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Sample records for human lung endothelial

  1. Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung.

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    Kaner, R J; Crystal, R G

    2001-04-01

    Based on assessment of mRNA expression, the lung is a major site of expression of the vascular endothelial growth factor (VEGF) gene, largely from type II alveolar epithelial cells. With the knowledge that VEGF can function to induce vascular leak, we hypothesized that to protect the lung from pulmonary edema, the VEGF produced in the lung must be compartmentalized from the pulmonary endothelium, and thus must be compartmentalized to the surface of the respiratory epithelium. To assess this hypothesis, we quantified the levels of VEGF in human respiratory epithelial lining fluid recovered by bronchoalveolar lavage from normal individuals. Strikingly, human respiratory epithelial lining fluid contains 11 +/- 5 ng/mL as quantified by ELISA, a 500-fold greater concentration than plasma (22 +/- 10 pg/mL, p Damocles sword" poised to induce lung endothelial permeability in conditions of acute lung injury when the integrity of the alveolar epithelial barrier is breached.

  2. Endocytic Pathways Used by Andes Virus to Enter Primary Human Lung Endothelial Cells.

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    Cheng-Feng Chiang

    Full Text Available Andes virus (ANDV is the major cause of hantavirus pulmonary syndrome (HPS in South America. Despite a high fatality rate (up to 40%, no vaccines or antiviral therapies are approved to treat ANDV infection. To understand the role of endocytic pathways in ANDV infection, we used 3 complementary approaches to identify cellular factors required for ANDV entry into human lung microvascular endothelial cells. We screened an siRNA library targeting 140 genes involved in membrane trafficking, and identified 55 genes required for ANDV infection. These genes control the major endocytic pathways, endosomal transport, cell signaling, and cytoskeleton rearrangement. We then used infectious ANDV and retroviral pseudovirions to further characterize the possible involvement of 9 of these genes in the early steps of ANDV entry. In addition, we used markers of cellular endocytosis along with chemical inhibitors of known endocytic pathways to show that ANDV uses multiple routes of entry to infect target cells. These entry mechanisms are mainly clathrin-, dynamin-, and cholesterol-dependent, but can also occur via a clathrin-independent manner.

  3. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

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    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  4. Particulate matter disrupts human lung endothelial barrier integrity via ROS- and p38 MAPK-dependent pathways.

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    Wang, Ting; Chiang, Eddie T; Moreno-Vinasco, Liliana; Lang, Gabriel D; Pendyala, Srikanth; Samet, Jonathan M; Geyh, Alison S; Breysse, Patrick N; Chillrud, Steven N; Natarajan, Viswanathan; Garcia, Joe G N

    2010-04-01

    Epidemiologic studies have linked exposure to airborne pollutant particulate matter (PM) with increased cardiopulmonary mortality and morbidity. The mechanisms of PM-mediated lung pathophysiology, however, remain unknown. We tested the hypothesis that PM, via enhanced oxidative stress, disrupts lung endothelial cell (EC) barrier integrity, thereby enhancing organ dysfunction. Using PM collected from Ft. McHenry Tunnel (Baltimore, MD), we assessed PM-mediated changes in transendothelial electrical resistance (TER) (a highly sensitive measure of barrier function), reactive oxygen species (ROS) generation, and p38 mitogen-activated protein kinase (MAPK) activation in human pulmonary artery EC. PM induced significant dose (10-100 microg/ml)- and time (0-10 h)-dependent EC barrier disruption reflected by reduced TER values. Exposure of human lung EC to PM resulted in significant ROS generation, which was directly involved in PM-mediated EC barrier dysfunction, as N-acetyl-cysteine (NAC, 5 mM) pretreatment abolished both ROS production and barrier disruption induced by PM. Furthermore, PM induced p38 MAPK activation and HSP27 phosphorylation, events that were both attenuated by NAC. In addition, PM-induced EC barrier disruption was partially prevented by the p38 MAP kinase inhibitor SB203580 (10 microM) as well as by reduced expression of either p38 MAPK beta or HSP27 (siRNA). These results demonstrate that PM induces ROS generation in human lung endothelium, resulting in oxidative stress-mediated EC barrier disruption via p38 MAPK- and HSP27-dependent pathways. These findings support a novel mechanism for PM-induced lung dysfunction and adverse cardiopulmonary outcomes.

  5. Particulate Matter Disrupts Human Lung Endothelial Barrier Integrity via ROS- and p38 MAPK–Dependent Pathways

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    Wang, Ting; Chiang, Eddie T.; Moreno-Vinasco, Liliana; Lang, Gabriel D.; Pendyala, Srikanth; Samet, Jonathan M.; Geyh, Alison S.; Breysse, Patrick N.; Chillrud, Steven N.; Natarajan, Viswanathan; Garcia, Joe G. N.

    2010-01-01

    Epidemiologic studies have linked exposure to airborne pollutant particulate matter (PM) with increased cardiopulmonary mortality and morbidity. The mechanisms of PM-mediated lung pathophysiology, however, remain unknown. We tested the hypothesis that PM, via enhanced oxidative stress, disrupts lung endothelial cell (EC) barrier integrity, thereby enhancing organ dysfunction. Using PM collected from Ft. McHenry Tunnel (Baltimore, MD), we assessed PM-mediated changes in transendothelial electrical resistance (TER) (a highly sensitive measure of barrier function), reactive oxygen species (ROS) generation, and p38 mitogen-activated protein kinase (MAPK) activation in human pulmonary artery EC. PM induced significant dose (10–100 μg/ml)- and time (0–10 h)-dependent EC barrier disruption reflected by reduced TER values. Exposure of human lung EC to PM resulted in significant ROS generation, which was directly involved in PM-mediated EC barrier dysfunction, as N-acetyl-cysteine (NAC, 5 mM) pretreatment abolished both ROS production and barrier disruption induced by PM. Furthermore, PM induced p38 MAPK activation and HSP27 phosphorylation, events that were both attenuated by NAC. In addition, PM-induced EC barrier disruption was partially prevented by the p38 MAP kinase inhibitor SB203580 (10 μM) as well as by reduced expression of either p38 MAPK β or HSP27 (siRNA). These results demonstrate that PM induces ROS generation in human lung endothelium, resulting in oxidative stress–mediated EC barrier disruption via p38 MAPK- and HSP27-dependent pathways. These findings support a novel mechanism for PM-induced lung dysfunction and adverse cardiopulmonary outcomes. PMID:19520919

  6. Transforming growth factor-β1 downregulates vascular endothelial growth factor-D expression in human lung fibroblasts via the Jun NH2-terminal kinase signaling pathway.

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    Cui, Ye; Osorio, Juan C; Risquez, Cristobal; Wang, Hao; Shi, Ying; Gochuico, Bernadette R; Morse, Danielle; Rosas, Ivan O; El-Chemaly, Souheil

    2014-03-20

    Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.

  7. Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function.

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    You-Hong Cheng

    Full Text Available Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012 and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3-68, CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05-0.5nM, which could be antagonized with AMD3100; ubiquitin (0.03-3μM was ineffective. In a human lung microvascular endothelial cell line (HULEC5a, CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3-68 was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3-68 to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3-68. Our findings

  8. Blocking of α1β1 and α2β1 adhesion molecules inhibits eosinophil migration through human lung microvascular endothelial cell monolayer

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    Stanisława Bazan-Socha

    2014-12-01

    Full Text Available In cell trafficking to the airways in asthma, among integrins the most important are those containing α4 and β2 subunits. We have previously shown that also blocking of collagen receptors, α1β1 and α2β1 integrins, inhibits transmigration of eosinophils of asthmatic subjects through a monolayer of skin microvascular endothelial cells seeded on collagen IV coated inserts. However, it was not clear whether this observation was limited to asthma or depended on the type of microvascular cell and collagen IV used as a base. In the current study we performed a transmigration assay using human lung microvascular endothelial cells seeded directly on a plastic surface as a base and blood cells isolated from 12 representatives of each of two groups, asthmatics and healthy donors, by gradient centrifugation, followed by immunomagnetic negative separation of eosinophils. Isolated eosinophils and peripheral blood mononuclear cells (PBMC were inhibited by snake venom-derived integrin antagonists including viperistatin and VP12, as inhibitors of α1β1 and α2β1 integrin, respectively, and VLO5 and VLO4, as inhibitors of α4β1 and α5β1 integrin, respectively. All snake venom-derived anti-adhesive proteins were effective in inhibiting eosinophil transmigration, whilst only VLO5 and VLO4 reduced PBMC mobility in this assay. This observation was similar in both groups of subjects studied. α1β1 and α2β1 integrins could be involved in transmigration of eosinophil to the inflammatory site. Migratory inhibition was observed in asthma subjects as well as in healthy donors, and did not depend on origin of endothelial cells or the extracellular matrix component used as a base.

  9. FOXF1 maintains endothelial barrier function and prevents edema after lung injury.

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    Cai, Yuqi; Bolte, Craig; Le, Tien; Goda, Chinmayee; Xu, Yan; Kalin, Tanya V; Kalinichenko, Vladimir V

    2016-04-19

    Multiple signaling pathways, structural proteins, and transcription factors are involved in the regulation of endothelial barrier function. The forkhead protein FOXF1 is a key transcriptional regulator of embryonic lung development, and we used a conditional knockout approach to examine the role of FOXF1 in adult lung homeostasis, injury, and repair. Tamoxifen-regulated deletion of both Foxf1 alleles in endothelial cells of adult mice (Pdgfb-iCreER/Foxf1(-/-)) caused lung inflammation and edema, leading to respiratory insufficiency and death. Deletion of a single Foxf1 allele made heterozygous Pdgfb-iCreER/Foxf1(+/-)mice more susceptible to acute lung injury. FOXF1 abundance was decreased in pulmonary endothelial cells of human patients with acute lung injury. Gene expression analysis of pulmonary endothelial cells with homozygous FOXF1 deletion indicated reduced expression of genes critical for maintenance and regulation of adherens junctions. FOXF1 knockdown in vitro and in vivo disrupted adherens junctions, enhanced lung endothelial permeability, and increased the abundance of the mRNA and protein for sphingosine 1-phosphate receptor 1 (S1PR1), a key regulator of endothelial barrier function. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that FOXF1 directly bound to and induced the transcriptional activity of the S1pr1 promoter. Pharmacological administration of S1P to injured Pdgfb-iCreER/Foxf1(+/-)mice restored endothelial barrier function, decreased lung edema, and improved survival. Thus, FOXF1 promotes normal lung homeostasis and repair, in part, by enhancing endothelial barrier function through activation of the S1P/S1PR1 signaling pathway. Copyright © 2016, American Association for the Advancement of Science.

  10. Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury.

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    Sugiyama, Michael G; Gamage, Asela; Zyla, Roman; Armstrong, Susan M; Advani, Suzanne; Advani, Andrew; Wang, Changsen; Lee, Warren L

    2015-12-04

    Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin α5β1, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.IMPORTANCE There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have

  11. Niclosamide enhances the antitumor effects of radiation by inhibiting the hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway in human lung cancer cells.

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    Xiang, Mei; Chen, Zihong; Yang, Donghong; Li, Haiwen; Zuo, Yufang; Li, Jingjing; Zhang, Wendian; Zhou, Hechao; Jiang, Danxian; Xu, Zumin; Yu, Zhonghua

    2017-08-01

    Lung cancer is one of the leading causes of cancer-associated mortality, worldwide. The overall survival rate remains low, but progress has been made in improving the diagnosis and treatment of lung cancer over the past decades. Niclosamide, a salicylanilide derivative used for the treatment of tapeworm infections, is safe, well tolerated, inexpensive and readily available. Previous studies have identified niclosamide as a potential anticancer agent. The present study demonstrated that niclosamide enhanced the effect of irradiation by inhibiting the hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway. These findings suggest that niclosamide may be a promising candidate for clinical evaluation as part of a combined regimen for the treatment of non-small cell lung cancer.

  12. Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

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    Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

    2017-08-01

    Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese

  13. Tumor endothelial inflammation predicts clinical outcome in diverse human cancers.

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    Sean P Pitroda

    Full Text Available Vascular endothelial cells contribute to the pathogenesis of numerous human diseases by actively regulating the stromal inflammatory response; however, little is known regarding the role of endothelial inflammation in the growth of human tumors and its influence on the prognosis of human cancers.Using an experimental model of tumor necrosis factor-alpha (TNF-α-mediated inflammation, we characterized inflammatory gene expression in immunopurified tumor-associated endothelial cells. These genes formed the basis of a multivariate molecular predictor of overall survival that was trained and validated in four types of human cancer.We report that expression of experimentally derived tumor endothelial genes distinguished pathologic tissue specimens from normal controls in several human diseases associated with chronic inflammation. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. This endothelial-derived signature predicted outcome independently of, but cooperatively with, standard clinical and pathological prognostic factors. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells.This study provides the first prognostic cancer gene signature derived from an experimental model of tumor-associated endothelial inflammation. These findings support the notion that activation of inflammatory pathways in non-malignant tumor-infiltrating endothelial cells contributes to tumor growth and progression in multiple human cancers. Importantly, these results identify endothelial-derived factors that could serve as potential targets for therapy in diverse human cancers.

  14. Arecoline is cytotoxic for human endothelial cells.

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    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. SIRT1 regulates endothelial Notch signaling in lung cancer.

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    Mian Xie

    Full Text Available BACKGROUND: Sirtuin 1 (SIRT1 acts as a key regulator of vascular endothelial homeostasis, angiogenesis, and endothelial dysfunction. However, the underlying mechanism for SIRT1-mediated lung carcinoma angiogenesis remains unknown. Herein, we report that the nicotinamide adenine dinucleotide 1 (NAD1-dependent deacetylase SIRT1 can function as an intrinsic negative modulator of Delta-like ligand 4 (DLL4/Notch signaling in Lewis lung carcinoma (LLC xenograft-derived vascular endothelial cells (lung cancer-derived ECs. PRINCIPAL FINDINGS: SIRT1 negatively regulates Notch1 intracellular domain (N1IC and Notch1 target genes HEY1 and HEY2 in response to Delta-like ligand 4 (DLL4 stimulation. Furthermore, SIRT1 deacetylated and repressed N1IC expression. Quantitative chromatin immunoprecipitation (qChIP analysis and gene reporter assay demonstrated that SIRT1 bound to one highly conserved region, which was located at approximately -500 bp upstream of the transcriptional start site of Notch1,and repressed Notch1 transcription. Inhibition of endothelial cell growth and sprouting angiogenesis by DLL4/Notch signaling was enhanced in SIRT1-silenced lung cancer-derived EC and rescued by Notch inhibitor DAPT. In vivo, an increase in proangiogenic activity was observed in Matrigel plugs from endothelial-specific SIRT1 knock-in mice. SIRT1 also enhanced tumor neovascularization and tumor growth of LLC xenografts. CONCLUSIONS: Our results show that SIRT1 facilitates endothelial cell branching and proliferation to increase vessel density and promote lung tumor growth through down-regulation of DLL4/Notch signaling and deacetylation of N1IC. Thus, targeting SIRT1 activity or/and gene expression may represent a novel mechanism in the treatment of lung cancer.

  16. Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes

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    Sarah Menzel

    2017-01-01

    Full Text Available In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

  17. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

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    Schmidt, Eric P; Yang, Yimu; Janssen, William J; Gandjeva, Aneta; Perez, Mario J; Barthel, Lea; Zemans, Rachel L; Bowman, Joel C; Koyanagi, Dan E; Yunt, Zulma X; Smith, Lynelle P; Cheng, Sara S; Overdier, Katherine H; Thompson, Kathy R; Geraci, Mark W; Douglas, Ivor S; Pearse, David B; Tuder, Rubin M

    2013-01-01

    Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α–responsive, heparan sulfate–specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue. PMID:22820644

  18. 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.

  19. Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

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    Sands, Michelle

    2011-01-25

    Abstract Background Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF) family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF) and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium. Methods Male Sprague Dawley rats were exposed to conditions of normoxia (21% O2) or hypoxia (10% O2) for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA), VEGFB, placenta growth factor (PlGF), VEGF receptor 1 (VEGFR1) and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay. Results Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic. Conclusions VEGFB and PlGF can either inhibit or potentiate the

  20. Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

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    McLoughlin Paul

    2011-01-01

    Full Text Available Abstract Background Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium. Methods Male Sprague Dawley rats were exposed to conditions of normoxia (21% O2 or hypoxia (10% O2 for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA, VEGFB, placenta growth factor (PlGF, VEGF receptor 1 (VEGFR1 and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay. Results Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic. Conclusions VEGFB and PlGF can either inhibit or

  1. Effects of Parietaria judaica pollen extract on human microvascular endothelial cells.

    Science.gov (United States)

    Taverna, Simona; Flugy, Anna; Colomba, Paolo; Barranca, Marilisa; De Leo, Giacomo; Alessandro, Riccardo

    2008-08-08

    Pollinosis from Parietaria judaica is one of the main causes of allergy in the Mediterranean area. The present study is designed to assess if P. judaica pollens contain bioactive compounds able to elicit a functional response in endothelial cells. We have demonstrated that addition of pollen extract to human lung microvascular endothelial cells (HMVEC-L) induces a modification of cell morphology, actin cytoskeletal rearrangements and an increase in endothelial cell permeability. We further showed that the treatment of endothelial cells with pollen extract causes an increase of E-selectin and VCAM-1 protein levels as well as an increase of IL-8 production. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of polymorphonuclear cells (PMNs) to HMVEC-L monolayer. Our results suggest for the first time that pollen affect directly endothelial cells (EC) modulating critical functions related to the inflammatory response.

  2. FIBROBLASTS, EPITHELIAL-CELLS, ENDOTHELIAL-CELLS AND SMOOTH-MUSCLE CELLS ARE MAJOR TARGETS OF HUMAN CYTOMEGALOVIRUS-INFECTION IN LUNG AND GASTROINTESTINAL TISSUES

    NARCIS (Netherlands)

    SINZGER, C; GREFTE, A; PLACHTER, B; GOUW, ASH; THE, TH; JAHN, G

    High titre replication of human cytomegalovirus (HCMV) in cell culture is restricted to primary human fibroblasts. During acute infection in vivo, HCMV nucleic acids and antigens have been found in various organs. Using only morphological criteria, inconsistent data have been reported about the cell

  3. Heterologous corneal endothelial cell transplantation--human corneal endothelial cell transplantation in Lewis rats.

    OpenAIRE

    Tchah, H.

    1992-01-01

    A heterologous corneal endothelial transplantation was attempted using human endothelial cells and a Lewis rat penetrating keratoplasty model. Cultured human endothelial cells were seeded to a Lewis rat cornea, which was denuded of its endothelium. When grafted into the syngeneic Lewis rat, the graft remained clear for at least five days, and then became opaque and edematous because of immune rejection reaction. In contrast, corneas denuded of their endothelium became opaque and edematous imm...

  4. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

    Science.gov (United States)

    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

    2016-12-15

    A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage

  5. Overexpression of both platelet-derived growth factor-BB and vascular endothelial growth factor-C and its association with lymphangiogenesis in primary human non-small cell lung cancer.

    Science.gov (United States)

    Liu, Jiannan; Liu, Chuanyong; Qiu, Liyun; Li, Juan; Zhang, Pei; Sun, Yuping

    2014-06-27

    Metastatic spread of tumor through lymphatic vasculature is an important adverse prognostic factor in a variety of human cancer and tumor lymphangiogenesis requires the interplay of several growth factors. Platelet-derived growth factor (PDGF)-BB and vascular endothelial growth factor (VEGF)-C are two important molecules involving in tumor metastasis and lymphangiogenesis. Therefore, the aim of this study was to investigate the coexpression of PDGF-BB and VEGF-C in primary human non-small cell lung cancer (NSCLC) and its association with lymphangiogenesis. Using immunohistochemical staining, PDGF-BB and VEGF-C expression were detected in 109 primary NSCLC tissues, while the lymphatic micro-vessel density (LMVD) was counted. Of 109 cases, PDGF-BB and VEGF-C overexpression was 66.97% (73/109) and 65.14% (71/109), respectively. 52 (47.7%) had overexpression of both PDGF-BB and VEGF-C (P+V+), 21 (19.3%) overexpression of PDGF-BB but low expression of VEGF-C (P+V-), 19(17.4%) overexpression of VEGF-C but low expression of PDGF-BB (P-V+) and 17(15.6%) low expression of both PDGF-BB and VEGF-C (P-V-). PDGF-BB expression was positively related to that of VEGF-C (r=0.451, p=0.034). LMVD in cases with P+V+was much higher than those with P-V- (p=0.004). In addition, the patients with P+V+were younger and also had larger tumor size, more likely lymph node metastasis and worse histological differentiation than those with P-V-. Moreover, the overall survival (OS) of patients with P+V+was shorter than those with P-V- (p=0.015). Coexpression of both PDGF-BB and VEGF-C was associated with lymphangiogenesis and poor prognosis in NSCLC, and might play a critical role in NSCLC progression. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2261801312571320.

