEC. E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2 and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use.

  13. Estetrol Modulates Endothelial Nitric Oxide Synthesis in Human Endothelial Cells.

    Science.gov (United States)

    Montt-Guevara, Maria Magdalena; Giretti, Maria Silvia; Russo, Eleonora; Giannini, Andrea; Mannella, Paolo; Genazzani, Andrea Riccardo; Genazzani, Alessandro David; Simoncini, Tommaso

    2015-01-01

    Estetrol (E4) is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO) is a key player for vascular function and disease during pregnancy and throughout aging in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVEC). E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2, and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use. PMID:26257704

  14. Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers

    DEFF Research Database (Denmark)

    Hersom, Maria; Helms, Hans Christian; Pretzer, Natasia;

    2016-01-01

    across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated...... in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe......]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin...

  15. Differential adhesion of tumor cells to capillary endothelial cells in vitro.

    OpenAIRE

    Alby, L; Auerbach, R

    1984-01-01

    Adhesion studies were carried out to determine the relative ability of glioma cells and ovary-derived teratoma cells to adhere to endothelial cells obtained from mouse brain capillaries (designated MBE cell line) or mouse ovaries (designated MOE cell line). The teratoma cells showed preferential adhesion to MOE cells, whereas the glioma cells showed preferential adhesion to the MBE cell line. In contrast, the glioma and teratoma cells adhered equally to L929 and 3T3 fibroblasts. A testicular ...

  16. Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

    Science.gov (United States)

    Ruud, Johan; Wilhelms, Daniel Björk; Nilsson, Anna; Eskilsson, Anna; Tang, Yan-Juan; Ströhle, Peter; Caesar, Robert; Schwaninger, Markus; Wunderlich, Thomas; Bäckhed, Fredrik; Engblom, David; Blomqvist, Anders

    2013-05-01

    Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

  17. Blood cells and endothelial barrier function.

    Science.gov (United States)

    Rodrigues, Stephen F; Granger, D Neil

    2015-01-01

    The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction. PMID:25838983

  18. Impairment of brain endothelial glucose transporter by methamphetamine causes blood-brain barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Murrin L Charles

    2011-03-01

    Full Text Available Abstract Background Methamphetamine (METH, an addictive psycho-stimulant drug with euphoric effect is known to cause neurotoxicity due to oxidative stress, dopamine accumulation and glial cell activation. Here we hypothesized that METH-induced interference of glucose uptake and transport at the endothelium can disrupt the energy requirement of the blood-brain barrier (BBB function and integrity. We undertake this study because there is no report of METH effects on glucose uptake and transport across the blood-brain barrier (BBB to date. Results In this study, we demonstrate that METH-induced disruption of glucose uptake by endothelium lead to BBB dysfunction. Our data indicate that a low concentration of METH (20 μM increased the expression of glucose transporter protein-1 (GLUT1 in primary human brain endothelial cell (hBEC, main component of BBB without affecting the glucose uptake. A high concentration of 200 μM of METH decreased both the glucose uptake and GLUT1 protein levels in hBEC culture. Transcription process appeared to regulate the changes in METH-induced GLUT1 expression. METH-induced decrease in GLUT1 protein level was associated with reduction in BBB tight junction protein occludin and zonula occludens-1. Functional assessment of the trans-endothelial electrical resistance of the cell monolayers and permeability of dye tracers in animal model validated the pharmacokinetics and molecular findings that inhibition of glucose uptake by GLUT1 inhibitor cytochalasin B (CB aggravated the METH-induced disruption of the BBB integrity. Application of acetyl-L-carnitine suppressed the effects of METH on glucose uptake and BBB function. Conclusion Our findings suggest that impairment of GLUT1 at the brain endothelium by METH may contribute to energy-associated disruption of tight junction assembly and loss of BBB integrity.

  19. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

    Directory of Open Access Journals (Sweden)

    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  20. Endothelial progenitor cells in cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    Poay; Sian; Sabrina; Lee; Kian; Keong; Poh

    2014-01-01

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells(EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vas-culogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk fac-tors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardio-vascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evalu-ate the challenges facing EPC research and how these may be overcome.

  1. Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

    OpenAIRE

    Meier, C.; Oelschlaeger, T A; Merkert, H; Korhonen, T K; Hacker, J

    1996-01-01

    Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epit...

  2. The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells

    OpenAIRE

    Jinju Wang; Runmin Guo; Yi Yang; Bradley Jacobs; Suhong Chen; Ifeanyi Iwuchukwu; Gaines, Kenneth J.; Yanfang Chen; Richard Simman; Guiyuan Lv; Keng Wu; Bihl, Ji C.

    2016-01-01

    Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by ...

  3. Blood cells and endothelial barrier function

    OpenAIRE

    Rodrigues, Stephen F.; Granger, D Neil

    2015-01-01

    The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of solub...

  4. THE RELATIONSHIP BETWEEN PERITUMORAL BRAIN EDEMA AND VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION IN PATIENTS WITH MENINGIOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To determine whether VEGF plays a role in the development of peritumoral brain edema. Methods 50 meningioma patients and their VEGF expression were studied. We took a mono- clonal antibody from mouse to VEGF to stain the tumor cells, the vascular endothelial cells and the interstitial cells. The severity of brain edema was evaluated according to CT or MR scans by the following equation: edema index = Vtumor+edema/Vtumor. The relationship between VEGF expression and edema index was analyzed statisti- cally. Results VEGF was expressed in meningioma tumor cells, which is usually concentrated at the pe- ripheral sites of the tumor. There was a positive linear correlation between the expression and the brain edema index. Conclusion VEGF may play a role in the development of peritumoral brain edema in meningioma patient.

  5. Endothelial Cell Response to Fusobacterium nucleatum.

    Science.gov (United States)

    Mendes, Reila Tainá; Nguyen, Daniel; Stephens, Danielle; Pamuk, Ferda; Fernandes, Daniel; Van Dyke, Thomas E; Kantarci, Alpdogan

    2016-07-01

    Vascular response is an essential aspect of an effective immune response to periodontal disease pathogens, as new blood vessel formation contributes to wound healing and inflammation. Gaining a greater understanding of the factors that affect vascular response may then contribute to future breakthroughs in dental medicine. In this study, we have characterized the endothelial cell response to the common bacterium Fusobacterium nucleatum, an important bridging species that facilitates the activity of late colonizers of the dental biofilm. Endothelial cells were infected with Fusobacterium nucleatum (strain 25586) for periods of 4, 12, 24, or 48 h. Cell proliferation and tube formation were analyzed, and expression of adhesion molecules (CD31 and CD34) and vascular endothelial growth factor (VEGF) receptors 1 and 2 was measured by fluorescence-activated cell sorter (FACS) analysis. Data indicate that F. nucleatum impaired endothelial cell proliferation and tube formation. The findings suggest that the modified endothelial cell response acts as a mechanism promoting the pathogenic progression of periodontal diseases and may potentially suggest the involvement of periodontopathogens in systemic diseases associated with periodontal inflammation. PMID:27185790

  6. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  7. Normal saline influences coagulation and endothelial function after traumatic brain injury and hemorrhagic shock in pigs

    DEFF Research Database (Denmark)

    Dekker, Simone E; Sillesen, Martin; Bambakidis, Ted;

    2014-01-01

    of endothelial activation (E-selectin, Intercellular adhesion molecule [ICAM]-1), coagulation activation (prothrombin fragment 1 + 2), and natural anticoagulation (activated protein C [aPC]) were determined in serum and brain whole cell lysates. RESULTS: Serum levels of aPC were greater in the NS group (203 ± 30......). Circulating ICAM-1 levels were increased in the NS group (151 ± 9 ng/mL) compared with the HEX (100 ± 9 ng/mL; P coagulation, natural......BACKGROUND: Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of trauma-related deaths. These insults disrupt coagulation and endothelial systems. This study investigated whether previously reported differences in lesion size and brain swelling during normal saline (NS...

  8. Optical Investigations of Endothelial Cell Motility

    DEFF Research Database (Denmark)

    Rossen, Ninna Struck

    A monolayer of endothelial cells lines the entire circulatory system and create a barrier between the circulatory system and the tissues. To create and maintain an intact barrier, the individual cells have to connect tightly with their neighbors, which causes a highly correlated motion between...

  9. Endothelial progenitor cells in hematologic malignancies.

    Science.gov (United States)

    Testa, Ugo; Saulle, Ernestina; Castelli, Germana; Pelosi, Elvira

    2016-01-01

    Studies carried out in the last years have improved the understanding of the cellular and molecular mechanisms controlling angiogenesis during adult life in normal and pathological conditions. Some of these studies have led to the identification of some progenitor cells that sustain angiogenesis through indirect, paracrine mechanisms (hematopoietic angiogenic cells) and through direct mechanisms, i.e., through their capacity to generate a progeny of phenotypically and functionally competent endothelial cells [endothelial colony forming cells (ECFCs)]. The contribution of these progenitors to angiogenetic processes under physiological and pathological conditions is intensively investigated. Angiogenetic mechanisms are stimulated in various hematological malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma, resulting in an increased angiogenesis that contributes to disease progression. In some of these conditions there is preliminary evidence that some endothelial cells could derive from the malignant clone, thus leading to the speculation that the leukemic cell derives from the malignant transformation of a hemangioblastic progenitor, i.e., of a cell capable of differentiation to the hematopoietic and to the endothelial cell lineages. Our understanding of the mechanisms underlying increased angiogenesis in these malignancies not only contributed to a better knowledge of the mechanisms responsible for tumor progression, but also offered the way for the discovery of new therapeutic targets. PMID:27583252

  10. Establishment of outgrowth endothelial cells from peripheral blood.

    Science.gov (United States)

    Martin-Ramirez, Javier; Hofman, Menno; van den Biggelaar, Maartje; Hebbel, Robert P; Voorberg, Jan

    2012-09-01

    Blood outgrowth endothelial cells (BOECs) are important tools when investigating diagnostic and therapeutic approaches for vascular disease. In this protocol, mononuclear cells are isolated from peripheral blood and plated on type I collagen at ∼135,000 cells per cm(2) in endothelial cell differentiation medium. On average, 0.34 colonies of endothelial cells per milliliter of blood can be obtained. Colonies of endothelial cells become visible after 14-28 d. Upon confluence, these rapidly expanding colonies can be passaged and have been shown to propagate up to 10(18)-fold. Isolated BOECs are phenotypically similar to vascular endothelial cells, as revealed by their cobblestone morphology, the presence of endothelial cell-specific Weibel-Palade bodies and the expression of endothelial cell markers such as VE-cadherin. The protocol presented here also provides a particularly useful tool for the ex vivo assessment of endothelial cell function from patients with different vascular abnormalities. PMID:22918388

  11. Radioprotection of human endothelial cells with amifostine

    Energy Technology Data Exchange (ETDEWEB)

    Andreopoulos, D.; Schleicher, U.M.; Ammon, J. [Technische Hochschule Aachen (Germany). Klinik fuer Radiotherapie - Onkologie; Cotarelo, C.L.; Hand, S. [Technische Hochschule Aachen (Germany). Inst. fuer Pathologie

    1999-11-01

    Materials and methods: We studied the effect of amifostine on radiation sensitivity of human endothelial cells and several tumor cell lines (HeLa, MIA PaCa-2 and BxPC-3). The cells were incubated in medium with a concentration of 1 {mu}g/{mu}l amifostine and after 1 hour irradiated with 10 or 20 Gy single dose. Proliferation index was measured by BrdU assay after another 8 and 24 hours. Results: The results show a higher proliferation rate of endothelial cells following radiation plus amifostine, compared with radiation alone. Amifostine induced an increase of proliferation in the control-non-irradiated human endothelial cells. After irradiation with 10 Gy single dose the proliferation of amifostine treated human endothelial cells was still higher. Amifostine exerts no apparent proliferative effect on the tumor cells. Conclusions: The results presented indicate that amifostine acts as an activation of proliferation of the human endothelial cells in a simple in-vitro system and indicate that amifostine supplementation prior to radiation therapy might exert a radioprotective effect to healthy tissue without spurring tumor growth. (orig.) [German] Material und Methode: Humane Endothelzellen und verschiedene Tumorzellinien (HeLa, MIA PaCa-2 and BxPC-3) wurden fuer eine Stunde mit 1 {mu}g/{mu}l Amifostin inkubiert und dann mit Dosen von 10 und 20 Gy bestrahlt. Die Proliferationsaktivitaet wurde mittels BrdU-Assay nach acht und 24 Stunden gemessen. Ergebnisse: Amifostin fuehrt zu einer verstaerkten Proliferation der unbestrahlten Endothelzellen. Nach der Bestrahlung mit 10 Gy Einzeitdosis zeigen die Endothelzellen mit Amifostin-Zusatz eine staerkere Proliferation als die Zellen ohne Amifostin. Ein protektiver Effekt auf die Tumorzellinien war nicht feststellbar. Schlussfolgerung: Die bisherigen Ergebnisse zeigen, dass Amifostin einen radioprotektiven Effekt auf humane Endothelzellen ausuebt und deren Proliferation stimuliert, ohne jedoch die Proliferation der Tumorzellen

  12. Circulating endothelial cells in cardiovascular disease.

    Science.gov (United States)

    Boos, Christopher J; Lip, Gregory Y H; Blann, Andrew D

    2006-10-17

    Quantification of circulating endothelial cells (CECs) in peripheral blood is developing as a novel and reproducible method of assessing endothelial damage/dysfunction. The CECs are thought to be mature cells that have detached from the intimal monolayer in response to endothelial injury and are a different cell population to endothelial progenitor cells (EPCs). The EPCs are nonleukocytes derived from the bone marrow that are believed to have proliferative potential and may be important in vascular regeneration. Currently accepted methods of CEC quantification include the use of immunomagnetic bead separation (with cell counting under fluorescence microscopy) and flow cytometry. Several recent studies have shown increased numbers of CECs in cardiovascular disease and its risk factors, such as unstable angina, acute myocardial infarction, stroke, diabetes mellitus, and critical limb ischemia, but no change in stable intermittent claudication, essential hypertension, or atrial fibrillation. Furthermore, CEC quantification at 48 h after acute myocardial infarction has been shown to be an accurate predictor of major adverse coronary events and death at both 1 month and 1 year. This article presents an overview of the pathophysiology of CECs in the setting of cardiovascular disease and a brief comparison with EPCs. PMID:17045885

  13. Endothelial cell transfection of ex vivo arteries

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Alexander Lohman, Adam Straub & Brant Isakson ### Abstract The vascular endothelium plays an essential role in regulating blood vessel tone, blood flow and blood pressure. Current vascular model systems for examination of endothelial cell biology and blood vessel physiology and pathology rely on cell culture and the generation of genetically modified animals. While these systems are advantageous for studying the endothelium, many cell culture models omit the contribution ...

  14. Histopathological aspects of endothelial dysfunction in the vessels of brain microcirculation in case of diabetic encephalopathy

    Directory of Open Access Journals (Sweden)

    Pashkovska N.V.

    2008-01-01

    Full Text Available The desquamation of endothelium of arteries, small veins and venules, the arteriolospasm and perivascular edematization of varying degrees of severity was established in histological preparations of different brain regions in case of diabetic encephalopathy. It was shown, that variation coefficients of optical density of stained nuclear chromatin of endotheliocytes in the vessels of brain microcirculation were reliably higher in case of diabetic encephalopathy as compared with corresponding indices of control group; this indicated the decrease of functional capability of these cells and the development of endothelial dysfunction.

  15. The control of vascular endothelial cell injury.

    Science.gov (United States)

    Murota, S; Morita, I; Suda, N

    1990-01-01

    The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity. PMID:2248437

  16. EGb761 provides a protective effect against Aβ1-42 oligomer-induced cell damage and blood-brain barrier disruption in an in vitro bEnd.3 endothelial model.

    Directory of Open Access Journals (Sweden)

    Wen-bin Wan

    Full Text Available Alzheimer's disease (AD is the most common form of senile dementia which is characterized by abnormal amyloid beta (Aβ accumulation and deposition in brain parenchyma and cerebral capillaries, and leads to blood-brain barrier (BBB disruption. Despite great progress in understanding the etiology of AD, the underlying pathogenic mechanism of BBB damage is still unclear, and no effective treatment has been devised. The standard Ginkgo biloba extract EGb761 has been widely used as a potential cognitive enhancer for the treatment of AD. However, the cellular mechanism underlying the effect remain to be clarified. In this study, we employed an immortalized endothelial cell line (bEnd.3 and incubation of Aβ(1-42 oligomer, to mimic a monolayer BBB model under conditions found in the AD brain. We investigated the effect of EGb761 on BBB and found that Aβ1-42 oligomer-induced cell injury, apoptosis, and generation of intracellular reactive oxygen species (ROS, were attenuated by treatment with EGb761. Moreover, treatment of the cells with EGb761 decreased BBB permeability and increased tight junction scaffold protein levels including ZO-1, Claudin-5 and Occludin. We also found that the Aβ(1-42 oligomer-induced upregulation of the receptor for advanced glycation end-products (RAGE, which mediates Aβ cytotoxicity and plays an essential role in AD progression, was significantly decreased by treatment with EGb761. To our knowledge, we provide the first direct in vitro evidence of an effect of EGb761 on the brain endothelium exposed to Aβ(1-42 oligomer, and on the expression of tight junction (TJ scaffold proteins and RAGE. Our results provide a new insight into a possible mechanism of action of EGb761. This study provides a rational basis for the therapeutic application of EGb761 in the treatment of AD.

  17. Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Deng Xiaolu

    2011-04-01

    Full Text Available Abstract Background Tumor necrosis factor-α (TNF-α, a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC barrier dysfunction. Methods Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL. RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER. p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability. Results We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER. Conclusions Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF

  18. Tissue factor expression by endothelial cells in sickle cell anemia.

    OpenAIRE

    Solovey, A; Gui, L; Key, N. S.; Hebbel, R.P.

    1998-01-01

    The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that s...

  19. Isolation of Murine Embryonic Hemogenic Endothelial Cells.

    Science.gov (United States)

    Fang, Jennifer S; Gritz, Emily C; Marcelo, Kathrina L; Hirschi, Karen K

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  20. Syncytin is involved in breast cancer-endothelial cell fusions

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Holck, S.; Christensen, I.J.;

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  1. Transition of mesenchymal stem/stromal cells to endothelial cells

    NARCIS (Netherlands)

    M. Crisan (Mihaela)

    2013-01-01

    textabstractMesenchymal stem/stromal cells (MSCs) are heterogeneous. A fraction of these cells constitute multipotent cells that can self-renew and mainly give rise to mesodermal lineage cells such as adipocytes, osteocytes and chondrocytes. The ability of MSCs to differentiate into endothelial cell

  2. Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

    Institute of Scientific and Technical Information of China (English)

    TAN YuZhen; ZHAO Yao; WANG HaiJie; ZHOU LiangFu; MAO Ying; LIU Rui; SHU Jia; WANG YongFei

    2008-01-01

    Human cerebral cavernous malformation (CM) is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type Ⅰ gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like struc-tures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to re-ceptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.

  3. The role of shear stress in Blood-Brain Barrier endothelial physiology

    Directory of Open Access Journals (Sweden)

    Puvenna Vikram

    2011-05-01

    Full Text Available Abstract Background One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs into a blood-brain barrier (BBB phenotype is shear stress (SS. With the use of a well established humanized dynamic in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions. Results Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc. was also upregulated. RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased

  4. Collective cell motion in endothelial monolayers

    International Nuclear Information System (INIS)

    Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior

  5. Cytokine production by endothelial cells infected with human T cell lymphotropic virus type I.

    OpenAIRE

    H. Takashima; Eguchi, K.; Kawakami, A; Kawabe, Y; Migita, K; Sakai, M; Origuchi, T; Nagataki, S.

    1996-01-01

    OBJECTIVE: To investigate the ability of human T cell lymphotropic virus type I (HTLV-I) to infect endothelial cells and induce cytokine production by these cells. METHODS: Human umbilical vein endothelial cells (HUVEC) were cocultured with HTLV-I infected T cell line (MT-2 cells) or uninfected T cell line (CEM cells). RESULTS: Following coculture with MT-2 cells, endothelial cells expressed HTLV-I specific core antigens. Endothelial cells cocultured with MT-2 cells produced significant amoun...

  6. Endothelial cell-derived interleukin-6 regulates tumor growth

    International Nuclear Information System (INIS)

    Endothelial cells play a complex role in the pathobiology of cancer. This role is not limited to the making of blood vessels to allow for influx of oxygen and nutrients required for the high metabolic demands of tumor cells. Indeed, it has been recently shown that tumor-associated endothelial cells secrete molecules that enhance tumor cell survival and cancer stem cell self-renewal. The hypothesis underlying this work is that specific disruption of endothelial cell-initiated signaling inhibits tumor growth. Conditioned medium from primary human dermal microvascular endothelial cells (HDMEC) stably transduced with silencing RNA for IL-6 (or controls) was used to evaluate the role of endothelial-derived IL-6 on the activation of key signaling pathways in tumor cells. In addition, these endothelial cells were co-transplanted with tumor cells into immunodefficient mice to determine the impact of endothelial cell-derived IL-6 on tumor growth and angiogenesis. We observed that tumor cells adjacent to blood vessels show strong phosphorylation of STAT3, a key mediator of tumor progression. In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells. Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells. Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells

  7. Fullerene derivatives protect endothelial cells against NO-induced damage

    Energy Technology Data Exchange (ETDEWEB)

    Lao Fang; Han Dong; Qu Ying; Liu Ying; Zhao Yuliang; Chen Chunying [CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 (China); Li Wei [CAS Key Laboratory for Nuclear Analytical Techniques, Institute of High Energy Physics (IHEP), Chinese Academy of Sciences, Beijing 100049 (China)], E-mail: chenchy@nanoctr.cn

    2009-06-03

    Functional fullerene derivatives have been demonstrated with potent antioxidation properties. Nitric oxide (NO) is a free radical that plays a part in leading to brain damage when it is accumulated to a high concentration. The possible scavenging activity of NO by the hydroxylated fullerene derivative C{sub 60}(OH){sub 22} and malonic acid derivative C{sub 60}(C(COOH){sub 2}){sub 2} was investigated using primary rat brain cerebral microvessel endothelial cells (CMECs). Results demonstrate that sodium nitroprusside (SNP), used as an NO donor, caused a marked decrease in cell viability and an increase in apoptosis. However, fullerene derivatives can remarkably protect against the apoptosis induced by NO assault. In addition, fullerene derivatives can also prevent NO-induced depolymerization of cytoskeleton and damage of the nucleus and accelerate endothelial cell repair. Further investigation shows that the sudden increase of the intercellular reactive oxygen species (ROS) induced by NO was significantly attenuated by post-treatment with fullerene derivatives. Our results suggest that functional fullerene derivatives are potential applications for NO-related disorders.

  8. Endothelial progenitor cells with Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    KONG Xiao-dong; ZHANG Yun; LIU Li; SUN Ning; ZHANG Ming-yi; ZHANG Jian-ning

    2011-01-01

    Background Endothelial dysfunction is thought to be critical events in the pathogenesis of Alzheimer's disease (AD).Endothelial progenitor cells (EPCs) have provided insight into maintaining and repairing endothelial function. To study the relation between EPCs and AD, we explored the number of circulating EPCs in patients with AD.Methods A total of 104 patients were recruited from both the outpatients and inpatients of the geriatric neurology department at General Hospital, rianjin Medical University. Consecutive patients with newly diagnosed AD (n=30),patients with vascular dementia (VaD, n=34), and healthy elderly control subjects with normal cognition (n=40) were enrolled after matching for age, gender, body mass index, medical history, current medication and Mini Mental State Examination. Middle cerebral artery flow velocity was examined with transcranial Doppler. Endothelial function was evaluated according to the level of EPCs, and peripheral blood EPCs was counted by flow cytometry.Results There were no significant statistical differences of clinical data in AD, VaD and control groups (P >0.05). The patients with AD showed decreased CD34-positive (CD34+) or CD133-positive (CD133+) levels compared to the control subjects, but there were no significant statistical differences in patients with AD. The patients with AD had significantly lower CD34+CD133+ EPCs(CD34 and CD133 double positive endothelial progenitor cells) than the control subjects (P <0.05). In the patients with AD, a lower CD34+CD133+ EPCs count was independently associated with a lower Mini-Mental State Examination score (r=0.514, P=0.004). Patients with VaD also showed a significant decrease in CD34+CD133+ EPCs levels, but this was not evidently associated with the Mini-Mental State Examination score. The changes of middle cerebral artery flow velocity were similar between AD and VaD. Middle cerebral artery flow velocity was decreased in the AD and VaD groups and significantly lower than

  9. Endothelial cells, tissue factor and infectious diseases

    Directory of Open Access Journals (Sweden)

    Lopes-Bezerra L.M.