  6. Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Hsuan [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Pan, Shiow-Lin; Wang, Jing-Chi; Teng, Che-Ming [National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Kuo, Sung-Hsin [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Department of Internal Medicine, Taipei (China); Cheng, Jason Chia-Hsien [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Graduate Institute of Clinical Medicine, Taipei (China)

    2014-12-15

    The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation. (orig.) [German] Die vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschliessend die endothelialen Zellen beeinflusst. Die durch Strahlentherapie induzierte Mikrolymphgefaessdichte (MLVD) im Tumor wurde in

  7. Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides.

    Science.gov (United States)

    Schweitzer, Kelly S; Hatoum, Hadi; Brown, Mary Beth; Gupta, Mehak; Justice, Matthew J; Beteck, Besem; Van Demark, Mary; Gu, Yuan; Presson, Robert G; Hubbard, Walter C; Petrache, Irina

    2011-12-01

    The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione

  8. Opposite effects of ANP receptors in attenuation of LPS-induced endothelial permeability and lung injury.

    Science.gov (United States)

    Xing, Junjie; Yakubov, Bakhtiyor; Poroyko, Valeriy; Birukova, Anna A

    2012-03-01

    Atrial natriuretic peptide (ANP) has been recently identified as a modulator of acute lung injury (ALI) induced by pro-inflammatory agonists. While previous studies tested effects of exogenous ANP administration, the role of endogenous ANP in the course of ALI remains unexplored. This study examined regulation of ANP and its receptors NPR-A, NPR-B and NPR-C by LPS and involvement of ANP receptors in the modulation of LPS-induced lung injury. Primary cultures of human pulmonary endothelial cells (EC) were used in the in vitro tests. Expression of ANP and its receptors was determined by quantitative RT-PCR analysis. Agonist-induced cytoskeletal remodeling was evaluated by immunofluorescence staining, and EC barrier function was characterized by measurements of transendothelial electrical resistance. In the murine model of ALI, LPS-induced lung injury was assessed by measurements of protein concentration and cell count in bronchoalveolar lavage fluid (BAL). LPS stimulation significantly increased mRNA expression levels of ANP and NPR-A in pulmonary EC. Pharmacological inhibition of NPR-A augmented LPS-induced EC permeability and blocked barrier protective effects of exogenous ANP on LPS-induced intercellular gap formation. In contrast, pharmacological inhibition of ANP clearance receptor NPR-C significantly attenuated LPS-induced barrier disruptive effects. Administration of NPR-A inhibitor in vivo exacerbated LPS-induced lung injury, whereas inhibition of NPR-C suppressed LPS-induced increases in BAL cell count and protein content. These results demonstrate for the first time opposite effects of NPR-A and NPR-C in the modulation of ALI and suggest a compensatory protective mechanism of endogenous ANP in the maintenance of lung vascular permeability in ALI. Copyright © 2011. Published by Elsevier Inc.

  9. Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels

    Directory of Open Access Journals (Sweden)

    Barleon Bernhard

    2010-07-01

    Full Text Available Abstract Background Postnatal endothelial progenitor cells (EPCs have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. Results In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels. Mouse lung microvascular endothelial cells (MLMVECs were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony. These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. Conclusion The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

  10. 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.

  11. Selective expression of erg isoforms in human endothelial cells.

    Science.gov (United States)

    Hewett, P W; Nishi, K; Daft, E L; Clifford Murray, J

    2001-04-01

    Erg and Fli-1 are closely related members of the ets family of transcription factors. There are at least five human Erg isoforms (Erg-1, Erg-2, Erg-3/p55(Erg), p49(Erg) and p38(Erg)) produced through differential mRNA splicing and alternative use of translational start codons. However, relatively little is known about the expression or function of these isoforms in vitro or their distribution in vivo. We used RT-PCR to screen a panel of primary and established human cell lines for erg and fli-1 consensus sequences. Whilst fli-1 was expressed in several human cell types, erg was detected mainly in endothelial cells. To identify which erg isoforms are expressed in endothelial cells we used RT-PCR, Northern blotting and 5'-RACE. Erg-3/p55(Erg) and p38(Erg)/p38(Erg)-like transcripts were detected in both microvascular and large vessel endothelial cells affinity-purified from different vascular beds. Moreover, these erg isoforms were present in both freshly isolated, and confluent endothelial cells following several passages in culture, indicating that endothelial erg expression in vitro may be broadly representative of that in vivo. The selective expression of the Erg-3/p55(Erg) and p38(Erg)/p38(Erg)-like isoforms in endothelial cells indicates their involvement in the regulation of endothelial-restricted genes.

  12. Heat Shock Protein A12B Protects Vascular Endothelial Cells Against Sepsis-Induced Acute Lung Injury in Mice

    Directory of Open Access Journals (Sweden)

    Yi Chen

    2017-05-01

    Full Text Available Background: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI during sepsis. Heat shock protein A12B (HSPA12B is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. Methods: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15: a sham operation group, a cecal ligation and puncture (CLP group, a HSPA12B siRNA-CLP group and a negative control (NC siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL-1β, tumor necrosis factor (TNF-α, and IL-6 and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. Results: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e

  13. Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.

    Science.gov (United States)

    Seedorf, Gregory; Metoxen, Alexander J; Rock, Robert; Markham, Neil; Ryan, Sharon; Vu, Thiennu; Abman, Steven H

    2016-06-01

    Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD. Copyright © 2016 the American Physiological Society.

  14. Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

    Directory of Open Access Journals (Sweden)

    Li Cui

    2014-06-01

    Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

  15. A Biohybrid Artificial Lung Prototype With Active Mixing of Endothelialized Microporous Hollow Fibers

    Science.gov (United States)

    Polk, Alexa A.; Maul, Timothy M.; McKeel, Daniel T.; Snyder, Trevor A.; Lehocky, Craig A.; Pitt, Bruce; Stolz, Donna Beer; Federspiel, William J.; Wagner, William R.

    2014-01-01

    Acute respiratory distress syndrome (ARDS) affects nearly 150,000 patients per year in the US, and is associated with high mortality (≈40%) and suboptimal options for patient care. Mechanical ventilation and extracorporeal membrane oxygenation are limited to short-term use due to ventilator-induced lung injury and poor bio-compatibility, respectively. In this report, we describe the development of a biohybrid lung prototype, employing a rotating endothelialized microporous hollow fiber (MHF) bundle to improve blood biocompatibility while MHF mixing could contribute to gas transfer efficiency. MHFs were surface modified with radio frequency glow discharge (RFGD) and protein adsorption to promote endothelial cell (EC) attachment and growth. The MHF bundles were placed in the biohybrid lung prototype and rotated up to 1,500 revolutions per minute (rpm) using speed ramping protocols to condition ECs to remain adherent on the fibers. Oxygen transfer, thrombotic deposition, and EC p-selectin expression were evaluated as indicators of biohybrid lung functionality and biocompatibility. A fixed aliquot of blood in contact with MHF bundles rotated at either 250 or 750 rpm reached saturating pO2 levels more quickly with increased rpm, supporting the concept that fiber rotation would positively contribute to oxygen transfer. The presence of ECs had no effect on the rate of oxygen transfer at lower fiber rpm, but did provide some resistance with increased rpm when the overall rate of mass transfer was higher due to active mixing. RFGD followed by fibronectin adsorption on MHFs facilitated near confluent EC coverage with minimal p-selectin expression under both normoxic and hyperoxic conditions. Indeed, even subconfluent EC coverage on MHFs significantly reduced thrombotic deposition adding further support that endothelialization enhances, blood biocompatibility. Overall these findings demonstrate a proof-of-concept that a rotating endothelialized MHF bundle enhances gas

  16. Cannabinoids inhibit angiogenic capacities of endothelial cells via release of tissue inhibitor of matrix metalloproteinases-1 from lung cancer cells.

    Science.gov (United States)

    Ramer, Robert; Fischer, Sascha; Haustein, Maria; Manda, Katrin; Hinz, Burkhard

    2014-09-15

    Cannabinoids inhibit tumor neovascularization as part of their tumorregressive action. However, the underlying mechanism is still under debate. In the present study the impact of cannabinoids on potential tumor-to-endothelial cell communication conferring anti-angiogenesis was studied. Cellular behavior of human umbilical vein endothelial cells (HUVEC) associated with angiogenesis was evaluated by Boyden chamber, two-dimensional tube formation and fibrin bead assay, with the latter assessing three-dimensional sprout formation. Viability was quantified by the WST-1 test. Conditioned media (CM) from A549 lung cancer cells treated with cannabidiol, Δ(9)-tetrahydrocannabinol, R(+)-methanandamide or the CB2 agonist JWH-133 elicited decreased migration as well as tube and sprout formation of HUVEC as compared to CM of vehicle-treated cancer cells. Inhibition of sprout formation was further confirmed for cannabinoid-treated A549 cells co-cultured with HUVEC. Using antagonists to cannabinoid-activated receptors the antimigratory action was shown to be mediated via cannabinoid receptors or transient receptor potential vanilloid 1. SiRNA approaches revealed a cannabinoid-induced expression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) as well as its upstream trigger, the intercellular adhesion molecule-1, to be causally linked to the observed decrease of HUVEC migration. Comparable anti-angiogenic effects were not detected following direct exposure of HUVEC to cannabinoids, but occurred after addition of recombinant TIMP-1 to HUVEC. Finally, antimigratory effects were confirmed for CM of two other cannabinoid-treated lung cancer cell lines (H460 and H358). Collectively, our data suggest a pivotal role of the anti-angiogenic factor TIMP-1 in intercellular tumor-endothelial cell communication resulting in anti-angiogenic features of endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-12-15

    Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

  18. Sustained Adenosine Exposure Causes Lung Endothelial Barrier Dysfunction via Nucleoside Transporter–Mediated Signaling

    Science.gov (United States)

    Newton, Julie; Hsiao, Vivian; Shamirian, Paul; Blackburn, Michael R.; Pedroza, Mesias

    2012-01-01

    Previous studies by our group as well as others have shown that acute adenosine exposure enhances lung vascular endothelial barrier integrity and protects against increased permeability lung edema. In contrast, there is growing evidence that sustained adenosine exposure has detrimental effects on the lungs, including lung edema. It is well established that adenosine modulates lung inflammation. However, little is known concerning the effect of sustained adenosine exposure on lung endothelial cells (ECs), which are critical to the maintenance of the alveolar–capillary barrier. We show that exogenous adenosine plus adenosine deaminase inhibitor caused sustained elevation of adenosine in lung ECs. This sustained adenosine exposure decreased EC barrier function, elevated cellular reactive oxygen species levels, and activated p38, JNK, and RhoA. Inhibition of equilibrative nucleoside transporters (ENTs) prevented sustained adenosine-induced p38 and JNK activation and EC barrier dysfunction. Inhibition of p38, JNK, or RhoA also partially attenuated sustained adenosine-induced EC barrier dysfunction. These data indicate that sustained adenosine exposure causes lung EC barrier dysfunction via ENT-dependent intracellular adenosine uptake and subsequent activation of p38, JNK, and RhoA. The antioxidant N-acetylcysteine and the NADPH inhibitor partially blunted sustained adenosine-induced JNK activation but were ineffective in attenuation of p38 activation or barrier dysfunction. p38 was activated exclusively in mitochondria, whereas JNK was activated in mitochondria and cytoplasm by sustained adenosine exposure. Our data further suggest that sustained adenosine exposure may cause mitochondrial oxidative stress, leading to activation of p38, JNK, and RhoA in mitochondria and resulting in EC barrier dysfunction. PMID:22744860

  19. Caspase-1 Activation Protects Lung Endothelial Barrier Function during Infection-Induced Stress

    Science.gov (United States)

    Alvarez, Diego F.; Housley, Nicole; Koloteva, Anna; Zhou, Chun; O’Donnell, Kristen

    2016-01-01

    Dysregulated activation of the inflammasome–caspase-1–IL-1β axis elicits damaging hyperinflammation during critical illnesses, such as pneumonia and sepsis. However, in critical illness models of Salmonella infection, burn, or shock, caspase-1 inhibition worsens outcomes. These paradoxical effects suggest that caspase-1 drives novel protective responses. Whether the protective effects of caspase-1 activation involve canonical immune cell and/or nonimmune cell responses is unknown. The objective of this study was to test the hypothesis that, in addition to its recognized proinflammatory function, caspase-1 initiates protective stress responses in nonimmune cells. In vivo, lung epithelial and endothelial barrier function and inflammation were assessed in mice infected with Pseudomonas aeruginosa in the presence or absence of a caspase-1 inhibitor. Lung endothelial barrier function was assessed ex vivo in isolated, perfused rat lungs infected with P. aeruginosa in the presence or absence of a caspase-1 inhibitor. Endothelial barrier function during P. aeruginosa infection was assessed in vitro in cultured rat wild-type pulmonary microvascular endothelial cells (PMVECs) or recombinant PMVECs engineered to decrease caspase-1 expression. We demonstrated in vivo that caspase-1 inhibition in P. aeruginosa–infected mice ameliorated hyperinflammation, but, counterintuitively, increased pulmonary edema. Ex vivo, caspase-1 inhibition increased pulmonary permeability in P. aeruginosa–infected isolated rat lungs. To uncouple caspase-1 from its canonical inflammatory role, we used cultured rat PMVECs in vitro and discovered that genetic knockdown of caspase-1 accelerated P. aeruginosa–induced barrier disruption. In conclusion, caspase-1 is a sentinel stress-response regulator that initiates proinflammatory responses and also initiates novel response(s) to protect PMVEC barrier function during pneumonia. PMID:27119735

  20. Proteomic mapping of the lung vascular endothelial cell surface in Schistosoma bovis-infected hamsters.

    Science.gov (United States)

    de la Torre-Escudero, Eduardo; Pérez-Sánchez, Ricardo; Manzano-Román, Raúl; Oleaga, Ana

    2014-06-25

    Schistosomes are blood trematodes that are perfectly adapted to living in their intravascular habitat and to achieve this they have developed mechanisms enabling them to evade the immune and haemostatic responses of the host and to regulate endothelial cell function to favour their own survival. The objective of this work was to analyse the changes induced by Schistosoma bovis schistosomula in the proteome expressed by infected hamsters, over 10 and 20 days, on the endothelial surface of their pulmonary vasculature. To accomplish this, we subjected the lungs of non-infected and S. bovis-infected hamsters to vascular perfusion with a biotin ester reactive. Analysis by liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) of endothelial surface proteins resulted in the identification of a total of 459 non-redundant proteins in the lung vasculature of infected and non-infected hamsters. Here we report the proteins identified, classified according to their biological function and cellular location, further analysing the differences in lung vascular proteomes between non-infected and S. bovis-infected hamsters for ten and twenty days. This work provides the first data on the vascular surface proteome of the lung after S. bovis infection and identifies some of the changes induced in it during infection. To identify the changes induced by schistosomula larvae of Schistosoma bovis in the proteome of the pulmonary vasculature of the host, we compared the proteins expressed on the vascular endothelium of the lungs of non-infected and infected hamsters over 10 and 20 days. Mass spectrometry analysis (LC-MS/MS) of the proteins isolated from the vascular endothelium resulted in the identification of a total of 459 non-redundant proteins in the lung of infected and non-infected hamsters. The proteins identified are classified according to their biological function and cellular location, further analysing the differences in lung vascular proteomes between non

  1. Expansion and cryopreservation of porcine and human corneal endothelial cells.

    Science.gov (United States)

    Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

    2017-08-01

    Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Human Endothelial Cell Models in Biomaterial Research.

    Science.gov (United States)

    Hauser, Sandra; Jung, Friedrich; Pietzsch, Jens

    2017-03-01

    Endothelial cell (EC) models have evolved as important tools in biomaterial research due to ubiquitously occurring interactions between implanted materials and the endothelium. However, screening the available literature has revealed a gap between material scientists and physiologists in terms of their understanding of these biomaterial-endothelium interactions and their relative importance. Consequently, EC models are often applied in nonphysiological experimental setups, or too extensive conclusions are drawn from their results. The question arises whether this might be one reason why, among the many potential biomaterials, only a few have found their way into the clinic. In this review, we provide an overview of established EC models and possible selection criteria to enable researchers to determine the most reliable and relevant EC model to use. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Effect of propionyl-L-carnitine on human endothelial cells

    NARCIS (Netherlands)

    Hinsbergh, V.W.M. van; Scheffer, M.A.

    1991-01-01

    A possible protective effect of propionyl-L-carnitine on human endothelial cells was studied both under basal culture conditions and in the presence of agents capable of influencing oxidative damage, such as glucose/glucose oxidase and oxidized low-density lipoproteins. Propionyl-L-carnitine had no

  4. Endothelial and lipoprotein lipases in human and mouse placenta

    DEFF Research Database (Denmark)

    Lindegaard, Marie L S; Olivecrona, Gunilla; Christoffersen, Christina

    2005-01-01

    Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharos...

  5. The Effects of Heart-Lung Bypass on Endothelial Function of Patients with Open Heart Surgery

    Directory of Open Access Journals (Sweden)

    Mohammad Borzouee

    2015-06-01

    Full Text Available Background: Endothelium is an essential organ for maintaining an adequate vascular tone and preventing the pathological process of atherosclerosis. Heart-lung machine is a mechanical support for maintenance of blood circulation during open heart surgery. It has been shown that flow of blood through this circuit can induce complement activation, endotoxines production, and release of many inflammatory mediators. Objectives: Cardiopulmonary Bypass (CPB has some detrimental effects on endothelial function. Flow Mediated Dilation (FMD is also a noninvasive method for evaluation of endothelial function. Patients and Methods: This study was conducted on 22 patients who were admitted in cardiac surgery ward for open heart operation and met the inclusion criteria of the study. Brachial artery FMD was measured the day before and 2 days after CPB. Results: The mean duration of CPB was 62.95 minutes. The mean percent of FMD changes was measured and compared before and after CPB (4.29 and 0.03 vs. 1.38 and 1.99, < 0.001. The results showed a significant relationship between CPB and bronchial endothelial function. Conclusions: This study revealed the influence of CPB on endothelial function. Yet, more studies are necessary to confirm this important issue, and decline in use of CPB is appreciated.

  6. High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

    Science.gov (United States)

    Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A

    2011-05-01

    Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.

  7. CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

    Directory of Open Access Journals (Sweden)

    Ifeanyi U Anidi

    Full Text Available Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS. To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our

  8. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

    Science.gov (United States)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

    1983-11-01

    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  9. Ponatinib reduces viability, migration, and functionality of human endothelial cells.

    Science.gov (United States)

    Gover-Proaktor, Ayala; Granot, Galit; Shapira, Saar; Raz, Oshrat; Pasvolsky, Oren; Nagler, Arnon; Lev, Dorit L; Inbal, Aida; Lubin, Ido; Raanani, Pia; Leader, Avi

    2017-06-01

    Tyrosine kinase inhibitors (TKIs) have revolutionized the prognosis of chronic myeloid leukemia. With the advent of highly efficacious therapy, the focus has shifted toward managing TKI adverse effects, such as vascular adverse events (VAEs). We used an in vitro angiogenesis model to investigate the TKI-associated VAEs. Our data show that imatinib, nilotinib, and ponatinib reduce human umbilical vein endothelial cells (HUVECs) viability. Pharmacological concentrations of ponatinib induced apoptosis, reduced migration, inhibited tube formation of HUVECs, and had a negative effect on endothelial progenitor cell (EPC) function. Furthermore, in HUVECs transfected with VEGF receptor 2 (VEGFR2), the effect of ponatinib on tube formation and on all parameters representing normal endothelial cell function was less prominent than in control cells. This is the first report regarding the pathogenesis of ponatinib-associated VAEs. The antiangiogenic effect of ponatinib, possibly mediated by VEGFR2 inhibition, as shown in our study, is another piece in the intricate puzzle of TKI-associated VAEs.

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

    Science.gov (United States)

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

  11. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    Science.gov (United States)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular pathophysiology associated with diabetic microangiopathy.

  12. 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.

  13. Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

    Science.gov (United States)

    Stratmann, Bernd; Engelbrecht, Britta; Espelage, Britta C; Klusmeier, Nadine; Tiemann, Janina; Gawlowski, Thomas; Mattern, Yvonne; Eisenacher, Martin; Meyer, Helmut E; Rabbani, Naila; Thornalley, Paul J; Tschoepe, Diethelm; Poschmann, Gereon; Stühler, Kai

    2016-11-29

    Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

  14. High glucose induced endothelial to mesenchymal transition in human umbilical vein endothelial cell.

    Science.gov (United States)

    Yu, Chun-Hong; Suriguga; Gong, Meng; Liu, Wen-Juan; Cui, Ning-Xuan; Wang, Ying; Du, Xin; Yi, Zong-Chun

    2017-06-01

    Studies have shown that endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis. The aim of this study was to investigate the influence of high glucose and related mechanism of MAPK inhibitor or specific antioxidant on the EndMT. In vitro human umbilical vein endothelial cells (HUVEC) were cultured with 11mM, 30mM, 60mM and 120mM glucose for 0, 24, 48, 72 and 168h. Endothelial cell morphology was observed with microscope, and RT-PCR was used to detect mRNA expression of endothelial markers VE-cadherin and CD31, mesenchymal markers α-SMA and collagen I, and transforming growth factor TGF-β1. Immunofluorescence staining was performed to detect the expression of CD31 and α-SMA. The concentration of TGF-β1 in the supernatant was detected by ELISA. ERK1/2 phosphorylation level was detected by Western blot analysis. High glucose induced EndMT and increased the TGF-β1 level in HUVEC cells. Cells in high glucose for 7 days showed a significant decrease in mRNA expression of CD31 and VE-cadherin, and a significant increase in that of α-SMA and collagen I, while lost CD31 staining and acquired α-SMA staining. ERK signaling pathway blocker PD98059 significantly attenuated the high glucose-induced increase in the ERK1/2 phosphorylation level. PD98059 and NAC both inhibited high glucose-induced TGF-β1 expression and attenuated EndMT marker protein synthesis. High glucose could induce HUVEC cells to undergo EndMT. NAC and ERK signaling pathway may play important role in the regulation of the TGF-β1 biosynthesis during high glucose-induced EndMT. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Dysregulation of Vascular Endothelial Progenitor Cells Lung-Homing in Subjects with COPD

    Directory of Open Access Journals (Sweden)

    Brittany M. Salter

    2016-01-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is characterized by fixed airflow limitation and progressive decline of lung function and punctuated by occasional exacerbations. The disease pathogenesis may involve activation of the bone marrow stimulating mobilization and lung-homing of progenitor cells. We investigated the hypothesis that lower circulating numbers of vascular endothelial progenitor cells (VEPCs are a consequence of increased lung-sequestration in COPD. Nonatopic, current or ex-smokers with diagnosed COPD and nonatopic, nonsmoking normal controls were enrolled. Blood and induced sputum extracted primitive hemopoietic progenitors (HPCs and VEPC were enumerated by flow cytometry. Migration and adhesive responses to fibronectin were assessed. In sputum, VEPC numbers were significantly greater in COPD compared to normal controls. In blood, VEPCs were significantly lower in COPD versus normal controls. There were no differences in HPC levels between the two groups in either compartment. Functionally, there was a greater migrational responsiveness of progenitors from COPD subjects to stromal cell-derived factor-1alpha (SDF-1α compared to normal controls. This was associated with greater numbers of CXCR4+ progenitors in sputum from COPD. Increased migrational responsiveness of progenitor cells may promote lung-homing of VEPC in COPD which may disrupt maintenance and repair of the airways and contribute to COPD disease pathogenesis.

  16. Endothelial and lipoprotein lipases in human and mouse placenta

    DEFF Research Database (Denmark)

    Lindegaard, Marie Louise Skakkebæk; Olivecrona, Gunilla; Christoffersen, Christina

    2005-01-01

    Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin...... protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse...... placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta....

  17. 3D map of the human corneal endothelial cell

    OpenAIRE

    Zhiguo He; Fabien Forest; Philippe Gain; Damien Rageade; Aurélien Bernard; Sophie Acquart; Michel Peoc’h; Dennis M. Defoe; Gilles Thuret

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexa...

  18. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    Science.gov (United States)

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

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

    Science.gov (United States)

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

    2017-11-01

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

  20. Vascular endothelial growth factor (VEGF) expression in the lung in toxic septic shock.

    Science.gov (United States)

    Manoilescu, Irina; Teleman, S; Cojocaru, Elena; Mihăilă, Doina; Plămădeală, P

    2011-01-01

    The need for reasoning with medical evidence the different types of shock, especially when there are medical and legal implications, has determined the search of biological markers of the shock. In the case of toxic septic shock, the most important markers to be used are: the cytokines, the tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6), procalcitonin, lactoferin and the vascular endothelial growth factor (VEGF). VEGF has an essential role in angiogenesis and vascular permeability. In our study group, we included 30 cases of different types of shock in which we studied the VEGF expression in the lungs. We added also 10 fragments of lung as control group. According to the etiology, the 30 cases of shock were: 15 with a toxic septic shock and 15 with a hemorrhagic shock. In all these cases we used the classical Hematoxylin and Eosin staining method and the immunohistochemical reactions for VEGF-A. Statistical analysis was performed using SPSS 13.0. The VEGF expression was decreased in all the cases of toxic septic shock, in the endothelium and also in the alveolar epithelium, compared to a high level of expression in other cases of shock and in the control lung. These data allow us to appreciate that VEGF has a different expression in different types of shock and in the normal lung. We observed a statistically significant difference between VEGF expression in toxic septic shock and hemorrhagic shock (p=0.000001). There is a similarity of VEGF expression between hemorrhagic shock and the control lungs (p=0.00001). An obviously low VEGF expression in the toxic septic shock represents a useful biological marker in the forensic medical cases.

  1. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    Science.gov (United States)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P pathophysiology associated with diabetic microangiopathy.

  2. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    Science.gov (United States)

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-09

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

  3. 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......, nano-scale 3D-imaging using focused ion beam/scanning electron microscopy (FIB/SEM), and single particle inductively coupled plasma-mass spectrometry (spICP-MS). HUVECs were cultured for 3 h or 24 h in medium with AuNPs in a concentration range of 1.25–10 μg ml−1. There was a concentration...

  4. Hemorrhagic shock activation of NLRP3 inflammasome in lung endothelial cells.

    Science.gov (United States)

    Xiang, Meng; Shi, Xiaolian; Li, Yuehua; Xu, Jia; Yin, Lianhua; Xiao, Guozhi; Scott, Melanie J; Billiar, Timothy R; Wilson, Mark A; Fan, Jie

    2011-11-01

    Hemorrhagic shock (HS) due to major trauma and surgery predisposes the host to the development of systemic inflammatory response syndrome (SIRS), including acute lung injury (ALI), through activating and exaggerating the innate immune response. IL-1β is a crucial proinflammatory cytokine that contributes to the development of SIRS and ALI. Lung endothelial cells (EC) are one important source of IL-1β, and the production of active IL-1β is controlled by the inflammasome. In this study, we addressed the mechanism underlying HS activation of the inflammasome in lung EC. We show that high mobility group box 1 acting through TLR4, and a synergistic collaboration with TLR2 and receptor for advanced glycation end products signaling, mediates HS-induced activation of EC NAD(P)H oxidase. In turn, reactive oxygen species derived from NAD(P)H oxidase promote the association of thioredoxin-interacting protein with the nucleotide-binding oligomerization domain-like receptor protein NLRP3 and subsequently induce inflammasome activation and IL-1β secretion from the EC. We also show that neutrophil-derived reactive oxygen species play a role in enhancing EC NAD(P)H oxidase activation and therefore an amplified inflammasome activation in response to HS. The present study explores a novel mechanism underlying HS activation of EC inflammasome and thus presents a potential therapeutic target for SIRS and ALI induced after HS.

  5. Increased Rho-kinase expression and activity and pulmonary endothelial dysfunction in smokers with normal lung function.

    Science.gov (United States)

    Duong-Quy, S; Dao, P; Hua-Huy, T; Guilluy, C; Pacaud, P; Dinh-Xuan, A T

    2011-02-01

    Endothelial dysfunction is one of the main consequences of the toxic effects of cigarette smoke on the vascular system. Increasing evidence suggests that the small G-protein RhoA and its downstream effectors, the Rho-kinases (ROCKs), are involved in systemic endothelial dysfunction induced by cigarette smoke. This study aimed to evaluate the role of the RhoA/ROCKs pathway in pulmonary artery endothelial function in current smokers with normal lung function. Lung tissues were obtained from nonsmokers and smokers who underwent lobectomy for lung carcinoma. Arterial relaxation in response to acetylcholine (ACh) was assessed in isolated pulmonary arterial rings. Protein expressions and activities of endothelial nitric oxide synthase (eNOS), ROCKs and the myosin phosphatase subunit 1 (MYPT-1) were sought. Relaxation in response to ACh was significantly lower in smokers as compared with nonsmokers (n = 8 in each group), consistent with reduced eNOS activity in the former compared with the latter. eNOS protein expression remained, however, the same in both groups. Expression of ROCKs, guanosine triphosphate-RhoA and phosphorylated MYPT-1 were significantly increased in smokers compared with controls. Pulmonary endothelial dysfunction is present in smokers whose lung function has not yet been impaired. Reduced activity of eNOS accounts at least in part for this endothelial dysfunction. Increased expression and activity of ROCKs accounts for another part through direct or indirect inhibition of the Rho-A/ROCKs pathway on nitric oxide synthesis and sustained pulmonary vasoconstriction through inhibition of myosin phosphatase.

  6. In vitro analysis of human periodontal microvascular endothelial cells.

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    Tsubokawa, Mizuki; Sato, Soh

    2014-08-01

    Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites

  7. Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

    Science.gov (United States)

    Kusuma, Rio Jati; Manca, Sonia; Friemel, Taylor; Sukreet, Sonal; Nguyen, Christopher; Zempleni, Janos

    2016-05-15

    Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues. Copyright © 2016 the American Physiological Society.

  8. Experimental sleep restriction causes endothelial dysfunction in healthy humans.

    Science.gov (United States)

    Calvin, Andrew D; Covassin, Naima; Kremers, Walter K; Adachi, Taro; Macedo, Paula; Albuquerque, Felipe N; Bukartyk, Jan; Davison, Diane E; Levine, James A; Singh, Prachi; Wang, Shihan; Somers, Virend K

    2014-11-25

    Epidemiologic evidence suggests a link between short sleep duration and cardiovascular risk, although the nature of any relationship and mechanisms remain unclear. Short sleep duration has also been linked to an increase in cardiovascular events. Endothelial dysfunction has itself been implicated as a mediator of heightened cardiovascular risk. We sought to determine the effect of 8 days/8 nights of partial sleep restriction on endothelial function in healthy humans. Sixteen healthy volunteers underwent a randomized study of usual sleep versus sleep restriction of two-thirds normal sleep time for 8 days/8 nights in a hospital-based clinical research unit. The main outcome was endothelial function measured by flow-mediated brachial artery vasodilatation (FMD). Those randomized to sleep restriction slept 5.1 hours/night during the experimental period compared with 6.9 hours/night in the control group. Sleep restriction was associated with significant impairment in FMD (8.6±4.6% during the initial pre-randomization acclimation phase versus 5.2±3.4% during the randomized experimental phase, P=0.01) whereas no change was seen in the control group (5.0±3.0 during the acclimation phase versus 6.73±2.9% during the experimental phase, P=0.10) for a between-groups difference of -4.40% (95% CI -7.00 to -1.81%, P=0.003). No change was seen in non-flow mediated vasodilatation (NFMD) in either group. In healthy individuals, moderate sleep restriction causes endothelial dysfunction. ClinicalTrials.gov. Unique identifier: NCT01334788. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  9. MALDI profiling of human lung cancer subtypes.

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    Angelo Gámez-Pozo

    Full Text Available BACKGROUND: Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree-based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non-small cell lung cancer histological subtypes. CONCLUSIONS/SIGNIFICANCE: A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer.

  10. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells▿

    OpenAIRE

    Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

    2011-01-01

    The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 μg/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barri...

  11. Epidermal Growth Factor Rescues Endothelial Dysfunction in Primary Human Tissues In Vitro.

    Science.gov (United States)

    Hastie, Roxanne; Tong, Stephen; Hannan, Natalie J; Brownfoot, Fiona; Cannon, Ping; Kaitu'u-Lino, Tu'uhevaha J

    2017-09-01

    Preeclampsia is a hypertensive disorder of pregnancy, responsible for over 60 000 maternal deaths annually. Endothelial dysfunction is a central aspect to its pathophysiology, and currently, no medical therapeutic is available for its treatment. In this study, we aim to investigate the effect of epidermal growth factor (EGF) on endothelial dysfunction using primary human tissues. We performed a number of in vitro assays that mimic the vascular endothelial dysfunction that occurs in preeclampsia. Epidermal growth factor reduced the expression of vascular cell adhesion molecule-1, a marker of endothelial dysfunction, after insult with tumor necrosis factor α (TNF-α) or serum from women with preeclampsia. Additionally, after TNF-α insult, EGF reduced tube disruption and the adhesion of monocytes to primary human umbilical vein endothelial cells (HUVECs). Our findings suggest that EGF reduces endothelial dysfunction in primary HUVECs. Epidermal growth factor may have potential as a novel peptide treatment for preeclampsia and other diseases where there is endothelial dysfunction.

  12. Cocaine enhances HIV-1 gp120-induced lymphatic endothelial dysfunction in the lung.

    Science.gov (United States)

    Zhang, Xuefeng; Jiang, Susan; Yu, Jinlong; Kuzontkoski, Paula M; Groopman, Jerome E

    2015-08-01

    Pulmonary complications are common in both AIDS patients and cocaine users. We addressed the cellular and molecular mechanisms by which HIV and cocaine may partner to induce their deleterious effects. Using primary lung lymphatic endothelial cells (L-LECs), we examined how cocaine and HIV-1 gp120, alone and together, modulate signaling and functional properties of L-LECs. We found that brief cocaine exposure activated paxillin and induced cytoskeletal rearrangement, while sustained exposure increased fibronectin (FN) expression, decreased Robo4 expression, and enhanced the permeability of L-LEC monolayers. Moreover, incubating L-LECs with both cocaine and HIV-1 gp120 exacerbated hyperpermeability, significantly enhanced apoptosis, and further impaired in vitro wound healing as compared with cocaine alone. Our studies also suggested that the sigma-1 receptor (Sigma-1R) and the dopamine-4 receptor (D4R) are involved in cocaine-induced pathology in L-LECs. Seeking clinical correlation, we found that FN levels in sera and lung tissue of HIV(+) donors were significantly elevated as compared to HIV(-) donors. Our in vitro data demonstrate that cocaine and HIV-1 gp120 induce dysfunction and damage of lung lymphatics, and suggest that cocaine use may exacerbate pulmonary edema and fibrosis associated with HIV infection. Continued exploration of the interplay between cocaine and HIV should assist the design of therapeutics to ameliorate HIV-induced pulmonary disorders within the drug using population. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  13. Shear stress mediates endothelial adaptations to exercise training in humans.

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    Tinken, Toni M; Thijssen, Dick H J; Hopkins, Nicola; Dawson, Ellen A; Cable, N Timothy; Green, Daniel J

    2010-02-01

    Although episodic changes in shear stress have been proposed as the mechanism responsible for the effects of exercise training on the vasculature, this hypothesis has not been directly addressed in humans. We examined brachial artery flow-mediated dilation, an index of NO-mediated endothelial function, in healthy men in response to an acute bout of handgrip exercise and across an 8-week period of bilateral handgrip training. Shear stress responses were attenuated in one arm by cuff inflation to 60 mm Hg. Similar increases were observed in grip strength and forearm volume and girth in both limbs. Acute bouts of handgrip exercise increased shear rate (P<0.005) and flow-mediated dilation percentage (P<0.05) in the uncuffed limb, whereas no changes were evident in the cuffed arm. Handgrip training increased flow-mediated dilation percentage in the noncuffed limb at weeks 2, 4, and 6 (P<0.001), whereas no changes were observed in the cuffed arm. Brachial artery peak reactive hyperemia, an index of resistance artery remodeling, progressively increased with training in the noncuffed limb (P<0.001 and 0.004); no changes were evident in the cuffed arm. Neither acute nor chronic shear manipulation during exercise influenced endothelium-independent glyceryl trinitrate responses. These results demonstrate that exercise-induced changes in shear provide the principal physiological stimulus to adaptation in flow-mediated endothelial function and vascular remodeling in response to exercise training in healthy humans.

  14. Fourier transform method to determine human corneal endothelial morphology

    Science.gov (United States)

    Masters, Barry R.; Lee, Yim-Kul; Rhodes, William T.

    1991-08-01

    The statistical evaluation of the size, shape, density and regularity of the cells in the human corneal endothelium is an important diagnostic technique. A method based on the Fourier transform of the cell boundaries was developed which can yield these statistical properties. The development of a hybrid optical/digital technique to obtain these statistical perimeters is our goal. The input images were tracings of human endothelial cell patterns. The optical Fourier transform of each image was obtained, and the radial projection and the angular correlation function were plotted versus distance and angle respectively. The average size of the cells was obtained from the first peak of the radial projection. The width of this peak is related to the coefficient of variation of the average cell size. The separation of the peaks in the normalized angular correlation plot is related to cell shape. This method is suitable for rapid analysis of large numbers of endothelial cell images. This technique may have potential for diagnostic ophthalmology.

  15. Transfusion-related acute lung injury: critical neutrophil activation by anti-HLA-A2 antibodies for endothelial permeability.

    Science.gov (United States)

    Khoy, Kathy; Nguyen, Minh Vu Chuong; Masson, Dominique; Bardy, Béatrice; Drouet, Christian; Paclet, Marie-Hélène

    2017-07-01

    Transfusion-related acute lung injury (TRALI) is a major complication of hemotherapy that may occur after the transfusion of any blood type component. Several clinical reports have suggested the presence of anti-HLA antibodies in the blood product. This study sought to examine the role of anti-HLA-A2 antibodies in polymorphonuclear neutrophil (PMN) activation and thus in endothelial permeability. PMN activation was assessed by both nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activity and reactive oxygen species (ROS) production. A coculture assay of EA.hy926 endothelial cells with PMNs or differentiated-PLB-985 cells, a model of neutrophil-like cells, was performed to estimate the impact of ROS on endothelial permeability. Anti-HLA-A2 antibodies significantly increased PMN activation, with subsequent endothelial dysfunction. Phagocyte NADPH oxidase (NOX2) activity was shown to be involved in this process and ROS themselves were demonstrated to induce VE-cadherin cleavage and endothelial permeability. Our data may support the existence of a critical anti-HLA-A2 antibody threshold for PMN activation, with NOX2 activity and subsequent endothelial permeability in the two-hit model of TRALI. © 2017 AABB.

  16. Circulating endothelial microparticles involved in lung function decline in a rat exposed in cigarette smoke maybe from apoptotic pulmonary capillary endothelial cells.

    Science.gov (United States)

    Liu, Hua; Ding, Liang; Zhang, Yanju; Ni, Songshi

    2014-06-01

    Plasma levels of endothelial microparticles (EMPs), small membrane vesicles, shed from activated or apoptotic endothelial cells are elevated in patients with COPD and in smokers with normal lung function. Whether plasma EMPs levels are elevated in a rat exposed in cigarette smoke, whether the elevated EMPs derived from pulmonary endothelial cell apoptosis, and the relationship between EMP and lung function are obscure. All 60 wister rats were divided into six groups, three groups of ten rats were exposed to cigarette smoke of ten non-filter cigarettes per day, 5 days a week, using a standard smoking machine (Beijing BeiLanBo Company, China) for a period of 2, 4 and 6 months (n=10, respectively). Age-matched three control groups were sham-smoked. Pulmonary function parameters, including the ratio of forced expiratory volume in 0.3 second over forced vital capacity (FEV0.3/FVC) and dynamic compliance (Cdyn), were tested at the end of each period (2, 4, 6 months). Blood samples were collected and platelet-free plasma was isolated. Then CD42b-/CD31+ EMPs were analysed by flow cytometry. In parallel, lungs were removed and Colocalization with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), Hoeschts and CD31 was performed to evaluate pulmonary capillaries-specific apoptosis and identify the origins of the EMPs. At 2, 4 and 6 months, in comparison with control groups, rats in cigarette smoke exposed groups had a significant increase in CD42b-/CD31+ EMPs (Pfunction indicated that FEV0.3/FVC (Pfunction in the rats exposed cigarette smoke. The increased EMPs may derive from pulmonary capillaries-specific apoptosis.