    2003-01-01

    Full Text Available Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

  10. Endoderm Generates Endothelial Cells during Liver Development

    Directory of Open Access Journals (Sweden)

    Orit Goldman

    2014-10-01

    Full Text Available Organogenesis requires expansion of the embryonic vascular plexus that migrates into developing organs through a process called angiogenesis. Mesodermal progenitors are thought to derive endothelial cells (ECs that contribute to both embryonic vasculogenesis and the subsequent organ angiogenesis. Here, we demonstrate that during development of the liver, which is an endoderm derivative, a subset of ECs is generated from FOXA2+ endoderm-derived fetal hepatoblast progenitor cells expressing KDR (VEGFR2/FLK-1. Using human and mouse embryonic stem cell models, we demonstrate that KDR+FOXA2+ endoderm cells developing in hepatic differentiation cultures generate functional ECs. This introduces the concept that ECs originate not exclusively from mesoderm but also from endoderm, supported in Foxa2 lineage-tracing mouse embryos by the identification of FOXA2+ cell-derived CD31+ ECs that integrate the vascular network of developing fetal livers.

  11. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  12. Production of soluble Neprilysin by endothelial cells

    International Nuclear Information System (INIS)

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17

  13. Endothelial Progenitor Cells Enter the Aging Arena.

    Directory of Open Access Journals (Sweden)

    Kate eWilliamson

    2012-02-01

    Full Text Available Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells.In this review, we discuss the current understanding of the cells termed ‘EPCs’, examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

  14. A Fluorescent Polymer Probe with High Selectivity toward Vascular Endothelial Cells for and beyond Noninvasive Two-Photon Intravital Imaging of Brain Vasculature.

    Science.gov (United States)

    Mettra, B; Appaix, F; Olesiak-Banska, J; Le Bahers, T; Leung, A; Matczyszyn, K; Samoc, M; van der Sanden, B; Monnereau, C; Andraud, C

    2016-07-13

    A chromophore-engineering strategy that relies on the introduction of a ground-state distortion in a quadrupolar chromophore was used to obtain a quasi-quadrupolar chromophore with red emission and large two-photon absorption (2PA) cross-section in polar solvents. This molecule was functionalized with water-solubilizing polymer chains. It constitutes not only a remarkable contrast agent for intravital two-photon microscopy of the functional cerebral vasculature in a minimally invasive configuration but presents intriguing endothelial staining ability that makes it a valuable probe for premortem histological staining. PMID:27267494

  15. Enhancing endothelial progenitor cell for clinical use

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Circulating endothelial progenitor cells (EPCs) havebeen demonstrated to correlate negatively with vascularendothelial dysfunction and cardiovascular risk factors.However, translation of basic research into the clinicalpractice has been limited by the lack of unambiguousand consistent definitions of EPCs and reduced EPCcell number and function in subjects requiring them forclinical use. This article critically reviews the definitionof EPCs based on commonly used protocols, their valueas a biomarker of cardiovascular risk factor in subjectswith cardiovascular disease, and strategies to enhanceEPCs for treatment of ischemic diseases.

  16. Estetrol Modulates Endothelial Nitric Oxide Synthesis in Human Endothelial Cells

    OpenAIRE

    Montt-Guevara, Maria Magdalena; Giretti, Maria Silvia; Russo, Eleonora; Giannini, Andrea; Mannella, Paolo; Genazzani, Andrea Riccardo; Genazzani, Alessandro David; Simoncini, Tommaso

    2015-01-01

    Estetrol (E4) is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO) is a key ...

  17. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    Science.gov (United States)

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  18. DIFFERENT RESPONSES OF CHORIOCAPILLARY ENDOTHELIAL CELLS AND RETINALCAPILLARY ENDOTHELIAL CELLS TO MITOGENIC AND VASOACTIVE FACTORS

    Institute of Scientific and Technical Information of China (English)

    李维业; 刘熙朴; MyronYanoff

    1994-01-01

    The reaponses of choriocapillary endothelial cells(CCE) and retinal capillary ondothelial cells (RCE) in cul-ture,in terms of phosphoinositide (PI) breakdown and cellular mitogenesis,to retinal pigment epithelial cell (RPE)-conditioned medium and vasoactive agents have been compared.RPE-conditioned medium did not induce PI breakdown in either type of cell.However,it stimulated DNA synthesis in CCE but not in RCE.Bradykinin (BDK)acted as both a fast signaling and a slow mitogenic factor on CCE,out BDK did not affect PI turnover or DNA synthesis in RCE.In contrast,thrombin stimulated PI turnover in RCE but not in CCE,though it did not in-duce 3H-thymidine incorporation into either type of cell.These differences in cellular functions between CCE and RCE following stimulation suggest that induction of DNA synthesis and recptor-mediated PI turnover by external factors is determined,at least in part,by the origin of the capillary endothelial cell.Therefore,extrapolation to CCE pathophysiology from experiments using endothelial cells from other capillary origins may not be valid.

  19. Ischemia-induced endothelial cell swelling and mitochondrial dysfunction are attenuated by dietary polyphenols in vitro

    Science.gov (United States)

    Polyphenols possess anti-oxidant and anti-inflammatory properties. Oxidative stress (OS) and inflammation have been implicated in the pathogenesis of cytotoxic brain edema in cerebral ischemia. In addition, OS and pro-inflammatory cytokines also damage the endothelial cells and the neurovascular uni...

  20. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    Science.gov (United States)

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  1. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; Kooten, TV; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  2. Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

    Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

  3. Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor)

    Energy Technology Data Exchange (ETDEWEB)

    Winkles, J.A.; Friesel, R.; Burgess, W.H.; Howk, R.; Mehlman, T.; Weinstein, R.; Maciag, T.

    1987-10-01

    The control of vascular endothelial and muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical cells also synthesize an HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with /sup 125/I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.

  4. Obstructive sleep apnea and endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Wang Q

    2013-10-01

    Full Text Available Qing Wang,1,* Qi Wu,2,* Jing Feng,3,4 Xin Sun5 1The Second Respiratory Department of the First People's Hospital of Kunming, Yunnan, People's Republic of China; 2Tianjin Haihe Hospital, Tianjin, People's Republic of China; 3Respiratory Department of Tianjin Medical University General Hospital, Tianjin, People's Republic of China; 4Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, NC, USA; 5Respiratory Department of Tianjin Haihe Hospital, Tianjin, People's Republic of China *These authors contributed equally to this work Background: Obstructive sleep apnea (OSA occurs in 4% of middle-aged men and 2% of middle-aged women in the general population, and the prevalence is even higher in specific patient groups. OSA is an independent risk factor for a variety of cardiovascular diseases. Endothelial injury could be the pivotal determinant in the development of cardiovascular pathology in OSA. Endothelial damage ultimately represents a dynamic balance between the magnitude of injury and the capacity for repair. Bone marrow–derived endothelial progenitor cells (EPCs within adult peripheral blood present a possible means of vascular maintenance that could home to sites of injury and restore endothelial integrity and normal function. Methods: We summarized pathogenetic mechanisms of OSA and searched for available studies on numbers and functions of EPCs in patients with OSA to explore the potential links between the numbers and functions of EPCs and OSA. In particular, we tried to elucidate the molecular mechanisms of the effects of OSA on EPCs. Conclusion: Intermittent hypoxia cycles and sleep fragmentation are major pathophysiologic characters of OSA. Intermittent hypoxia acts as a trigger of oxidative stress, systemic inflammation, and sympathetic activation. Sleep fragmentation is associated with a burst of sympathetic activation and systemic inflammation. In most studies, a reduction in circulating EPCs has

  5. Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

    OpenAIRE

    Avraham, H; Cowley, S; Chi, S. Y.; Jiang, S.; Groopman, J E

    1993-01-01

    Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor ...

  6. Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system.

    Science.gov (United States)

    Konradt, Christoph; Ueno, Norikiyo; Christian, David A; Delong, Jonathan H; Pritchard, Gretchen Harms; Herz, Jasmin; Bzik, David J; Koshy, Anita A; McGavern, Dorian B; Lodoen, Melissa B; Hunter, Christopher A

    2016-01-01

    An important function of the blood-brain barrier is to exclude pathogens from the central nervous system, but some microorganisms benefit from the ability to enter this site. It has been proposed that Toxoplasma gondii can cross biological barriers as a motile extracellular form that uses transcellular or paracellular migration, or by infecting a host cell that then crosses the blood-brain barrier. Unexpectedly, analysis of acutely infected mice revealed significant numbers of free parasites in the blood and the presence of infected endothelial cells in the brain vasculature. The use of diverse transgenic parasites combined with reporter mice and intravital imaging demonstrated that replication in and lysis of endothelial cells precedes invasion of the central nervous system, and highlight a novel mechanism for parasite entry to the central nervous system. PMID:27572166

  7. Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

    OpenAIRE

    Caterina Mammi; Donatella Pastore; Lombardo, Marco F; Francesca Ferrelli; Massimiliano Caprio; Claudia Consoli; Manfredi Tesauro; Lucia Gatta; Massimo Fini; Massimo Federici; Paolo Sbraccia; Giulia Donadel; Alfonso Bellia; Giuseppe M Rosano; Andrea Fabbri

    2011-01-01

    BACKGROUND: The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Human umbilical vein endothelial cel...

  8. 循环内皮细胞与放射性脑损伤相关性实验研究%Correlation between circulating endothelial cells and radiation-induced brain injury

    Institute of Scientific and Technical Information of China (English)

    马代远; 谭榜宪; 李祥攀; 阮林; 甘浪舸; 韦力

    2008-01-01

    Objective To investigate the correlation between circulating endothelial cells (CECs) and radiation-induced brain injury. Methods One hundred and eight SD rats were randomly divided into control group, single-dose 10 Gy 60Co irradiation group and single-dose 30 Gy group. The neurobehavioral changes were observed by Morris water labyrinth at 1 week, 1 month and 3 months after irradiation. The CECs in right ventricular blood were counted after Morrie water test. Hippocamp ultramicrostructure and GFAP positive cell were detected after perfusion of encephalon. Results Neuobehavior change: at 1 month and 3 months after irradiation the swim latency was significantly prolonged (30 Gy group>10 Gy group>control group, P 10 Gy group> 30 Gy group, P 10 Gy group>control group, P10 Gy group>control group, P<0.05). Good correlations between the numbers of CECs and the swim lantency (r10 Gy=0.97, P=0.034; r30 Gy=0.95, P=0.013),and the numbers of GFAP positive cells(r10 Gy=0.94, P=0.037; r30 Gy=0.96, P=0.027) were demonstrated.Conclusion The changes of the CECs numbers are definitely correlated to radiation-induced brain injury, which is more with irradiation dose and duration.%目的 探讨循环内皮细胞(circulating endothelial cegs,CECs)与放射性脑损伤相关性.方法 108只SD大鼠信封法随机分成对照组、实验组(10 Gy组、30 Gy组),分别于照射后1周、1月和3月每组随机抽取9只进行Morris水迷宫行为测试,心脏取血计数CECs,取脑观察海马形态和结构.结果 神经行为改变:照射1和3个月后平台潜伏期延长、穿越平台象限时间及次数明显减少.海马齿状回形态改变:照射1和3个月后实验组神经胶质酸性蛋白(glial fibfillary acidic protein,GFAP)阳性细胞数量明显高于对照组,30 Gy组高于10 Gy组;照射1和3个月后超微结构变化为毛细血管内皮细胞变薄、内皮细胞吞饮小泡明显增多、内皮细胞间紧密连接破坏,毛细血管基膜外星形胶质细胞

  9. In Vitro Guidance of Dental Pulp Cells by Nd:YAG Laser-Irradiated Endothelial Cells

    OpenAIRE

    Masuda, Yoshiko Murakami; Yamada, Yoshishige; Kimura, Yuichi

    2012-01-01

    Objective: After endothelial cells were ablated by neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation, we investigated the response of pulp cells by examining the expression of transforming growth factor beta-1 (TGF-β1). Background data: The reaction of stimulated blood vessels is related to the initiation of dentinogenesis. After artificial injury of endothelial cells, pulp cells migrate to the site of the injured endothelial cells. Materials and methods: Rat aortic endothelial cel...

  10. Silencing of directional migration in roundabout4 knockdown endothelial cells

    Directory of Open Access Journals (Sweden)

    Roberts David D

    2008-11-01

    Full Text Available Abstract Background Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4, the predominant Robo in endothelial cells using small interfering RNA technology in vitro. Results Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells. Conclusion This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.

  11. Variations in mass transfer to single endothelial cells.

    Science.gov (United States)

    Van Doormaal, Mark A; Zhang, Ji; Wada, Shigeo; Shaw, James E; Won, Doyon; Cybulsky, Myron I; Yip, Chris M; Ethier, C Ross

    2009-06-01

    Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.

  12. Radiation-induced apoptosis in microvascular endothelial cells.

    OpenAIRE

    Langley, R. E.; Bump, E A; Quartuccio, S. G.; Medeiros, D.; Braunhut, S. J.

    1997-01-01

    The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary en...

  13. Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Guang-fa WANG; Shao-yu WU; Jin-jun RAO; Lin L(U); Wei XU; Jian-xin PANG; Zhong-qiu LIU; Shu-guang WU; Jia-jie ZHANG

    2009-01-01

    Aim: Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin,an aglycon of geniposide, inhibits endothelial exocytosis.Methods: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.Results: Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.Conclusion: Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

  14. A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells

    DEFF Research Database (Denmark)

    Claessens, Antoine; Adams, Yvonne; Ghumra, Ashfaq;

    2012-01-01

    .029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.......Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human...... of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann-Whitney test, P = 0...

  15. Listeria monocytogenes Virulence Factors That Stimulate Endothelial Cells

    OpenAIRE

    Drevets, Douglas A.

    1998-01-01

    Listeria monocytogenes infection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function. Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-type L. monocytogenes or L. monocytogenes mutants; then surface expression of E-selectin and neutro...

  16. Paclitaxel Induces Thrombomodulin Downregulation in Human Aortic Endothelial Cells

    OpenAIRE

    Wang, Huang-Joe; Lu, Te-Ling; Huang, Haimei; Huang, Huey-Chun

    2011-01-01

    Patients with paclitaxel-eluting stents are at risk of developing stent thrombosis upon premature discontinuation of dual antiplatelet therapy. In this study, we set out to clarify whether paclitaxel can modulate thrombomodulin expression in human aortic endothelial cells. Human aortic endothelial cells were stimulated with paclitaxel. Methoxyphenyl tetrazolium inner salt cell viability assay, Western blot analysis, real-time polymerase chain reaction, and immunohistochemical assay were perfo...

  17. Nitric oxide modulates lipopolysaccharide-induced endothelial platelet endothelial cell adhesion molecule expression via interleukin-10.

    Science.gov (United States)

    Hebeda, C B; Teixeira, S A; Tamura, E K; Muscará, M N; de Mello, S B V; Markus, R P; Farsky, S H P

    2011-08-01

    We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 µg/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions. PMID:21564091

  18. Phagocytosis by glomerular endothelial cells in infection-related glomerulopathy.

    Science.gov (United States)

    van Velthuysen, M L; Mayen, A E; Prins, F A; de Heer, E; Bruijn, J A; Fleuren, G J

    1994-01-01

    Glomerulonephritis in BALB/c mice following infection with Trypanosoma brucei is characterized by albuminuria and glomerular deposition of immunoglobulins. Electron-dense deposits are present in the mesangium, as well as subendothelially and subepithelially along the glomerular capillary wall. In this study the nature of intracytoplasmic, electron-dense, round structures observed in glomerular endothelial cells was investigated by immunoelectron-microscopy and enzyme histochemistry. The presence of these structures was related in time with the development of proteinuria. Mice from the C57BL10 strain, which upon infection develop glomerular immune complexes without proteinuria, were examined as well. The results demonstrated that the first endothelial changes, occurring 3-4 weeks after infection, were swelling of endothelial cells containing intracytoplasmic, electron-dense, round structures. These changes were seen prior to the onset of proteinuria, and were not present in glomeruli of mice that did not develop proteinuria. The endothelial granules were shown to contain immunoglobulins and typical lysosomal enzymes, providing evidence for phagocytosis by the glomerular endothelial cells. Liver endothelial cells did not show comparable changes. Thus, local phagocytosis by glomerular endothelial cells is shown to be a specific event in the development of glomerular disease. PMID:7800204

  19. Endothelial cell tumor growth is Ape/ref-1 dependent.

    Science.gov (United States)

    Biswas, Ayan; Khanna, Savita; Roy, Sashwati; Pan, Xueliang; Sen, Chandan K; Gordillo, Gayle M

    2015-09-01

    Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

  20. High-density lipoprotein endocytosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Stefanie; Fruhwürth; Margit; Pavelka; Robert; Bittman; Werner; J; Kovacs; Katharina; M; Walter; Clemens; Rhrl; Herbert; Stangl

    2013-01-01

    AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

  1. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.

    Science.gov (United States)

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells. PMID:26977592

  2. Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jason Tran

    Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  3. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  4. Alk1 controls arterial endothelial cell migration in lumenized vessels.

    Science.gov (United States)

    Rochon, Elizabeth R; Menon, Prahlad G; Roman, Beth L

    2016-07-15

    Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs. PMID:27287800

  5. Lonidamine Causes Inhibition of Angiogenesis-Related Endothelial Cell Functions

    Directory of Open Access Journals (Sweden)

    Donatella Del Bufalo

    2004-09-01

    Full Text Available The aim of this study was to assess whether lonidamine (LND interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 μg/ml. In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secretion of matrix metalloproteinase-2 and metalloproteinase-9 by endothelial cells. Vessel formation in a matrigel plug was also reduced by LND. The viability, migration, invasion, and matrix metalloproteinase production of different tumor cell lines were not affected by low doses of LND (1-10 μg/ml, whereas 50 μg/ml LND, which corresponds to the dose used in clinical management of tumors, triggered apoptosis both in endothelial and tumor cells. Together, these data demonstrate that LND is a compound that interferes with endothelial cell functions, both at low and high doses. Thus, the effect of LND on endothelial cell functions, previously undescribed, may be a significant contributor to the antitumor effect of LND observed for clinical management of solid tumors.

  6. Breast cancer cells stimulate osteoprotegerin (OPG production by endothelial cells through direct cell contact

    Directory of Open Access Journals (Sweden)

    Holen Ingunn

    2009-07-01

    Full Text Available Abstract Background Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment. Results It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on ανβ3 ligation and NFκB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFβ had no effect on HuDMEC OPG levels. Conclusion These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve ανβ3 ligation and NFκB activation.

  7. Endothelial cell pseudopods and angiogenesis of breast cancer tumors

    Directory of Open Access Journals (Sweden)

    Sun LuZhe

    2005-05-01

    Full Text Available Abstract Background A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse. Once the tumor grew to 35 mm3, it developed a well-vascularized capsule. Histological sections of tumors greater than 35 mm3 were stained with PAS, with CD-31 antibody (an endothelial cell maker, or with hypoxia inducible factor 1α antibody (HIF. The extent of blood vessel and endothelial cell pseudopod volume density was measured by ocular grid intercept counting in the PAS stained slides. Results The tumor area within 100–150 μm of the well-vascularized capsule had few blood vessels and only occasional endothelial cell pseudopods, whereas the area greater than 150 μm from the capsule had more blood vessels, capillaries, and a three-fold increase in volume density of pseudopods sprouting from the capillary endothelial cells. This subcortical region, rich in pseudopods, some of which were observed to have vacuoles/lumens, was strongly positive for presence of HIF. In some larger tumors, pseudopods were observed to insinuate for mm distances through hypoxic regions of the tumor. Conclusion The positive correlation between presence of HIF and the increased extent of pseudopods suggests volume density measure of the latter as a quantifiable marker of tumor hypoxia. Apparently, hypoxic regions of the tumor produce HIF leading to production of vascular endothelial growth factors that stimulate sprouting of capillary endothelial cells and formation of endothelial cell pseudopods.

  8. Mitochondrial function in vascular endothelial cell in diabetes

    OpenAIRE

    Pangare, Meenal; Makino, Ayako

    2012-01-01

    Micro- and macrovascular complications are commonly seen in diabetic patients and endothelial dysfunction contributes to the development and progression of the complications. Abnormal functions in endothelial cells lead to the increase in vascular tension and atherosclerosis, followed by systemic hypertension as well as increased incident of ischemia and stroke in diabetic patients. Mitochondria are organelles serving as a source of energy production and as regulators of cell survival (e.g., ...

  9. Lonidamine Causes Inhibition of Angiogenesis-Related Endothelial Cell Functions

    OpenAIRE

    Donatella Del Bufalo; Daniela Trisciuoglio; Marco Scarsella; Giulia D'Amati; Antonio Candiloro; Angela Iervolino; Carlo Leonetti; Gabriella Zupi

    2004-01-01

    The aim of this study was to assess whether lonidamine (LND) interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 μg/ml). In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secreti...

  10. Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells

    Science.gov (United States)

    Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Brazier, Andrew Jay

    2016-01-01

    ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. PMID:27406562

  11. Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

    OpenAIRE

    Lu, T T; Yan, L G; Madri, J. A.

    1996-01-01

    Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell sprea...

  12. Traction Forces of Endothelial Cells under Slow Shear Flow

    Science.gov (United States)

    Perrault, Cecile M.; Brugues, Agusti; Bazellieres, Elsa; Ricco, Pierre; Lacroix, Damien; Trepat, Xavier

    2015-01-01

    Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress. PMID:26488643

  13. 自体血管内皮祖细胞治疗缺血缺氧性脑损伤**★%Autologous endothelial progenitor cells for treatment of ischemic/hypoxic brain injury

    Institute of Scientific and Technical Information of China (English)

    崔立玲; 黄国志; 陈镇洲; 郭阳

    2013-01-01

    BACKGROUND: Fol owing ischemic/hypoxic brain injury, neurogenesis and neurofunctional recovery are closely related to vascular formation and plasticity in ischemic region. Vascular endothelial progenitor cel s participate in vascular formation and repair in postnatal ischemic tissue, promote the recanalization of blood flow and the supply of nutritive substances such as oxygen, providing microenvironment for neurofunctional recovery. OBJECTIVE: To evaluate the feasibility, efficacy and safety of use of autologous vascular endothelial progenitor cel s in the treatment of ischemic/hypoxic brain injury and investigate a new method for improving the neurological function of patients with ischemic/hypoxic brain injury. METHODS: A computer-based online retrieval of PubMed, ScienceDirect, Springerlink and CNKI databases was performed for papers describing use of vascular endothelial progenitor cel s in the treatment of ischemic/hypoxic brain injury using the key words “EPCs, endothelial progenitor cel , stroke” in English and Chinese. In the same research filed, papers that published recently or in high impact factor journals were selected. A total of 43 papers were suitable for final analysis. RESULTS AND CONCLUSION: Fol owing ischemic/hypoxic brain injury, neurogenesis and neurofunctional recovery are closely related to vascular formation and plasticity in ischemic region. Vascular endothelial progenitor cel s participate in vascular formation and repair in postnatal ischemic tissue, promote the recanalization of blood flow and the supply of nutritive substances such as oxygen, providing microenvironment for neurofunctional recovery. The use of autologous vascular endothelial progenitor cel s in the treatment of ischemic/hypoxic brain injury is feasible, safe and effective. Nevertheless, a larger number of biological and animal experiments are needed for providing theoretical evidence for clinical application of autologous vascular endothelial progenitor cel s.% 

  14. The endothelin system in breast tumour–endothelial cell interactions

    Directory of Open Access Journals (Sweden)

    Julia Botha

    2011-01-01

    Full Text Available The role of endothelin-1 (ET-1 and its receptors (ET-RA and ET-RB in tumour development and progression involves complex interactions. ET-1, produced by tumours and associated cells like endothelial cells, functions in an autocrine and paracrine manner to promote tumour angiogenesis. Thus, we hypothesised that endothelin, released into the tumour milieu by both tumours and the tumour vasculature, would influence angiogenesis. Therefore, this preliminary study aimed to investigate changes in ET1, ET-RA and ET-RB in breast tumour and microvascular endothelial cultures when each cell type was exposed directly to the other (co-culture model as well as to the conditioned-medium metabolites of the other (challenge model. ET-1 secretion was measured by an enzyme-linked immunosorbent assay and ET-1, ET-RA and ET-RB expression investigated by the linked streptavidin–biotin method. In challenge experiments, endothelial metabolites significantly increased secretion of breast tumour ET-1. Tumour metabolites promoted endothelial membrane projections with no effect on ET-1 secretion. ET-1 and its receptors were immunolocalised in both cell types, including in projections. Increasing cancer cell conditioned medium resulted in decreased endothelial ET-RA and increased ET-RB staining. Co-cultures demonstrated ET proteins in projections of both cell types as well as at heterogeneous contact points. The findings support a role for the endothelin system in endothelial cell and breast cancer cell invasion. It is tempting to consider that early endothelial and tumour cell alterations may be promoted by ET-1 produced by both cell types. Further work is required that will examine localised cellular gene expression of the endothelin system as well as its pro-invasive and angiogenic effects in breast cancer models.

  15. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2.

    Science.gov (United States)

    Eum, Sung Yong; Jaraki, Dima; András, Ibolya E; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs.

  16. Lack of vimentin impairs endothelial differentiation of embryonic stem cells.

    Science.gov (United States)

    Boraas, Liana C; Ahsan, Tabassum

    2016-01-01

    The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM -/- ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM -/- EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM -/- EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro. PMID:27480130

  17. The effects of glucocorticoids on cultured human endothelial cells.

    Science.gov (United States)

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man. PMID:646949

  18. Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

    OpenAIRE

    Douglas, G; van Kampen, E.; Hale, AB; McNeill, E; Patel, J.; Crabtree, MJ; Ali, Z; Hoerr, RA; Alp, NJ; Channon, KM

    2013-01-01

    Aims Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Methods and results Endothelial cell repopulation was assessed en face in stented arteries in ApoE−/− mice with end...

  19. Endothelial Cells and Astrocytes: A Concerto en Duo in Ischemic Pathophysiology

    Directory of Open Access Journals (Sweden)

    Vincent Berezowski

    2012-01-01

    Full Text Available The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

  20. Prehospital resuscitation with hypertonic saline-dextran modulates inflammatory, coagulation and endothelial activation marker profiles in severe traumatic brain injured patients

    OpenAIRE

    Morrison Laurie J; Baker Andrew J; Crnko Naomi T; Rhind Shawn G; Shek Pang N; Scarpelini Sandro; Rizoli Sandro B

    2010-01-01

    Abstract Background Traumatic brain injury (TBI) initiates interrelated inflammatory and coagulation cascades characterized by wide-spread cellular activation, induction of leukocyte and endothelial cell adhesion molecules and release of soluble pro/antiinflammatory cytokines and thrombotic mediators. Resuscitative care is focused on optimizing cerebral perfusion and reducing secondary injury processes. Hypertonic saline is an effective osmotherapeutic agent for the treatment of intracranial ...

  1. 脑微血管内皮细胞与周细胞共培养构建体外血脑屏障模型%Establishment of an in vitro blood-brain harrier modal by coculturing brain microvascular endothelial cells and pericytes

    Institute of Scientific and Technical Information of China (English)

    鹿文葆; 秦伟伟; 张秋菊; 李宏伟; 刘淑英; 修瑞娟

    2012-01-01

    目的 应用原代培养的大鼠脑微血管内皮细胞(brain microvascular endothelial cell,BMVEC)与脑周细胞共培养建立可模拟在体状态的稳定体外血脑屏障(blood-brain barrier,BBB)模型.方法 原代分离、纯化培养大鼠BMVEC和周细胞,通过免疫细胞化学染色方法鉴定分离的细胞,应用Transwell插槽(孔径0.4μm)共培养构建体外BBB模型,经4h渗漏试验、紧密连接蛋白鉴定、跨内皮电阻检测以及通透性试验评价其屏障功能,比较共培养模型与单纯BMVEC模型膜两侧电阻值差异以及对小分子荧光素钠(sodium fluorescein,Na-F)通透性的差异.结果 融合的BMVEC单层呈现典型的鹅卵石样外观,脑周细胞呈典型的不规则外形并具有重叠生长等特性.免疫双标法鉴定显示,脑周细胞阳性表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和神经元-胶质抗原2(neuron-glial antigen2,NG2);共培养模型内皮细胞融合后,液面渗漏试验呈阳性;免疫细胞化学染色显示,内皮细胞间形成连续而致密的紧密连接;与BMVEC模型相比,共培养模型跨内皮细胞电阻[( 190.762±10.326)Ω/cm2对(96.503±8.012)Ω/cm2;t=- 24.489,P<0.01]显著增高,通透性显著降低(为单内皮模型的56.149% ±3.572%;t=19.330,P<0.01).结论 原代分离大鼠BMVEC 和周细胞共培养体外模型的形态、结构及屏障功能具备BBB的基本特征,为研究BBB提供了一种有用工具.%Objective To establish a stable in vitro model of blood-brain barrier (BBB) simulating in vivo state using the primary-cultured rat brain microvascular endothelial cells (BMVECs) and pericytes.Methods The primary rat BMVECs and pericytes were isolated,purified and cultured.The isolated cells were identified by immunocytochemical staining method.An in vitro model of BBB was constructed using Transwell inserts (pore size 0.4 μm) coculture.Its barrier function was evaluated by the 4-hour leakage test,tight junction

  2. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

    Directory of Open Access Journals (Sweden)

    Maskomani Silambarasan

    2016-04-01

    Full Text Available Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose and at various time intervals (6, 12, 24 and 48 h. miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis.

  3. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction.

    Science.gov (United States)

    Silambarasan, Maskomani; Tan, Jun Rong; Karolina, Dwi Setyowati; Armugam, Arunmozhiarasi; Kaur, Charanjit; Jeyaseelan, Kandiah

    2016-01-01

    Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose) and at various time intervals (6, 12, 24 and 48 h). miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a) to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis. PMID:27070575

  4. Endothelial Progenitor Cells Promote Directional Three-Dimensional Endothelial Network Formation by Secreting Vascular Endothelial Growth Factor

    OpenAIRE

    Yoshinori Abe; Yoshiyuki Ozaki; Junichi Kasuya; Kimiko Yamamoto; Joji Ando; Ryo Sudo; Mariko Ikeda; Kazuo Tanishita

    2013-01-01

    Endothelial progenitor cell (EPC) transplantation induces the formation of new blood-vessel networks to supply nutrients and oxygen, and is feasible for the treatment of ischemia and cardiovascular diseases. However, the role of EPCs as a source of proangiogenic cytokines and consequent generators of an extracellular growth factor microenvironment in three-dimensional (3D) microvessel formation is not fully understood. We focused on the contribution of EPCs as a source of proangiogenic cytoki...

  5. Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization

    NARCIS (Netherlands)

    Westenbrink, B. Daan; Lipsic, Erik; van der Meer, Peter; van der Harst, Pirn; Oeseburg, Hisko; Sarvaas, Gideon J. Du Marchie; Koster, Johan; Voors, Adriaan A.; van Veldhuisen, Dirk J.; van Gilst, Wiek H.; Schoemaker, Regien G.

    2007-01-01

    Aims Erythropoietin (EPO) improves cardiac function and induces neovascutarization in chronic heart failure (CHF), although the exact mechanism has not been elucidated. We studied the effects of EPO on homing and incorporation of endothelial progenitor cells (EPC) into the myocardial microvasculatur

  6. Fructose Induces the Inflammatory Molecule ICAM-1 in Endothelial Cells

    OpenAIRE

    Glushakova, Olena; Kosugi, Tomoki; Roncal, Carlos; Mu, Wei; Heinig, Marcelo; Cirillo, Pietro; Sánchez-Lozada, Laura G.; Richard J Johnson; Nakagawa, Takahiko

    2008-01-01

    Epidemiologic studies have linked fructose intake with the metabolic syndrome, and it was recently reported that fructose induces an inflammatory response in the rat kidney. Here, we examined whether fructose directly stimulates endothelial inflammatory processes by upregulating the inflammatory molecule intercellular adhesion molecule-1 (ICAM-1). When human aortic endothelial cells were stimulated with physiologic concentrations of fructose, ICAM-1 mRNA and protein expression increased in a ...

  7. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    Science.gov (United States)

    Rudnicki, Martina; Tripodi, Gustavo L; Ferrer, Renila; Boscá, Lisardo; Pitta, Marina G R; Pitta, Ivan R; Abdalla, Dulcineia S P

    2016-07-01

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2',7'-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation. PMID:27108791

  8. Berberine protects vascular endothelial cells in hypertensive rats

    OpenAIRE

    Wang, Yang; Ding, Yun

    2015-01-01

    Objective: This study is to investigate the effect and mechanism of berberine on vascular endothelial cell injury. Methods: The isolated aortic endothelial cells were divided into negative control group, spontaneous hypertension group, and berberine group (1.25, 2.5, and 5 μmol/L berberine). CCK-8 assay was performed to detect cell proliferation. Annexin V-FITC flow cytometry and Hochest33342/PI staining were used to measure cell apoptosis. Expression of TLR4, Myd88, and NF-κB was detected wi...

  9. Metformin improves endothelial function in aortic tissue and microvascular endothelial cells subjected to diabetic hyperglycaemic conditions.

    Science.gov (United States)

    Ghosh, Suparna; Lakshmanan, Arun P; Hwang, Mu Ji; Kubba, Haidar; Mushannen, Ahmed; Triggle, Chris R; Ding, Hong

    2015-12-01

    The cellular mechanisms whereby metformin, the first line drug for type 2 diabetes (T2DM), mediates its antidiabetic effects remain elusive, particularly as to whether metformin has a direct protective action on the vasculature. This study was designed to determine if a brief 3-h exposure to metformin protects endothelial function against the effects of hyperglycaemia. We investigated the protective effects of metformin on endothelial-dependent vasodilatation (EDV) in thoracic aortae from T2DM db/db mice and on high glucose (HG, 40 mM) induced changes in endothelial nitric oxide synthase (eNOS) signaling in mouse microvascular endothelial cells (MMECs) in culture. Exposure of aortae from db+/? non-diabetic control mice to high glucose (HG, 40 mM) containing Krebs for 3-h significantly (Pmetformin; metformin also improved ACh-induced EDV in aortae from diabetic db/db mice. Immunoblot analysis of MMECs cultured in HG versus NG revealed a significant reduction of the ratio of phosphorylated (p-eNOS)/eNOS and p-Akt/Akt, but not the expression of total eNOS or Akt. The 3-h exposure of MMECs to metformin significantly (Pmetformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt.

  10. Antiangiogenic effects of melatonin in endothelial cell cultures.

    Science.gov (United States)

    Alvarez-García, Virginia; González, Alicia; Alonso-González, Carolina; Martínez-Campa, Carlos; Cos, Samuel

    2013-05-01

    Endothelial cells represent one of the critical cellular elements in tumor microenvironment playing a crucial role in the growth and progression of cancer through controlling angiogenesis. Vascular endothelial growth factor (VEGF) produced from tumor cells is essential for the expansion of breast cancer and may function in both paracrine and autocrine manners to promote proliferation, growth, survival and migration of endothelial cells. Since melatonin regulates tumor microenvironment by decreasing the secretion of VEGF by malignant epithelial cells and also regulates VEGF expression in human breast cancer cells, the aim of the present study was to investigate the anti-angiogenic activity of melatonin against the pro-angiogenic effects of breast cancer cells. In this work, we demonstrate that melatonin strongly inhibited the proliferation as well as invasion/migration of human umbilical vein endothelial cells (HUVECs). Melatonin disrupted tube formation and counteracted the VEGF-stimulated tubular network formation by HUVEC. In addition, conditioned media collected from human breast cancer cells were angiogenically active and stimulated tubule length formation and this effect was significantly counteracted by the addition of anti-VEGF or melatonin. Melatonin also disintegrated preformed capillary network. All these findings demonstrate that melatonin may play a role in the paracrine interactions that take place between malignant epithelial cells and proximal endothelial cells. Melatonin could be important in reducing endothelial cell proliferation, invasion, migration and tube formation, through a downregulatory action on VEGF. Taken together, our findings suggest that melatonin could potentially be beneficial as an antiangiogenic agent in breast cancer with possible future clinical applications. PMID:23473980

  11. [Pulmonary arterial hypertension, bone marrow, endothelial cell precursors and serotonin].

    Science.gov (United States)

    Ayme-Dietrich, Estelle; Banas, Sophie M; Monassier, Laurent; Maroteaux, Luc

    2016-01-01

    Serotonin and bone-marrow-derived stem cells participate together in triggering pulmonary hypertension. Our work has shown that the absence of 5-HT2B receptors generates permanent changes in the composition of the blood and bone-marrow in the myeloid lineages, particularly in endothelial cell progenitors. The initial functions of 5-HT2B receptors in pulmonary arterial hypertension (PAH) are restricted to bone-marrow cells. They contribute to the differentiation/proliferation/mobilization of endothelial progenitor cells from the bone-marrow. Those bone-marrow-derived cells have a critical role in the development of pulmonary hypertension and pulmonary vascular remodeling. These data indicate that bone-marrow derived endothelial progenitors play a key role in the pathogenesis of PAH and suggest that interactions involving serotonin and bone morphogenic protein type 2 receptor (BMPR2) could take place at the level of the bone-marrow. PMID:27687599

  12. Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities.

    Science.gov (United States)

    Aman, Jurjan; Weijers, Ester M; van Nieuw Amerongen, Geerten P; Malik, Asrar B; van Hinsbergh, Victor W M

    2016-08-01

    Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches. PMID:27343194

  13. Infection of human endothelial cells by Japanese encephalitis virus: increased expression and release of soluble HLA-E.

    Directory of Open Access Journals (Sweden)

    Shwetank

    Full Text Available Japanese encephalitis virus (JEV is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP. In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-α as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-α and IFN-β as well as the dsRNA analog, poly (I:C. Both IFN-β and TNF-α further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

  14. Characterization of vascular endothelial progenitor cells from chicken bone marrow

    Directory of Open Access Journals (Sweden)

    Bai Chunyu

    2012-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

  15. Acrylamide induces accelerated endothelial aging in a human cell model.

    Science.gov (United States)

    Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

    2015-09-01

    Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

  16. Endothelial cells regulate neural crest and second heart field morphogenesis

    Directory of Open Access Journals (Sweden)

    Michal Milgrom-Hoffman

    2014-07-01

    Full Text Available Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio–craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1 in the mesoderm results in early embryonic lethality, severe deformation of the cardio–craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1 along with changes in the extracellular matrix (ECM composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio–craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

  17. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

    Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin;

    2015-01-01

    Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy......-dependent increase of AuNPs inside cells measured by flow cytometry, spICP-MS and 3D confocal microscopy. The latter also showed that AuNPs were located in the cytosol. This was supported by FIB/SEM, showing that AuNPs were located in membrane enclosures in the cytoplasm as single particles or agglomerates of 2...

  18. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    Energy Technology Data Exchange (ETDEWEB)

    Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

    2013-12-01

    When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

  19. Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells.

    Science.gov (United States)

    Horrillo, Angélica; Porras, Gracia; Ayuso, Matilde S; González-Manchón, Consuelo

    2016-08-01

    Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl(-/-) mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury. PMID:27289182

  20. Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Zhang Lili

    2007-04-01

    Full Text Available Abstract Background Hepatitis B virus (HBV replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is trans-infected into extrahepatic tissues such as HBV associated myocarditis remains largely unknown. Results In this study, we showed that human cord blood endothelial progenitor cells (EPCs, but not human umbilical vein endothelial cells (HUVECs could be effectively infected by uptake of HBV in vitro. Exposure of EPCs with HBV resulted in HBV DNA and viral particles were detected in EPCs at day 3 after HBV challenge, which were peaked around day 7 and declined in 3 weeks. Consistently, HBV envelope surface and core antigens were first detected in EPCs at day 3 after virus challenge and were retained to be detectable for 3 weeks. In contrast, HBV covalently closed circular DNA was not detected in EPCs at any time after virus challenge. Intravenous transplantation of HBV-treated EPCs into myocardial infarction and acute renal ischemia mouse model resulted in incorporation of HBV into injured heart, lung, and renal capillary endothelial tissues. Conclusion These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured endothelial tissues. The findings might provide a novel mechanism for HBV-associated myocarditis and other HBV-related extrahepatic diseases as well.

  1. Human neural stem cell-induced endothelial morphogenesis requires autocrine/paracrine and juxtacrine signaling

    Science.gov (United States)

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

    Transplanted neural stem cells (NSC) interact with the host brain microenvironment. A neovascularization is commonly observed in the vicinity of the cell deposit, which is correlated with behavioral improvements. To elucidate the signaling mechanisms between human NSCs and endothelial cells (ECs), these were cocultured in an in vitro model in which NSC-induced endothelial morphogenesis produced a neurovascular environment. Soluble (autocrine/paracrine) and contact–mediated (juxtacrine) signaling molecules were evaluated for two conditionally immortalized fetal NSC lines derived from the cortical anlage (CTXOE03) and ganglionic eminence (STROC05), as well as an adult EC line (D3) derived from the cerebral microvasculature of a hippocampal biopsy. STROC05 were 4 times as efficient to induce endothelial morphogenesis compared to CTXOE03. The cascade of reciprocal interactions between NSCs and ECs in this process was determined by quantifying soluble factors, receptor mapping, and immunocytochemistry for extracellular matrix molecules. The mechanistic significance of these was further evaluated by pharmacological blockade. The sequential cell-specific regulation of autocrine/paracrine and juxtacrine signaling accounted for the differential efficiency of NSCs to induce endothelial morphogenesis. These in vitro studies shed new light on the reciprocal interactions between NSCs and ECs, which are pivotal for our mechanistic understanding of the efficacy of NSC transplantation. PMID:27374240

  2. Experiment Study of Effect of Perfiuorohexyloctane on Corneal Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Ding; Chunfang Li; Lin Lu; Guanguang Feng; Huling Zheng

    2001-01-01

    Purpose: To investigate the effect of Perfluorohexyloctane (F6H8)on corneal endothelial celIs(CEC) of rabbit eyes. Methods: Fifteen New Zealand white rabbits were devided into two groups:experimental group(F6H8) and control group(BSS) . All rabbits underwent anterior chamber injection of 0. 15ml F6H8 or BSS. Slit-lamp biomicroscopy and corneal endothelium photography were performed pre-operatively and postoperatively. Histopathological examination and Transmission electron microscopy(TEM) were done after the rabbits were sacrificed. Results: All the corneas were clear. Since 4 weeks after operation, the endothelial cells were markedly irregular in size and shape and the number of endothelial cells was markedly decreased. Multilayered retrocorneal membranes (RCM)grew gradually 2 weeks after surgery. Vacuolar degeneration was seen in some endothelial cells. Nuclear degeneration and edema of plasma were seen in TEM. Conclusion: Corneal endothelial cell degenerated after contacting with F6H8 for 2 ~4weeks. As a silicone solvent, it should be removed completely after injection. We don't recommend it to be used as a new intraocular temponade. Eye Science 2001: 17:21 ~ 26.

  3. Opioid-induced proliferation of vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Sandra Leo

    2009-05-01

    Full Text Available Sandra Leo1,2, Rony Nuydens1, Theo F Meert11Pain and Neurology, CNS Department, Johnson and Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V, Beerse, Belgium; 2Laboratory of Biological Psychology, University of Leuven, Leuven, BelgiumAbstract: Angiogenesis is an important issue in cancer research and opioids are often used to treat pain in cancer patients. Therefore it is important to know if the use of opioids is associated with an aberrant stimulation of tumor growth triggered by the stimulation of angiogenesis in cancer patients. Some studies in the literature have suggested the presence of the μ3 opioid receptor, known as the receptor for many opioids, on endothelial cells, which are key players in the process of angiogenesis. In this study we used endothelial cells known to express the μ3 opioid receptor (MOR3, to evaluate the effects of morphine on angiogenesis. We first investigated the effect of morphine on the proliferation of endothelial cells. We showed that morphine is able to stimulate vascular endothelial cell proliferation in vitro. This effect of morphine is mediated by the mitogen-activated protein kinase (MAPK pathway as pre-treatment with PD98059 inhibited this excessive proliferation. Because previous studies indicated nitric oxide (NO as a downstream messenger we investigated the role of NO in the aberrant proliferation of endothelial cells. Our data could not confirm these findings using intracellular NO measurements and quantitative fluorescence microscopy. The potential use and pitfalls of opioids in cancer patients is discussed in light of these negative findings. Keywords: endothelial cells, morphine, cell proliferation, MAPK, nitric oxide, μ3 opioid receptor, angiogenesis

  4. Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue.

    Science.gov (United States)

    Ugele, B; Lange, F

    2001-01-01

    Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary. PMID:11573814

  5. Endothelial differentiation gene-1, a new downstream gene is involved in RTEF-1 induced angiogenesis in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Ping He

    Full Text Available Related Transcriptional Enhancer Factor-1 (RTEF-1 has been suggested to induce angiogenesis through regulating target genes. Whether RTEF-1 has a direct role in angiogenesis and what specific genes are involved in RTEF-1 driven angiogenisis have not been elucidated. We found that over-expressing RTEF-1 in Human dermal microvascular endothelial cells-1 (HMEC-1 significantly increased endothelial cell aggregation, growth and migration while the processes were inhibited by siRNA of RTEF-1. In addition, we observed that Endothelial differentiation gene-1 (Edg-1 expression was up-regulated by RTEF-1 at the transcriptional level. RTEF-1 could bind to Edg-1 promoter and subsequently induce its activity. Edg-1 siRNA significantly blocked RTEF-1-driven increases in endothelial cell aggregation in a Matrigel assay and retarded RTEF-1-induced endothelial cell growth and migration. Pertussis Toxin (PTX, a Gi/Go protein sensitive inhibitor, was found to inhibit RTEF-1 driven endothelial cell aggregation and migration. Our data demonstrates that Edg-1 is a potential target gene of RTEF-1 and is involved in RTEF-1-induced angiogenesis in endothelial cells. Gi/Go protein coupled receptor pathway plays a role in RTEF-1 driven angiogenesis in endothelial cells.

  6. New insights in endothelial and smooth muscle cell communication.

    Science.gov (United States)

    Conejo, Víctor Arana; De Haro, Roberto; Sosa-Melgarejo, Jorge; Méndez, José D

    2007-01-01

    Based on immunohistochemical techniques against connexins and the intercellular flux of staining molecules, it has previously been shown that electrotonic communication occurs among endothelial and vascular smooth muscle cells, this due to the presence of myoendothelial gap junctions. The aim of this study was to evaluate the density of myoendothelial contacts in the left coronary and internal mammary arteries as well as in the left saphenous vein by means of electron microscopy, the distance between both cells participating in an myoendothelial contact with a semi-automatic image analysis system and the presence of homocellular and heterocellular gap junctions between endothelial and smooth muscle cells by using the immunohistochemical technique and confocal microscopy in thoracic aorta were also analyzed. The results are that all blood vessels studied present myoendothelial contacts, while density studies show that they are more abundant in the saphenous vein. The myoendothelial contact distance is constant and in no case the cytoplasmic processes reach the plasma membrane of the partner cell toward which they are advanced. Homocellular gap junctions were found between smooth muscle cells and between endothelial cells. Heterocellular gap junctions were absent, evidencing the possibility that signaling molecules between endothelial and smooth muscle cells may be transferred through plasma membranes as was once thought and not necessarily by electrotonic communication. PMID:17383847

  7. Effect of Antioxidants on Endothelial Cell Reactive Oxygen Species (ROI) Generation and Adhesion of Leukocytes to Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Huang Qian; Michael Grafe; Kristoph Graf; Hans Lehmkuhl; Eckart Fleck

    2000-01-01

    Objective To investigate whether antioxidants inhibit adhesion of leukocytes to endothelium and furthermore, whether all antioxidants regulate NF-κB activation through a redox sensitive mechanism. Methods The effect of the antioxidative substances pyrrolidin dithiocarbamat (PDTC),dichloroisocumarin (DCI), chrysin and probucol on the endothelial leukocyte adhesion were examined under near physiological flow conditions. The antioxidative activity of antioxidants was measured in a DCF fluorescence assay with flow cytometry. The activation of NF-κB in endothelial cells was investigated in a gel shift assay. Results PDTC and probucol did not show an inhibitory effect to the formation of intracellular H2O2 in TNFct activated human vascular endothelial cells (HUVEC) . Chrysin showed a moderate effect.DCI showed a strong antioxidative effect. In contrast,PDTC and chrysin inhibited the adhesion of HL 60 cells to TNFa-stimulated HUVEC. DCI and probucol did not have influence on the adhesion within the area of the examined shear stresses. Only PDTC inhibited the TNFα-induced activation of NF-kB in endothelial cells.Conclusion The inhibition of the endothelial leukocyte adhesion by antioxidative substances is not to be explained by its antioxidative characteristics only. The inhibitory effect of PDTC on NF-kB activation was probably not related to its antioxidative properties.

  8. Reprogramming Cells for Brain Repair

    OpenAIRE

    McKinnon, Randall D.; Alyx T. Guarino

    2013-01-01

    At present there are no clinical therapies that can repair traumatic brain injury, spinal cord injury or degenerative brain disease. While redundancy and rewiring of surviving circuits can recover some lost function, the brain and spinal column lack sufficient endogenous stem cells to replace lost neurons or their supporting glia. In contrast, pre-clinical studies have demonstrated that exogenous transplants can have remarkable efficacy for brain repair in animal models. Mesenchymal stromal c...

  9. Recovery of Corneal Endothelial Cells from Periphery after Injury.

    Directory of Open Access Journals (Sweden)

    Sang Ouk Choi

    Full Text Available Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.To investigate the recovery process of corneal endothelial cells (CECs from corneal endothelial injury.Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group. Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.

  10. Melatonin modulates aromatase activity and expression in endothelial cells.

    Science.gov (United States)

    Alvarez-García, Virginia; González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

    2013-05-01

    Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real‑time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. We conclude that melatonin inhibits aromatase activity and expression in HUVECs by regulating gene expression of specific aromatase promoter regions, thereby reducing the local production of estrogens. PMID:23450505

  11. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, Claudia R.; Grosso, Mariela F. del [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Behar, Moni [Instituto de Física, UFRGS, Porto Alegre, RS (Brazil); García Bermúdez, Gerardo, E-mail: ggb@tandar.cnea.gov.ar [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Escuela de Ciencia y Tecnología, UNSAM (Argentina)

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  12. Mechanical property quantification of endothelial cells using scanning acoustic microscopy

    Science.gov (United States)

    Shelke, A.; Brand, S.; Kundu, T.; Bereiter-Hahn, J.; Blase, C.