  17. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells▿

    Science.gov (United States)

    Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

    2011-01-01

    The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 μg/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ≥100 μg/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 μg/ml do not decrease cell viability, intracameral voriconazole concentrations of ≥100 μg/ml may increase the risk of corneal endothelial damage. PMID:21768517

  18. Erythropoietin receptor expression is a potential prognostic factor in human lung adenocarcinoma.

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    Anita Rózsás

    Full Text Available Recombinant human erythropoietins (rHuEPOs are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR signaling in human non-small cell lung cancer (NSCLC also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III-IV adenocarcinoma (ADC and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC proliferation was determined by 5-bromo-2'-deoxy-uridine (BrdU incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.

  19. A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

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    Stephen W Waldo

    Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

  20. Successful transplantation of in vitro expanded human corneal endothelial precursors to corneal endothelial surface using a nanocomposite sheet

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    Parikumar P

    2011-01-01

    Full Text Available Background: Though the transplantation of in vitro expanded human corneal endothelial precursors in animal models of endothelial damage by injecting into the anterior chamber has been reported, the practical difficulties of accomplishing such procedure in human patients have been a hurdle to clinical translation. Here we report the successful transplantation of in vitro expanded human corneal precursor cells to an animal eye using a transparent Nano-composite sheet and their engraftment.Materials and Methods: Human Corneal endothelial cells (HCEC were isolated from human cadaver eyes with informed consent and expanded in the lab using a sphere forming assay in a novel Thermoreversible Gelation Polymer (TGP for 26 days. HCEC obtained by sphere forming assay were seeded in a novel Nano-composite sheet, which was made of PNIPA-NC gels by in-situ, free-radical polymerization of NIPA monomer in the presence of exfoliated clay (synthetic hectorite “Laponite XLG” uniformly dispersed in aqueous media. After a further seven days in vitro culture of HCEC in the Nano-composite sheet, cells were harvested and transplanted on cadaver-bovine eyes (n=3. The cells were injected between the corneal endothelial layer and the Nano-composite sheet that had been placed prior to the injection in close proximity to the endothelial layer. After three hours, the transplanted Nano-composite sheets were removed from the bovine eyes and subjected to microscopic examination. The corneas were subjected to Histo-pathological studies along with controls. Results: HCEC formed sphere like colonies in TGP which expressed relevant markers as confirmed by RT-PCR. Microscopic studies of the Nanosheets and histopathological studies of the cornea of the Bull’s eye revealed that the HCEC got engrafted to the corneal endothelial layer of the bovine eyes with no remnant cells in the Nanosheet. Conclusion: Transplantation of in vitro expanded donor human corneal endothelial cells

  1. Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells

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    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.

  2. 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

  3. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

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    Suhaila Muid

    2016-07-01

    Full Text Available 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

  4. Double suicide genes selectively kill human umbilical vein endothelial cells

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    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

  5. Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions

    DEFF Research Database (Denmark)

    Bartels, Emil D; Nielsen, John E; Lindegaard, Marie Louise Skakkebæk

    2007-01-01

    Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies...

  6. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells

    DEFF Research Database (Denmark)

    Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex

    2012-01-01

    The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined...

  7. Modeling serotonin uptake in the lung shows endothelial transporters dominate over cleft permeation

    Science.gov (United States)

    Bassingthwaighte, James B.

    2013-01-01

    A four-region (capillary plasma, endothelium, interstitial fluid, cell) multipath model was configured to describe the kinetics of blood-tissue exchange for small solutes in the lung, accounting for regional flow heterogeneity, permeation of cell membranes and through interendothelial clefts, and intracellular reactions. Serotonin uptake data from the Multiple indicator dilution “bolus sweep” experiments of Rickaby and coworkers (Rickaby DA, Linehan JH, Bronikowski TA, Dawson CA. J Appl Physiol 51: 405–414, 1981; Rickaby DA, Dawson CA, and Linehan JH. J Appl Physiol 56: 1170–1177, 1984) and Malcorps et al. (Malcorps CM, Dawson CA, Linehan JH, Bronikowski TA, Rickaby DA, Herman AG, Will JA. J Appl Physiol 57: 720–730, 1984) were analyzed to distinguish facilitated transport into the endothelial cells (EC) and the inhibition of tracer transport by nontracer serotonin in the bolus of injectate from the free uninhibited permeation through the clefts into the interstitial fluid space. The permeability-surface area products (PS) for serotonin via the inter-EC clefts were ∼0.3 ml·g−1·min−1, low compared with the transporter-mediated maximum PS of 13 ml·g−1·min−1 (with Km = ∼0.3 μM and Vmax = ∼4 nmol·g−1·min−1). The estimates of serotonin PS values for EC transporters from their multiple data sets were similar and were influenced only modestly by accounting for the cleft permeability in parallel. The cleft PS estimates in these Ringer-perfused lungs are less than half of those for anesthetized dogs (Yipintsoi T. Circ Res 39: 523–531, 1976) with normal hematocrits, but are compatible with passive noncarrier-mediated transport observed later in the same laboratory (Dawson CA, Linehan JH, Rickaby DA, Bronikowski TA. Ann Biomed Eng 15: 217–227, 1987; Peeters FAM, Bronikowski TA, Dawson CA, Linehan JH, Bult H, Herman AG. J Appl Physiol 66: 2328–2337, 1989) The identification and quantitation of the cleft pathway conductance from these

  8. Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

    Directory of Open Access Journals (Sweden)

    Hannah J Levis

    Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

  9. Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

    Science.gov (United States)

    Levis, Hannah J; Peh, Gary S L; Toh, Kah-Peng; Poh, Rebekah; Shortt, Alex J; Drake, Rosemary A L; Mehta, Jodhbir S; Daniels, Julie T

    2012-01-01

    Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

  10. Expression of Fascin-1 on human lung cancer and paracarcinoma tissue and its relation to clinicopathological characteristics in patients with lung cancer

    Science.gov (United States)

    Zhao, Wei; Gao, Jing; Wu, Jing; Liu, Qiu-hong; Wang, Zhi-gang; Li, Hui-ling; Xing, Li-hua

    2015-01-01

    Background Lung cancer poses a severe threat to human life. Biomarkers of cancers are helpful in the diagnosis and treatment of patients with cancers. Biomarkers of lung cancers are rare, and thus deserve further research. Objective The objective of the present study was to explore the expression of Fascin-1 in human lung cancer and paracarcinoma tissue, its correlation with clinicopathological characteristics in patients with lung cancer, and study the possible relationship between Fascin-1 expression and clinical–biological behavior of lung cancer. Method This study used the MaxVision two-step immunohistochemical detection method to detect Fascin-1 expression in 84 of lung cancer and paracarcinoma tissues. This study set the expression of Fascin-1 in vascular endothelial cells as the positive control, and used phosphate buffered saline (replacing the primary antibodies) as negative control. Result Of all the 84 lung cancer tissues and paracarcinoma tissues, positive expression of the Fascin-1 protein were detected in 78 cases (92.9%) and 27 cases (32.1%), respectively, and the difference was statistically significant (P0.05). The survival times of the patients with different Fascin-1 protein-positive expressions in lung cancer tissues were statistically significant (P>0.05), while the survival times of the patients with different Fascin-1 protein-positive expressions in paracarcinoma tissues were not statistically significant (P>0.05). Conclusion In lung cancer, Fascin-1 expression was closely related to tumor invasion and metastasis, and the difference in expression of Fascin-1 had a significant effect on the survival time of the lung cancer patients. Therefore, Fascin-1 might be expected to serve as a possible potential biomarker of lung cancer. PMID:26451116

  11. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver

    NARCIS (Netherlands)

    Filali, Ebtisam El; Hiralall, Johan K.; van Veen, Henk A.; Stolz, Donna B.; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and

  12. Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells.

    Science.gov (United States)

    Tancharoen, Waleephan; Aungsuchawan, Sirinda; Pothacharoen, Peraphan; Markmee, Runchana; Narakornsak, Suteera; Kieodee, Junjira; Boonma, Nonglak; Tasuya, Witoon

    2017-03-01

    Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. Endothelial cell changes are associated with pulmonary edema and respiratory distress in mice infected with the WA1 human Babesia parasite.

    Science.gov (United States)

    Hemmer, R M; Wozniak, E J; Lowenstine, L J; Plopper, C G; Wong, V; Conrad, P A

    1999-06-01

    A C3H/HeN mouse model was established to study the pathogenesis of the human babesial parasites, WA1 and Babesia microti. To evaluate the course of parasitemia and the associated lesions, mice were inoculated intraperitoneally with either WA1-infected, B. microti-infected, or uninfected hamster red blood cells. WA1-infected mice developed dyspnea and moderate parasitemias, after which death occurred. Babesia microti-infected mice experienced low parasitemias with no apparent morbidity or mortality. WA1-infected mice were thrombocytopenic but not anemic. Hemograms for B. microti-infected mice were similar to controls. Postmortem examination of WA1-infected mice revealed prominent lesions in the lungs, including pulmonary edema and intravascular margination of leukocytes. No pulmonary changes were detected in B. microti-infected mice. Blood gas measurements of WA1-infected mice showed reduced oxygen saturation and pH, and increased carbonic acid compared to controls, indicating hypoxia and respiratory acidosis. Ultrastructure studies of WA1-infected lungs showed hypertrophied endothelial cells containing transcellular channels associated with protein-rich intra-alveolar fluid. Endothelial cell activation was demonstrated by an upregulation of intercellular adhesion molecule-1 in the lungs of WA1-infected mice. The results suggest that recruitment of inflammatory cells to the lungs in WA1-infected mice induces endothelial cell alterations, leading to pulmonary edema and acute respiratory failure.

  14. Adhesion Molecule Expression in Human Endothelial Cells under Simulated Microgravity

    Science.gov (United States)

    Rudimov, E. G.; Andreeva, E. R.; Buravkova, L. B.

    2013-02-01

    High gravisensitivity of endothelium is now well recognized. Therefore, the microgravity can be one of the main factors affecting the endothelium in space flight. In this work we studied the effects of gravity vector randomization (3D-clinorotation in RPM) on the viability of endothelial cells from human umbilical vein (HUVEC) and the expression of adhesion molecules on its surface. After RPM exposure, HUVEC conditioning medium was collected for cytokines evaluation, a part of vials was used for immunocytochemistry and other one - for cytofluorimetric analysis of ICAM-I, VCAM-I, PECAM-I, E-selectin, Endoglin, VE-cadherin expression. The viability of HUVEC and constitutive expression of EC marker molecules PECAM-I and Endoglin were similar in all experimental groups both after 6 and 24 hrs of exposure. There were no differences in ICAM-I and E-selectin expression on HUVEC in 3 groups after 6 hrs of exposure. 24 hrs incubation has provoked decrease in ICAM-I and E-selectin expression. Thus, gravity vector randomization can lead to the disruption of ECs monolayer.

  15. Silicon microgrooves for contact guidance of human aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Sara Fernández-Castillejo

    2017-03-01

    Full Text Available Background: Micro- and nanoscale substrates have been fabricated in order to study the influence of the topography on the cellular response. The aim of this work was to prepare different collagen-coated silicon substrates displaying grooves and ridges to mimic the aligned and elongated endothelium found in linear vessels, and to use them as substrates to study cell growth and behaviour.Results: The influence of groove-shaped substrates on cell adhesion, morphology and proliferation were assessed, by comparing them to flat silicon substrates, used as control condition. Using human aortic endothelial cells, microscopy images demonstrate that the cellular response is different depending on the silicon surface, when it comes to cell adhesion, morphology (alignment, circularity and filopodia presence and proliferation. Moreover, these structures exerted no cytotoxic effect.Conclusion: The results suggest that topographical patterning influences cell response. Silicon groove substrates can be used in developing medical devices with microscale features to mimic the endothelium in lineal vessels.

  16. Structural and molecular regulation of lung maturation by intratracheal vascular endothelial growth factor administration in the normally grown and placentally restricted fetus.

    Science.gov (United States)

    McGillick, Erin V; Orgeig, Sandra; Morrison, Janna L

    2016-03-01

    Inhibition of hypoxia signalling leads to respiratory distress syndrome (RDS), whereas administration of vascular endothelial growth factor (VEGF), the most widely characterized hypoxia responsive factor, protects from RDS. In the lung of the chronically hypoxaemic placentally restricted (PR) fetus, there is altered regulation of hypoxia signalling. This leads to reduced surfactant maturation in late gestation and provides evidence for the increased risk of RDS in growth restricted neonates at birth. We evaluated the effect of recombinant human VEGF administration with respect to bypassing the endogenous regulation of hypoxia signalling in the lung of the normally grown and PR sheep fetus. There was no effect of VEGF administration on fetal blood pressure or fetal breathing movements. We examined the effect on the expression of genes regulating VEGF signalling (FLT1 and KDR), angiogenesis (ANGPT1, AQP1, ADM), alveolarization (MMP2, MMP9, TIMP1, COL1A1, ELN), proliferation (IGF1, IGF2, IGF1R, MKI67, PCNA), inflammation (CCL2, CCL4, IL1B, TNFA, TGFB1, IL10) and surfactant maturation (SFTP-A, SFTP-B, SFTP-C, SFTP-D, PCYT1A, LPCAT, LAMP3, ABCA3). Despite the effects of PR on the expression of genes regulating airway remodelling, inflammatory signalling and surfactant maturation, there were very few effects of VEGF administration on gene expression in the lung of both the normally grown and PR fetus. There were, however, positive effects of VEGF administration on percentage tissue, air space and numerical density of SFTP-B positive alveolar epithelial cells in fetal lung tissue. These results provide evidence for the stimulatory effects of VEGF administration on structural maturation in the lung of both the normally grown and PR fetus. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  17. Rho inhibition by lovastatin affects apoptosis and DSB repair of primary human lung cells in vitro and lung tissue in vivo following fractionated irradiation

    Science.gov (United States)

    Ziegler, Verena; Henninger, Christian; Simiantonakis, Ioannis; Buchholzer, Marcel; Ahmadian, Mohammad Reza; Budach, Wilfried; Fritz, Gerhard

    2017-01-01

    Thoracic radiotherapy causes damage of normal lung tissue, which limits the cumulative radiation dose and, hence, confines the anticancer efficacy of radiotherapy and impacts the quality of life of tumor patients. Ras-homologous (Rho) small GTPases regulate multiple stress responses and cell death. Therefore, we investigated whether pharmacological targeting of Rho signaling by the HMG-CoA-reductase inhibitor lovastatin influences ionizing radiation (IR)-induced toxicity in primary human lung fibroblasts, lung epithelial and lung microvascular endothelial cells in vitro and subchronic mouse lung tissue damage following hypo-fractionated irradiation (4x4 Gy). The statin improved the repair of radiation-induced DNA double-strand breaks (DSBs) in all cell types and, moreover, protected lung endothelial cells from IR-induced caspase-dependent apoptosis, likely involving p53-regulated mechanisms. Under the in vivo situation, treatment with lovastatin or the Rac1-specific small molecule inhibitor EHT1864 attenuated the IR-induced increase in breathing frequency and reduced the percentage of γH2AX and 53BP1-positive cells. This indicates that inhibition of Rac1 signaling lowers IR-induced residual DNA damage by promoting DNA repair. Moreover, lovastatin and EHT1864 protected lung tissue from IR-triggered apoptosis and mitigated the IR-stimulated increase in regenerative proliferation. Our data document beneficial anti-apoptotic and genoprotective effects of pharmacological targeting of Rho signaling following hypo-fractionated irradiation of lung cells in vitro and in vivo. Rac1-targeting drugs might be particular useful for supportive care in radiation oncology and, moreover, applicable to improve the anticancer efficacy of radiotherapy by widening the therapeutic window of thoracic radiation exposure. PMID:28796249

  18. Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs.

    Directory of Open Access Journals (Sweden)

    Mansoor A Syed

    Full Text Available The role of vascular endothelial growth factor (VEGF-induced 3 different nitric oxide synthase (NOS isoforms in lung development and injury in the newborn (NB lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.We studied NB wild type (WT, lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG, VEGFTG treated with a NOS1 inhibitor (L-NIO, VEGFTG x NOS2-/- and VEGFTG x NOS3+/- mice in room air (RA for 7 postnatal (PN days. Lung morphometry (chord length, vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin, cell proliferation (Ki67, vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG were evaluated.VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2-/- and VEGFTG x NOS3+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.

  19. Numerical Simulation of Particle Deposition in the Human Lungs

    OpenAIRE

    Gengenbach, Thomas

    2012-01-01

    We model, simulate and calculate breathing and particle depositions in the human lungs. We review the theory and discretization of fluid mechanics, the anatomy, physiology and measuring methods of lungs. A new model is introduced and investigated with a sensitivity analysis using the singular value decomposition. Particle depositions are simulated in patient-specific and schematized human lungs and compared to the particle deposition in a multiplicative model of subsequent bifurcations.

  20. Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Three-Dimensional Microphysiological Systems.

    Science.gov (United States)

    Kurokawa, Yosuke K; Yin, Rose T; Shang, Michael R; Shirure, Venktesh S; Moya, Monica L; George, Steven C

    2017-08-01

    Microphysiological systems (MPS), or "organ-on-a-chip" platforms, aim to recapitulate in vivo physiology using small-scale in vitro tissue models of human physiology. While significant efforts have been made to create vascularized tissues, most reports utilize primary endothelial cells that hinder reproducibility. In this study, we report the use of human induced pluripotent stem cell-derived endothelial cells (iPS-ECs) in developing three-dimensional (3D) microvascular networks. We established a CDH5-mCherry reporter iPS cell line, which expresses the vascular endothelial (VE)-cadherin fused to mCherry. The iPS-ECs demonstrate physiological functions characteristic of primary endothelial cells in a series of in vitro assays, including permeability, response to shear stress, and the expression of endothelial markers (CD31, von Willibrand factor, and endothelial nitric oxide synthase). The iPS-ECs form stable, perfusable microvessels over the course of 14 days when cultured within 3D microfluidic devices. We also demonstrate that inhibition of TGF-β signaling improves vascular network formation by the iPS-ECs. We conclude that iPS-ECs can be a source of endothelial cells in MPS providing opportunities for human disease modeling and improving the reproducibility of 3D vascular networks.

  1. A nanoengineered peptidic delivery system with specificity for human brain capillary endothelial cells

    DEFF Research Database (Denmark)

    Wu, Linping; Moghimi, Seyed Moein

    2016-01-01

    avidity of the majority of the so-called ‘brain-specific’ nanoparticles to the brain capillary endothelial cells has been poor, even during in vitro conditions. We have addressed this issue and designed a versatile peptidic nanoplatform with high binding avidity to the human cerebral capillary endothelial...... cells. This was achieved by selecting an appropriate phage-derived peptide with high specificity for human brain capillary endothelial cells, which following careful structural modifications spontaneously formed a nanoparticle-fiber network. The peptidic network was characterized fully and its uptake...... by the human brain capillary endothelial cell line hCMEC/D3 was confirmed by live-cell fluorescent microscopy and quantified by flow cytometry. Recognition and internalization was medicated by two receptors leading to endolysosomal accumulation. Furthermore, the network was capable of delivering functional si...

  2. Role of pulmonary microvascular endothelial cell apoptosis in murine sepsis-induced lung injury in vivo.

    Science.gov (United States)

    Gill, Sean E; Rohan, Marta; Mehta, Sanjay

    2015-09-16

    Sepsis remains a common and serious condition with significant morbidity and mortality due to multiple organ dysfunction, especially acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Sepsis-induced ALI is characterized by injury and dysfunction of the pulmonary microvasculature and pulmonary microvascular endothelial cells (PMVEC), resulting in enhanced pulmonary microvascular sequestration and pulmonary infiltration of polymorphonuclear leukocytes (PMN) as well as disruption of the normal alveolo-capillary permeability barrier with leak of albumin-rich edema fluid into pulmonary interstitium and alveoli. The role of PMVEC death and specifically apoptosis in septic pulmonary microvascular dysfunction in vivo has not been established. In a murine cecal ligation/perforation (CLP) model of sepsis, we quantified and correlated time-dependent changes in pulmonary microvascular Evans blue (EB)-labeled albumin permeability with (1) PMVEC death (propidium iodide [PI]-staining) by both fluorescent intravital videomicroscopy (IVVM) and histology, and (2) PMVEC apoptosis using histologic fluorescent microscopic assessment of a panel of 3 markers: cell surface phosphatidylserine (detected by Annexin V binding), caspase activation (detected by FLIVO labeling), and DNA fragmentation (TUNEL labeling). Compared to sham mice, CLP-sepsis resulted in pulmonary microvascular barrier dysfunction, quantified by increased EB-albumin leak, and PMVEC death (PI+ staining) as early as 2 h and more marked by 4 h after CLP. Septic PMVEC also exhibited increased presence of all 3 markers of apoptosis (Annexin V+, FLIVO+, TUNEL+) as early as 30 mins--1 h after CLP-sepsis, which all similarly increased markedly until 4 h. The time-dependent changes in septic pulmonary microvascular albumin-permeability barrier dysfunction were highly correlated with PMVEC death (PI+; r = 0.976, p pulmonary microvascular dysfunction, including both albumin-permeability barrier dysfunction and

  3. HABITUAL FLAVONOID INTAKE AND ENDOTHELIAL FUNCTION IN HEALTHY HUMANS

    Science.gov (United States)

    Fisher, Naomi DL; Hurwitz, Shelley; Hollenberg, Norman K

    2013-01-01

    Objective Endothelial function, as measured by non-invasive techniques, is known to vary widely within populations. Our study was designed to test the hypothesis that this variation is determined in large part by a person’s habitual dietary intake of flavonoids. Methods This was an analytical study examining the relationship between endothelial function and dietary flavonoids in 19 healthy older adults (mean age 72 years). The study took place in the inpatient Clinical Research Center of the Brigham and Women’s Hospital. Habitual flavonoid intake was assessed via a focused food frequency questionnaire. Endothelial function, measured as the reactive hyperemia response to one dose of flavonoid-rich cocoa, was recorded with a plethysmograpic device via peripheral arterial tonometry (PAT). Results Background flavonoid intake and the reactive hyperemia (RH)-PAT response were significantly correlated (r=0.7, p=0.001); subjects with higher habitual flavonoid intake showed a significantly greater RH-PAT response than did lower consumers. PAT response to cocoa was also significantly correlated with simultaneous flavanol concentration in the blood (r=0.5, p=0.03). Conclusion Individual variation in endothelial function among healthy older people, measured as PAT response to flavonoid-rich cocoa, is highly dependent upon usual daily flavonoid consumption. These data raise the possibility that the consumption of fruits and vegetables dictates basal endothelial function, likely related to their flavonoid content and influence on nitric oxide. PMID:23378455

  4. Vascular endothelial growth factor blockade alters magnetic resonance imaging biomarkers of vascular function and decreases barrier permeability in a rat model of lung cancer brain metastasis.

    Science.gov (United States)

    Pishko, Gregory L; Muldoon, Leslie L; Pagel, Michael A; Schwartz, Daniel L; Neuwelt, Edward A

    2015-02-17

    Blockade of vascular endothelial growth factor (VEGF) to promote vascular normalization and inhibit angiogenesis has been proposed for the treatment of brain metastases; however, vascular normalization has not been well-characterized in this disease. We investigated the effect of treatment with bevacizumab anti-VEGF antibody on magnetic resonance imaging (MRI) biomarkers of brain tumor vascular characteristics in comparison to small molecule delivery in a rat model of human lung cancer brain metastasis. Athymic rats with A549 human lung adenocarcinoma intracerebral xenografts underwent MRI at 11.75 T before and one day after treatment with bevacizumab (n = 8) or saline control (n = 8) to evaluate tumor volume, free water content (edema), blood volume and vascular permeability (Ktrans). One day later, permeability to 14C-aminoisobutyric acid (AIB) was measured in tumor and brain to assess the penetration of a small drug-like molecule. In saline control animals, tumor volume, edema and permeability increased over the two day assessment period. Compared to controls, bevacizumab treatment slowed the rate of tumor growth (P = 0.003) and blocked the increase in edema (P = 0.033), but did not alter tumor blood volume. Bevacizumab also significantly reduced Ktrans (P = 0.033) and AIB passive permeability in tumor (P = 0.04), but not to peritumoral tissue or normal brain. Post-treatment Ktrans correlated with AIB levels in the bevacizumab-treated rats but not in the saline controls. The correlation of an MRI biomarker for decreased vascular permeability with decreased AIB concentration in tumor after antiangiogenic treatment suggests that bevacizumab partially restored the normal low permeability characteristics of the blood-brain barrier in a model of human lung cancer brain metastasis.

  5. Forkhead box O-1 modulation improves endothelial insulin resistance in human obesity.

    Science.gov (United States)

    Karki, Shakun; Farb, Melissa G; Ngo, Doan T M; Myers, Samantha; Puri, Vishwajeet; Hamburg, Naomi M; Carmine, Brian; Hess, Donald T; Gokce, Noyan

    2015-06-01

    Increased visceral adiposity has been closely linked to insulin resistance, endothelial dysfunction, and cardiometabolic disease in obesity, but pathophysiological mechanisms are poorly understood. We sought to investigate mechanisms of vascular insulin resistance by characterizing depot-specific insulin responses and gain evidence that altered functionality of transcription factor forkhead box O-1 (FOXO-1) may play an important role in obesity-related endothelial dysfunction. We intraoperatively collected paired subcutaneous and visceral adipose tissue samples from 56 severely obese (body mass index, 43 ± 7 kg/m(2)) and 14 nonobese subjects during planned surgical operations, and characterized depot-specific insulin-mediated responses using Western blot and quantitative immunofluorescence techniques. Insulin signaling via phosphorylation of FOXO-1 and consequent endothelial nitric oxide synthase stimulation was selectively impaired in the visceral compared with subcutaneous adipose tissue and endothelial cells of obese subjects. In contrast, tissue actions of insulin were preserved in nonobese individuals. Pharmacological antagonism with AS1842856 and biological silencing using small interfering RNA-mediated FOXO-1 knockdown reversed insulin resistance and restored endothelial nitric oxide synthase activation in the obese. We observed profound endothelial insulin resistance in the visceral adipose tissue of obese humans which improved with FOXO-1 inhibition. FOXO-1 modulation may represent a novel therapeutic target to diminish vascular insulin resistance. In addition, characterization of endothelial insulin resistance in the adipose microenvironment may provide clues to mechanisms of systemic disease in human obesity. © 2015 American Heart Association, Inc.

  6. Constitutively active endothelial Notch4 causes lung arteriovenous shunts in mice

    Science.gov (United States)

    Jelin, Eric B.; Ng, Jennifer; Wu, Jianfeng; Carlson, Timothy R.; Wu, Xiaoqing; Looney, Mark R.; Wang, Rong A.

    2010-01-01

    Lung arteriovenous (AV) shunts or malformations cause significant morbidity and mortality in several distinct clinical syndromes. For most patients with lung AV shunts, there is still no optimal treatment. The underlying molecular and cellular etiology for lung AV shunts remains elusive, and currently described animal models have insufficiently addressed this problem. Using a tetracycline-repressible system, we expressed constitutively active Notch4 (Notch4*) specifically in the endothelium of adult mice. More than 90% of mice developed lung hemorrhages and respiratory insufficiency and died by 6–7 wk after gene expression began. Vascular casting and fluorescent microsphere analysis showed evidence of lung AV shunts in affected mice. Cessation of Notch4* expression reversed these pathophysiological effects. Assessment of the vascular morphology revealed enlarged, tortuous vessels in the lungs that resembled arteriovenous malformations. By using whole lung organ culture, we demonstrated the effects of constitutively active Notch4 on the lung vasculature to be a primary lung phenomenon. Together, our results indicate the importance of Notch signaling in maintaining the lung vasculature and offer a new, reliable model with which to study the pathobiology of lung arteriovenous shunts and malformations. PMID:19933399

  7. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

    Directory of Open Access Journals (Sweden)

    Jesintha Navaratnam

    2015-09-01

    Full Text Available Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented.

  8. Human endothelial progenitor cells internalize high-density lipoprotein.

    Directory of Open Access Journals (Sweden)

    Kaemisa Srisen

    Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal

  9. Establishment and long-term culture of human cystic fibrosis endothelial cells.

    Science.gov (United States)

    Plebani, Roberto; Tripaldi, Romina; Lanuti, Paola; Recchiuti, Antonio; Patruno, Sara; Di Silvestre, Sara; Simeone, Pasquale; Anile, Marco; Venuta, Federico; Prioletta, Marco; Mucilli, Felice; Del Porto, Paola; Marchisio, Marco; Pandolfi, Assunta; Romano, Mario

    2017-11-01

    Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.

  10. Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells.

    Science.gov (United States)

    Unger, R E; Peters, K; Wolf, M; Motta, A; Migliaresi, C; Kirkpatrick, C J

    2004-09-01

    We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps between individual fibers. Higher attachment and growth coverage was obtained if nets were first coated with gelatin, fibronectin or collagen type I. Proinflammatory markers of endothelial cells on the fibers exhibited a non-activated state and LPS-stimulated cells exhibited activation of these markers. Furthermore, a typical PECAM-1 localization at cell-cell contacts was observed. Scanning electron microscopic examination of fibroin nets after removal of cells did not demonstrate any changes to the fibroin structure. HUVEC and HDMEC on fibroin nets embedded in collagen type I gels formed microvessel-like structures. Thus, silk fibroin nets are a highly endothelial cell-compatible scaffolding material that support the growth, normal and inducible cell functions and angiogenesis potential of human endothelial cells in vitro similar to that observed in vivo.

  11. Inhaled nitric oxide decreases pulmonary endothelial nitric oxide synthase expression and activity in normal newborn rat lungs

    Directory of Open Access Journals (Sweden)

    Thông Hua-Huy

    2016-02-01

    Full Text Available Inhaled nitric oxide (iNO is commonly used in the treatment of very ill pre-term newborns. Previous studies showed that exogenous NO could affect endothelial NO synthase (eNOS activity and expression in vascular endothelial cell cultures or adult rat models, but this has never been fully described in newborn rat lungs. We therefore aimed to assess the effects of iNO on eNOS expression and activity in newborn rats. Rat pups, post-natal day (P 0 to P7, and their dams were placed in a chamber containing NO at 5 ppm (iNO-5 ppm group or 20 ppm (iNO-20 ppm group, or in room air (control group. Rat pups were sacrificed at P7 and P14 for evaluation of lung eNOS expression and activity. At P7, eNOS protein expression in total lung lysates, in bronchial and arterial sections, was significantly decreased in the iNO-20 ppm versus control group. At P14, eNOS expression was comparable among all three groups. The amounts of eNOS mRNA significantly differed at P7 between the iNO-20 ppm and control groups. NOS activity decreased in the iNO-20 ppm group at P7 and returned to normal levels at P14. There was an imbalance between superoxide dismutase and NOS activities in the iNO-20 ppm group at P7. Inhalation of NO at 20 ppm early after birth decreases eNOS gene transcription, protein expression and enzyme activity. This decrease might account for the rebound phenomenon observed in patients treated with iNO.

  12. Comparison of Endothelial Differentiation Capacities of Human and Rat Adipose-Derived Stem Cells.

    Science.gov (United States)

    Orbay, Hakan; Devi, Kamaljit; Williams, Priscilla A; Dehghani, Tima; Silva, Eduardo A; Sahar, David E

    2016-12-01

    The authors compared the endothelial differentiation capacities of human and rat adipose-derived stem cells to determine whether human adipose-derived stem cells can be a source of endothelial cells clinically. Human and rat adipose-derived stem cells were harvested and characterized with flow cytometry and trilineage differentiation. Cells from passages III through V were fed with endothelial cell differentiation medium for up to 3 weeks. Cells were harvested after 1, 2, and 3 weeks, and endothelial differentiation was evaluated with quantitative reverse-transcriptase polymerase chain reaction, flow cytometry, and angiogenic sprouting assays. Both human and rat adipose-derived stem cells were CD90, CD44, and CD31 before differentiation. The cells were successfully differentiated into adipogenic, osteogenic, and chondrogenic lineages. Expression of endothelial cell-specific genes peaked at the second week of differentiation in both human and rat cells. The fold changes in expression of CD31, vascular endothelial growth factor receptor-1, nitric oxide synthase, and von Willebrand factor genes at week 2 were 0.4 ± 0.1, 34.7 ± 0.3, 2.03 ± 0.25, and 12.5 ± 0.3 respectively, in human adipose-derived stem cells; and 1.5 ± 1.01, 21.6 ± 1.7, 17.9 ± 0.6, and 11.2 ± 1.3, respectively, in rat cells. The percentages of CD31 cells were 0.2, 0.64, and 1.6 in human cell populations and 0.5, 5.91, and 11.5 in rat cell populations at weeks 1, 2, and 3, respectively. Rat adipose-derived stem cell-derived endothelial cells displayed enhanced sprouting capability compared with the human cells. Human adipose-derived stem cells responded less strongly to EGM-2MV endothelial differentiation medium than did the rat cells. Still, the human cells have the potential to become a clinical source of endothelial cells with modifications in the differentiation conditions.

  13. Regional differences in alveolar density in the human lung are related to lung height.

    Science.gov (United States)

    McDonough, John E; Knudsen, Lars; Wright, Alexander C; Elliott, W Mark; Ochs, Matthias; Hogg, James C

    2015-06-01

    The gravity-dependent pleural pressure gradient within the thorax produces regional differences in lung inflation that have a profound effect on the distribution of ventilation within the lung. This study examines the hypothesis that gravitationally induced differences in stress within the thorax also influence alveolar density in terms of the number of alveoli contained per unit volume of lung. To test this hypothesis, we measured the number of alveoli within known volumes of lung located at regular intervals between the apex and base of four normal adult human lungs that were rapidly frozen at a constant transpulmonary pressure, and used microcomputed tomographic imaging to measure alveolar density (number alveoli/mm3) at regular intervals between the lung apex and base. These results show that at total lung capacity, alveolar density in the lung apex is 31.6 ± 3.4 alveoli/mm3, with 15 ± 6% of parenchymal tissue consisting of alveolar duct. The base of the lung had an alveolar density of 21.2 ± 1.6 alveoli/mm3 and alveolar duct volume fraction of 29 ± 6%. The difference in alveolar density can be negated by factoring in the effects of alveolar compression due to the pleural pressure gradient at the base of the lung in vivo and at functional residual capacity.

  14. Growth hormone-releasing peptide ghrelin inhibits homocysteine-induced endothelial dysfunction in porcine coronary arteries and human endothelial cells.

    Science.gov (United States)

    Hedayati, Nasim; Annambhotla, Suman; Jiang, Jun; Wang, Xinwen; Chai, Hong; Lin, Peter H; Yao, Qizhi; Chen, Changyi

    2009-01-01

    Ghrelin, a novel growth hormone-releasing peptide, is implicated to play a protective role in cardiovascular tissues. However, it is not clear whether ghrelin protects vascular tissues from injury secondary to risk factors such as homocysteine (Hcy). This study investigated the effect and potential mechanisms of ghrelin on Hcy-induced endothelial dysfunction. Porcine coronary artery rings were incubated for 24 hours with ghrelin (100 ng/mL), Hcy (50 microM), or ghrelin plus Hcy. Endothelial vasomotor function was evaluated using the myograph tension model. The response to the thromboxane A(2)analog U46619, bradykinin, and sodium nitroprusside was analyzed. Endothelial nitric oxide synthase (eNOS) expression was determined using real-time polymerase chain reaction and immunohistochemistry staining, and superoxide anion production was documented lucigenin-enhanced chemiluminescence analysis. Human coronary artery endothelial cells (HCAECs) were treated with different concentrations of Hcy, ghrelin, or antighrelin receptor antibody for 24 hours, and eNOS protein levels were determined by Western blot analysis. Maximal contraction with U46619 and endothelium-independent vasorelaxation with sodium nitroprusside were not different among the four groups. However, endothelium-dependent vasorelaxation with bradykinin (10(-6) M) was significantly reduced by 34% with Hcy compared with controls (P ghrelin to Hcy had a protective effect, with 61.6% relaxation, which was similar to controls (64.7%). Homocysteine significantly reduced eNOS expression, whereas ghrelin cotreatment effectively restored eNOS expression to the control levels. Superoxide anion levels, which were increased by 100% with Hcy, returned to control levels with ghrelin cotreatment. Ghrelin also effectively blocked the Hcy-induced decrease of eNOS protein levels in HCAECs in a concentration-dependent manner. Antighrelin receptor antibody effectively inhibited the effect of ghrelin. Ghrelin has a protective

  15. Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition.

    Science.gov (United States)

    Suzuki, Toshio; Tada, Yuji; Gladson, Santhi; Nishimura, Rintaro; Shimomura, Iwao; Karasawa, Satoshi; Tatsumi, Koichiro; West, James

    2017-10-16

    Pulmonary fibrosis is a late manifestation of acute respiratory distress syndrome (ARDS). Sepsis is a major cause of ARDS, and its pathogenesis includes endotoxin-induced vascular injury. Recently, endothelial-to-mesenchymal transition (EndMT) was shown to play an important role in pulmonary fibrosis. On the other hand, dipeptidyl peptidase (DPP)-4 was reported to improve vascular dysfunction in an experimental sepsis model, although whether DPP-4 affects EndMT and fibrosis initiation during lipopolysaccharide (LPS)-induced lung injury is unclear. The aim of this study was to investigate the anti-EndMT effects of the DPP-4 inhibitor vildagliptin in pulmonary fibrosis after systemic endotoxemic injury. A septic lung injury model was established by intraperitoneal injection of lipopolysaccharide (LPS) in eight-week-old male mice (5 mg/kg for five consecutive days). The mice were then treated with vehicle or vildagliptin (intraperitoneally, 10 mg/kg, once daily for 14 consecutive days from 1 day before the first administration of LPS.). Flow cytometry, immunohistochemical staining, and quantitative polymerase chain reaction (qPCR) analysis was used to assess cell dynamics and EndMT function in lung samples from the mice. Lung tissue samples from treated mice revealed obvious inflammatory reactions and typical interstitial fibrosis 2 days and 28 days after LPS challenge. Quantitative flow cytometric analysis showed that the number of pulmonary vascular endothelial cells (PVECs) expressing alpha-smooth muscle actin (α-SMA) or S100 calcium-binding protein A4 (S100A4) increased 28 days after LPS challenge. Similar increases in expression were also confirmed by qPCR of mRNA from isolated PVECs. EndMT cells had higher proliferative activity and migration activity than mesenchymal cells. All of these changes were alleviated by intraperitoneal injection of vildagliptin. Interestingly, vildagliptin and linagliptin significantly attenuated EndMT in the absence of immune

  16. Expression of vascular endothelial growth factor and its two receptors in normal human endometrium

    Institute of Scientific and Technical Information of China (English)

    王海燕; 陈贵安

    2003-01-01

    Objectives: We try to demonstrate the expression of vascular endothelial growthfactor (VEGF) and its receptors, flt-1 and KDR, in normal human emdometrium duringthe menstrual cycle.Methods: Immunohistochemical method was used to observe the expression ofVEGF and its two receptors in emdometrium throughout the normal menstrual cyclemeanwhile the isoforms of VEGF were also detected by Western blot analysis. The en-dothelial cells of micro-vessels were marked with Ⅷ factor antibody.Results: VEGF and its receptors existed in endometrial glandular, stromal and vas-cular endothelial cells of human endometrium. Their expressions were higher in the mid-secretory phase of menstrual cycle and highest at menstruation. VEGF121 and VEGF165were the predominant isoforms in normal human endometrium.Conclusion: The expression of VEGF and its two receptors showed cycle-dependentin human endometrium, probably involved in embryonic implantation and endometrialproliferation and differentiation.

  17. Human Lung Tissue Transcriptome : Influence of Sex and Age

    NARCIS (Netherlands)

    Dugo, Matteo; Cotroneo, Chiara E.; Lavoie-Charland, Emilie; Incarbone, Matteo; Santambrogio, Luigi; Rosso, Lorenzo; van den Berge, Maarten; Nickle, David; Pare, Peter D.; Bosse, Yohan; Dragani, Tommaso A.; Colombo, Francesca

    2016-01-01

    Background Sex and age strongly influence the pathophysiology of human lungs, but scarce information is available about their effects on pulmonary gene expression. Methods We followed a discovery-validation strategy to identify sex-and age-related transcriptional differences in lung. Results We

  18. Green tea polyphenol induces significant cell death in human lung ...

    African Journals Online (AJOL)

    Green tea polyphenol induces significant cell death in human lung cancer cells. ... Tropical Journal of Pharmaceutical Research ... (8-OHdG), and apoptosis based on 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were evaluated in non-small cell lung cancer (NSCLC) cell lines, namely, H1155, ...

  19. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-05-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.

  20. Responses of human endothelial cells to pathogenic and non-pathogenic Leptospira species.

    Directory of Open Access Journals (Sweden)

    Denise G Martinez-Lopez

    2010-12-01

    Full Text Available Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.