    2012-04-01

    The mechanical properties of cells reflect dynamic changes of cellular organization which occur during physiologic activities like cell movement, cell volume regulation or cell division. Thus the study of cell mechanical properties can yield important information for understanding these physiologic activities. Endothelial cells form the thin inner lining of blood vessels in the cardiovascular system and are thus exposed to shear stress as well as tensile stress caused by the pulsatile blood flow. Endothelial dysfunction might occur due to reduced resistance to mechanical stress and is an initial step in the development of cardiovascular disease like, e.g., atherosclerosis. Therefore we investigated the mechanical properties of primary human endothelial cells (HUVEC) of different age using scanning acoustic microscopy at 1.2 GHz. The HUVECs are classified as young (tD 90 h) cells depending upon the generation time for the population doubling of the culture (tD). Longitudinal sound velocity and geometrical properties of cells (thickness) were determined using the material signature curve V(z) method for variable culture condition along spatial coordinates. The plane wave technique with normal incidence is assumed to solve two-dimensional wave equation. The size of the cells is modeled using multilayered (solid-fluid) system. The propagation of transversal wave and surface acoustic wave are neglected in soft matter analysis. The biomechanical properties of HUVEC cells are quantified in an age dependent manner.

  13. Is manual counting of corneal endothelial cell density in eye banks still acceptable? The French experience

    OpenAIRE

    Thuret, G; Manissolle, C; Acquart, S.; Petit, J-C Le; Maugery, J; Campos-Guyotat, L; Doughty, M J; Gain, P

    2003-01-01

    Aim: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant.

  14. Improved endothelialization of titanium vascular implants by extracellular matrix secreted from endothelial cells.

    Science.gov (United States)

    Tu, Qiufen; Zhao, Yuancong; Xue, Xiaoqing; Wang, Jin; Huang, Nan

    2010-12-01

    A variety of metals have been widely used in construction of cardiovascular implants (CVIs), such as artificial heart valves, ventricular pumps, and vascular stents. Although great effects have been put into rigorous anticoagulation, late thrombosis still occurred due to inferior blood and cell compatibility. Natural endothelium is popularly regarded as the only substance that has long-term anticoagulant ability. So, establishment of a compact endothelial cell (EC) monolayer on CVIs surface is a guarantee for their long-term potency. In the work described here, titanium (Ti) disks were coated with extracellular matrix (ECM) directly secreted by human umbilical vein endothelial cells (HUVECs), so as to help ECs proliferate and migrate and to improve their endothelialization in vivo. Deposition of ECM on Ti disks was detected by immunofluorescence microscopy, diffuse reflectance Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. The surface topography and wettability of the Ti disks significantly changed after ECM deposition. Most importantly, it was found that ECM deposition inhibited platelet adhesion, stimulated EC proliferation, increased EC migration speed in vitro, and eventually accelerated the re-cellularization speed of Ti disks in vivo. These important results render it reasonable and feasible to modify CVIs with ECM secreted from ECs for improving their long-term potency. PMID:20666613

  15. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    International Nuclear Information System (INIS)

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  16. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    Energy Technology Data Exchange (ETDEWEB)

    Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  17. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  18. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  19. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Infante, Teresa [SDN-Foundation, Institute of Diagnostic and Nuclear Development, IRCCS, 80143 Naples (Italy); Cesario, Elena [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy); Gallo, Michele; Fazioli, Flavio [Division of Skeletal Muscles Oncology Surgery, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); De Chiara, Annarosaria [Anatomic Pathology Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Tutucci, Cristina; Apice, Gaetano [Medical Oncology of Bone and Soft Sarcoma tissues Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Nigris, Filomena de, E-mail: filomena.denigris@unina2.it [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy)

    2013-04-11

    Cooperation between endothelial cells and bone in bone remodelling is well established. In contrast, bone microvasculature supporting the growth of primary tumors and metastasis is poorly understood. Several antiangiogenic agents have recently been undergoing trials, although an extensive body of clinical data and experimental research have proved that angiogenic pathways differ in each tumor type and stage. Here, for the first time, we characterize at the molecular and functional level tumor endothelial cells from human bone sarcomas at different stages of disease and with different histotypes. We selected a CD31{sup +} subpopulation from biopsies that displayed the capability to grow as adherent cell lines without vascular endothelial growth factor (VEGF). Our findings show the existence in human primary bone sarcomas of highly proliferative endothelial cells expressing CD31, CD44, CD105, CD146 and CD90 markers. These cells are committed to develop capillary-like structures and colony formation units, and to produce nitric oxide. We believe that a better understanding of tumor vasculature could be a valid tool for the design of an efficacious antiangiogenic therapy as adjuvant treatment of sarcomas.

  20. Prostacyclin mediates endothelial COX-2-dependent neuroprotective effects during excitotoxic brain injury

    Directory of Open Access Journals (Sweden)

    An Y

    2014-05-01

    Full Text Available Ying An,1,2 Natalya Belevych,1,2 Yufen Wang,1,2 Hao Zhang,1 Jason S Nasse,3 Harvey Herschman,4 Qun Chen,1,2 Andrew Tarr,1,2 Xiaoyu Liu,1,2 Ning Quan1,21Institute for Behavior Medicine Research, 2Department of Oral Biology, College of Dentistry, 3Neuroscience Graduate Studies Program, The Ohio State University, Columbus, OH, USA; 4Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USAAbstract: In a previous study, we found that intracerebral administration of excitotoxin (RS-(tetrazole-5yl glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2flox/flox. In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2 in wild type, but not in endothelial COX-2 deleted mice. Inhibition of PGI2 synthase exacerbated brain lesions induced by the excitotoxin in wild type, but not in endothelial COX-2 deleted mice. Administration of a PGI2 agonist reduced neural damage in both wild type and endothelial COX-2 deleted mice. Increased PGI2 synthase expression was found in infiltrating neutrophils. In an ex vivo assay, PGI2 reduced the excitotoxin-induced calcium influx into neurons, suggesting a cellular mechanism for PGI2 mediated neuroprotection. These results reveal that PGI2 mediates endothelial COX-2 dependent neuroprotection.Keywords: neural injury, prostaglandins, neutrophil, conditional COX-2 deletion, PGI2

  1. Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes%大鼠脑微血管内皮细胞与周细胞、星形胶质细胞共培养建立体外血脑屏障模型

    Institute of Scientific and Technical Information of China (English)

    查雨锋; 傅晓钟; 张顺; 罗敏; 欧瑜; 董永喜; 王爱民; 王永林

    2015-01-01

    目的:应用原代培养的大鼠脑微血管内皮细胞(brain-microvessel endothelial cells,BMECs )与脑微血管周细胞(brain-microvessel pericytes,BMPC )、星形胶质细胞(astro-cytes,AS)共培养建立可模拟在体状态的体外血脑屏障(blood-brain barrier,BBB)模型。方法原代分离、纯化和培养大鼠BMECs、BMPC和AS,通过细胞形态学和免疫细胞化学染色方法鉴定原代培养的细胞,应用Millicell细胞培养插(孔径0.4μm)建立5种不同类型的体外BBB模型,经跨内皮电阻值(transendothelial electrical resistance,TEER)、荧光素钠通透性(sodium fluorescent,Na-FLU )、碱性磷酸酶(AKP)和γ-谷氨酰转肽酶(γ-GT1)的表达测定以及阳性药在体内和体外BBB通透量的相似性,比较评价其屏障功能。结果原代培养的BMECs呈典型的铺路卵石样结构,BMPC胞体较大且呈分枝状,AS 有细长突触,胞质较浅;免疫细胞化学染色证实原代细胞为目标细胞;BMECs与BMPC、AS共培养后TEER值可达(478±25)Ω·cm2,Na-FLU 的表观渗透系数为[(8.23±0.78)×10-6]cm·s-1,AKP和γ-GT1表达分别为(6.90±0.27)金氏单位· g-1 Pro,(4.39±0.32)μg·g-1 Pro;阳性药在体外BBB的表观渗透系数(apparent permeability coefficient,Papp )与在体数据具有较好的相关性(R2=0.92)。结论原代培养的大鼠BMECs与BMPC、AS共培养建立的体外BBB模型在形态、结构及屏障功能方面具备BBB的基本特征,为研究BBB的生理学、病理学以及筛选化合物提供了一种有用工具。%Aim To establish in vitro blood-brain barrier (BBB) model with characteristics of simulation of in vivo BBB by primi-tive co-culture of brain-microvessel endothelial cells (BMECs) with brain-microvessel pericytes (BMPC)and astrocytes (AS). Methods BMECs,BMPC and AS from SD rats were primitively isolated,purified and cultured,and then

  2. High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Tang Rining

    2012-09-01

    Full Text Available Abstract Background Vascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs via the endothelial-to-mesenchymal transition (EndMT could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs. Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes. Methods HAECs were divided into three groups: a normal glucose (NG group, HG group (30 mmol/L, and mannitol (5.5 mmol/L NG + 24.5 mmol/L group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1. The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining. Results The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P  Conclusions Our

  3. Preclinical pulmonary capillary endothelial dysfunction is present in brain dead subjects.

    Science.gov (United States)

    Glynos, Constantinos; Athanasiou, Chariclea; Kotanidou, Anastasia; Korovesi, Ioanna; Kaziani, Katerina; Livaditi, Olga; Dimopoulou, Ioanna; Maniatis, Nikolaos A; Tsangaris, Iraklis; Roussos, Charis; Armaganidis, Apostolos; Orfanos, Stylianos E

    2013-04-01

    Pulmonary endothelium is a major metabolic organ affecting pulmonary and systemic vascular homeostasis. Brain death (BD)-induced physiologic and metabolic derangements in donors' lungs, in the absence of overt lung pathology, may cause pulmonary dysfunction and compromise post-transplant graft function. To explore the impact of BD on pulmonary endothelium, we estimated pulmonary capillary endothelium-bound (PCEB)-angiotensin converting enzyme (ACE) activity, a direct and quantifiable index of pulmonary endothelial function, in eight brain-dead patients and ten brain-injured mechanically ventilated controls. No subject suffered from acute lung injury or any other overt lung pathology. Applying indicator-dilution type techniques, we measured single-pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic, biologically inactive, and highly specific for ACE substrate (3)H-benzoyl-Phe-Ala-Pro, under first order reaction conditions, and calculated lung functional capillary surface area (FCSA). Substrate %M (35 ± 6.8%) and v (0.49 ± 0.13) in BD patients were decreased as compared to controls (55.9 ± 4.9, P = 0.033 and 0.9 ± 0.15, P = 0.033, respectively), denoting decreased pulmonary endothelial enzyme activity at the capillary level; FCSA, a reflection of endothelial enzyme activity per vascular bed, was also decreased (BD patients: 1,563 ± 562 mL/min vs 4,235 ± 559 in controls; P = 0.003). We conclude that BD is associated with subtle pulmonary endothelial injury, expressed by decreased PCEB-ACE activity. The applied indicator-dilution type technique provides direct and quantifiable indices of pulmonary endothelial function at the bedside that may reveal the existence of preclinical lung pathology in potential lung donors. PMID:24015344

  4. METABOLIC CAPACITY REGULATES IRON HOMEOSTATIS IN ENDOTHELIAL CELLS

    Science.gov (United States)

    The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuat...

  5. Apoptosis and calcification of vascular endothelial cell under hyperhomocysteinemia.

    Science.gov (United States)

    Fang, Kuaifa; Chen, Zhujun; Liu, Meng; Peng, Jian; Wu, Pingsheng

    2015-01-01

    In recent years, it is found that increase in Hcy level in blood can directly or indirectly cause vascular endothelial cell injury and induce vascular calcification. However, the mechanism of vascular endothelial cell injury and vascular calcification has not been studied thoroughly. This paper carried out experiment for research aiming at discussing the effect and action mechanism of Hhcy on endothelial cells and vascular calcification. Firstly, human umbilical vein endothelial cells (HUVECs) were cultured and then intervened by Hcy of different concentrations (0, 0.01, 0.1, 1.0, 3.0, 5.0 mmol/L) and at different action time (3, 6, 12, 24 h). Then apoptosis rate and reactive oxygen were detected by flow cytometry. At the same time, the model for the culture of rat vascular calcification was set up and induced into Hhcy so as to detect the total plasma Hcy level and judge vascular calcification degree. The results showed that with the increase in Hcy concentration and extension of action period, the apoptosis rate and generation of reactive oxygen of HUVECs all significantly increased, and the differences were all statistically significant (P animal calcification model, mass of black particle deposition was seen after Von Kossa staining of rat vessels in calcification group. Compared with the control group, the vascular calcium content, alkaline phosphatase activity and osteocalcin content in calcification group all increased (P benefits on clinical prevention works. PMID:25476479

  6. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Science.gov (United States)

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  7. Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

    Science.gov (United States)

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

    2013-11-01

    Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

  8. High glucose augments stress-induced apoptosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Hui Tian

    2009-01-01

    Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. Eahy 926 endothelial cells were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide (H2O2) or tumour necrosis factor- α (TNF- α ), following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of Eahy 926 cells to apoptosis in the presence of 500 μmol/L H2O2, above that induced in normal glucose (P<0.02). A reduction of H2O2- and TNF- α -induced apoptosis occurred in both high and low glucose after treatment with dexametha-sone (P<0.05). Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.

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    Lifescience Database Archive (English)

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  13. File list: Pol.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Oth.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: ALL.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  17. File list: Oth.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  1. File list: His.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  4. File list: ALL.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Unc.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Oth.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Unc.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: His.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: His.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Unc.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.CDV.20.AllAg.Brachiocephalic_endothelial_cells hg19 Unclassified Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.CDV.20.AllAg.Brachiocephalic_endothelial_cells.bed ...

  12. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

    DEFF Research Database (Denmark)

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje;

    2012-01-01

    with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape (‘rounding up’). Increased expression of apoptotic markers indicated...

  13. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    János Haskó

    2014-05-01

    Full Text Available During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB. The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2; therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A, GPR18 (transcriptional variant 1 and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A, GPR18 (transcriptional variants 1 and 2, GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

  14. Endothelial induced EMT in breast epithelial cells with stem cell properties

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla;

    2011-01-01

    endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal......Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether...

  15. Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells.

    Science.gov (United States)

    Nowatzki, Jenifer; de Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Célia; Nader, Helena Bonciani; Trindade, Edvaldo S; Franco, Célia Regina C

    2010-09-15

    Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.

  16. The effects of TSH on human vascular endothelial cells and smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    田利民

    2014-01-01

    Objective To study the effect of thyroid-stimulating hormone(TSH)on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used

  17. Endothelial cell markers reflecting endothelial cell dysfunction in patients with mixed connective tissue disease

    OpenAIRE

    Soltész Pál (1961-) (belgyógyász, kardiológus); Bereczki Dániel (1960-) (neurológus); Szodoray Péter (1973-) (belgyógyász, orvos); Magyar Mária Tünde (1970-) (neurológus); Dér Henrietta (1977-) (orvos); Csípő István (1953-) (vegyész); Hajas Ágota Helga (1985-) (orvos); Paragh György (1953-) (belgyógyász, kardiológus, endokrinológus, lipidológus, sürgősségi orvostani szakorvos, belgyógyászati angiológiai minősített orvos); Szegedi Gyula (1936-2013) (belgyógyász, immunológus); Bodolay Edit (1950-) (belgyógyász, allergológus és klinikai immunológus)

    2010-01-01

    Introduction The aim of the present study was to investigate the association between cardiovascular risk factors and endothelial dysfunction in patients with mixed connective tissue disease (MCTD) and to determine which biomarkers are associated with atherosclerotic complications, such as cardiovascular disease. Methods Fifty MCTD patients and 38 healthy age-matched and sex-matched controls were enrolled in this study. In order to describe endothelial dysfunction, we assessed flow-mediated di...

  18. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  19. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Bruno Lorusso

    2015-01-01

    Full Text Available Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs. Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.

  20. Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization.

    Science.gov (United States)

    Seda, Robinson; Li, David S; Fowlkes, J Brian; Bull, Joseph L

    2015-12-01

    Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.

  1. Influence of mild hypothermia on vascular endothelial growth factor and infarct volume in brain tissues after cerebral ischemia in rats

    Institute of Scientific and Technical Information of China (English)

    Fei Ye; Gangming Xi; Biyong Qin; Shifeng Wang; Chengyan Li

    2006-01-01

    : All the rats were anesthetized by intraperitoneal injection overdose sodium pentobarbital 7 days postoperatively, and then the heads were cut down to harvest brain. The brain tissues were placed into -20 ℃ refrigerator for 20 minutes, coronal sections of 2 mm were prepared. The infarct sites were not stained, whereas normal brain tissues were stained as red. The infarct volumes were calculated by using MPLAS-500 multimedia color pathological image&word analytical system. ③ Counting positive cells of vascular endothelial growth factor protein: The brains were harvested by cutting heads, then coronal sections of 2 mm were prepared. Routine dehydration, hyalinization, wax immersion and embedding were performed, then the detected with SP immunohistochemistry, the kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. The cells whose cytoplasm was yellow-brown were positive ones, a single sample as a unit, peri-ischemic site and ischemic core were selected, and the corresponding sites in controlateral hemisphere were taken as controls. Five visual fields were selected from each site to be observed under microscope, the cells were counted, and the average number of positive cells was calculated in each group. The numbers of positive cells were determined with the image analytical apparatus.MAIN OUTCOME MEASURES: Number of the positive cells of vascular endothelial growth factor protein; Infarct volume of rat brain tissue.RESULTS: All the 20 rats were involved in the analysis of results. ① Number of positive cells of vascular endothelial growth factor protein in brain tissue: It was obviously lower in the mild hypothermia group than in the normal temperature group [(24.02±5.05), (36.07±2.69) cells/high power visual field, P < 0.01]. ② Comparison of infarct volume of brain tissue: After MCAO, it was obviously smaller in the mild hypothermia group than in the normal temperature group [(153.25±23.14), (253.45±36.21) mm3, P< 0

  2. An Important Method in the Investigation of Vascular Pathologies: Endothelial Cell Culture

    Directory of Open Access Journals (Sweden)

    Yusufhan Yazır

    2012-12-01

    Full Text Available Endothelial cells line the interior surface of blood vessels and form an interface between circulating blood in the lumen and the rest of the vessel wall. Endothelial cells are involved in many aspects of vascular biology, including barrier function, vasoconstriction, coagulation and inflamation. The endothelial cells in different organs have different functions and surface phenotype. These cells express prostoglandin-I2, platelet activating factor, collagen, endothelin-1, laminin, fibronectin and growth factors including platelet derived growth factor, fibroblast growth factor. İn the cell culture, cells can be isolated, maintened and proliferate in the laboratory conditions. The techniques of the cell culture have allowed scientists to use the cells in vitro for experimental studies, such as the production of vaccine, antibody and enzime, drug research, cell-cell interactions. Human umbilical vein endothelial cell is a good source for endothelial cell, because it is cheaper, easy to find and has the basic features of the normal endothelial cells.

  3. Adiponectin promotes endothelial progenitor cell number and function

    OpenAIRE

    Shibata, Rei; Skurk, Carsten; Ouchi, Noriyuki; Galasso, Gennaro; Kondo, Kazuhisa; Ohashi, Taiki; Shimano, Masayuki; Kihara, Shinji; Murohara, Toyoaki; Walsh, Kenneth

    2008-01-01

    Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells ...

  4. Endothelial cell pseudopods and angiogenesis of breast cancer tumors

    OpenAIRE

    Sun LuZhe; Short Nicholas; Cameron Ivan L; Hardman W Elaine

    2005-01-01

    Abstract Background A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse). Once the tumor grew to 35 mm3, it developed a well-vascularized capsule. Histological sections of tumors greater than...

  5. Characterization of calcium signals provoked by lysophosphatidylinositol in human microvascular endothelial cells.

    Science.gov (United States)

    Al Suleimani, Y M; Hiley, C R

    2016-01-01

    The lipid molecule, lysophosphatidylinositol (LPI), is hypothesised to form part of a novel lipid signalling system that involves the G protein-coupled receptor GPR55 and distinct intracellular signalling cascades in endothelial cells. This work aimed to study the possible mechanisms involved in LPI-evoked cytosolic Ca(2+) mobilization in human brain microvascular endothelial cells. Changes in intracellular Ca(2+) concentrations were measured using cell population Ca(2+) assay. LPI evoked biphasic elevation of intracellular calcium concentration, a rapid phase and a sustained phase. The rapid phase was attenuated by the inhibitor of PLC (U 73122), inhibitor of IP(3) receptors, 2-APB and the depletor of endoplasmic reticulum Ca(2+) store, thapsigargin. The sustained phase, on the other hand, was enhanced by U 73122 and abolished by the RhoA kinase inhibitor, Y-27632. In conclusion, the Ca(2+) signal evoked by LPI is characterised by a rapid phase of Ca(2+) release from the endoplasmic reticulum, and requires activation of the PLC-IP(3) signalling pathway. The sustained phase mainly depends on RhoA kinase activation. LPI acts as novel lipid signalling molecule in endothelial cells, and elevation of cytosolic Ca(2+) triggered by it may present an important intracellular message required in gene expression and controlling of vascular tone. PMID:26596318

  6. Apoptosis of endothelial cells of cerebral basilar arteries in symptomatic cerebral vasospasm rabbit models Electron microscopic observation

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Recent researchers report that vasospasm is caused by that, on one hand, damage of endothelial cells reduces synthesis and liberation of vessel dilator; on the other hand, defluxion of endothelial cells directly exposure vascular smooth muscles in active materials of vasoconstriction in blood.OBJECTIVE: To study whether apoptosis of cerebrovascular cells occurs in symptomatic cerebral vasospasm (CVS) rabbit models by using transmission electron microscope.DESIGN: Contrast observation.SETTINGS: The Fifth Endemic Area, the 89 Hospital of Chinese PLA; Minimally Invasive Neurosurgical Center, Tangdu Hospital, the Fourth Military Medical University of Chinese PLA.MATERIALS: A total of 24 New Zealand rabbits, of either sex, weighing 2.4 - 3.0 kg, of clear grade, were selected from the Experimental Animal Center of the Fourth Military Medical University of Chinese PLA.JEM-2000EX transmission electron microscope was made in Japan.METHODS: The experiment was carried out in the Laboratory of Anatomy (National Key Laboratory), the Fourth Military Medical University of Chinese PLA from April 2001 to April 2002. ① Preparation of symptomatic CVS models: Eighteen animals which were successfully modeled were randomly divided into experimental group (n =13) and control group (n =5). Animals in the experimental group were poured with blood into cavitas subarachnoidealis; while, animals in the control group were poured with the same volume of saline into cavitas subarachnoidealis. At the 5th day injection, three rabbits selected from the experimental group were anesthetized and perfused into left ventricle. And then, aorta pectoralis and caval vein were blocked by using ring clamp. Cranium was rapidly cut open to obtain cerebral basilar artery and a few of brain tissues. Both of them were fixed for 8 hours. Two rabbits selected from the control group were perfused with the same method to obtain basilar artery and brain tissues and fix. ② After fixation by using optic

  7. Signal transduction pathways in mast cell granule-mediated endothelial cell activation

    Directory of Open Access Journals (Sweden)

    Luqi Chi

    2003-01-01

    Full Text Available Background: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8.

  8. Nitrative Stress Participates in Endothelial Progenitor Cell Injury in Hyperhomocysteinemia

    Science.gov (United States)

    Dong, Yu; Sun, Qi; Liu, Teng; Wang, Huanyuan; Jiao, Kun; Xu, Jiahui; Liu, Xin; Liu, Huirong; Wang, Wen

    2016-01-01

    In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that

  9. Nitrative Stress Participates in Endothelial Progenitor Cell Injury in Hyperhomocysteinemia.

    Science.gov (United States)

    Dong, Yu; Sun, Qi; Liu, Teng; Wang, Huanyuan; Jiao, Kun; Xu, Jiahui; Liu, Xin; Liu, Huirong; Wang, Wen

    2016-01-01

    In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that

  10. Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

    Science.gov (United States)

    Headley, Colwyn A; DiSilvestro, David; Bryant, Kelsey E; Hemann, Craig; Chen, Chun-An; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A

    2016-03-15

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

  11. Synthesis of an endothelial cell mimicking surface containing thrombomodulin and endothelial protein C receptor

    Science.gov (United States)

    Kador, Karl Erich

    Synthetic materials for use in blood contacting applications have been studied for many years with limited success. One of the main areas of need for these materials is the design of synthetic vascular grafts for use in the hundreds of thousands of patients who have coronary artery bypass grafting, many without suitable veins for autologous grafts. The design of these grafts is constrained by two common modes of failure, the formation of intimal hyperplasia (IH) and thrombosis. IH formation has been previously linked to a mismatching of the mechanical properties of the graft and has been overcome by creating grafts using materials whose compliance mimics that of the native artery. Several techniques and surface modification have been designed to limit thrombosis on the surface of synthetic materials. One which has shown the greatest promise is the immobilization of Thrombomodulin (TM), a protein found on the endothelial cell membrane lining native blood vessels involved in the activation of the anticoagulant Protein C (PC). While TM immobilization has been shown to arrest thrombin formation and limit fibrous formations in in-vitro and in-vivo experiments, it has shown to be transport limiting under arterial flow. On the endothelial cell surface, TM is co-localized with Endothelial Protein C Receptor (EPCR), which increases PC transport onto the cell surface and increases PC activation via TM between 20-100 fold. This dissertation will describe the chemical modification of medical grade polyurethane (PU), whose compliance has been shown to match that of native arteries. This modification will enable the immobilization of two proteins on an enzymatically relevant scale estimated at less than 10 nm. This dissertation will further describe the immobilization of the proteins TM and EPCR, and analyze the ability of a surface co-immobilized with these proteins to activate the anticoagulant PC. Finally, it will compare the ability of this co-immobilized surface to delay

  12. Vascular Endothelial Growth Factor Increases during Blood-Brain Barrier-Enhanced Permeability Caused by Phoneutria nigriventer Spider Venom

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    Monique C. P. Mendonça

    2014-01-01

    Full Text Available Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV causes blood-brain barrier breakdown (BBBb. The PNV-induced excitotoxicity results from disturbances on Na+, K+ and Ca2+ channels and glutamate handling. The vascular endothelial growth factor (VEGF, beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.

  13. Coniferyl Aldehyde Ameliorates Radiation Intestine Injury via Endothelial Cell Survival

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Ye Ji; Jung, Myung Gu; Lee, Yoonjin; Lee, Haejune [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yunsil [Ewha Woman' s Univ., Seoul (Korea, Republic of); Ko, Younggyu [Korea Univ., Seoul (Korea, Republic of)

    2014-05-15

    Cancer treatments related gastrointestinal toxicity has also been recognized as a significant economic burden. Especially, extensive apoptosis of microvascular endothelial cell of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Coniferyl aldehyde (CA) is phenolic compounds isolated from cork stoppers, and one of the major pyrolysis products of lignin. Shi H. was support for the empirical use of CA as a medicinal food for cardiovascular diseases. CA has positive effect in broad way but there is no consequence in radiation induced intestine damage. Here, we investigate effect of CA on small intestine after abdominal IR to mice in this study. In this study, CA increased the survival rate in C3H mice against 13.5 Gy abdominal IR. We found CA protects small intestine via preventing endothelial cell apoptosis and enhancing their angiogenic activity. CA also showed protective effect on crypt cell survival. Endothelial cell survival may affect crypt cell protection against IR. From this data, we concluded that CA is effective for protection against abdominal radiation injury. CA could ameliorate side-effect of radiation therapy.

  14. Junctional communication is induced in migrating capillary endothelial cells.

    Science.gov (United States)

    Pepper, M S; Spray, D C; Chanson, M; Montesano, R; Orci, L; Meda, P

    1989-12-01

    Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration. PMID:2592412

  15. Morphological and protein profile comparison of large vessel and microvascular endothelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Beer, D.M.; Kim, J.S.; Carson, M.P.; Haudeuschild, C.C.; Patton, W.F.; Jacobson, B.S.

    1986-05-01

    Bovine adrenal medulla (AmMEC) and brain (BrMEC) microvessel endothelial cells, and bovine aortic (BAE) endothelial cells were isolated and cultured under identical conditions using a modification of a technique previously described for BrMEC. The cells were isolated and passaged under conditions minimizing cell surface alterations. Primary cultures were confluent in 4-6 days at a plating density in the region of 10/sup 4/ cells/cm/sup 2/. BAEs maintained a cobblestone morphology and a denser monolayer than MECs in primary and passaged cells whether the cells were passaged using Pancreatin, Trypsin-EDTA, or Collagenase-EDTA. MECs were initially elongate and became more like BAEs with passaging. BAEs and AmMECs were examined for differences in whole cell, Triton extracted cytoskeleton and plasma membrane (PM) protein profiles by two-dimensional gel electrophoresis. Cells were labeled with /sup 35/S-methionine and PM by lactoperoxidase catalyzed iodination. Though for the most part protein patterns were similar, several proteins in the PM and cytoskeletal preparations differed. A significant difference in the isoelectric forms of proteins with the same molecular weight was observed in the PM.

  16. Mesangial cell integrin αvβ8 provides glomerular endothelial cell cytoprotection by sequestering TGF-β and regulating PECAM-1.

    Science.gov (United States)

    Khan, Shenaz; Lakhe-Reddy, Sujata; McCarty, Joseph H; Sorenson, Christine M; Sheibani, Nader; Reichardt, Louis F; Kim, Jane H; Wang, Bingcheng; Sedor, John R; Schelling, Jeffrey R

    2011-02-01

    Integrins are heterodimeric receptors that regulate cell adhesion, migration, and apoptosis. Integrin αvβ8 is most abundantly expressed in kidney and brain, and its major ligand is latent transforming growth factor-β (TGF-β). Kidney αvβ8 localizes to mesangial cells, which appose glomerular endothelial cells and maintain glomerular capillary structure by mechanical and poorly understood paracrine mechanisms. To establish kidney αvβ8 function, mice with homozygous Itgb8 deletion (Itgb8(-/-)) were generated on outbred and C57BL/6 congenic backgrounds. Most Itgb8(-/-) mice died in utero, and surviving Itgb8(-/-) mice failed to gain weight, and rarely survived beyond 6 weeks. A renal glomerular phenotype included azotemia and albuminuria, as well as increased platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which was surprisingly not associated with conventional functions, such as endothelial cell hyperplasia, hypertrophy, or perivascular inflammation. Itgb8(-/-) mesangial cells demonstrated reduced latent TGF-β binding, resulting in bioactive TGF-β release, which stimulated glomerular endothelial cell apoptosis. Using PECAM-1 gain and loss of function strategies, we show that PECAM-1 provides endothelial cytoprotection against mesangial cell TGF-β. These results clarify a singular mechanism of mesangial-to-endothelial cell cross-talk, whereby mesangial cell αvβ8 homeostatically arbitrates glomerular microvascular integrity by sequestering TGF-β in its latent conformation. Under pathological conditions associated with decreased mesangial cell αvβ8 expression and TGF-β secretion, compensatory PECAM-1 modulation facilitates glomerular endothelial cell survival.

  17. Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells.

    Science.gov (United States)

    Brizzi, M F; Garbarino, G; Rossi, P R; Pagliardi, G L; Arduino, C; Avanzi, G C; Pegoraro, L

    1993-06-01

    Proliferation and functional activation of endothelial cells within a tissue site of inflammation are regulated by humoral factors released by cells, such as T lymphocytes and monocytes, infiltrating the perivascular space. In the present study we investigated the effects of interleukin 3 (IL-3), an activated T lymphocyte-derived cytokine, on cultured human umbilical vein endothelial cells (HUVEC). Proliferative activity, evaluated both by estimation of the fraction of cells in the S phase and by direct cell count demonstrated that IL-3, at the dose of 25 ng/ml, enhances more than threefold both DNA synthesis and cell proliferation above baseline control conditions. Binding studies with radioiodinated ligand demonstrated that HUVEC constitutively express a smaller number of IL-3 binding sites (approximately 99 binding sites per cell, with an apparent Kd of 149 pM). Accordingly, molecular analysis showed the presence of transcripts for both alpha and beta subunits of the IL-3 receptor. Functional activation of endothelial cells was evaluated by the expression of the endothelial-leukocyte adhesion molecule 1 (ELAM-1) transcript and by leukocyte adhesion. The ELAM-1 gene transcript was clearly detectable 4 h after IL-3 addition and started to decrease after 12 h. Moreover, IL-3-induced ELAM-1 transcription was followed by enhanced adhesion of neutrophils and CD4+ T cells to HUVEC. The findings that IL-3 can stimulate both proliferation and functional activation of endothelial cells suggest that this cytokine can be involved in sustaining the process of chronic inflammation.

  18. Regulation of Neuronal Stem Cell Proliferation in the Hippocampus by Endothelial Ceramide

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    Anne Gulbins

    2016-08-01

    Full Text Available Background/Aims: Major depressive disorder is one of the most common diseases in western countries. The disease is mainly defined by its psychiatric symptoms. However, the disease has also many symptoms outside the central nervous system, in particular cardiovascular symptoms. Recent studies demonstrated that the acid sphingomyelinase/ceramide system plays an important role in the development of major depressive disorder and functions as a target of antidepressants. Methods: Here, we investigated (i whether ceramide accumulates in endothelial cells in the neurogenetic zone of the hippocampus after glucocorticosterone-mediated stress, (ii whether ceramide is released into the extracellular space of the hippocampus and (iii whether extracellular ceramide inhibits neuronal proliferation. Ceramide was determined in endothelial cell culture supernatants or extracellular hippocampus extracts by a kinase assay. Endothelial ceramide in the hippocampus was analyzed by confocal microscopy of brain sections stained with Cy3-labelled anti-ceramide antibodies and FITC-Isolectin B4. Neuronal proliferation was measured by incubation of pheochromocytoma neuronal cells with culture supernatants and extracellular hippocampus extracts. Results: Treatment of cultured endothelial cells with glucocorticosterone induces a release of ceramide into the supernatant. Likewise, treatment of mice with glucocorticosterone triggers a release of ceramide into the extracellular space of the hippocampus. The release of ceramide is inhibited by concomitant treatment with the antidepressant amitriptyline, which also inhibits the activity of the acid sphingomyelinase. Studies employing confocal microscopy revealed that ceramide is formed and accumulates exclusively in endothelial cells in the hippocampus of stressed mice, a process that was again prevented by co-application of amitriptyline. Ceramide released in the culture supernatant or into the extracellular space of the

  19. The Glycoprofile Patterns of Endothelial Cells in Usual Interstitial Pneumonia

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    A Barkhordari

    2014-09-01

    Full Text Available [THIS ARTICLE HAS BEEN RETRACTED FOR DUPLICATE PUBLICATION] Background: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists.Objective: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP compared to those found in normal tissue.Methods: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a panel of 27 biotinylated lectins and an avidin-peroxidase revealing system.Results: High expression of several classes of glycan was seen de novo in capillary endothelial cells from patients with UIP including small complex and bi/tri-antennary bisected complex N-linked sequences bolund by Concanavalin A and erythro-phytohemagglutinin, respectively, GalNAca1 residues bound by Helix pomatia and Maclura pomifera agglutinins, and L-fucosylated derivatives of type II glycan chains recognized by Ulex europaeus agglutinin-I. Glycans bound by agglutinins from Lycopersicon esculentum (β1,4GlcNAc and Wisteria floribunda (GalNAc as well as GlcNAc oligomers bound by Phytolacca americana and succinylated Wheat Germ agglutinin were also seen in the capillary endothelial cells of UIP. In contrast, L-fucosylated derivatives of type I glycan chains were absent in cells from cases of UIP when Anguilla anguilla agglutinin was applied, unlike the situation in normal tissue.Conclusion: These results may indicate existence of two distinct populations of endothelial cell in UIP with markedly different patterns of glycosylation, reflecting a pattern of differentiation and angiogenesis, which is not detectable morphologically.

  20. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Science.gov (United States)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  1. Reprogramming Cells for Brain Repair

    Directory of Open Access Journals (Sweden)

    Randall D. McKinnon

    2013-08-01

    Full Text Available At present there are no clinical therapies that can repair traumatic brain injury, spinal cord injury or degenerative brain disease. While redundancy and rewiring of surviving circuits can recover some lost function, the brain and spinal column lack sufficient endogenous stem cells to replace lost neurons or their supporting glia. In contrast, pre-clinical studies have demonstrated that exogenous transplants can have remarkable efficacy for brain repair in animal models. Mesenchymal stromal cells (MSCs can provide paracrine factors that repair damage caused by ischemic injury, and oligodendrocyte progenitor cell (OPC grafts give dramatic functional recovery from spinal cord injury. These studies have progressed to clinical trials, including human embryonic stem cell (hESC-derived OPCs for spinal cord repair. However, ESC-derived allografts are less than optimal, and we need to identify a more appropriate donor graft population. The cell reprogramming field has developed the ability to trans-differentiate somatic cells into distinct cell types, a technology that has the potential to generate autologous neurons and glia which address the histocompatibility concerns of allografts and the tumorigenicity concerns of ESC-derived grafts. Further clarifying how cell reprogramming works may lead to more efficient direct reprogram approaches, and possibly in vivo reprogramming, in order to promote brain and spinal cord repair.

  2. “Decoding” Angiogenesis: New Facets Controlling Endothelial Cell Behavior

    Science.gov (United States)

    Sewduth, Raj; Santoro, Massimo M.

    2016-01-01

    Angiogenesis, the formation of new blood vessels, is a unique and crucial biological process occurring during both development and adulthood. A better understanding of the mechanisms that regulates such process is mandatory to intervene in pathophysiological conditions. Here we highlight some recent argument on new players that are critical in endothelial cells, by summarizing novel discoveries that regulate notorious vascular pathways such as Vascular Endothelial Growth Factor (VEGF), Notch and Planar Cell Polarity (PCP), and by discussing more recent findings that put metabolism, redox signaling and hemodynamic forces as novel unforeseen facets in angiogenesis. These new aspects, that critically regulate angiogenesis and vascular homeostasis in health and diseased, represent unforeseen new ground to develop anti-angiogenic therapies. PMID:27493632

  3. Inhibition of endothelial cell apoptosis by netrin-1 during angiogenesis.

    Science.gov (United States)

    Castets, Marie; Coissieux, Marie-May; Delloye-Bourgeois, Céline; Bernard, Laure; Delcros, Jean-Guy; Bernet, Agnès; Laudet, Vincent; Mehlen, Patrick

    2009-04-01

    Netrin-1 was recently proposed to play an important role in embryonic and pathological angiogenesis. However, data reported led to the apparently contradictory conclusions that netrin-1 is either a pro- or an antiangiogenic factor. Here, we reconcile these opposing observations by demonstrating that netrin-1 acts as a survival factor for endothelial cells, blocking the proapoptotic effect of the dependence receptor UNC5B and its downstream death signaling effector, the serine/threonine kinase DAPK. The netrin-1 effect on blood vessel development is mimicked by caspase inhibitors in ex vivo assays, and the inhibition of caspase activity, the silencing of the UNC5B receptor, and the silencing of DAPK are each sufficient to rescue the vascular sprouting defects induced by netrin-1 silencing in zebrafish. Thus, the proapoptotic effect of unbound UNC5B and the survival effect of netrin-1 on endothelial cells finely tune the angiogenic process. PMID:19386270

  4. Mechanism of neuronal versus endothelial cell uptake of Alzheimer's disease amyloid beta protein.

    Directory of Open Access Journals (Sweden)

    Karunya K Kandimalla

    Full Text Available Alzheimer's disease (AD is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of beta-amyloid (Abeta proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Abeta proteins due to their inefficient clearance at the blood-brain-barrier (BBB, places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Abeta proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT mouse brain slices treated with fluorescein labeled Abeta40 (F-Abeta40 demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Abeta proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Abeta40 or F-Abeta42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Abeta40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Abeta40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Abeta proteins and help explain the vulnerability of cortical

  5. The Glycoprofile Patterns of Endothelial Cells in Usual Interstitial Pneumonia

    OpenAIRE

    Barkhordari, A.; CJP Jones; RW Stoddart; SF McClure; McClure, J; S Rahimi Moghadam

    2014-01-01

    [THIS ARTICLE HAS BEEN RETRACTED FOR DUPLICATE PUBLICATION] Background: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists.Objective: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP) compared to those found in normal tissue.Methods: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a pan...

  6. Endothelial progenitor cells regenerate infracted myocardium with neovascularisation development ☆

    OpenAIRE

    M.T. Abd El Aziz; Abd El Nabi, E.A.; Abd El Hamid, M.; D. Sabry; Atta, H.M.; L.A. Rahed; A. Shamaa; Mahfouz, S.; Taha, F.M.; S. Elrefaay; Gharib, D.M.; Elsetohy, Khaled A

    2013-01-01

    We achieved possibility of isolation, characterization human umbilical cord blood endothelial progenitor cells (EPCs), examination potency of EPCs to form new blood vessels and differentiation into cardiomyoctes in canines with acute myocardial infarction (AMI). EPCs were separated and cultured from umbilical cord blood. Their phenotypes were confirmed by uptake of double stains dioctadecyl tetramethylindocarbocyanine-labeled acetylated LDL and FITC-labeled Ulex europaeus agglutinin 1 (DILDL-...

  7. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

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    Narumon Sopapornamorn

    2008-03-01

    Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer’s test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand

  8. Effect of Intracameral Use of Dexamethasone on Corneal Endothelial Cells

    International Nuclear Information System (INIS)

    Objective: To evaluate the effect of intracameral dexamethasone on corneal endothelium. Study Design: Quasi experimental study. Place and Duration of Study: Layton Rehmatulla Benevolent Trust Eye Hospital, Lahore, from May 2011 to January 2012. Methodology: Study subjects were adults of either gender with senile cataract who underwent phacoemulsification. They were divided in two groups, each had 110 patients. Group-A received subconjunctival injection of dexamethasone (2 mg/0.5 ml) at the end of surgery while group-B received intracameral injection of dexamethasone (0.4 mg/0.1 ml) at the end of surgery. Endothelial cell count was performed by specular microscopy pre-operatively and postoperatively at first week, first month and three months. Outcome measures included changes in endothelial cell count. Results were compared using t-test for means. Results: There were 55 (50%) males and 55 (50%) females in group-A and 44 (40%) males and 66 (60%) females in group-B. In group-A, there were 66 (60%) right and 44 (40%) left eyes while group-B had 62 (56.36%) right and 48 (43.63%) left eyes. Mean age in group-A was 55.17 A +- 5.93 years and 54.87 A +- 5.55 years in group-B. Mean phacoemulsification time in group-A was 1.92 A +- 0.63 minutes and 1.82 A +- 0.54 minutes in group-B. After 3 months, in group-A, there was 7.55 A +- 1.19% endothelial cell loss while in group-B, there was 7.63 A +- 1.10% endothelial cell loss. The difference between the two groups was not statistically significant (p=0.614). Conclusion: Use of intracameral dexamethasone at the end of cataract surgery is safe for corneal endothelium. (author)

  9. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    Science.gov (United States)

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  10. Effect of endothelial progenitor cells in neovascularization and their application in tumor therapy

    Institute of Scientific and Technical Information of China (English)

    DONG Fang; HA Xiao-qin

    2010-01-01

    Objective To review the effect of endothelial progenitor cells in neovascularization as well as their application to the therapy of tumors.Data sources The data used in this review were mainly from PubMed for relevant English language articles published from 1997 to 2009. The search term was "endothelial progenitor cells".Study selection Articles regarding the role of endothelial progenitor cells in neovascularization and their application to the therapy of tumors were selected.Results Endothelial progenitor cells isolated from bone marrow, umbilical cord blood and peripheral blood can proliferate, mobilize and differentiate into mature endothelial cells. Experiments suggest endothelial progenitor cells take part in forming the tumor vascular through a variety of mechanisms related to vascular endothelial growth factor, matrix metalloproteinases, chemokine stromal cell-derived factor 1 and its receptor C-X-C receptor-4, erythropoietin, Notchsignal pathway and so on. Evidence demonstrates that the number and function change of endothelial progenitor cells in peripheral blood can be used as a biomarker of the response of cancer patients to anti-tumor therapy and predict the prognosis and recurrence. In addition, irradiation temporarily increased endothelial cells number and decreased the endothelial progenitor cell counts in animal models. Meanwhile, in preclinical experiments, therapeutic gene-modified endothelial progenitor cells have been approved to attenuate tumor growth and offer a novel strategy for cell therapy and gene therapy of cancer.Conclusions Endothelial progenitor cells play a particular role in neovascularization and have attractively potential prognostic and therapeutic applications to malignant tumors. However, a series of problems, such as the definitive biomarkers of endothelial progenitor cells, their interrelationship with radiotherapy and their application in cell therapy and gene therapy of tumors, need further investigation.

  11. Shear-Induced Nitric Oxide Production by Endothelial Cells.

    Science.gov (United States)

    Sriram, Krishna; Laughlin, Justin G; Rangamani, Padmini; Tartakovsky, Daniel M

    2016-07-12

    We present a biochemical model of the wall shear stress-induced activation of endothelial nitric oxide synthase (eNOS) in an endothelial cell. The model includes three key mechanotransducers: mechanosensing ion channels, integrins, and G protein-coupled receptors. The reaction cascade consists of two interconnected parts. The first is rapid activation of calcium, which results in formation of calcium-calmodulin complexes, followed by recruitment of eNOS from caveolae. The second is phosphorylation of eNOS by protein kinases PKC and AKT. The model also includes a negative feedback loop due to inhibition of calcium influx into the cell by cyclic guanosine monophosphate (cGMP). In this feedback, increased nitric oxide (NO) levels cause an increase in cGMP levels, so that cGMP inhibition of calcium influx can limit NO production. The model was used to predict the dynamics of NO production by an endothelial cell subjected to a step increase of wall shear stress from zero to a finite physiologically relevant value. Among several experimentally observed features, the model predicts a highly nonlinear, biphasic transient behavior of eNOS activation and NO production: a rapid initial activation due to the very rapid influx of calcium into the cytosol (occurring within 1-5 min) is followed by a sustained period of activation due to protein kinases. PMID:27410748

  12. In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

    Science.gov (United States)

    Zaniboni, Andrea; Bernardini, Chiara; Bertocchi, Martina; Zannoni, Augusta; Bianchi, Francesca; Avallone, Giancarlo; Mangano, Chiara; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2015-09-01

    Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes. PMID:26135800

  13. Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase.

    Science.gov (United States)

    Lee, Kwon Ho; Lee, Sang Ki; Kim, Hyo Shin; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Eun Ji; Lee, Ji Young; Park, Myoung Soo; Chang, Seok Jong; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2009-12-01

    The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

  14. Effects of verteporfin-mediated photodynamic therapy on endothelial cells

    Science.gov (United States)

    Kraus, Daniel; Chen, Bin

    2015-03-01

    Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.

  15. Endothelialization of Magnetic Graft Materials using SPION-labeled Endothelial Cells

    Science.gov (United States)

    Newman, Brant R.; Dragomir-Daescu, Dan; Harbuzariu, Adriana; McIntosh, Malcolm; Harburn, J. Jonathan; Parakka, Anthony; Kalra, Manju; Holmes, David; Simari, Robert D.; Sandhu, Gurpreet S.

    2010-12-01

    Seeding vascular grafts with autologous endothelial cells (EC) has been shown to improve in vivo patency, but high cost and development time have prevented widespread clinical use. A technique for loading EC with superparamagnetic iron-oxide nanospheres (SPIONs) was recently described. SPION-loaded EC experience magnetic attractive forces in the presence of sufficient magnetic field gradients. Using a multi-factorial design of experiments approach, the quantity and spatial distribution of magnetizable metal particles within a poly (ether urethane) matrix were systematically varied to produce unique material specimens. Specimens were seeded with SPION-loaded ECs, and cell coverage was quantified at various post-seeding time intervals using micrographic image analysis. The effects of changing design parameters on cell capture and sustained cell viability on magnetic substrates were statistically examined. Magnetized ferrites and samarium cobalt demonstrated cell capture, though cytotoxicity prevented sustained cell growth. Cobalt chromium substrates showed effective cell capture and growth to near complete confluence for up to one month.

  16. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    International Nuclear Information System (INIS)

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO2) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO2 nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO2 nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells

  17. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, Claudia, E-mail: Claudia.Strobel@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany); Oehring, Hartmut [Jena University Hospital – Friedrich Schiller University Jena, Institute of Anatomy II (Germany); Herrmann, Rudolf [University of Augsburg, Department of Physics (Germany); Förster, Martin [Jena University Hospital – Friedrich Schiller University Jena, Department of Internal Medicine I, Division of Pulmonary Medicine and Allergy/Immunology (Germany); Reller, Armin [University of Augsburg, Department of Physics (Germany); Hilger, Ingrid, E-mail: ingrid.hilger@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany)

    2015-05-15

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO{sub 2}) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO{sub 2} nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO{sub 2} nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  18. Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny

    NARCIS (Netherlands)

    Moonen, Jan-Renier A. J.; Krenning, Guido; Brinker, Marja G. L.; Koerts, Jasper A.; van Luyn, Marja J. A.; Harmsen, Martin C.