  1. CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

    Science.gov (United States)

    Greene, Jennifer A.; Portillo, Jose-Andres C.; Lopez Corcino, Yalitza; Subauste, Carlos S.

    2015-01-01

    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. PMID:26710229

  2. 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.

  3. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I

    2006-01-01

    Objective Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which...... directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... results. Results Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10 000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation...

  4. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Hoegh, A M; Tannetta, D; Sargent, I

    2006-01-01

    OBJECTIVE: Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which...... directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. DESIGN: Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... results. RESULTS: Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10,000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation...

  5. 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.

  6. Vasculogenesis, angiogenesis and the molecular organisation of endothelial junctions in the early human placenta.

    Science.gov (United States)

    Leach, Lopa; Babawale, Michael O; Anderson, Mark; Lammiman, Michael

    2002-01-01

    Vasculogenesis and angiogenesis are regulated by the capacity of endothelial cells to adhere to each other and form new tubes. The presence and role of junctional adhesion molecules during physiological vasculogenesis is unknown. Using ultrastructural and immunocytochemical approaches, we compared the junctional phenotype of developing vessels of the first-trimester human placenta with vessels in the last trimester; the latter include newly formed terminal capillaries and the quiescent vascular bed. First-trimester placental vessels contained the adherens junctional molecules, vascular endothelial cadherin and alpha- and beta-catenin but lacked plakoglobin, the component of fully differentiated adherens junctions. Furthermore, these vessels did not contain the transmembrane tight junctional molecules occludin and claudin-1 and -2. This profile reflects the phenotype of terminal capillaries but differs from large vessels of the full-term placenta. Electron microscopic studies revealed that endothelial tight junctions are present in the first-trimester placenta. Thus, occludin and claudin-1 appear to play no part in the formation of endothelial tight junctions, but are a later requirement. In the early placenta, the predominant growth factor appears to be vascular endothelial growth factor (VEGF), whilst at term, angiopoietin-1 was present in large vessels, with intense angiopoietin-2 immunofluorescence (and VEGF) located in terminal villous capillaries. Thus, endothelial junctions in the human placenta possess two distinct molecular phenotypes, i.e. stable or dynamic, dependent on maturity and plasticity. These distinct phenotypes may be influenced by the angiopoietins/VEGF present in the placenta. Copyright 2002 S. Karger AG, Basel

  7. Role of Rutin on Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Azizah Ugusman

    2014-01-01

    Full Text Available Nitric oxide (NO, produced by endothelial nitric oxide synthase (eNOS, is a major antiatherogenic factor in the blood vessel. Oxidative stress plays an important role in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Decreased availability of endothelial NO promotes the progression of endothelial dysfunction and atherosclerosis. Rutin is a flavonoid with multiple cardiovascular protective effects. This study aimed to investigate the effects of rutin on eNOS and NO production in cultured human umbilical vein endothelial cells (HUVEC. HUVEC were divided into four groups: control; oxidative stress induction with 180 μM H2O2; treatment with 300 μM rutin; and concomitant induction with rutin and H2O2 for 24 hours. HUVEC treated with rutin produced higher amount of NO compared to control (P<0.01. In the oxidative stress-induced HUVEC, rutin successfully induced cells’ NO production (P<0.01. Rutin promoted NO production in HUVEC by inducing eNOS gene expression (P<0.05, eNOS protein synthesis (P<0.01, and eNOS activity (P<0.05. Treatment with rutin also led to increased gene and protein expression of basic fibroblast growth factor (bFGF in HUVEC. Therefore, upregulation of eNOS expression by rutin may be mediated by bFGF. The results showed that rutin may improve endothelial function by augmenting NO production in human endothelial cells.

  8. GLP-1 inhibits VEGFA-mediated signaling in isolated human endothelial cells and VEGFA-induced dilation of rat mesenteric arteries

    DEFF Research Database (Denmark)

    Rotbøl, Cecilie Egholm; Khammy, Makhala Michell; Dalsgaard, Thomas

    2016-01-01

    to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries...

  9. Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function.

    Science.gov (United States)

    Stevens, T; Creighton, J; Thompson, W J

    1999-07-01

    Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells (PAECs); however, the mechanisms responsible for this intrinsic feature of PMVECs are unknown. Because cAMP improves endothelial barrier function, we hypothesized that differences in enzyme regulation of cAMP synthesis and/or degradation uniquely establish an elevated content in PMVECs. PMVECs possessed 20% higher basal cAMP concentrations than did PAECs; however, increased content was accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs, responsiveness to beta-adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and responsiveness to phosphodiesterase inhibition (rolipram) was increased compared with those in PAECs. Although both types of endothelial cells express calcium-inhibited adenylyl cyclase, constitutive PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at elevated levels compared with PAECs, which likely contribute to enhanced microvascular barrier function.

  10. Gene expression microarray data from human microvascular endothelial cells supplemented with a low concentration of niacin

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    Jennifer M. Hughes-Large

    2016-03-01

    Full Text Available The systemic lipid modifying drug, niacin, can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions, possibly through activation of niacin receptors “Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity” (Hughes-Large et al. 2014. Here we provide accompanying data collected using Affymetrix GeneChip microarrays to identify changes in gene expression in human microvascular endothelial cells treated with 10 μM niacin. Statistical analyses of robust multi-array average (RMA values revealed that only 16 genes exhibited greater than 1.3-fold differential expression. Of these 16, only 5 were identified protein coding genes, while 3 of the remaining 11 genes appeared to be small nuclear/nucleolar RNAs. Altered expression of EFCAB4B, NAP1L2, and OR13C8 was confirmed by real time quantitative PCR.

  11. Nicotine promotes vascular endothelial growth factor secretion by human trophoblast cells under hypoxic conditions and improves the proliferation and tube formation capacity of human umbilical endothelial cells.

    Science.gov (United States)

    Zhao, Hongbo; Wu, Lanxiang; Wang, Yahui; Zhou, Jiayi; Li, Ruixia; Zhou, Jiabing; Wang, Zehua; Xu, Congjian

    2017-04-01

    Pre-eclampsia, characterized as defective uteroplacental vascularization, remains the major cause of maternal and fetal mortality and morbidity. Previous epidemiological studies demonstrated that cigarette smoking reduced the risk of pre-eclampsia. However, the molecular mechanism remains elusive. In the present study, it is demonstrated that a low dose of nicotine decreased soluble vascular endothelial growth factor receptor 1 (sFlt1) secretion in human trophoblast cells under hypoxic conditions. Nicotine was then observed to promote vascular endothelial growth factor (VEGF) secretion by reducing sFlt1 secretion and increasing VEGF mRNA transcription. Further data showed that nicotine enhanced hypoxia-mediated hypoxia-inducible factor-1α (HIF-1α) expression and HIF-1α small interfering RNA abrogated nicotine-induced VEGF secretion, indicating that HIF-1α may be responsible for nicotine-mediated VEGF transcription under hypoxic conditions. Moreover, conditioned medium from human trophoblast cells treated with nicotine under hypoxic conditions promoted the proliferation and tube formation capacity of human umbilical endothelial cells (HUVEC) by promoting VEGF secretion. These findings indicate that nicotine may promote VEGF secretion in human trophoblast cells under hypoxic conditions by reducing sFlt1 secretion and up-regulating VEGF transcription and improve the proliferation and tube formation of HUVEC cells, which may contribute to elucidate the protective effect of cigarette smoking against pre-eclampsia. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium

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    Jie Shi Chua

    2013-03-01

    Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet’s membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.

  13. Data from a comparative proteomic analysis of tumor-derived lung-cancer CD105+ endothelial cells

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    Hongwei Jin

    2016-06-01

    Full Text Available Increasing evidence indicates that tumor-derived endothelial cells (TECs are more relevant for the study of tumor angiogenesis and for screening antiangiogenic drugs than normal ECs (NECs. In this data article, high-purity (>98% primary CD105+ NECs and TECs purified from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS. All the identified proteins were categorized functionally by Gene Ontology (GO analysis, and gene-pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG. Finally, protein–protein interaction networks were also built. The proteomics and bioinformatics data presented here provide novel insights into the molecular characteristics and the early modulation of the TEC proteome in the tumor microenvironment.

  14. Reduced Dimensional Modeling of the Entire Human Lung

    OpenAIRE

    Ismail, Mahmoud

    2015-01-01

    The entire human lung, including the pulmonary circulation, the lower respiratory tract and the oxygen exchange interface, was modeled using a novel multi-scale approach. This novel lung model very closely represents the actual human anatomy and moreover reproduces its physiological behavior. For the first time, the simulated results provide evidence for local phenomena such as volume competition between neighboring acini, volutrauma and hypoxia at a level never achieved before. Die gesamt...

  15. Lipoteichoic acid from Staphylococcus aureus induces lung endothelial cell barrier dysfunction: role of reactive oxygen and nitrogen species.

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    Amy Barton Pai

    Full Text Available Tunneled central venous catheters (TCVCs are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. Staphylococcus aureus biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA, a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2. The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from Staphylococcus aureus induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS. The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam((3CSK((4 induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS activation (as measured by the p-eNOS(ser1177:p-eNOS(thr495 ratio. The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.

  16. Upregulation of transmembrane endothelial junction proteins in human cerebral cavernous malformations.

    Science.gov (United States)

    Burkhardt, Jan-Karl; Schmidt, Dörthe; Schoenauer, Roman; Brokopp, Chad; Agarkova, Irina; Bozinov, Oliver; Bertalanffy, Helmut; Hoerstrup, Simon P

    2010-09-01

    Cerebral cavernous malformations (CCMs) are among the most prevalent cerebrovascular malformations, and endothelial cells seem to play a major role in the disease. However, the underlying mechanisms, including endothelial intercellular communication, have not yet been fully elucidated. In this article, the authors focus on the endothelial junction proteins CD31, VE-cadherin, and occludin as important factors for functional cell-cell contacts known as vascular adhesion molecules and adherence and tight junctions. Thirteen human CCM specimens and 6 control tissue specimens were cryopreserved and examined for the presence of VE-cadherin, occludin, and CD31 by immunofluorescence staining. Protein quantification was performed by triplicate measurements using western blot analysis. Immunofluorescent analyses of the CCM sections revealed a discontinuous pattern of dilated microvessels and capillaries as well as increased expression of occludin, VE-cadherin, and CD31 in the intima and in the enclosed parenchymal tissue compared with controls. Protein quantification confirmed these findings by showing upregulation of the levels of these proteins up to 2-6 times. A protocol enabling the molecular and morphological examination of the intercellular contact proteins in human CCM was validated. The abnormal and discontinuous pattern in these endothelial cell-contact proteins compared with control tissue explains the loose intercellular junctions that are considered to be one of the causes of CCM-associated bleeding or transendothelial oozing of erythrocytes. Despite the small number of specimens, this study demonstrates for the first time a quantitative analysis of endothelial junction proteins in human CCM.

  17. Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model.

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    Natalia Vázquez

    Full Text Available Corneal keratoplasty (penetrating or lamellar using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world's population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of

  18. Endothelial Differentiation of Human Adipose-Derived Stem Cells on Polyglycolic Acid/Polylactic Acid Mesh

    Science.gov (United States)

    Deng, Meng; Gu, Yunpeng; Liu, Zhenjun; Qi, Yue; Ma, Gui E.; Kang, Ning

    2015-01-01

    Adipose-derived stem cell (ADSC) is considered as a cell source potentially useful for angiogenesis in tissue engineering and regenerative medicine. This study investigated the growth and endothelial differentiation of human ADSCs on polyglycolic acid/polylactic acid (PGA/PLA) mesh compared to 2D plastic. Cell adhesion, viability, and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination while endothelial differentiation was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings, and gene expression analysis of endothelial related markers. Results showed hADSCs gained a mature endothelial phenotype with a positive ratio of 21.4 ± 3.7% for CD31+/CD34− when induced in 3D mesh after 21 days, which was further verified by the expressions of a comprehensive range of endothelial related markers, whereas hADSCs in 2D induced and 2D/3D noninduced groups all failed to differentiate into endothelial cells. Moreover, compared to 2D groups, the expression for α-SMA was markedly suppressed in 3D cultured hADSCs. This study first demonstrated the endothelial differentiation of hADSCs on the PGA/PLA mesh and pointed out the synergistic effect of PGA/PLA 3D culture and growth factors on the acquisition of mature characteristic endothelial phenotype. We believed this study would be the initial step towards the generation of prevascularized tissue engineered constructs. PMID:26106426

  19. Endothelial Differentiation of Human Adipose-Derived Stem Cells on Polyglycolic Acid/Polylactic Acid Mesh

    Directory of Open Access Journals (Sweden)

    Meng Deng

    2015-01-01

    Full Text Available Adipose-derived stem cell (ADSC is considered as a cell source potentially useful for angiogenesis in tissue engineering and regenerative medicine. This study investigated the growth and endothelial differentiation of human ADSCs on polyglycolic acid/polylactic acid (PGA/PLA mesh compared to 2D plastic. Cell adhesion, viability, and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination while endothelial differentiation was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings, and gene expression analysis of endothelial related markers. Results showed hADSCs gained a mature endothelial phenotype with a positive ratio of 21.4 ± 3.7% for CD31+/CD34− when induced in 3D mesh after 21 days, which was further verified by the expressions of a comprehensive range of endothelial related markers, whereas hADSCs in 2D induced and 2D/3D noninduced groups all failed to differentiate into endothelial cells. Moreover, compared to 2D groups, the expression for α-SMA was markedly suppressed in 3D cultured hADSCs. This study first demonstrated the endothelial differentiation of hADSCs on the PGA/PLA mesh and pointed out the synergistic effect of PGA/PLA 3D culture and growth factors on the acquisition of mature characteristic endothelial phenotype. We believed this study would be the initial step towards the generation of prevascularized tissue engineered constructs.

  20. Double-stranded RNA attenuates the barrier function of human pulmonary artery endothelial cells.

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    Zoltán Bálint

    Full Text Available Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs. The effect of natural and synthetic double-stranded RNA (dsRNA on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca(2+ homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca(2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca(2+-ATPase (SERCA which is involved in the regulation of the intracellular Ca(2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.

  1. Depleted energy charge and increased pulmonary endothelial permeability induced by mitochondrial complex I inhibition are mitigated by coenzyme Q1 in the isolated perfused rat lung.

    Science.gov (United States)

    Bongard, Robert D; Yan, Ke; Hoffmann, Raymond G; Audi, Said H; Zhang, Xiao; Lindemer, Brian J; Townsley, Mary I; Merker, Marilyn P

    2013-12-01

    Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 μM). Compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 µmol · g(-1) dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 to 38.62 ± 3.14 µmol · 15 min(-1) perfusion · g(-1) dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone coenzyme Q1 (CoQ1; 50 μM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H-quinone oxidoreductase 1 or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 to 0.156 ± 0.037 ml · min(-1) · cm H2O(-1) · g(-1) dry lung, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate:pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low-molecular-weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency. Published by Elsevier Inc.

  2. Depleted energy charge and increased pulmonary endothelial permeability induced by mitochondrial complex I inhibition are mitigated by coenzyme Q1 in the isolated perfused rat lung

    Science.gov (United States)

    Bongard, Robert D.; Yan, Ke; Hoffmann, Raymond G.; Audi, Said H.; Zhang, Xiao; Lindemer, Brian J.; Townsley, Mary I.; Merker, Marilyn P.

    2013-01-01

    Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing the isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 uM). As compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 (SEM) μmol·gram−1 dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 (SEM) to 38.62 ± 3.14 μmol·15 min−1 perfusion·gm−1 dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone, coenzyme Q1 (CoQ1; 50 μM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H:quinone oxidoreductase 1 (NQO1) or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 (SEM) to 0.156 ± 0.037 (SEM) ml·min−1·cm H2O−1·gm−1 dry lung weight, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate/pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low molecular weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency. PMID:23912160

  3. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Science.gov (United States)

    Zhao, Runzhen; Liang, Xinrong; Zhao, Meimi; Liu, Shan-Lu; Huang, Yao; Idell, Steven; Li, Xiumin; Ji, Hong-Long

    2014-01-01

    Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  4. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

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    Runzhen Zhao

    Full Text Available Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC, cystic fibrosis transmembrane conductance regulator (CFTR, and aquaporin 5 (AQP5 proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI and II (ATII-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3 was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  5. Coagulopathy, catecholamines, and biomarkers of endothelial damage in experimental human endotoxemia and in patients with severe sepsis

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Berg, Ronan M G; Windeløv, Nis A

    2013-01-01

    The aim of this study was to investigate associations between circulating catecholamines, endothelial damage, and coagulopathy in experimental human endotoxemia and septic patients.......The aim of this study was to investigate associations between circulating catecholamines, endothelial damage, and coagulopathy in experimental human endotoxemia and septic patients....

  6. Lung-homing of endothelial progenitor cells and airway vascularization is only partially dependant on eosinophils in a house dust mite-exposed mouse model of allergic asthma.

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    Nirooya Sivapalan

    Full Text Available Asthmatic responses involve a systemic component where activation of the bone marrow leads to mobilization and lung-homing of progenitor cells. This traffic may be driven by stromal cell derived factor-1 (SDF-1, a potent progenitor chemoattractant. We have previously shown that airway angiogenesis, an early remodeling event, can be inhibited by preventing the migration of endothelial progenitor cells (EPC to the lungs. Given intranasally, AMD3100, a CXCR4 antagonist that inhibits SDF-1 mediated effects, attenuated allergen-induced lung-homing of EPC, vascularization of pulmonary tissue, airway eosinophilia and development of airway hyperresponsiveness. Since SDF-1 is also an eosinophil chemoattractant, we investigated, using a transgenic eosinophil deficient mouse strain (PHIL whether EPC lung accumulation and lung vascularization in allergic airway responses is dependent on eosinophilic inflammation.Wild-type (WT BALB/c and eosinophil deficient (PHIL mice were sensitized to house dust mite (HDM using a chronic exposure protocol and treated with AMD3100 to modulate SDF-1 stimulated progenitor traffic. Following HDM challenge, lung-extracted EPCs were enumerated along with airway inflammation, microvessel density (MVD and airway methacholine responsiveness (AHR.Following Ag sensitization, both WT and PHIL mice exhibited HDM-induced increase in airway inflammation, EPC lung-accumulation, lung angiogenesis and AHR. Treatment with AMD3100 significantly attenuated outcome measures in both groups of mice. Significantly lower levels of EPC and a trend for lower vascularization were detected in PHIL versus WT mice.This study shows that while allergen-induced lung-homing of endothelial progenitor cells, increased tissue vascularization and development lung dysfunction can occur in the absence of eosinophils, the presence of these cells worsens the pathology of the allergic response.

  7. Muscle contraction induced arterial shear stress increases endothelial nitric oxide synthase phosphorylation in humans.

    Science.gov (United States)

    Casey, Darren P; Ueda, Kenichi; Wegman-Points, Lauren; Pierce, Gary L

    2017-10-01

    We determined if local increases in brachial artery shear during repetitive muscle contractions induce changes in protein expression of endothelial nitric oxide synthase (eNOS) and/or phosphorylated (p-)eNOS at Ser1177, the primary activation site on eNOS, in endothelial cells (ECs) of humans. Seven young male subjects (25 ± 1 yr) performed 20 separate bouts (3 min each) of rhythmic forearm exercise at 20% of maximum over a 2-h period. Each bout of exercise was separated by 3 min of rest. An additional six male subjects (24 ± 1 yr) served as time controls (no exercise). ECs were freshly isolated from the brachial artery using sterile J-wires through an arterial catheter at baseline and again after the 2-h exercise or time control period. Expression of eNOS or p-eNOS Ser1177 in ECs was determined via immunofluorescence. Brachial artery mean shear rate was elevated compared with baseline and the time control group throughout the 2-h exercise protocol (P 0.05 for both). Our novel results suggest that elevations in brachial artery shear increase eNOS Ser1177 phosphorylation in the absence of changes in total eNOS in ECs of young healthy male subjects, suggesting that this model is sufficient to alter posttranslational modification of eNOS activity in vivo in humans.NEW & NOTEWORTHY Elevations in brachial artery shear in response to forearm exercise increased endothelial nitric oxide synthase Ser1177 phosphorylation in brachial artery endothelial cells of healthy humans. Our present study provides the first evidence in humans that muscle contraction-induced increases in conduit arterial shear lead to in vivo posttranslational modification of endothelial nitric oxide synthase activity in endothelial cells. Copyright © 2017 the American Physiological Society.

  8. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

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    Shumei Man

    2008-01-01

    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  9. Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma.