    2010-01-01

    Reciprocal plasticity exists between endothelial and mesenchymal lineages. For instance, mature endothelial cells adopt a smooth muscle-like phenotype through transforming growth factor beta-1 (TGF beta 1)-driven endothelial-to-mesenchymal transdifferentiation (EndMT). Peripheral blood contains circ

  19. Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

    Science.gov (United States)

    Nojiri, Takashi; Hosoda, Hiroshi; Tokudome, Takeshi; Miura, Koichi; Ishikane, Shin; Otani, Kentaro; Kishimoto, Ichiro; Shintani, Yasushi; Inoue, Masayoshi; Kimura, Toru; Sawabata, Noriyoshi; Minami, Masato; Nakagiri, Tomoyuki; Funaki, Soichiro; Takeuchi, Yukiyasu; Maeda, Hajime; Kidoya, Hiroyasu; Kiyonari, Hiroshi; Shioi, Go; Arai, Yuji; Hasegawa, Takeshi; Takakura, Nobuyuki; Hori, Megumi; Ohno, Yuko; Miyazato, Mikiya; Mochizuki, Naoki; Okumura, Meinoshin; Kangawa, Kenji

    2015-01-01

    Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells. PMID:25775533

  20. Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

    Science.gov (United States)

    Hofmann, Nicola; Sun, Huan; Chatterjee, Anamika; Saha, Debapriya; Glasmacher, Birgit

    2015-10-01

    A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation. PMID:26419006

  1. An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.

    Science.gov (United States)

    Martinelli, Roberta; Carman, Christopher V

    2015-12-24

    Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

  2. Induction of procoagulant activity on human endothelial cells by Streptococcus pneumoniae.

    OpenAIRE

    Geelen, S; Bhattacharyya, C; Tuomanen, E

    1992-01-01

    The inflammatory response in infection caused by gram-negative organisms involves induction of procoagulant activity (PCA) on human endothelial cells. Although infections caused by gram-positive organisms are also associated with fibrin formation and thrombosis, the bacterial determinants inducing PCA are unknown. This study shows that intact pneumococci and the pneumococcal cell wall efficiently induce PCA on human endothelial cells. Upon exposure of endothelial cells to pneumococci, PCA was...

  3. Angiostatin binds ATP synthase on the surface of human endothelial cells

    OpenAIRE

    Moser, Tammy L.; Stack, M. Sharon; Asplin, Iain; Enghild, Jan J; Højrup, Peter; Everitt, Lorraine; Hubchak, Susan; Schnaper, H. William; Pizzo, Salvatore V.

    1999-01-01

    Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin...

  4. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Efthalia Kerasioti

    2016-01-01

    Full Text Available Excessive production of reactive oxygen species (ROS may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP from tert-butyl hydroperoxide- (tBHP- induced oxidative stress in endothelial cells (EA.hy926 were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS, protein carbonyls (CARB, and oxidized glutathione (GSSG were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress.

  5. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells.

    Science.gov (United States)

    Kerasioti, Efthalia; Stagos, Dimitrios; Georgatzi, Vasiliki; Bregou, Erinda; Priftis, Alexandros; Kafantaris, Ioannis; Kouretas, Dimitrios

    2016-01-01

    Excessive production of reactive oxygen species (ROS) may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP) from tert-butyl hydroperoxide- (tBHP-) induced oxidative stress in endothelial cells (EA.hy926) were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH) and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS), protein carbonyls (CARB), and oxidized glutathione (GSSG) were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL(-1) increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress. PMID:27127549

  6. In vitro differentiation of human adipose-derived mesenchymal stem cells into endothelial-like cells

    Institute of Scientific and Technical Information of China (English)

    GUAN Lidong; SHI Shuangshuang; PEI Xuetao; LI Shaoqing; WANG Yunfang; YUE Huimin; LIU Daqing; HE Lijuan; BAI Cixian; YAN Fang; NAN Xue

    2006-01-01

    The neovascularization of ischemic tissue is a crucial initial step for the functional rehabilitation and wound healing. However, the short of seed cell candidate for the foundation of vascular network is still a big issue. Human adipose tissue derived mesenchymal stem cells (hADSCs), which possess multilineage potential, are capable of adipogenic, osteogenic, and chondrogenic differentiation. We examined whether this kind of stem cells could differentiate into endothelial-like cells and participate in blood vessel formation, and whether they could be used as an ideal cell source for therapeutic angiogenesis in ischemic diseases or vascularization of tissue constructs. The results showed that hADSCs, grown under appropriately induced conditions, displayed characteristics similar to those of vessel endothelium. The differentiated cells expressed endothelial cell markers CD34 and vWF, and had high metabolism of acetylated low-density lipoprotein and prostacyclin. In addition, the induced cells were able to form tube-like structures when cultured on matrigel. Our data indicated that induced hADSCs could exhibit characteristics of endothelial cells. Therefore, these cells, as a source of human endothelial cells, may find many applications in such realms as engineering blood vessels, endothelial cell transplantation for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  7. In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

    Science.gov (United States)

    Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

    2016-03-01

    The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

  8. In Vivo Vascularization of Endothelial Cells Derived from Bone Marrow Mesenchymal Stem Cells in SCID Mouse Model

    OpenAIRE

    Allameh Abdolamir; Jazayeri Maryam; Adelipour Maryam

    2016-01-01

    Objective In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods We induced human mesenchymal stem cells (MSCs) to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM) gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor...

  9. YAP regulates S-phase entry in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Zhewei Shen

    Full Text Available The Hippo pathway regulates cell proliferation and apoptosis through the Yes-associated protein (YAP transcriptional activator. YAP has a well-described role in promoting cell proliferation and survival, but the precise mechanisms and transcriptional targets that underlie these properties are still unclear and likely context-dependent. We found, using siRNA-mediated knockdown, that YAP is required for proliferation in endothelial cells but not HeLa cells. Specifically, YAP is required for S-phase entry and its absence causes cells to accumulate in G1. Microarray analysis suggests that YAP mediates this effect by regulating the transcription of genes involved in the assembly and/or firing of replication origins and homologous recombination of DNA. These findings thus provide insight into the molecular mechanisms by which YAP regulates cell cycle progression.

  10. Endothelial cell compatibility of trovafloxacin and levofloxacin for intravenous use.

    Science.gov (United States)

    Armbruster, C; Robibaro, B; Griesmacher, A; Vorbach, H

    2000-04-01

    Levofloxacin and trovafloxacin have excellent activity against a variety of Gram-positive and Gram-negative organisms resistant to the established agents. One local side-effect closely related to the use of parenteral fluoroquinolones is phlebitis. To evaluate the effect of trovafloxacin and levofloxacin on endothelial cell viability, intracellular levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) levels were measured using high-performance liquid chromatography. Trovafloxacin at concentrations of 2 and 1 mg/mL reduced the intracellular ATP content from 12.5 +/- 1.7 to 1.9 +/- 0.3 nmol/10(6) cells and 9.3 +/- 0.8 nmol/10(6) cells, respectively, within 60 min. In addition, ADP, GTP and GDP levels were extensively depleted. Levofloxacin at concentrations of 5 and 2.5 mg/mL led to a significant ATP decline from 12.5 +/- 1.7 to 2.3 +/- 0.2 nmol/10(6) cells and 10.3 +/- 0.9 nmol/10(6) cells, respectively, within 60 min. These data indicate that infusions of high doses of trovafloxacin or levofloxacin are not compatible with maintenance of endothelial cell function. Commercial preparations have to be diluted and should be administered into large veins.

  11. Flow-induced Expression and Phosphorylation of VASPin Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Muller; SYLYAINE; Jean-FranoisSYOLTZ

    2005-01-01

    1 Introduction It is well known that mechanical forces have important influence on endothelial cells, in particular, on cytoskeleton reorganization. VASP (vasodilator stimulated phosphoprotein) is a 46 KD actin associated protein. It is a member of Ena/VASP protein family and composed of EVH1, proline-rich and EVH2 domains. It is considered as an important component of the sub-cellular regions where remodelling of the actin cytoskeleton takes place, such as the front of spreading lamellipodia in motile cell...

  12. Endothelial progenitor cells: what use for the cardiologist?

    Directory of Open Access Journals (Sweden)

    Siddique Aurangzeb

    2010-02-01

    Full Text Available Abstract Endothelial Progenitor Cells (EPC were first described in 1997 and have since been the subject of numerous investigative studies exploring the potential of these cells in the process of cardiovascular damage and repair. Whilst their exact definition and mechanism of action remains unclear, they are directly influenced by different cardiovascular risk factors and have a definite role to play in defining cardiovascular risk. Furthermore, EPCs may have important therapeutic implications and further understanding of their pathophysiology has enabled us to explore new possibilities in the management of cardiovascular disease. This review article aims to provide an overview of the vast literature on EPCs in relation to clinical cardiology.

  13. Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

    Institute of Scientific and Technical Information of China (English)

    Shaobo Hu; Zifang Song; Qichang Zheng; Jun Nie

    2009-01-01

    Objective: To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods: Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers,and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion 6me, attachment time and recovery after cryopreservation. Results: Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of singleendothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion: The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin

  14. Galectin-3 induces pulmonary artery endothelial cell morphogenesis and angiogenesis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li; LI Yu-mei; WANG Xiao-yan; ZHU Da-ling

    2016-01-01

    AIM:Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis .Ga-lectin-3, a multifunctional protein of an expanding family of β-galactoside-binding animal lectins , is the major nonintegrin cellular laminin-binding protein , and is implicated in a variety of biologic events , such as inflammation and angiogenesis .Because galectin-3 expression was shown to participate in mediating tumor angiogenesis and initiate signaling cascades in several diseases .We hypothe-sized that galectin-3 may promote pulmonary vascular endothelial neovascularization .METHODS:Hypoxic and MCT rat model of pul-monary artery remodeling was used .The mRNA and protein levels of galectin-3 in rats were measured by in situ hybrization and West-ern blot analysis.Endothelial cell (EC) proliferation, migration and tube formation were measured using MTT , cell scratch and Matri-gel assays, respectively.Protein expression was quantitated by Western blot analysis .LC 3A/B staining was detected with cellular im-munofluorescence staining .RESULTS:We found that galectin-3 was localized on the intima and adventitial wall .Galectin-3 was in-creased after rat hypoxia and MCT administration .Galectin-3 promoted EC proliferation , migration and tube formation , while its roles were reversed by RNA interference.Galectin-3 induced Atg 5, Beclin-1, LAMP-2, and LC 3A/B expression increases.Galectin-3 al-so increased LC 3A/B staining in ECs.Akt/mTOR and GSK-3βsignaling pathways were activated after galectin-3 treated ECs using its specific phosphorylation antibodies , while blocked it with LY294002 inhibited cell autophagy and EC dynamic alterations induced by galectin-3.CONCLUSION:These findings demonstrate that galectin-3 can induce an Akt signaling cascade leading to cell autoph-agy, and then the differentiation and angiogenesis of pulmonary artery endothelial cells .

  15. Growth factor-and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

    Institute of Scientific and Technical Information of China (English)

    Philip V.Peplow

    2014-01-01

    Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes speciifc growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endotheli-al progenitor cells migrate and home to speciifc sites following ischemic stroke via growth factor/cytokine gradients. Some growth factors are less stable under acidic conditions of tissue isch-emia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovas-cularization following ischemic stroke.

  16. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    Directory of Open Access Journals (Sweden)

    Rui-Li Zhang

    Full Text Available The recombinant Treponema pallidum protein Tp0965 (rTp0965, one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.

  17. Prune melanoidins protect against oxidative stress and endothelial cell death.

    Science.gov (United States)

    Posadino, Anna Maria; Cossu, Annalisa; Piga, Antonio; Madrau, Monica Assunta; Del Caro, Alessandra; Colombino, Maria; Paglietti, Bianca; Rubino, Salvatore; Iaccarino, Ciro; Crosio, Claudia; Sanna, Bastiano; Pintus, Gianfranco

    2011-06-01

    The health-promoting effects of fruit and vegetable consumption are thought to be due to phytochemicals contained in fresh plant material. Whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed plums (prunes) were isolated and their presence confirmed by hydroxymethylfurfural content and browning index. Oxidative-induced endothelial cell (EC) damage is the trigger for the development of cardiovascular diseases (CVD); therefore the potential protective effect of prune melanoidins on hydrogen peroxide-induced oxidative cell damage was investigated on human endothelial ECV304 cells. Cytoplasmic and mitochondrial redox status was assessed by using the novel, redox-sensitive, ratiometric fluorescent protein sensor (roGFP), while mitochondrial membrane potential (MMP) was investigated with the fluorescent dye, JC-1. Treatment of ECV304 cells with hydrogen peroxide dose-dependently induced both mitochondrial and cytoplasmic oxidation, in addition to MMP dissipation, with ensuing cell death. Pretreatment of ECV304 with prune melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide elicited phenomena, clearly indicating that these polymers protect human EC against oxidative stress.

  18. Vascular endothelial growth factors enhance the permeability of the mouse blood-brain barrier.

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    Shize Jiang

    Full Text Available The blood-brain barrier (BBB impedes entry of many drugs into the brain, limiting clinical efficacy. A safe and efficient method for reversibly increasing BBB permeability would greatly facilitate central nervous system (CNS drug delivery and expand the range of possible therapeutics to include water soluble compounds, proteins, nucleotides, and other large molecules. We examined the effect of vascular endothelial growth factor (VEGF on BBB permeability in Kunming (KM mice. Human VEGF165 was administered to treatment groups at two concentrations (1.6 or 3.0 µg/mouse, while controls received equal-volume saline. Changes in BBB permeability were measured by parenchymal accumulation of the contrast agent Gd-DTPA as assessed by 7 T magnetic resonance imaging (MRI. Mice were then injected with Evans blue, sacrificed 0.5 h later, and perfused transcardially. Brains were removed, fixed, and sectioned for histological study. Both VEGF groups exhibited a significantly greater signal intensity from the cerebral cortex and basal ganglia than controls (P<0.001. Evans blue fluorescence intensity was higher in the parenchyma and lower in the cerebrovasculature of VEGF-treated animals compared to controls. No significant brain edema was observed by diffusion weighted MRI (DWI or histological staining. Exogenous application of VEGF can increase the permeability of the BBB without causing brain edema. Pretreatment with VEGF may be a feasible method to facilitate drug delivery into the CNS.

  19. Endothelial cell motility, coordination and pattern formation during vasculogenesis.

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    Czirok, Andras

    2013-01-01

    How vascular networks assemble is a fundamental problem of developmental biology that also has medical importance. To explain the organizational principles behind vascular patterning, we must understand how can tissue level structures be controlled through cell behavior patterns like motility and adhesion that, in turn, are determined by biochemical signal transduction processes? We discuss the various ideas that have been proposed as mechanisms for vascular network assembly: cell motility guided by extracellular matrix alignment (contact guidance), chemotaxis guided by paracrine and autocrine morphogens, and multicellular sprouting guided by cell-cell contacts. All of these processes yield emergent patterns, thus endothelial cells can form an interconnected structure autonomously, without guidance from an external pre-pattern.

  20. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

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    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  1. Transplanted microvascular endothelial cells promote oligodendrocyte precursor cell survival in ischemic demyelinating lesions.

    Science.gov (United States)

    Iijima, Keiya; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Puentes, Sandra; Imai, Hideaki; Yoshimoto, Yuhei; Mikuni, Masahiko; Ishizaki, Yasuki

    2015-11-01

    We previously showed that transplantation of brain microvascular endothelial cells (MVECs) greatly stimulated remyelination in the white matter infarct of the internal capsule (IC) induced by endothelin-1 injection and improved the behavioral outcome. In the present study, we examined the effect of MVEC transplantation on the infarct volume using intermittent magnetic resonance image and on the behavior of oligodendrocyte lineage cells histochemically. Our results in vivo show that MVEC transplantation reduced the infarct volume in IC and apoptotic death of oligodendrocyte precursor cells (OPCs). These results indicate that MVECs have a survival effect on OPCs, and this effect might contribute to the recovery of the white matter infarct. The conditioned-medium from cultured MVECs reduced apoptosis of cultured OPCs, while the conditioned medium from cultured fibroblasts did not show such effect. These results suggest a possibility that transplanted MVECs increased the number of OPCs through the release of humoral factors that prevent their apoptotic death. Identification of such humoral factors may lead to the new therapeutic strategy against ischemic demyelinating diseases. PMID:26212499

  2. Functional and gene expression analysis of hTERT overexpressed endothelial cells

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    Haruna Takano

    2008-09-01

    Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

  3. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    Science.gov (United States)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  4. In Vivo Vascularization of Endothelial Cells Derived from Bone Marrow Mesenchymal Stem Cells in SCID Mouse Model

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    Allameh Abdolamir

    2016-07-01

    Full Text Available Objective In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods We induced human mesenchymal stem cells (MSCs to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor (vWF, vascular endothelial growth factor (VEGF receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency (SCID mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry (DIHC staining. Results Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin (H&E staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site. Conclusion The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis.

  5. Effects of blood products on inflammatory response in endothelial cells in vitro.

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    Martin Urner

    Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

  6. ECM-Dependence of Endothelial Progenitor Cell Features.

    Science.gov (United States)

    Siavashi, Vahid; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Vafaei, Rana; Sariri, Reyhaneh

    2016-08-01

    Preserving self-renewal, multipotent capacity, and large-scale expansion of highly functional progenitor cells, including endothelial progenitor cells (EPCs), is a controversial issue. These current limitations, therefore, raise the need of developing promising in vitro conditions for prolonged expansion of EPCs without loss of their stemness feature. In the current study, the possible role of three different natural extracellular substrates, including collagen, gelatin, and fibronectin, on multiple parameters of EPCs such as cell morphology, phenotype, clonogenic, and vasculogenic properties was scrutinized. Next, EPCs from GFP-positive mice were pre-expanded on each of these ECM substrates and then systemically transplanted into sublethaly irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed considerable promise for fibronectin for EPC expansion with maintenance of stemness characteristics, whereas gelatin and collagen matrices directed the cells toward a mature endothelial phenotype. Transplantation of EPCs pre-expanded on fibronectin resulted in widespread distribution and appropriate engraftment to various tissues with habitation in close association with the microvasculature. In addition, fibronectin pre-expanded cells were gradually enriched in the bone marrow after transplantation, resulting in marrow repopulation and hematologic recovery, leading to improved survival of recipient mice whereas gelatin- and collagen-expanded cells failed to reconstitute the bone marrow. This study demonstrated that, cell characteristics of in vitro expanded EPCs are determined by the subjacent matrix. Fibronectin-expanded EPCs are heralded as a source of great promise for bone marrow reconstitution and neo-angiogenesis in therapeutic bone marrow transplantation. J. Cell. Biochem. 117: 1934-1946, 2016. © 2016 Wiley Periodicals, Inc. PMID:26756870

  7. Endothelial monolayers on collagen-coated nanofibrous membranes: cell-cell and cell-ECM interactions.

    Science.gov (United States)

    Kang, Donggu; Kim, Jeong Hwa; Jeong, Young Hun; Kwak, Jong-Young; Yoon, Sik; Jin, Songwan

    2016-06-01

    Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems. PMID:27186924

  8. Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Bingong; ZENG Qiutang; WANG Hongxiang; MAO Xiaobo

    2006-01-01

    To isolate and culture adipose stromal cells (ASCs), and study the effect of cytokines secreted by ASCs on endothelial cells, human adipose tissue was digested with collagenase type Ⅰ solution and ASCs were derived by culture. The cells surface phenotype was examined by flow cytometry. ELISA was used to detect the secretion of VEGF, HGF, SDF-1 α and RT-PCR was employed to detect the expression of their mRNA. Then the ASC medium was utilized to culture human umbilical vein endothelial cells ECV304. Cells were counted by hemacytometer to determine the proliferation and Annexin V/PI was employed for the examination of the apoptosis rate of ECV304. ASCs were derived by culture and expressed CD34, CD105 while they did not express CD31 or CD45. ASCs secreted cytokines such as VEGF, HGF and SDF-1 α so the ASC medium could stimulate proliferation and counteract apoptosis of endothelial cells (P<0.05). Bcl-2 mRNA was also found to be up-regulated in the endothelial cells. It is concluded that ASCs can secrete cytokines and has significant effect on the proliferation of endothelial cells and apoptosis.

  9. Synergism of matrix stiffness and vascular endothelial growth factor on mesenchymal stem cells for vascular endothelial regeneration.

    Science.gov (United States)

    Wingate, Kathryn; Floren, Michael; Tan, Yan; Tseng, Pi Ou Nancy; Tan, Wei

    2014-09-01

    Mesenchymal stem cells (MSCs) hold tremendous potential for vascular tissue regeneration. Research has demonstrated that individual factors in the cell microenvironment such as matrix elasticity and growth factors regulate MSC differentiation to vascular lineage. However, it is not well understood how matrix elasticity and growth factors combine to direct the MSC fate. This study examines the combined effects of matrix elasticity and vascular endothelial growth factor (VEGF) on both MSC differentiation into endothelial lineage and MSC paracrine signaling. MSCs were seeded in soft nanofibrous matrices with or without VEGF, and in Petri dishes with or without VEGF. Only MSCs seeded in three-dimensional soft matrices with VEGF showed significant increases in the expression of endothelial markers (vWF, eNOS, Flt-1, and Flk-1), while eliminating the expression of smooth muscle marker (SM-α-actin). MSCs cultured in VEGF alone on two-dimensional dishes showed increased expression of both early-stage endothelial and smooth muscle markers, indicating immature vascular differentiation. Furthermore, MSCs cultured in soft matrices with VEGF showed faster upregulation of endothelial markers compared with MSCs cultured in VEGF alone. Paracrine signaling studies found that endothelial cells cultured in the conditioned media from MSCs differentiated in the soft matrix and VEGF condition exhibited increased migration and formation of capillary-like structures. These results demonstrate that VEGF and soft matrix elasticity act synergistically to guide MSC differentiation into mature endothelial phenotype while enhancing paracrine signaling. Therefore, it is critical to control both mechanical and biochemical factors to safely regenerate vascular tissues with MSCs.

  10. Glycoconjugates and Related Molecules in Human Vascular Endothelial Cells

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    Norihiko Sasaki

    2013-01-01

    Full Text Available Vascular endothelial cells (ECs form the inner lining of blood vessels. They are critically involved in many physiological functions, including control of vasomotor tone, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and immunity. It is considered that impairment of EC functions leads to the development of vascular diseases. The carbohydrate antigens carried by glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans mainly present on the cell surface serve not only as marker molecules but also as functional molecules. Recent studies have revealed that the carbohydrate composition of the EC surface is critical for these cells to perform their physiological functions. In this paper, we consider the expression and functional roles of endogenous glycoconjugates and related molecules (galectins and glycan-degrading enzymes in human ECs.

  11. Exogenous endothelial cells as accelerators of hematopoietic reconstitution

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    Mizer J

    2012-11-01

    Full Text Available Abstract Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic growth factors. Advantages of this approach would include: a ability to regulate secretion of cytokines based on biological need; b long term, localized production of growth factors, alleviating need for systemic administration of factors that possess unintended adverse effects; and c potential to actively repair the hematopoietic stem cell niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal stem cells (MSC in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.

  12. Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells.

    Science.gov (United States)

    Zhao, Jing; Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Morrell, Nicholas W; Lever, Andrew M L

    2016-01-01

    Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm(2). The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair. PMID:27413378

  13. Carnosine facilitates nitric oxide production in endothelial f-2 cells.

    Science.gov (United States)

    Takahashi, Satoru; Nakashima, Yukiko; Toda, Ken-Ichi

    2009-11-01

    We examined the effect of carnosine (beta-alanyl-histidine) on nitric oxide (NO) production and endothelial NO synthase (eNOS) activation in endothelial F-2 cells. Carnosine enhanced NO production in a dose-dependent manner, and the stimulatory effect of carnosine was observed at concentrations exceeding 5 mM. The carnosine-stimulated NO production was inhibited by N(G)-nitro-L-arginine methyl ester, but not by N(G)-nitro-D-arginine methyl ester. In contrast, beta-alanine, histidine (carnosine components) and anserine (N-methyl carnosine) failed to increase NO production. Carnosine had no effect on NO production for the initial 5 min, but thereafter resulted in a gradual increase in NO production up to 15 min. Carnosine did not induce phosphorylation of eNOS at Ser1177. The carnosine-induced increase in NO production was observed even when extracellular Ca2+ was depleted by ethylene glycol bis(2-aminoethyl ether)-N,N,N'-N'-tetraacetic acid however, the effect was abolished upon depletion of intracellular Ca2+ by BAPTA. After F-2 cells were incubated with carnosine for 4 min, intracellular Ca2+ concentration gradually increased. The carnosine-induced increase in intracellular Ca2+ concentration occurred even in the absence of extracellular Ca2+. These results indicate that carnosine facilitates NO production in endothelial F-2 cells. It is also suggested that eNOS is activated by Ca2+, which might be released from intracellular Ca2+ stores in response to carnosine. PMID:19881293

  14. Glucocorticoids induce transactivation of tight junction genes occludin and claudin-5 in retinal endothelial cells via a novel cis-element.

    Science.gov (United States)

    Felinski, Edward A; Cox, Amy E; Phillips, Brett E; Antonetti, David A

    2008-06-01

    Tight junctions between vascular endothelial cells help to create the blood-brain and blood-retinal barriers. Breakdown of the retinal tight junction complex is problematic in several disease states including diabetic retinopathy. Glucocorticoids can restore and/or preserve the endothelial barrier to paracellular permeability, although the mechanism remains unclear. We show that glucocorticoid treatment of primary retinal endothelial cells increases content of the tight junction proteins occludin and claudin-5, co-incident with an increase in barrier properties of endothelial monolayers. The glucocorticoid receptor antagonist RU486 reverses both the glucocorticoid-stimulated increase in occludin content and the increase in barrier properties. Transcriptional activity from the human occludin and claudin-5 promoters increases in retinal endothelial cells upon glucocorticoid treatment, and is dependent on the glucocorticoid receptor (GR) as demonstrated by siRNA. Deletion analysis of the occludin promoter reveals a 205bp sequence responsible for the glucocorticoid response. However, this region does not possess a canonical glucocorticoid response element and does not bind to the GR in a chromatin immunoprecipitation (ChIP) assay. Mutational analysis of this region revealed a novel 40bp occludin enhancer element (OEE), containing two highly conserved regions of 10 and 13 base pairs, that is both necessary and sufficient for glucocorticoid-induced gene expression in retinal endothelial cells. These data suggest a novel mechanism for glucocorticoid induction of vascular endothelial barrier properties through increased occludin and claudin-5 gene expression. PMID:18501346

  15. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  16. Circulating endothelial cells and microparticles as prognostic markers in advanced non-small cell lung cancer.

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    Tania Fleitas

    Full Text Available BACKGROUND: Circulating endothelial cells and microparticles have prognostic value in cancer, and might be predictors of response to chemotherapy and antiangiogenic treatments. We have investigated the prognostic value of circulating endothelial cells and microparticles in patients treated for advanced non-small cell lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood samples were obtained from 60 patients before first line, platinum-based chemotherapy +/- bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Circulating endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Phosphatidylserine-positive microparticles were evaluated by flow cytometry. Microparticle-mediated procoagulant activity was measured by the endogen thrombin generation assay. RESULTS: pre- and posttreatment levels of markers were higher in patients than in controls (p<0.0001. Elevated levels of microparticles were associated with longer survival. Elevated pretreatment levels of circulating endothelial cells were associated with shorter survival. CONCLUSIONS/SIGNIFICANCE: Circulating levels of microparticles and circulating endothelial cells correlate with prognosis, and could be useful as prognostic markers in patients with advanced non-small cell lung cancer.

  17. Cationic Nanocylinders Promote Angiogenic Activities of Endothelial Cells

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    Jung Bok Lee

    2016-01-01

    Full Text Available Polymers have been used extensively taking forms as scaffolds, patterned surface and nanoparticle for regenerative medicine applications. Angiogenesis is an essential process for successful tissue regeneration, and endothelial cell–cell interaction plays a pivotal role in regulating their tight junction formation, a hallmark of angiogenesis. Though continuous progress has been made, strategies to promote angiogenesis still rely on small molecule delivery or nuanced scaffold fabrication. As such, the recent paradigm shift from top-down to bottom-up approaches in tissue engineering necessitates development of polymer-based modular engineering tools to control angiogenesis. Here, we developed cationic nanocylinders (NCs as inducers of cell–cell interaction and investigated their effect on angiogenic activities of human umbilical vein endothelial cells (HUVECs in vitro. Electrospun poly (l-lactic acid (PLLA fibers were aminolyzed to generate positively charged NCs. The aninolyzation time was changed to produce two different aspect ratios of NCs. When HUVECs were treated with NCs, the electrostatic interaction of cationic NCs with negatively charged plasma membranes promoted migration, permeability and tubulogenesis of HUVECs compared to no treatment. This effect was more profound when the higher aspect ratio NC was used. The results indicate these NCs can be used as a new tool for the bottom-up approach to promote angiogenesis.

  18. Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

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    Caterina Oriana Aragona

    2016-01-01

    Full Text Available Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina pectoris” or “myocardial infarction”; “stroke” or “cerebrovascular disease”; “homocysteine”; “C-reactive protein”; “vitamin D”. Study Selection. Database hits were evaluated against explicit inclusion criteria. From 927 database hits, 43 quantitative studies were included. Data Syntheses. EPC count has been suggested for cardiovascular risk estimation in the clinical practice, since it is currently accepted that EPCs can work as proangiogenic support cells, maintaining their importance as regenerative/reparative potential, and also as prognostic markers. Conclusions. EPCs showed an important role in identifying cardiovascular risk conditions, and to suggest their evaluation as predictor of outcomes appears to be reasonable in different defined clinical settings. Due to their capability of proliferation, circulation, and the development of functional progeny, great interest has been directed to therapeutic use of progenitor cells in atherosclerotic diseases. This trial is registered with registration number: Prospero CRD42015023717.

  19. Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).

    OpenAIRE

    Winkles, J A; Friesel, R; Burgess, W H; Howk, R; Mehlman, T; Weinstein, R.; T. MACIAG

    1987-01-01

    The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, exp...

  20. Visualization of brain tumor using I-123-vascular endothelial growth factor scintigraphy

    International Nuclear Information System (INIS)

    Full text: Aim:Vascular endothelial growth factor (VEGF) is a major angiogenic factor. VEGF receptors have been shown to be overexpressed in a variety of tumor vessels including glioblastoma, which may provide the molecular basis for a successful use of radiolabeled VEGF as tumor angiogenesis tracer. In this study we investigated the usefulness of 1231- VEGF as angiogenesis tracer for imaging brain tumors in vivo. Methods and Results: SPECT examinations were performed 30 minutes and 18 hours after intravenous application of 1231-VEGF (191 ± 15 MBq) in 20 patients with brain tumor. Glioblastomas were visualized in 7 of 8 patients (88 %) shortly after application of 1231- VEGF and were still clearly shown 18 hours post injection. Negative scan results were obtained in one patient with a small glioblastoma size (diameter <2.0 cm) and in 3 patients with benign glioma as well as in 5 patients with glioblastoma after receiving radiotherapy and for chemotherapy. Weak positive results were obtained in 3 patients with brain lymphoma or other tumors. No side effects were observed in patients after administration of 1231- VEG F. Conclusion: Our results indicate that 1231- VEGF scintigraphy may be useful to visualize the angiogenesis of brain tumors and to monitor the treatment response.

  1. Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Jielu Tan; Xiangrong Zheng; Shanshan Zhang; Yujia Yang; Xia Wang; Xiaohe Yu; Le Zhong

    2014-01-01

    Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge-nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into ifve groups: (1) sham operation (control), (2) cerebral palsy model alone or with (3) phosphate-buffered saline, (4) vascular en-dothelial growth factor 165 + neural stem cells, or (5) neural stem cells alone. hTe cerebral palsy model was established by ligating the letf common carotid artery followed by exposure to hypox-ia. Phosphate-buffered saline, vascular endothelial growth factor + neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. Atfer transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas-cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for ifnding water and the ifnding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. hTese ifndings indicate that the transplantation of vascu-lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deifcits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.

  2. DNA damage in human endothelial cells after irradiation in anoxia

    International Nuclear Information System (INIS)

    Endothelial cells and fibroblasts have been reported to respond differently to oxidative stress. Both the effects of high oxygen tension and radiation involve the action of free radicals. DNA damage (single strand breaks, SSB, and double strand breaks, DSB) was assayed in human umbilical cord vein (HUV) cells and in Chinese hamster fibroblasts (V79) after irradiation under oxic or anoxic conditions. The cells were exposed to single doses in the range of 2-18 Gy of γ-radiation from 60Co. Significantly more DNA damage was induced in the V79 cells than in the HUV cells. As a consequence, a higher oxygen enhancement ratio was obtained for the HUV cells (6.3) as compared to the V79 cells (2.8). The repair of SSB was slower in the HUV cells than in the V79 cells, irrespective of oxic state. For the higher doses, the damage remaining at 60 min after anoxic irradiation, i.e. DSB, was only detected in the V79 cells. (orig.)

  3. Endothelial cell chimerism by fluorescence in situ hybridization in gender mismatched renal allograft biopsies

    Institute of Scientific and Technical Information of China (English)

    BAI Hong-wei; SHI Bing-yi; QIAN Ye-yong; NA Yan-qun; ZENG Xuan; ZHONG Ding-rong; LU Min; ZOU Wan-zhong; WU Shi-fei

    2007-01-01

    Background The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ can be of recipient origin after transplantation. In this study, we tested whether endothelial chimerism correlated with the graft rejection and cold ischemia.Methods We studied the biopsy samples from 34 renal transplants of female recipients who received the kidney from a male donor for the presence of endothelial cells of recipient origin. We examined the tissue sections of renal biopsy samples by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes using a biotinylated Y chromosome probe and digoxigenin labelled X chromosome probe, and then analyzed the relationship between the endothelial cell chimerism and the rejection and cold ischemia.Results Endothelial chimerism was common and irrespective of rejections (P>0.05). The cold ischemic time of chimerism group was longer than no chimerism group ((14.83±4.03) hours vs (11.27±3.87) hours, P<0.05).Conclusions There is no correlation between the percentage of recipient endothelial cells in vascular endothelial cells and the type of graft rejection. The endothelium damaged by ischemic injury might be repaired by the endothelial cells from the recipient.

  4. Thalidomide effect in endothelial cell of acute radiation proctitis

    Institute of Scientific and Technical Information of China (English)

    Ki-Tae Kim; Hiun-Suk Chae; Jin-Soo Kim; Hyung-Keun Kim; Young-Seok Cho; Whang Choi; Kyu-Yong Choi; Sang-Young Rho; Suk-Jin Kang

    2008-01-01

    AIM: To determine whether thalidomide prevents microvascular injury in acute radiation proctitis in white rats. METHODS: Fourteen female Wistar rats were used:six in the radiation group,six in the thalidomide group,and two in normal controls.The radiation and thalidomide groups were irradiated at the pelvic area using a single 30 Gy exposure.The thalidomide (150 mg/kg) was injected into the peritoneum for 7 d from the day of irradiation.All animals were sacrificed and the rectums were removed on day 8 after irradiation.The microvessels of resected specimens were immunohistochemically stained with thrombomodulin (TM),yon Willebrand Factor (vWF),and vascular endothelial growth factor (VEGF).RESULTS: The microscopic scores did not differ significantly between the radiation and thalidomide groups,but both were higher than in the control group.Expression of TM was significantly lower in the endothelial cells (EC) of the radiation group than in the control and thalidomide groups (P < 0.001).The number of capillaries expressing vWF in the EC was higher in the radiation group (15.3 ± 6.8) than in the control group (3.7 ± 1.7),and the number of capillaries expressing vWF was attenuated by thalidomide (10.8 ± 3.5,P < 0.001).The intensity of VEGF expression in capillaries was greater in the radiation group than in the control group and was also attenuated by thalidomide (P = 0.003).CONCLUSION: The mechanisms of acute radiationinduced proctitis in the rats are related to endothelial cell injury of microvessel,which may be attenuated with thalidomide.

  5. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

    OpenAIRE

    Patsch, Christoph; Challet-Meylan, Ludivine; Eva C Thoma; Urich, Eduard; Heckel, Tobias; O’Sullivan, John F.; Grainger, Stephanie J.; Kapp, Friedrich G.; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H. C.; He, Wei; Pan, Wei; Prummer, Michael

    2015-01-01

    The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of ei...

  6. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    International Nuclear Information System (INIS)

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  7. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  8. Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

    OpenAIRE

    Sukumar, Piruthivi; Viswambharan, Hema; Imrie, Helen; Cubbon, Richard M.; Yuldasheva, Nadira; Gage, Matthew; Galloway, Stacey; Skromna, Anna; Kandavelu, Parkavi; Santos, Celio X.; Gatenby, V. Kate; Smith, Jessica; Beech, David J; Wheatcroft, Stephen B.; Channon, Keith M.

    2013-01-01

    Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which cou...

  9. Expression and Function of the Homeostatic Molecule Del-1 in Endothelial Cells and the Periodontal Tissue

    OpenAIRE

    Jieun Shin; Hosur, Kavita B.; Kalyani Pyaram; Ravi Jotwani; Shuang Liang; Triantafyllos Chavakis; George Hajishengallis

    2013-01-01

    Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interf...

  10. Optical studies of oxidative stress in pulmonary artery endothelial cells

    Science.gov (United States)

    Ghanian, Zahra; Sepehr, Reyhaneh; Eis, Annie; Kondouri, Ganesh; Ranji, Mahsa

    2015-03-01

    Reactive oxygen species (ROS) play an essential role in facilitating signal transduction processes within the cell and modulating the injuries. However, the generation of ROS is tightly controlled both spatially and temporally within the cell, making the study of ROS dynamics particularly difficult. This study present a novel protocol to quantify the dynamic of the mitochondrial superoxide as a precursor of reactive oxygen species. To regulate the mitochondrial superoxide level, metabolic perturbation was induced by administration of potassium cyanide (KCN). The presented method was able to monitor and measure the superoxide production rate over time. Our results demonstrated that the metabolic inhibitor, potassium cyanide (KCN) induced a significant increase in the rate of superoxide production in mitochondria of fetal pulmonary artery endothelial cells (FPAEC). Presented method sets the stage to study different ROS mediated injuries in vitro.

  11. Modulation of the sis Gene Transcript during Endothelial Cell Differentiation in vitro

    Science.gov (United States)

    Jaye, Michael; McConathy, Evelyn; Drohan, William; Tong, Benton; Deuel, Thomas; Maciag, Thomas

    1985-05-01

    Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.

  12. Biological behaviour and role of endothelial progenitor cells in vascular diseases

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qiu-hua; SHE Ming-peng

    2007-01-01

    Obiective To review the biological behaviour of endothelial progenitor cells and their role in vascular diseases.Data sources The data used in this review were mainly from Medline and PubMed for relevant English language articles published from 1985 to March 2007.The search term was "endothelial progenitor cells".Study selection Articles about the biological behaviour of endothelial progenitor cells and their roles in the pathogenesis of vascular diseases such as atherogenesis were used.Results Progenitor cells in bone marrow,peripheral blood and adventitia can differentiate into mature endothelial cells (ECs).The progenitor cells,which express certain surface markers including AC133,CD34 and KDR,enable restoration of the microcirculation and ECs when injury or ischaemia occurs.Endothelial progenitor cells used in experimental models and clinical trials for ischaemic syndromes could restore endothelial integrity and inhibit neointima development.Moreover,their number and functional properties are influenced by certain cytokines and atherosclerotic risk factors.Impairment of the progenitor cells might limit the regenerative capacity,even lead to the development of atherosclerosis or other vascular diseases.Conclusions Endothelial progenitor cells have a particular role in prevention and treatment of certain cardiovascular diseases.However,many challenges remain in understanding differentiation of endothelial progenitor cells,their mobilization and revascularization.

  13. The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells.

    Science.gov (United States)

    Cha, Min-Ji; Choi, Eunhyun; Lee, Seahyoung; Song, Byeong-Wook; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-02-15

    In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3β (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.

  14. Endothelial-mural cell signaling in vascular development and angiogenesis.

    Science.gov (United States)

    Gaengel, Konstantin; Genové, Guillem; Armulik, Annika; Betsholtz, Christer

    2009-05-01

    Mural cells are essential components of blood vessels and are necessary for normal development, homeostasis, and organ function. Alterations in mural cell density or the stable attachment of mural cells to the endothelium is associated with several human diseases such as diabetic retinopathy, venous malformation, and hereditary stroke. In addition mural cells are implicated in regulating tumor growth and have thus been suggested as potential antiangiogenic targets in tumor therapy. In recent years our knowledge of mural cell function and endothelial-mural cell signaling has increased dramatically, and we now begin to understand the mechanistic basis of the key signaling pathways involved. This is mainly thanks to sophisticated in vivo experiments using a broad repertoire of genetic technologies. In this review, we summarize the five currently best understood signaling pathways implicated in mural cell biology. We discuss PDGFB/PDGFRbeta- dependent pericyte recruitment, as well as the role of angiopoietins and Tie receptors in vascular maturation. In addition, we highlight the effects of sphingosine-1-phosphate signaling on adherens junction assembly and vascular stability, as well as the role of TGF-beta-signaling in mural cell differentiation. We further reflect recent data suggesting an important function for Notch3 signaling in mural cell maturation.

  15. CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jennifer A Greene

    Full Text Available CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina.

  16. Mast cells in mammalian brain.

    Science.gov (United States)

    Dropp, J J

    1976-01-01

    Mast cells, which had until recently been believed to be not present in the mammalian brain, were studied in the brains of 29 mammalian species. Although there was considerable intraspecific and interspecific variation, mast cells were most numerous within the leptomeninges (especially in those overlying the cerebrum and the dorsal thalamus - most rodents, most carnivores, chimpanzees, squirrel monkeys and elephant), the cerebral cortex (most rodents, tiger, fox, chimpanzee, tarsier, and elephant) and in many nuclei of the dorsal thalamus (most rodents, tiger, lion, and fox). In some mammals, mast cells were also numerous in the stroma of the telencephalic choroid plexuses (chimpanzee, squirrel monkey), the putamen and the claustrum (chimpanzee), the subfornical organ (pack rat, tiger, chimpanzee), the olfactory peduncles (hooded rat, albino rat), the stroma of the diencephalic choroid plexus (lion, chimpanzee, squirrel monkey), the pineal organ (chimpanzee, squirrel monkey), some nuclei of the hypothalamus (tiger), the infundibulum (hooded rat, tiger, fox) the area postrema (pack rat, chinchilla, lion, spider monkey, chimpanzee, fox) and some nuclei and tracts of the metencephalon and the myelencephalon (tiger). Neither the sex of the animal nor electrolytic lesions made in the brains of some of the animals at various times prior to sacrifice appeared to effect the number and the distribution of mast cells. Age-related changes in mast cell number and distribution were detected in the albino rat. PMID:961335

  17. Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

    Science.gov (United States)

    Melhado, Caroline; Sanford, Gary; Harris-Hooker, Sandra

    1997-01-01

    Many of the physiological changes of the cardiovascular system during space flight may originate from the dysfunction of basic biological mechanisms caused by microgravity. The weightlessness affects the system when blood and other fluids move to the upper body causing the heart to enlarge to handle the increased blood flow to the upper extremities and decrease circulating volume. Increase arterial pressure triggers baroreceptors which signal the brain to adjust heart rate. Hemodynarnic studies indicate that the microgravity-induced headward fluid redistribution results in various cardiovascular changes such as; alteration of vascular permeability resulting in lipid accumulation in the lumen of the vasculature and degeneration of the the vascular wall, capillary alteration with extensive endothelial invagination. Achieving a true microgravity environment in ground based studies for prolonged periods is virtually impossible. The application of vector-averaged gravity to mammalian cells using horizontal clinostat produces alterations of cellular behavior similar to those observed in microgravity. Similarly, the low shear, horizontally rotating bioreactor (originally designed by NASA) also duplicates several properties of microgravity. Additionally, increasing gravity, i.e., hypcrgravity is easily achieved. Hypergravity has been found to increase the proliferation of several different cell lines (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. The effect of altered gravity on cells maybe similar to those of other physical forces, i.e. shear stress. Previous studies examining laminar flow and shear stress on endothelial cells found that the cells elongate, orient with the direction of flow, and reorganize their F-actin structure, with concomitant increase in cell stiffness. These studies suggest that alterations in the gravity environment will change the behavior of most cells, including

  18. Endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty versus penetrating keratoplasty in Asian eyes

    Directory of Open Access Journals (Sweden)

    Ang M

    2012-04-01

    Full Text Available Marcus Ang1,2, Jodhbir S Mehta1–4, Arundhati Anshu1,2, Hon Kiat Wong5, Hla M Htoon2, Donald Tan1–31Singapore National Eye Centre, 2Singapore Eye Research Institute, 3Department of Ophthalmology, National University Health Systems, 4Department of Clinical Sciences, Duke-NUS Graduate Medical School, 5Department of Ophthalmology, Tan Tock Seng Hospital, SingaporeBackground: The purpose of this study was to compare endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty (DSAEK and penetrating keratoplasty in Asian eyes.Methods: This was a retrospective study of patients from our prospective Singapore Corneal Transplant Study cohort who received corneal transplantation in 2006–2008. We compared eyes that underwent DSAEK or penetrating keratoplasty for Fuchs’ endothelial dystrophy or pseudophakic and aphakic bullous keratopathy. Clinical data, and donor and recipient characteristics were recorded. Of 241 patients who met our inclusion criteria, 68 underwent DSAEK and 173 underwent penetrating keratoplasty. The main outcome measure was endothelial cell loss at 1 year. Secondary outcome measures were graft survival and visual outcomes at 1-year follow-up.Results: There were no significant differences in baseline characteristics of patients between the treatment groups. Percent endothelial cell loss at 1-year follow-up was greater in penetrating keratoplasty eyes (40.9% ± 2.9% compared with DSAEK eyes (22.4% ± 2.3%; P < 0.001. DSAEK-treated eyes had significantly superior uncorrected visual acuity (mean difference = 0.42 ± 0.0059; P < 0.001 and best spectacle-corrected visual acuity (mean difference = 0.14 ± 0.032; P < 0.001 as compared with penetrating keratoplasty-treated eyes. Penetrating keratoplasty-treated eyes had worse astigmatism as compared with DSAEK-treated eyes (-3.0 ± 2.1 versus -1.7 ± 0.8; P < 0.001. Graft survival at 1 year was comparable in both groups, ie, 66/68 (97.0% DSAEK-treated eyes

  19. Effect of essential fatty acids on glucose-induced cytotoxicity to retinal vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Shen Junhui

    2012-07-01

    Full Text Available Abstract Background Diabetic retinopathy is a major complication of dysregulated hyperglycemia. Retinal vascular endothelial cell dysfunction is an early event in the pathogenesis of diabetic retinopathy. Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by docosahexaenoic acid (DHA, 22:6 ω-3 and eicosapentaenoic acid (EPA, 20:5 ω-3. The influence of dietary omega-3 PUFA on brain zinc metabolism has been previously implied. Zn2+ is essential for the activity of Δ6 desaturase as a co-factor that, in turn, converts essential fatty acids to their respective long chain metabolites. Whether essential fatty acids (EFAs α-linolenic acid and linoleic acid have similar beneficial effect remains poorly understood. Methods RF/6A cells were treated with different concentrations of high glucose, α-linolenic acid and linoleic acid and Zn2+. The alterations in mitochondrial succinate dehydrogenase enzyme activity, cell membrane fluidity, reactive oxygen species generation, SOD enzyme and vascular endothelial growth factor (VEGF secretion were evaluated. Results Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by both linoleic acid (LA and α-linolenic acid (ALA, while the saturated fatty acid, palmitic acid was ineffective. A dose–response study with ALA showed that the activity of the mitochondrial succinate dehydrogenase enzyme was suppressed at all concentrations of glucose tested to a significant degree. High glucose enhanced fluorescence polarization and microviscocity reverted to normal by treatment with Zn2+ and ALA. ALA was more potent that Zn2+. Increased level of high glucose caused slightly increased ROS generation that correlated with corresponding decrease in SOD activity. ALA suppressed ROS generation to a significant degree in a dose dependent fashion and raised SOD activity significantly. ALA suppressed

  20. Staphylococcal SSL5 Binding to Human Leukemia Cells Inhibits Cell Adhesion to Endothelial Cells and Platelets

    Directory of Open Access Journals (Sweden)

    Annemiek M. E. Walenkamp

    2010-01-01

    Full Text Available Bacterial proteins provide promising tools for novel anticancer therapies. Staphylococcal superantigen-like 5 (SSL5 was recently described to bind P-selectin glycoprotein ligand-1 (PSGL-1 on leukocytes and to inhibit neutrophil rolling on a P-selectin surface. As leukocytes and tumor cells share many characteristics in migration and dissemination, we explored the potential of SSL5 as an antagonist of malignant cell behavior. Previously, it was demonstrated that rolling of human HL-60 leukemia cells on activated endothelial cells was mediated by P-selectin. In this study, we show that SSL5 targets HL-60 cells. Binding of SSL5 was rapid and without observed toxicity. Competition of SSL5 with the binding of three anti-PSGL-1 antibodies and P-selectin to HL-60 cells identified PSGL-1 as the ligand on HL-60 cells. Presence of sialyl Lewis x epitopes on PSGL-1 was crucial for its interaction with SSL5. Importantly, SSL5 not only inhibited the interaction of HL-60 cells with activated endothelial cells but also with platelets, which both play an important role in growth and metastasis of cancers. These data support the concept that SSL5 could be a lead in the search for novel strategies against hematological malignancies.

  1. Divergent responses of different endothelial cell types to infection with Candida albicans and Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Kati Seidl

    Full Text Available Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.

  2. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    Science.gov (United States)

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis. PMID:26321757

  3. Pro-angiogenic Hematopoietic Progenitor Cells and Endothelial Colony Forming Cells in Pathological Angiogenesis of Bronchial and Pulmonary Circulation

    OpenAIRE

    Duong, Heng; Erzurum, Serpil; Asosingh, Kewal

    2011-01-01

    Dysregulation of angiogenesis is a common feature of many disease processes. Vascular remodeling is believed to depend on the participation of endothelial progenitor cells, but the identification of endothelial progenitors in postnatal neovascularization remains elusive. Current understanding posits a role for circulating pro-angiogenic hematopoietic cells, which interact with local endothelial cells to establish an environment that favors angiogenesis in physiologic and pathophysiologic resp...

  4. Improved culture-based isolation of differentiating endothelial progenitor cells from mouse bone marrow mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Haruki Sekiguchi

    Full Text Available Numerous endothelial progenitor cell (EPC-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT cells and floating (FL cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL but not fast attached (AT BMMNCs in culture are EPC-rich population in mouse.

  5. Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner.50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did.Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  6. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke. PMID:27038754

  7. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.