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    G-Andre Banat

    Full Text Available Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+, cytotoxic-T cells (CD8+, T-helper cells (CD4+, B cells (CD20+, macrophages (CD68+, mast cells (CD117+, mononuclear cells (CD11c+, plasma cells, activated-T cells (MUM1+, B cells, myeloid cells (PD1+ and neutrophilic granulocytes (myeloperoxidase+ compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.

  10. Follistatin is a novel biomarker for lung adenocarcinoma in humans.

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    Fangfang Chen

    Full Text Available Follistatin (FST, a single chain glycoprotein, is originally isolated from follicular fluid of ovary. Previous studies have revealed that serum FST served as a biomarker for pregnancy and ovarian mucinous tumor. However, whether FST can serve as a biomarker for diagnosis in lung adenocarcinoma of humans remains unclear.The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80, which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma (60%, 24/40 using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis.These data indicate that lung adenocarcinoma cells can secret FST into serum, which may be beneficial to the survival of adenocarcinoma cells by neutralizing activin A action. Thus, FST can serve as a promising biomarker for diagnosis of lung adenocarcinoma and a useful biotherapy target for lung adenocarcinoma.

  11. Follistatin Is a Novel Biomarker for Lung Adenocarcinoma in Humans

    Science.gov (United States)

    Feng, Ye; Liu, Haiyan; Sun, Yang; Liu, Zhonghui; Ge, Jingyan; Cui, Xueling

    2014-01-01

    Background Follistatin (FST), a single chain glycoprotein, is originally isolated from follicular fluid of ovary. Previous studies have revealed that serum FST served as a biomarker for pregnancy and ovarian mucinous tumor. However, whether FST can serve as a biomarker for diagnosis in lung adenocarcinoma of humans remains unclear. Methods and Results The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80), which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma (60%, 24/40) using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis. Conclusions These data indicate that lung adenocarcinoma cells can secret FST into serum, which may be beneficial to the survival of adenocarcinoma cells by neutralizing activin A action. Thus, FST can serve as a promising biomarker for diagnosis of lung adenocarcinoma and a useful biotherapy target for lung adenocarcinoma. PMID:25347573

  12. Integrin α6β4 identifies human distal lung epithelial progenitor cells with potential as a cell-based therapy for cystic fibrosis lung disease.

    Directory of Open Access Journals (Sweden)

    Xiaopeng Li

    Full Text Available To develop stem/progenitor cell-based therapy for cystic fibrosis (CF lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4(+ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4(+ epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4(- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4(+ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs serotypes, AAV2 and AAV8, capable of transducing α6β4(+ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4(+ epithelial cells significantly rescued defects in Cl(- transport. Therefore, targeting the α6β4(+ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

  13. Early gene response of human brain endothelial cells to Listeria monocytogenes

    Science.gov (United States)

    The gene expression of human brain microvascular endothelial cells (HBMEC) to Listeria monocytogenes at 4 hour infection was analyzed. Four hours after infection, the expression of 456 genes of HBMEC had changed (p<0.05). We noted that many active genes were involved in the formyl-methionylleucylph...

  14. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus

    2013-01-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...

  15. UP-REGULATION OF ANTITHROMBOTIC ECTONUCLEOTIDASES BY ASPIRIN IN HUMAN ENDOTHELIAL-CELLS IN-VITRO

    NARCIS (Netherlands)

    CHEUNG, PK; VISSER, J; BAKKER, WW

    1994-01-01

    Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP

  16. Effects of phthalates on the human corneal endothelial cell line B4G12

    DEFF Research Database (Denmark)

    Krüger, Tanja; Cao, Yi; Kjærgaard, Søren K.

    2012-01-01

    Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2...

  17. Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles

    NARCIS (Netherlands)

    van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H

    OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a

  18. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  19. Ex Vivo Perfusion Treatment of Infection in Human Donor Lungs.

    Science.gov (United States)

    Nakajima, D; Cypel, M; Bonato, R; Machuca, T N; Iskender, I; Hashimoto, K; Linacre, V; Chen, M; Coutinho, R; Azad, S; Martinu, T; Waddell, T K; Hwang, D M; Husain, S; Liu, M; Keshavjee, S

    2016-04-01

    Ex vivo lung perfusion (EVLP) is a platform to treat infected donor lungs with antibiotic therapy before lung transplantation. Human donor lungs that were rejected for transplantation because of clinical concern regarding infection were randomly assigned to two groups. In the antibiotic group (n = 8), lungs underwent EVLP for 12 h with high-dose antibiotics (ciprofloxacin 400 mg or azithromycin 500 mg, vancomycin 15 mg/kg, and meropenem 2 g). In the control group (n = 7), lungs underwent EVLP for 12 h without antibiotics. A quantitative decrease in bacterial counts in bronchoalveolar lavage (BAL) was found in all antibiotic-treated cases but in only two control cases. Perfusate endotoxin levels at 12 h were significantly lower in the antibiotic group compared with the control group. EVLP with broad-spectrum antibiotic therapy significantly improved pulmonary oxygenation and compliance and reduced pulmonary vascular resistance. Perfusate endotoxin levels at 12 h were strongly correlated with levels of perfusates tumor necrosis factor α, IL-1β and macrophage inflammatory proteins 1α and 1β at 12 h. In conclusion, EVLP treatment of infected donor lungs with broad-spectrum antibiotics significantly reduced BAL bacterial counts and endotoxin levels and improved donor lung function. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

  20. Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.

    Science.gov (United States)

    Olszewska-Pazdrak, Barbara; Hein, Travis W; Olszewska, Paulina; Carney, Darrell H

    2009-05-01

    Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

  1. Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils.

    Science.gov (United States)

    Patel, K D; Zimmerman, G A; Prescott, S M; McEver, R P; McIntyre, T M

    1991-02-01

    The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell-dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence.

  2. [Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].

    Science.gov (United States)

    Wang, Wei-rong; Lin, Rong; Yang, Yu-cong; Gan, Wei-jie; Liu, Jun-tian; Lü, She-min

    2005-12-01

    To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells. The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively. Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry. The recombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.

  3. A HISTOLOGICAL STUDY OF HUMAN LUNG

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    Darshana Bora

    2016-11-01

    Full Text Available BACKGROUND The lungs are the essential organ of respiration. Maturation of lung is divided into four stages pseudoglandular, canalicular, terminal sac and alveolar. By 16 weeks, all major elements have formed except those involved with gas exchange. Respiration is not possible; hence, foetuses born during this period are unable to survive. By 26 weeks, the terminal sacs are lined by squamous epithelial cells and scattered among them are round secretary epithelial cells, which secrete surfactant. Respiratory distress syndrome affects 2% live newborn infants, premature are more susceptible. Surfactant deficiency is the major cause of RDS. Sufficient alveolar sac and surfactant should be present to permit survival of a prematurely born infant. Keeping this in view, the present study was done to study the microstructure of lungs in different age groups. MATERIALS AND METHODS The study was carried out in the Department of Anatomy, Assam Medical College and Hospital, Dibrugarh, for a period of one year. The study was carried out in specimens, which was collected from adult cadavers obtained for routine dissection of undergraduate students and also from the Department of Forensic Medicine. Specimens was also collected from perinatal cadavers from the Department of Obstetrics and Gynaecology, Assam Medical College and Hospital, Dibrugarh. The study has been carried out on three primary groups- Group 1, Group 2 and Group 3 according to the age. RESULTS In each of the groups, we have studied the right-sided and left-sided lungs separately and studied their histological parameters (presence/absence of pseudostratified columnar, columnar, cuboidal and squamous epithelium in the bronchial tree (lung. The results and observations obtained in the present study are compared with established findings of other workers. CONCLUSION Foetuses born prematurely at 24 to 26 weeks after fertilisation may survive if given intensive care; however, they suffer from

  4. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-01-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

  5. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells.

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-11-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1'-dioctadecyl-3,3,3',3'-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration.

  6. Nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, protects against Shiga toxin cytotoxicity in human microvascular endothelial cells.

    Science.gov (United States)

    Ohmi, K; Kiyokawa, N; Sekino, T; Suzuki, T; Mimori, K; Taguchi, T; Nakajima, H; Katagiri, Y U; Fujimoto, J; Nakao, H; Takeda, T

    2001-01-01

    Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome. The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder. Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs). The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1. However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1. NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs. The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

  7. Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Fridriksdottir, Agla J R; Kjartansson, Jens

    2007-01-01

    uptake of low-density lipoprotein, and had E-selectin induced upon treatment with tumor necrosis factor-alpha. The first signs of senescence in passage 14 were accompanied by gain of trisomy 11. At passage 18 cells showed chromosomal aberrations and growth arrest as revealed by beta......Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits......-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis....

  8. DPP-4 inhibition protects human umbilical vein endothelial cells from hypoxia-induced vascular barrier impairment

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    Naoko Hashimoto

    2017-09-01

    Full Text Available Dipeptidyl peptidase-4 (DPP-4 inhibitors are relatively new class of anti-diabetic drugs. Some protective effects of DPP-4 on cardiovascular disease have been described independently from glucose-lowering effect. However, the detailed mechanisms by which DPP-4 inhibitors exert on endothelial cells remain elusive. The purpose of this research was to determine the effects of DPP-4 inhibitor on endothelial barrier function. Human umbilical vein endothelial cells (HUVECs were cultured and exposed to hypoxia in the presence or absence of Diprotin A, a DPP-4 inhibitor. Immunocytochemistry of vascular endothelial (VE- cadherin showed that jagged VE-cadherin staining pattern induced by hypoxia was restored by treatment with Diprotin A. The increased level of cleaved β-catenin in response to hypoxia was significantly attenuated by Diprotin A, suggesting that DPP-4 inhibition protects endothelial adherens junctions from hypoxia. Subsequently, we found that Diprotin A inhibited hypoxia-induced translocation of NF-κB from cytoplasm to nucleus through decreasing TNF-α expression level. Furthermore, the tube formation assay showed that Diprotin A significantly restored hypoxia-induced decrease in number of tubes by HUVECs. These results suggest that DPP-4 inhibitior protects HUVECs from hypoxia-induced barrier impairment.

  9. Transcriptional Remodeling of Ion Channel Subunits by Flow Adaptation in Human Coronary Artery Endothelial Cells

    Science.gov (United States)

    Kefaloyianni, Eirini; Coetzee, William A.

    2011-01-01

    Endothelial cells (ECs) are constantly exposed to blood flow-induced shear forces in the vessels and this is a major determinant of endothelial function. Ion channels have a major role in endothelial function and in the control of vascular tone. We hypothesized that shear force is a general regulator of ion channel expression, which will have profound effects on endothelial function. We examined this hypothesis using large-scale quantitative real-time RT-PCR. Human coronary artery ECs were exposed to two levels of flow-induced shear stress for 24 h, while control cells were grown under static conditions. The expression of ion channel subunits was compared between control and flow-adapted cells. We used primers against 55 ion channel and exchanger subunits and were able to detect 54 subunits. Five dyn/cm2 of shear induced downregulation of 1 (NCX1) and upregulation of 18 subunits, including KCa2.2, KCa2.3, CX37, Kv1.5 and HCN2. Fifteen dyn/cm2 of shear stress induced the expression of 30 ion channel subunits, including KCa2.3, KCa2.2, CX37, Kir2.3 and KCa3.1. Our data demonstrate that substantial remodeling of endothelial ion channel subunit expression occurs with flow adaptation and suggest that altered ion channel expression may significantly contribute to vascular pathology associated with flow-induced alterations. PMID:21389733

  10. Oxidative stress and apoptosis induced by iron oxide nanoparticles in cultured human umbilical endothelial cells.

    Science.gov (United States)

    Zhu, Mo-Tao; Wang, Yun; Feng, Wei-Yue; Wang, Bing; Wang, Meng; Ouyang, Hong; Chai, Zhi-Fang

    2010-12-01

    Recent epidemiologic researches indicate that exposure to ultrafine particles (nanoparticles) is an independent risk factor for several cardiovascular diseases. The induction of endothelial injuries is hypothesized to be an attractive mechanism involved in these cardiovascular diseases. To investigate this hypothesis, the widely used iron nanomaterials, ferric oxide (Fe2O3) and ferriferrous oxide (Fe3O4) nanoparticles were incubated with human umbilical endothelial cells (ECV304 cells) at different concentrations of 2, 20, 100 microg/mL. The cell viability, the rate of apoptosis, the apoptotic nuclear morphology and the mitochondria membrane potential were measured to estimate the cell necrosis and apoptosis caused by the nanoparticle exposure. The stimulation of superoxide anion (O2*-) and nitric oxide (NO) were examined to evaluate the stress responses of endothelial cells. Our results indicated that both the Fe2O3 and Fe3O4 nanoparticles could generate oxidative stress as well as the significant increase of nitric oxide in ECV304 cells. The loss of mitochondria membrane potential and the apoptotic chromatin condensation in the nucleus were observed as the early signs of apoptosis. It is inferred the stress response might be an important mechanism involving in endothelial cells apoptosis and death, and these injuries in endothelial cells might play a key role in downstream cardiovascular diseases such as atheroscelerosis, hypertension and myocardial infarction (MI).

  11. Recombinant human soluble thrombomodulin attenuates FK506-induced endothelial dysfunction through prevention of Akt inactivation.

    Science.gov (United States)

    Eguchi, Ryoji; Fujimori, Yoshihiro; Okada, Masaya; Tamaki, Hiroya; Wakabayashi, Ichiro; Ogawa, Hiroyasu

    2014-04-15

    Thrombomodulin (TM), a transmembrane glycoprotein on vascular endothelial cells, is a naturally occurring anticoagulant. Recombinant human soluble TM (rTM), composed of the extracellular domain of TM, also shows anti-coagulant and anti-inflammatory activity, but the effects of rTM on microangiopathy remain unclear. We reported that FK506 induced endothelial dysfunction through inactivation of Akt and extracellular-regulated kinase 1/2 using a three-dimensional culture blood vessel model. In the present study, we examined the effects of rTM on FK506-induced endothelial dysfunction. We found that rTM suppressed FK506-induced endothelial cell death, but not the breakdown of capillary-like tube structures. rTM prevented FK506-induced inactivation of Akt, but not of extracellular-regulated kinase 1/2. Akt inhibition by LY294002 abrogated the preventive effect of rTM on FK506-induced Akt inactivation and the suppressive effect of rTM on FK506-induced cell death. These results suggest that rTM attenuates FK506-induced endothelial dysfunction through prevention of Akt inactivation. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation.

    Science.gov (United States)

    Parekh, Mohit; Ferrari, Stefano; Sheridan, Carl; Kaye, Stephen; Ahmad, Sajjad

    2016-02-01

    The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance: The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field. ©AlphaMed Press.

  13. Human Lung Mononuclear Phagocytes in Health and Disease

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    Anna Smed-Sörensen

    2017-05-01

    Full Text Available The lungs are vulnerable to attack by respiratory insults such as toxins, allergens, and pathogens, given their continuous exposure to the air we breathe. Our immune system has evolved to provide protection against an array of potential threats without causing collateral damage to the lung tissue. In order to swiftly detect invading pathogens, monocytes, macrophages, and dendritic cells (DCs—together termed mononuclear phagocytes (MNPs—line the respiratory tract with the key task of surveying the lung microenvironment in order to discriminate between harmless and harmful antigens and initiate immune responses when necessary. Each cell type excels at specific tasks: monocytes produce large amounts of cytokines, macrophages are highly phagocytic, whereas DCs excel at activating naïve T cells. Extensive studies in murine models have established a division of labor between the different populations of MNPs at steady state and during infection or inflammation. However, a translation of important findings in mice is only beginning to be explored in humans, given the challenge of working with rare cells in inaccessible human tissues. Important progress has been made in recent years on the phenotype and function of human lung MNPs. In addition to a substantial population of alveolar macrophages, three subsets of DCs have been identified in the human airways at steady state. More recently, monocyte-derived cells have also been described in healthy human lungs. Depending on the source of samples, such as lung tissue resections or bronchoalveolar lavage, the specific subsets of MNPs recovered may differ. This review provides an update on existing studies investigating human respiratory MNP populations during health and disease. Often, inflammatory MNPs are found to accumulate in the lungs of patients with pulmonary conditions. In respiratory infections or inflammatory diseases, this may contribute to disease severity, but in cancer patients this may

  14. Bortezomib induces autophagic death in proliferating human endothelial cells

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    Belloni, Daniela; Veschini, Lorenzo [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy); Foglieni, Chiara [Department of Cardiology, IRCCS H San Raffaele, Milan (Italy); Dell' Antonio, Giacomo [Department of Pathology, IRCCS H San Raffaele, Milan (Italy); Caligaris-Cappio, Federico [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy); Universita Vita-Salute IRCCS H San Raffaele, Milan (Italy); Ferrarini, Marina [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy); Ferrero, Elisabetta, E-mail: elisabetta.ferrero@hsr.it [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy)

    2010-04-01

    The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

  15. Selective secretion of microRNAs from lung cancer cells via extracellular vesicles promotes CAMK1D-mediated tube formation in endothelial cells.

    Science.gov (United States)

    Lawson, James; Dickman, Christopher; MacLellan, Sara; Towle, Rebecca; Jabalee, James; Lam, Stephen; Garnis, Cathie

    2017-10-13

    Extracellular vesicles (EVs) are key signaling mediators between cancer cells and their supporting stroma, and regulate critical processes such as invasion, metastases, and angiogenesis. We have identified a subset of miRNAs (miR-142-3p, miR-143-3p, miR-145-5p, miR-150-5p, miR-223-3p, miR-451a, miR-486-5p, miR-605-5p) that are enriched in lung adenocarcinoma extracellular vesicles compared to the donor cells from which they were derived. Two well-known tumor suppressors, miR-143-3p and miR-145-5p, were also enriched in serum samples collected during surgery from blood vessels draining directly from lung adenocarcinoma tumor beds. Recently, both miRNAs were found to promote neoangiogenesis in endothelial cells in mouse models of lung adenocarcinoma through targeting of CAMK1D, an inhibitory kinase that can impair angiogenesis when over-expressed. We show that the transfer of miR-143-3p and miR-145-5p within extracellular vesicles from lung adenocarcinoma cells to endothelial cells reduces the levels of CAMK1D and increases tube formation by endothelial cells. This finding suggests that transfer of miRNAs within extracellular vesicles is a method of communication between cancer and endothelial cells which promotes angiogenesis while simultaneously removing tumor suppressive miRNAs within the tumor cells, thus driving tumorigenesis.

  16. Inhibition of TGF-β signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine.

    Directory of Open Access Journals (Sweden)

    Naoki Okumura

    Full Text Available Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+/K(+-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

  17. Human brain microvascular endothelial cells resist elongation due to shear stress.

    Science.gov (United States)

    Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

    2015-05-01

    Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Vasomotor control in mice overexpressing human endothelial nitric oxide synthase.

    Science.gov (United States)

    van Deel, Elza D; Merkus, Daphne; van Haperen, Rien; de Waard, Monique C; de Crom, Rini; Duncker, Dirk J

    2007-08-01

    Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.

  19. Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

    Science.gov (United States)

    Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

    2011-01-01

    Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

  20. Anti-thrombotic and pro-fibrinolytic effects of levosimendan in human endothelial cells in vitro.

    Science.gov (United States)

    Krychtiuk, Konstantin A; Kaun, Christoph; Hohensinner, Philipp J; Stojkovic, Stefan; Seigner, Jacqueline; Kastl, Stefan P; Zuckermann, Andreas; Eppel, Wolfgang; Rauscher, Sabine; de Martin, Rainer; Maurer, Gerald; Huber, Kurt; Wojta, Johann; Speidl, Walter S

    2017-03-01

    Levosimendan is an inodilator for the treatment of acute decompensated heart failure (HF). Data from clinical studies suggest that levosimendan is particularly effective in HF due to myocardial infarction. After acute revascularization, no reflow-phenomenon is a common complication that may lead to pump failure and cardiogenic shock. Our aim was to examine whether levosimendan interferes with the pro-thrombotic phenotype of activated endothelial cells in vitro. Human heart microvascular endothelial cells (HHMEC) and human umbilical vein endothelial cells (HUVEC) were treated with interleukin-1β (IL-1β) (200U/mL) or thrombin (5U/mL) and co-treated with or without levosimendan (0.1-10μM) for 2-24h. In addition, flow experiments were performed. Effects on plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression and activity were measured by rt-PCR, specific ELISA and flow cytometry. Treatment with IL-1β or thrombin significantly increased the expression of PAI-1 and TF in endothelial cells. Co-treatment with levosimendan strongly attenuated the effects of IL-1β and thrombin on PAI-1 and TF mRNA by up to 50% and 45%, in a dose- and time-dependent manner. Similar results were obtained under flow conditions. Furthermore, co-treatment with levosimendan dampened the antigen production of PAI-1 and the surface expression of TF by 35% and 45%, respectively. Additionally, levosimendan diminished both TF and PAI-1 activity. Levosimendan down-regulates the expression of the pro-thrombotic and anti-fibrinolytic biomolecules TF and PAI-1 in activated human endothelial cells. Our findings may, at least in part, explain some of the beneficial effects of levosimendan after myocardial reperfusion. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Interleukin-33 induces urokinase in human endothelial cells--possible impact on angiogenesis.