  8. Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis

    OpenAIRE

    Xiao-ming Wu; Yan-bin Gao; Fang-qiang Cui; Na Zhang

    2016-01-01

    The interaction between glomerular endothelial cells (GECs) and glomerular mesangial cells (GMCs) is an essential aspect of diabetic nephropathy (DN). Therefore, understanding how GECs communicate with GMCs in the diabetic environment is crucial for the development of new targets for the prevention and treatment of DN. Exosomes, nanometer-sized extracellular membrane vesicles secreted by various cell types, play important roles in cell-to-cell communication via the transfer of mRNA, microRNA ...

  9. Caveolin-1 Deficiency Induces Spontaneous Endothelial-to-Mesenchymal Transition in Murine Pulmonary Endothelial Cells in Vitro

    OpenAIRE

    Li, Zhaodong; Wermuth, Peter J.; Benn, Bryan S.; Lisanti, Michael P.; Jimenez, Sergio A.

    2013-01-01

    It was previously demonstrated that transforming growth factor β (TGF-β) induces endothelial-to-mesenchymal transition (EndoMT) in murine lung endothelial cells (ECs) in vitro. Owing to the important role of caveolin-1 (CAV1) in TGF-β receptor internalization and TGF-β signaling, the participation of CAV1 in the induction of EndoMT in murine lung ECs was investigated. Pulmonary ECs were isolated from wild-type and Cav1 knockout mice using immunomagnetic methods with sequential anti-CD31 and a...

  10. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  11. Protection of Vascular Endothelial Growth Factor to Brain Edema Following Intracerebral Hemorrhage and Its Involved Mechanisms: Effect of Aquaporin-4.

    Directory of Open Access Journals (Sweden)

    Heling Chu

    Full Text Available Vascular endothelial growth factor (VEGF has protective effects on many neurological diseases. However, whether VEGF acts on brain edema following intracerebral hemorrhage (ICH is largely unknown. Our previous study has shown aquaporin-4 (AQP4 plays an important role in brain edema elimination following ICH. Meanwhile, there is close relationship between VEGF and AQP4. In this study, we aimed to test effects of VEGF on brain edema following ICH and examine whether they were AQP4 dependent. Recombinant human VEGF165 (rhVEGF165 was injected intracerebroventricularly 1 d after ICH induced by microinjecting autologous whole blood into striatum. We detected perihemotomal AQP4 protein expression, then examined the effects of rhVEGF165 on perihemotomal brain edema at 1 d, 3 d, and 7 d after injection in wild type (AQP4(+/+ and AQP4 knock-out (AQP4(-/- mice. Furthermore, we assessed the possible signal transduction pathways activated by VEGF to regulate AQP4 expression via astrocyte cultures. We found perihemotomal AQP4 protein expression was highly increased by rhVEGF165. RhVEGF165 alleviated perihemotomal brain edema in AQP4(+/+ mice at each time point, but had no effect on AQP4(-/- mice. Perihemotomal EB extravasation was increased by rhVEGF165 in AQP4(-/- mice, but not AQP4(+/+ mice. RhVEGF165 reduced neurological deficits and increased Nissl's staining cells surrounding hemotoma in both types of mice and these effects were related to AQP4. RhVEGF165 up-regulated phospharylation of C-Jun amino-terminal kinase (p-JNK and extracellular signal-regulated kinase (p-ERK and AQP4 protein in cultured astrocytes. The latter was inhibited by JNK and ERK inhibitors. In conclusion, VEGF reduces neurological deficits, brain edema, and neuronal death surrounding hemotoma but has no influence on BBB permeability. These effects are closely related to AQP4 up-regulation, possibly through activating JNK and ERK pathways. The current study may present new insights to

  12. Double suicide genes selectively kill human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  13. Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules

    OpenAIRE

    1991-01-01

    Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their ...

  14. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

    Science.gov (United States)

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a

  15. Recent advances in understanding the roles of vascular endothelial cells in allergic inflammation.

    Science.gov (United States)

    Shoda, Tetsuo; Futamura, Kyoko; Orihara, Kanami; Emi-Sugie, Maiko; Saito, Hirohisa; Matsumoto, Kenji; Matsuda, Akio

    2016-01-01

    Allergic disorders commonly involve both chronic tissue inflammation and remodeling caused by immunological reactions to various antigens on tissue surfaces. Due to their anatomical location, vascular endothelial cells are the final responders to interact with various exogenous factors that come into contact with the epithelial surface, such as pathogen-associated molecular patterns (PAMPs) and antigens. Recent studies have shed light on the important roles of endothelial cells in the development and exacerbation of allergic disorders. For instance, endothelial cells have the greatest potential to produce several key molecules that are deeply involved in allergic inflammation, such as periostin and thymus and activation-regulated chemokine (TARC/CCL17). Additionally, endothelial cells were recently shown to be important functional targets for IL-33--an essential regulator of allergic inflammation. Notably, almost all endothelial cell responses and functions involved in allergic inflammation are not suppressed by corticosteroids. These corticosteroid-refractory endothelial cell responses and functions include TNF-α-associated angiogenesis, leukocyte adhesion, IL-33-mediated responses and periostin and TARC production. Therefore, these unique responses and functions of endothelial cells may be critically involved in the pathogenesis of various allergic disorders, especially their refractory processes. Here, we review recent studies, including ours, which have elucidated previously unknown pathophysiological roles of vascular endothelial cells in allergic inflammation and discuss the possibility of endothelium-targeted therapy for allergic disorders.

  16. Corneal endothelial cell changes associated with cataract surgery in patients with type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Hugod, Mikkel; Storr-Paulsen, Allan; Norregaard, Jens Christian;

    2011-01-01

    To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation.......To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation....

  17. Relationship between protecitve effect of probucol on endothelial cells and asymmetrical dimethylarginine levels

    Institute of Scientific and Technical Information of China (English)

    Jun-linJIANG; Xiao-hongZHANG; Han-wuDENG; Yuan-JianLI

    2004-01-01

    AIM: To investigate the relationship between protective effect of probucol on endothelial cells and endogenous nitric oxide synthase inhibitor levels. METHODS: Endothelial cells were treated with oxidative-low density lipoprotein (ox-LDL) (100 rag/L) or lysophosphatidyl choline (LPC) (5 mg/L) for 48 h, and the release of lactate dehydrogenase (LDH), levels of nitric oxide (NO),

  18. Ethanolamine metabolism in cultured bovine aortic endothelial cells

    International Nuclear Information System (INIS)

    The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells. Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine

  19. INSULIN INDUCES NITRIC OXIDE PRODUCTION IN BOVINEAORTIC ENDOTHELIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To examine the effects of insulin on cell proliferation, nitric oxide (NO) release and nitric oxide synthase (NOS) gene expression in bovine aortic endothelial cells ( BAEC ) . Methods The mi togenesis was assessed by MTT method; the products of NO in the culture media, by Griess reaction; and the levels of NOS mRNA in BAEC , by RT/PCR tech nique. Results BAEC were not responsive to the growth-promoting effects of insulin. Stimulation with insulin resulted a dose-dependent rise of NO in the culture supernatants 2h later, with a maximum at 12~24h and a decline at 24h. This rise was inhibited by an inhibitor of NOS (L-NAME). NOS mRNA increased slightly in BAEC without statistical significance. Conelu sion The study suggested that the insulin-induced NO release might be caused directly by NOS activation.

  20. Inhibitory Effect of the Punica granatum Fruit Extract on Angiotensin-II Type I Receptor and Thromboxane B2 in Endothelial Cells Induced by Plasma from Preeclamptic Patients

    OpenAIRE

    Widya Kusumawati; Kusnarman Keman; Setyawati Soeharto

    2016-01-01

    This study aims to evaluate whether the Punica granatum fruit extract modulates the Angiotensin-II Type I receptor (AT1-R) and thromboxane B2 level in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluence, endothelial cells were divided into five groups, which included endothelial cells exposed to 2% plasma from normal pregnancy (NP), endothelial cells exposed to 2% plasma from preeclampti...

  1. Circulating endothelial progenitor cells in kidney transplant patients.

    Directory of Open Access Journals (Sweden)

    Giovana S Di Marco

    Full Text Available BACKGROUND: Kidney transplantation (RTx leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs. METHODS: We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males. RTx patients received a calcineurin inhibitor (CNI-based (65% or a CNI-free therapy (35% and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1 levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+ and CD26+ cells were determined by flow cytometry. RESULTS: Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1--a cytokine responsible for EPC mobilization--is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1. CONCLUSIONS: We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in

  2. The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis

    International Nuclear Information System (INIS)

    Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model. Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system. Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg-/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg-/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner. Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain

  3. Endothelial progenitor cells display clonal restriction in multiple myeloma

    Directory of Open Access Journals (Sweden)

    Dai Kezhi

    2006-06-01

    Full Text Available Abstract Background In multiple myeloma (MM, increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI patterns in female patients by a human androgen receptor assay (HUMARA. In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. Methods A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH. Results In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele in 64% (n = 7. In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele. In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5 of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status

  4. Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

    Directory of Open Access Journals (Sweden)

    Ye-Ji Jeong

    Full Text Available Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA, an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

  5. Cell-based monitoring of cancer : Circulating tumor and endothelial cells

    NARCIS (Netherlands)

    J. Kraan (Jaco)

    2015-01-01

    markdownabstractThis thesis aimed to optimize the predictive and prognostic information that can be obtained from Circulating Tumor cells (CTC) and Circulating Endothelial Cells (CEC) in whole blood by improving and standardization of their detection and characterization methods in patients with sol

  6. File list: ALL.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  10. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema

    DEFF Research Database (Denmark)

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten;

    2011-01-01

    Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor....... Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression...... in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p

  11. Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ulisse Garbin

    2008-01-01

    Full Text Available The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L for 24 hours and oxidative stress was induced by the addition of oxidized (ox-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin; proteins linked to inflammation (IL-6 and TNFalpha, thrombotic state (tissue factor, PAI-1 and uPA, hypertension such as endothelin-1 (ET-1, and vascular remodeling such as metalloproteinases (MMP-2, MMP-9 and protease inhibitor (TIMP-1. The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

  12. CD34+ cells represent highly functional endothelial progenitor cells in murine bone marrow.

    Directory of Open Access Journals (Sweden)

    Junjie Yang

    Full Text Available BACKGROUND: Endothelial progenitor cells (EPCs were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. METHODOLOGY/PRINCIPAL FINDINGS: CD34(+ cells, c-Kit(+/Sca-1(+/Lin(- (KSL cells, c-Kit(+/Lin(- (KL cells and Sca-1(+/Lin(- (SL cells were isolated from mouse bone marrow mononuclear cells (BMMNCs using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34(+ cells showed the lowest EPC colony forming activity, CD34(+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. CONCLUSION: These findings suggest that mouse CD34(+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

  13. Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis.

    Science.gov (United States)

    Derricks, Kelsey E; Trinkaus-Randall, Vickery; Nugent, Matthew A

    2015-09-01

    Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and individual cell responses. Endothelial cells on soft substrates, 4 kPa, were the most responsive to VEGF, whereas cells on the 125 kPa substrates exhibited an attenuated response. Magnitude of activation, not the quantity of cells responding or the number of local maximums each cell experienced distinguished the responses. Individual cell analysis, across all treatments, identified two unique cell clusters. One cluster, containing most of the cells, exhibited minimal or slow calcium release. The remaining cell cluster had a rapid, high magnitude VEGF activation that ultimately defined the population based average calcium response. Interestingly, at low doses of VEGF, the high responding cell cluster contained smaller cells on average, suggesting that cell shape and size may be indicative of VEGF-sensitive endothelial cells. This study provides a new analytical tool to quantitatively analyze individual cell signaling response kinetics, that we have used to help uncover outcomes that are hidden within the average. The ability to selectively identify highly VEGF responsive cells within a population may lead to a better understanding of the specific phenotypic characteristics that define cell responsiveness, which could provide new insight for the development of targeted anti- and pro-angiogenic therapies.

  14. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

    Directory of Open Access Journals (Sweden)

    Cai-Guo Yu

    2016-01-01

    Full Text Available Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on “VEGF uncoupling with nitric oxide” and “competitive angiopoietin 1/angiopoietin 2” mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics.

  15. Modeling human endothelial cell transformation in vascular neoplasias

    Directory of Open Access Journals (Sweden)

    Victoria W. Wen

    2013-09-01

    Full Text Available Endothelial cell (EC-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, which responds poorly to current treatments and has a very high mortality rate. The development of treatments that are more effective for these disorders will be expedited by insight into the processes that promote abnormal proliferation and malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by the rarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a central role in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This review describes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes that underpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through the introduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed in relation to their relevance as models of vascular neoplasia.

  16. High precision measurement of electrical resistance across endothelial cell monolayers.

    Science.gov (United States)

    Tschugguel, W; Zhegu, Z; Gajdzik, L; Maier, M; Binder, B R; Graf, J

    1995-05-01

    Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 omega cm2) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric field across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 omega resistance changes of the endothelium on top of a 21 omega series resistance of the support and bathing fluid layers. Histamine (10 microM) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 microM) was less effective, PAF (380 nM) and LTC4 (1 microM) had no effect. TEER was also reduced by the calcium ionophore A-23187 (10 microM). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.

  17. Late Release of Circulating Endothelial Cells and Endothelial Progenitor Cells after Chemotherapy Predicts Response and Survival in Cancer Patients

    Directory of Open Access Journals (Sweden)

    Jeanine M. Roodhart

    2010-01-01

    Full Text Available We and others have previously demonstrated that the acute release of progenitor cells in response to chemotherapy actually reduces the efficacy of the chemotherapy. Here, we take these data further and investigate the clinical relevance of circulating endothelial (progenitor cells (CE(PCs and modulatory cytokines in patients after chemotherapy with relation to progression-free and overall survival (PFS/OS. Patients treated with various chemotherapeutics were included. Blood sampling was performed at baseline, 4 hours, and 7 and 21 days after chemotherapy. The mononuclear cell fraction was analyzed for CE(PC by FACS analysis. Plasma was analyzed for cytokines by ELISA or Luminex technique. CE(PCs were correlated with response and PFS/OS using Cox proportional hazard regression analysis. We measured CE(PCs and cytokines in 71 patients. Only patients treated with paclitaxel showed an immediate increase in endothelial progenitor cell 4 hours after start of treatment. These immediate changes did not correlate with response or survival. After 7 and 21 days of chemotherapy, a large and consistent increase in CE(PC was found (P < .01, independent of the type of chemotherapy. Changes in CE(PC levels at day 7 correlated with an increase in tumor volume after three cycles of chemotherapy and predicted PFS/OS, regardless of the tumor type or chemotherapy. These findings indicate that the late release of CE(PC is a common phenomenon after chemotherapeutic treatment. The correlation with a clinical response and survival provides further support for the biologic relevance of these cells in patients' prognosis and stresses their possible use as a therapeutic target.

  18. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion.

    OpenAIRE

    Klein, L M; Lavker, R M; Matis, W L; Murphy, G F

    1989-01-01

    To understand better the role of mast cell secretory products in the genesis of inflammation, a system was developed for in vitro degranulation of human mast cells in skin organ cultures. Within 2 hr after morphine sulfate-induced degranulation, endothelial cells lining microvessels adjacent to affected mast cells expressed an activation antigen important for endothelial-leukocyte adhesion. Identical results were obtained when other mast cell secretagogues (anti-IgE, compound 48/80, and calci...

  19. Surface-enhanced Raman spectroscopy of the endothelial cell membrane.

    Directory of Open Access Journals (Sweden)

    Simon W Fogarty

    Full Text Available We applied surface-enhanced Raman spectroscopy (SERS to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.

  20. The effect of 193 nm excimer laser radiation on the human corneal endothelial cell density

    Energy Technology Data Exchange (ETDEWEB)

    Isager, P.; Hjortdal, J.Oe.; Ehlers, N. [Aarhus Univ. Hospital, Dept. of Ophthalmology, Aarhus (Denmark)

    1996-06-01

    The effect of 193 nm excimer laser radiation on human corneal endothelial cell density was examined. Fifty-five eyes from 35 patients underwent photorefractive keratectomy for myopia. Photomicrographs of the endothelium were taken a short time before the operation and on an average of 7 months postoperatively with a specular microscope. The average endothelial cell densities were preoperatively 3375 {+-} 266 cells/mm{sup 2} (means {+-} SD) and postoperatively 3348 {+-} 287 cells/mm{sup 2}, corresponding to a fall of 27 cells/mm{sup 2} (N = 55). This fall in endothelial cell density was not statistically significant. A significant correlation between the change in cell density and age of the patient was found, with older patients losing more cells (N = 35, 2p < 0.05). The magnification of the specular microscope was found to change with corneal thickness. The importance of correcting the endothelial cell densities for corneal thickness is discussed. (au) 14 refs.

  1. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

    Science.gov (United States)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

    2016-03-01

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  2. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

    Science.gov (United States)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

    2016-03-03

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  3. Curcuma oil reduces endothelial cell-mediated inflammation in postmyocardial ischemia/reperfusion in rats.

    Science.gov (United States)

    Manhas, Amit; Khanna, Vivek; Prakash, Prem; Goyal, Dipika; Malasoni, Richa; Naqvi, Arshi; Dwivedi, Anil K; Dikshit, Madhu; Jagavelu, Kumaravelu

    2014-09-01

    Endothelial cells initiated inflammation persisting in postmyocardial infarction needs to be controlled and moderated for avoiding fatal complications. Curcuma oil (C.oil, Herbal Medicament), a standardized hexane soluble fraction of Curcuma longa has possessed neuroprotective effect. However, its effect on myocardial ischemia/reperfusion (MI/RP) and endothelial cells remains incompletely defined. Here, using in vivo rat MI/RP injury model and in vitro cellular approaches using EA.hy926 endothelial cells, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and myograph, we provide evidence that with effective regimen and preconditioning of rats with C.oil (250 mg/kg, PO), before and after MI/RP surgery protects rats from MI/RP-induced injury. C.oil treatment reduces left ventricular ischemic area and endothelial cell-induced inflammation, specifically in the ischemic region (*P < 0.0001) and improved endothelial function by reducing the expression of proinflammatory genes and adhesion factors on endothelial cells both in vitro and in vivo. Furthermore, mechanistic studies have revealed that C.oil reduced the expression of adhesion factors like E-selectin (#P = 0.0016) and ICAM-1 ($P = 0.0069) in initiating endothelial cells-induced inflammation. In line to the real-time polymerase chain reaction expression data, C.oil reduced the adhesion of inflammatory cells to endothelial cells as assessed by the interaction of THP-1 monocytes with the endothelial cells using flow-based adhesion and under inflammatory conditions. These studies provide evidence that salutary effect of C.oil on MI/RP could be achieved with pretreatment and posttreatment of rats, C.oil reduced MI/RP-induced injury by reducing the endothelial cell-mediated inflammation, specifically in the ischemic zone of MI/RP rat heart.

  4. Diabetes alters intracellular calcium transients in cardiac endothelial cells.

    Directory of Open Access Journals (Sweden)

    Abdul Q Sheikh

    Full Text Available Diabetic cardiomyopathy (DCM is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+](i homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+](i homeostasis due to altered sarcoplasmic reticulum Ca(2+ ATPase (SERCA and sodium-calcium exchanger (NCX activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO, elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+ regulatory mechanisms in cardiac endothelial cells (CECs remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+](i homeostasis in CECs in the rat model (streptozotocin-induced of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+](i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+ ATPase (PMCA and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+](i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.

  5. Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells.

    Science.gov (United States)

    Abbasian, Nima; Burton, James O; Herbert, Karl E; Tregunna, Barbara-Emily; Brown, Jeremy R; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J; Goodall, Alison H; Bevington, Alan

    2015-09-01

    Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. PMID:25745026

  6. Hydrogel Surfaces to Promote Attachment and Spreading of Endothelial Progenitor Cells

    OpenAIRE

    Camci-Unal, Gulden; Nichol, Jason William; Bae, Hojae; Tekin, Halil; Bischoff, Joyce; Khademhosseini, Ali

    2012-01-01

    Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA-based hydrogels provides an attractiv...

  7. Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

    OpenAIRE

    Rong Liu; Hua Liu; Yonju Ha; Tilton, Ronald G.; Wenbo Zhang

    2014-01-01

    Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protei...

  8. Regulation of Thrombomodulin Expression and Release in Human Aortic Endothelial Cells by Cyclic Strain

    OpenAIRE

    Martin, Fiona A.; Alisha McLoughlin; Rochfort, Keith D.; Colin Davenport; Murphy, Ronan P.; Cummins, Philip M.

    2014-01-01

    Background and Objectives Thrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis. Methods This ...

  9. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema

    DEFF Research Database (Denmark)

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten;

    2011-01-01

    (VEGF) is an endothelial cell-specific mitogen and angiogen. VEGF-A protein, which is identical to vascular permeability factor, is a regulator of angiogenesis. In this study, 101 patients with meningiomas, and possible co-factors to PTBE, such as meningioma subtypes and tumor location, were examined...... positively correlated to the PTBE (p = 0.038). If VEGF is responsible for the formation of PTBE, the edema may be treated with the anti-VEGF drug Bevacizumab (Avastin), which has been shown to reduce PTBE in patients with glioblastoma multiforme....

  10. Brain tumor stem cell dancing

    Directory of Open Access Journals (Sweden)

    Giuseppina Bozzuto

    2014-09-01

    Full Text Available Background. Issues regarding cancer stem cell (CSC movement are important in neurosphere biology as cell-cell or cell-environment interactions may have significant impacts on CSC differentiation and contribute to the heterogeneity of the neurosphere. Aims. Despite the growing body of literature data on the biology of brain tumor stem cells, floating CSC-derived neurospheres have been scarcely characterized from a morphological and ultrastructural point of view. Results. Here we report a morphological and ultrastructural characterization performed by live imaging and scanning electron microscopy. Glioblastoma multiforme (GBM CSC-derived neurospheres are heterogeneous and are constituted by cells, morphologically different, capable of forming highly dynamic structures. These dynamic structures are regulated by not serendipitous cell-cell interactions, and they synchronously pulsate following a cyclic course made of "fast" and "slow" alternate phases. Autocrine/paracrine non canonical Wnt signalling appears to be correlated with the association status of neurospheres. Conclusions. The results obtained suggest that GBM CSCs can behave both as independents cells and as "social" cells, highly interactive with other members of its species, giving rise to a sort of "multicellular organism".

  11. Oct-4+/Tenascin C+ neuroblastoma cells serve as progenitors of tumor-derived endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Annalisa Pezzolo; Silvia Deaglio; Fabio Malavasi; Vito Pistoia; Federica Parodi; Danilo Marimpietri; Lizzia Raffaghello; Claudia Cocco; Angela Pistorio; Manuela Mosconi; Claudio Gambini; Michele Cillj

    2011-01-01

    Neuroblastoma (NB)-associated endothelial microvessels (EMs) may be lined by tumor-derived endothelial cells (TECs),that are genetically unstable and chemoresistant.Here we have addressed the identification of TEC progenitors in NB by focusing on Octamer-binding transcription factor 4 (Oct-4) as a putative marker.Oct-4+ cells were detected in primary NB samples (n = 23),metastatic bone marrow aspirates (n = 10),NB cell lines (n = 4),and orthotopic tumors (n = 10) formed by the HTLA-230 NB cell line in immunodeficient mice.Most Oct-4+ cells showed a perivascular distribution,with 5% of them homing in perinecrotic areas.All Oct-4+ cells were tumor-derived since they shared amplification of MYCN oncogene with malignant cells.Perivascular Oct-4+ cells expressed stem cellrelated,neural progenitor-related and NB-related markers,including surface Tenascin C (TNC),that was absent from perinecrotic Oct-4+ cells and bulk tumor cells.TNC+ but not TNC- HTLA-230 cells differentiated in vitro into endothelial-like cells expressing vascular-endothellal-cadherin,prostate-specific membrane antigen and CD31 upon culture in medium containing vascular endothelial growth factor (VEGF).TNC+ but not TNC- HTLA-230 cells formed neurospheres when cultured in serum-free medium.Both cell fractions were tumorigenic,but only tumors formed by TNC+ cegs contained EMs fined by TECs.In conclusion,we have identified in NB tumors two putative niches containing Oct-4+ tumor cells.Oct-4+/TNC+ perivascular NB cells displayed a high degree of plasticity and served as progenitors of TECs.Therapeutic targeting of Oct4+/TNC+ progenitors may counteract the contribution of NB-derived ECs to tumor relapse and chemoresistance.

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    Lifescience Database Archive (English)

    Full Text Available His.CDV.20.AllAg.Aortic_valve_endothelial_cells hg19 Histone Cardiovascular Aortic valve... endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.20.AllAg.Aortic_valve_endothelial_cells.bed ...

  2. File list: His.CDV.10.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.CDV.10.AllAg.Aortic_valve_endothelial_cells hg19 Histone Cardiovascular Aortic valve... endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.10.AllAg.Aortic_valve_endothelial_cells.bed ...

  3. File list: Oth.CDV.10.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.10.AllAg.Aortic_valve_endothelial_cells hg19 TFs and others Cardiovascular Aortic valve... endothelial cells SRX285599 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.10.AllAg.Aortic_valve_endothelial_cells.bed ...

  4. File list: DNS.CDV.10