    Science.gov (United States)

    Stojkovic, S; Kaun, C; Heinz, M; Krychtiuk, K A; Rauscher, S; Lemberger, C E; de Martin, R; Gröger, M; Petzelbauer, P; Huk, I; Huber, K; Wojta, J; Demyanets, S

    2014-06-01

    Urokinase-type plasminogen activator (u-PA) plays a pivotal role in extracellular proteolysis and is thought to be critically involved in the modulation of angiogenesis. Interleukin (IL)-33 is a member of the IL-1 cytokine family, which is thought to act as danger signal that is released from cells after injury. IL-33 is involved in the pathogenesis of various inflammatory diseases and previously was shown to induce angiogenesis and inflammatory activation of endothelial cells. We investigated the impact of IL-33 on u-PA in endothelial cells as a new possible function for IL-33. We could demonstrate that IL-33 upregulated u-PA mRNA expression and protein production in human coronary artery and human umbilical vein endothelial cells in a time- and concentration-dependent manner via interaction with its receptor ST2 and activation of the nuclear factor-κB pathway but independent of autocrine IL-1-induced effects. The hydroxymethylglutaryl-coenzyme A reductase inhibitor simvastatin abrogated the IL-33-induced increase in u-PA, thus providing further evidence for pleiotropic effects of statins. IL-33 induced u-PA-dependent capillary-like tube formation and vessel sprouting. In human carotid atherosclerotic plaques (n = 16), u-PA mRNA positively correlated with IL-33 mRNA expression (r = 0.780, P < 0.001). Furthermore, IL-33 and u-PA protein were detected in endothelial cells in these samples using fluorescence immunohistochemistry. We hypothesize that IL-33, representing a danger signal that is released after tissue damage, in addition to its role in the inflammatory activation of endothelial cells, is involved in u-PA-driven angiogenesis, a process that has been shown before to be linked to inflammation in various pathologies. © 2014 International Society on Thrombosis and Haemostasis.

  2. Effect of polyhexanide and gentamycin on human osteoblasts and endothelial cells.

    Science.gov (United States)

    Ince, Akif; Schütze, Norbert; Hendrich, Christian; Jakob, Franz; Eulert, Jochen; Löhr, Jochen F

    2007-03-10

    Infection of total joint replacements is painful, disabling and difficult to treat because of the increasing bacterial resistance against antibiotics. In view of this, antiseptics show limited bacterial tolerance and have a broad-spectrum antimicrobial activity. However, the application of antiseptics to bone is insufficiently studied in literature. Therefore, we investigated the biocompatibility of the antiseptic polyhexanide with bone related cells and asked whether supplementation to bone cement is appropriate in the management of total arthroplasty infections. We performed an in vitro study with immortalised human foetal osteoblast cells (hFOB 1.19) and human endothelial cells (EAhy 926). The cultured cells were exposed to media containing various concentrations of gentamicin (12.5-800 microg/ml) and polyhexanide (0.0006-0.01%) for six hours. We measured the phase-contrast microscopy images, the cell viability, cell number and the alkaline phosphatase activity as a parameter for osteogenic function. The exposure of hFOB and endothelial cells to polyhexanide showed a severe reduction of viability and cell number. Gentamicin did not have negative effects on hFOB and endothelial cell number and viability. The alkaline phosphatase activity of hFOB showed a significant decrease after exposure to polyhexanide and gentamicin. The viability and the cell number of endothelial cells seem more negatively affected by polyhexanide than the parameters of the hFOB-cells. The exposure of human osteoblasts and endothelial cells to polyhexanide at concentrations with questionable antibacterial activity resulted in severe cell damage whereas exposure to high dosed gentamicin did not. These results raise questions as to the feasibility of using antiseptics in bone cement for the treatment of total arthroplasty infections. Further in vivo studies are necessary to show the in vivo relevance of these in vitro findings.

  3. Action of shiga toxin type-2 and subtilase cytotoxin on human microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    María M Amaral

    Full Text Available The hemolytic uremic syndrome (HUS associated with diarrhea is a complication of Shiga toxin (Stx-producing Escherichia coli (STEC infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF and platelet/endothelial cell adhesion molecule 1 (PECAM-1. HGEC also expressed the globotriaosylceramide (Gb3 receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

  4. Direct Reprogramming of Human Dermal Fibroblasts Into Endothelial Cells Using ER71/ETV2.

    Science.gov (United States)

    Lee, Sangho; Park, Changwon; Han, Ji Woong; Kim, Ju Young; Cho, Kyuwon; Kim, Eun Jae; Kim, Sangsung; Lee, Shin-Jeong; Oh, Se Yeong; Tanaka, Yoshiaki; Park, In-Hyun; An, Hyo Jae; Shin, Claire Min; Sharma, Shraya; Yoon, Young-Sup

    2017-03-03

    Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR+ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process. © 2016 American Heart Association, Inc.

  5. Antiangiogenic therapy in lung cancer: focus on vascular endothelial growth factor pathway.

    LENUS (Irish Health Repository)

    Korpanty, Grzegorz

    2010-01-01

    Lung cancer (LC) is a leading cause of death worldwide. Recent advances in chemotherapeutic agents have not yielded any significant improvement in the prognosis of patients with LC. The five-year survival rate for all combined disease stages remains about 15%. For this reason, new therapies such as those that inhibit tumor angiogenesis or block activity of growth factor receptors are of special interest in this group of patients. In this review we will summarize the most recent clinical data on biologic therapies that inhibit tumor angiogenesis in LC, focusing on those that are most clinically relevant.

  6. Vitamin D modulates prostaglandin E2 synthesis and degradation in human lung fibroblasts.

    Science.gov (United States)

    Liu, Xiangde; Nelson, Amy; Wang, Xingqi; Farid, Maha; Gunji, Yoko; Ikari, Jun; Iwasawa, Shun; Basma, Hesham; Feghali-Bostwick, Carol; Rennard, Stephen I

    2014-01-01

    Vitamin D insufficiency has been increasingly recognized in the general population worldwide and has been associated with several lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and respiratory tract infections. Fibroblasts play a critical role in tissue repair and remodeling, which is a key feature of COPD and asthma. Fibroblasts modulate tissue repair by producing and modifying extracellular matrix components and by releasing mediators that act as autocrine or paracrine modulators of tissue remodeling. The current study was designed to investigate if vitamin D alters fibroblast release of key autocrine/paracrine repair factors. First, we demonstrated that human fetal lung (HFL)-1 cells express the vitamin D receptor (VDR) and that vitamin D, 25-hydroxyvitamin D [25(OH)D], or 1,25-dihydroxyvitamin D [1,25(OH)2D] induce VDR nuclear translocation and increase VDR-DNA binding activity. We next demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D significantly reduced prostaglandin (PG)E2 production by human lung fibroblasts (HFL-1) but had no effect on transforming growth factor β1, vascular endothelial growth factor, or fibronectin production. Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited IL-1β-induced microsomal PGE synthase (mPGES)-1 expression; in contrast, all three forms of vitamin D stimulated 15-hydroxy PG dehydrogenase, an enzyme that degrades PGE2. Cyclooxygenase-1 and -2 and the other two PGE2 synthases (mPGES-2 and cytosolic PGE synthase) were not altered by vitamin D, 25(OH)D, or 1,25(OH)2D. Finally, the effect of PGE2 inhibition by 25(OH)D was observed in adult lung fibroblasts. These findings suggest that vitamin D can regulate PGE2 synthesis and degradation and by this mechanism can modulate fibroblast-mediated tissue repair function.

  7. Impact of Statins on Gene Expression in Human Lung Tissues.

    Directory of Open Access Journals (Sweden)

    Jérôme Lane

    Full Text Available Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that alter the synthesis of cholesterol. Some studies have shown a significant association of statins with improved respiratory health outcomes of patients with asthma, chronic obstructive pulmonary disease and lung cancer. Here we hypothesize that statins impact gene expression in human lungs and may reveal the pleiotropic effects of statins that are taking place directly in lung tissues. Human lung tissues were obtained from patients who underwent lung resection or transplantation. Gene expression was measured on a custom Affymetrix array in a discovery cohort (n = 408 and two replication sets (n = 341 and 282. Gene expression was evaluated by linear regression between statin users and non-users, adjusting for age, gender, smoking status, and other covariables. The results of each cohort were combined in a meta-analysis and biological pathways were studied using Gene Set Enrichment Analysis. The discovery set included 141 statin users. The lung mRNA expression levels of eighteen and three genes were up-regulated and down-regulated in statin users (FDR < 0.05, respectively. Twelve of the up-regulated genes were replicated in the first replication set, but none in the second (p-value < 0.05. Combining the discovery and replication sets into a meta-analysis improved the significance of the 12 up-regulated genes, which includes genes encoding enzymes and membrane proteins involved in cholesterol biosynthesis. Canonical biological pathways altered by statins in the lung include cholesterol, steroid, and terpenoid backbone biosynthesis. No genes encoding inflammatory, proteases, pro-fibrotic or growth factors were altered by statins, suggesting that the direct effect of statin in the lung do not go beyond its antilipidemic action. Although more studies are needed with specific lung cell types and different classes and doses of statins, the improved health outcomes and survival

  8. Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration

    Science.gov (United States)

    Fazal, Fabeha; Bijli, Kaiser M.; Murrill, Matthew; Leonard, Antony; Minhajuddin, Mohammad; Anwar, Khandaker N.; Finkelstein, Jacob N.; Watterson, D. Martin; Rahman, Arshad

    2013-01-01

    The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation. PMID:23555849

  9. Effect of high-glucose conditions on human periodontal ligament endothelial cells: in vitro analysis.

    Science.gov (United States)

    Maruyama, Kosuke; Sato, Soh

    2017-01-01

    Endothelial cells participate in key aspects of vascular biology, such as maintenance of capillary permeability and regulation of inflammation. According to previous reports, endothelial cells have revealed highly specific characteristics depending on the organs and tissues. In particular, periodontal endothelial cells have a higher permeability than vascular endothelial cells of other types of tissue. Periodontal disease is not only a chronic disease in oral, but also affect the entire body. Diabetes and periodontal disease are closely related, with periodontal disease even been referred to as the sixth complication of disease. However, no reports have investigated the pathophysiology of microvascular in periodontal tissue once diabetes has developed. Therefore, the aim of the present study is to investigate changes in the properties of human periodontal endothelial cells (HPDLECs) that were cultured under high-glucose conditions. We isolated HPDLECs from human periodontal ligament cells. HPDLECs were cultured under high-glucose (5.5, 11.0, 22.0 mM) and investigated proliferation, apoptosis, tube formation and the expression of cell adhesion molecules. A 5.5 mM (100 mg/dl) control was used in this study. HPDLECs stimulated with high glucose concentration exhibited suppression of cell proliferation and an increased percentage of apoptosis-positive cells. This results suggested that apoptosis was caused by TNF-α expression. The expression levels cell adhesion molecules increased. These results suggest that when HPDLECs are stimulated with a high glucose concentrations, PKC in the intracellular cell substrate is activated, increasing the expression of intercellular and vascular adhesion molecules. Thus, the results of this study demonstrate that diabetes exacerbates periodontal disease.

  10. Biomaterials trigger endothelial cell activation when co-incubated with human whole blood.

    Science.gov (United States)

    Herklotz, Manuela; Hanke, Jasmin; Hänsel, Stefanie; Drichel, Juliane; Marx, Monique; Maitz, Manfred F; Werner, Carsten

    2016-10-01

    Endothelial cell activation resulting from biomaterial contact or biomaterial-induced blood activation may in turn also affect hemostasis and inflammatory processes in the blood. Current in vitro hemocompatibility assays typically ignore these modulating effects of the endothelium. This study describes a co-incubation system of human whole blood, biomaterial and endothelial cells (ECs) that was developed to overcome this limitation. First, human endothelial cells were characterized in terms of their expression of coagulation- and inflammation-relevant markers in response to various activators. Subsequently, their capacity to regulate hemostasis as well as complement and granulocyte activation was monitored in a hemocompatibility assay. After blood contact, quiescent ECs exhibited anticoagulant and anti-inflammatory properties. When they were co-incubated with surfaces exhibiting pro-coagulant or pro-inflammatory characteristics, the ECs down-regulated coagulation but not complement or leukocyte activation. Analysis of intracellular levels of the endothelial activation markers E-selectin and tissue factor showed that co-incubation with model surfaces and blood significantly increased the activation state of ECs. Finally, the coagulation- and inflammation-modulating properties of the ECs were tested after blood/biomaterial exposure. Pre-activation of ECs by biomaterials in the blood induced a pro-coagulant and pro-inflammatory state of the ECs, wherein the pro-coagulant response was higher for biomaterial/blood pre-activated ECs than for TNF-α-pre-activated cells. This work provides evidence that biomaterials, even without directly contacting the endothelium, affect the endothelial activation state with and have consequences for plasmatic and cellular reactions in the blood. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Heparanase mediates vascular endothelial growth factor gene transcription in high-glucose human retinal microvascular endothelial cells

    Science.gov (United States)

    Wang, Jingwei; Leng, Xuan; Hu, Yijun; Shen, Huangxuan; Song, Xin

    2017-01-01

    Purpose To observe the nuclear expression and interaction of heparanase and RNA polymerase II (RNA Pol II), an enzyme that catalyzes the transcription of DNA in eukaryotic cells) in human retinal microvascular endothelial cells (HRECs) under high glucose condition and to investigate the association of heparanase with the transcription activity of the vascular endothelial growth factor (VEGF) gene promoter. Methods Cultured HRECs were maintained for 3 days in media with high or normal glucose. The expressions of heparanase and RNA Pol II in each group were analyzed with immunofluorescence. Co-immunoprecipitation was applied to detect the interaction of heparanase and Pol II proteins. Cells in both groups were used for chromatin immunoprecipitation (ChIP) with anti-heparanase and anti-RNA Pol II antibodies to identify high-confidence heparanase-binding regions across the entire VEGF gene promoter. Moreover, real-time PCR was used to demonstrate the interaction between heparanase and the VEGF gene promoter region. Results The immunofluorescence studies showed that the nuclear expression of heparanase was intense in high-glucose HRECs but faint in the normal group; RNA Pol II in the nucleus was also intense in high glucose HRECs, and the distribution of heparanase was consistent with that of RNA Pol II. The co-immunoprecipitation data showed that heparanase combined with RNA Pol II in HRECs cells treated with high glucose, and the molecular size of HPA interacted with RNA Pol II was 50 kDa, while no combination of two proteins was evident in normal HRECs cells. Real-time PCR–based ChIP results showed that the high-confidence HPA-binding region was −1155 to −1018 (containing hypoxia response element) in the VEGF gene promoter, and the cells treated with high glucose showed increases in heparanase and RNA Pol II in the VEGF gene promoter region compared with the normal glucose treated cells (t = –3.244, p = 0.032; t = –6.096, p = 0.004, respectively

  12. p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, Katsuya; Oka, Kiyomasa [Kringle Pharma Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Matsumoto, Kunio [Division of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934 (Japan); Nakamura, Toshikazu, E-mail: nakamura@casi.osaka-u.ac.jp [Kringle Pharma Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

  13. Effects of depolarizing or non-depolarizing preservation solutions on human endothelial cells during cold hypoxia.

    Science.gov (United States)

    Hidalgo, M A; Mann, D J; Fuller, B J; Green, C J

    1996-02-01

    1. Hypothermic storage of whole organs flushed with a preservation solution is common practice in clinical transplantation. This procedure leaves vascular endothelial cells in direct contact with the preservation solution during the length of the cold ischaemic period. 2. Aiming to study the effects of organ preservation on vascular endothelium, we subjected cultures of human umbilical vein endothelial cells to hypoxic and hypothermic storage conditions in vitro for 3 or 16 h. Four preservation solutions with different levels of sodium and potassium were tested. Morphometric analysis and 51Cr leakage index were used to assess monolayer continuity, cell viability and membrane integrity. 3. Hypothermic storage resulted in severe changes in endothelial cell morphology with formation of intercellular gaps that destroyed monolayer continuity after only 3h. Cellular blebbing was a common feature in seriously damaged cells. 4. Morphometric analysis and 51Cr leakage results correlated well. No significant differences between the solutions tested were found after 3h of hypothermic hypoxic storage. After 16h, viability and monolayer continuity were significantly better preserved (Mann-Whitney, P storage, vascular endothelial cells appeared morphologically deformed and poorly attached in vitro. Lactobionate-based preservation solutions were more effective in preserving viability and continuity. Protection of vascular endothelium under cold hypoxic conditions could be a critical factor in successfully preserving organs for transplantation.

  14. eNOS-dependent antisenscence effect of a calcium channel blocker in human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Toshio Hayashi

    Full Text Available Senescence of vascular endothelial cells is an important contributor to the pathogenesis of age-associated vascular disorders such as atherosclerosis. We investigated the effects of antihypertensive agents on high glucose-induced cellular senescence in human umbilical venous endothelial cells (HUVECs. Exposure of HUVECs to high glucose (22 mM for 3 days increased senescence-associated- β-galactosidase (SA-β-gal activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. The calcium channel blocker nifedipine, but not the β1-adrenergic blocking agent atenolol or the angiotensin-converting enzyme inhibitor perindopril, reduced SA-β-gal positive cells and prevented a decrease in telomerase activity in a high-glucose environment. This beneficial effect of nifedipine was associated with reduced reactive oxygen species (ROS and increased endothelial nitric oxide synthase (eNOS activity. Thus, nifedipine prevented high glucose-induced ROS generation and increased basal eNOS phosphorylation level at Ser-1177. Treatment with N (G-nitro-L-arginine (L-NAME and transfection of small interfering RNA (siRNA targeting eNOS eliminated the anti-senscence effect of nifedipine. These results demonstrate that nifedipine can prevent endothelial cell senescence in an eNOS-dependent manner. The anti-senescence action of nifedipine may represent a novel mechanism by which it protects against atherosclerosis.

  15. Urea immunoliposome inhibits human vascular endothelial cell proliferation for hemangioma treatment

    Science.gov (United States)

    2013-01-01

    Background Urea injection has been used in hemangioma treatment as sclerotherapy. It shrinks vascular endothelial cells and induces degeneration, necrosis, and fibrosis. However, this treatment still has disadvantages, such as lacking targeting and difficulty in controlling the urea dosage. Thus, we designed a urea immunoliposome to improve the efficiency of treatment. Methods The urea liposome was prepared by reverse phase evaporation. Furthermore, the urea immunoliposome was generated by coupling the urea liposome with a vascular endothelial growth factor receptor (VEGFR) monoclonal antibody using the glutaraldehyde cross-linking method. The influence of the urea immunoliposome on cultured human hemangioma vascular endothelial cells was observed preliminarily. Results Urea immunoliposomes showed typical liposome morphology under a transmission electron microscope, with an encapsulation percentage of 54.4% and a coupling rate of 36.84% for anti-VEGFR. Treatment with the urea immunoliposome significantly inhibited the proliferation of hemangioma vascular endothelial cells (HVECs) in a time- and dose-dependent manner. Conclusions The urea immunoliposome that we developed distinctly and persistently inhibited the proliferation of HVECs and is expected to be used in clinical hemangioma treatment. PMID:24266957

  16. Asbestos fibers in human lung: forensic significance

    Energy Technology Data Exchange (ETDEWEB)

    Ehrenreich, T.; Selikoff, I.J.

    1981-03-01

    Asbestos is a fibrous mineral which, because of its unique properties, has innumerable applications in many industries and is used in a large variety of consumer products. It has become ubiquitous and is woven, literally and figuratively, into the fabric of our present-day civilization. However, its presence is sometimes unknown and unsuspected by those who are exposed to asbestos by virtue of occupation or environment and inhale its fibers. Exposed workers and even urban dwellers may have a variable lung burden of asbestos fibers. There is indisputable clinical, pathological, experimental and epidemiological proof that, after varying periods of latency, asbestos may cause benign and malignant disease often leading to disability or death. Forensic investigation of suspected asbestos-related deaths includes a life-time occupational history, a complete autopsy, and identification of the asbestos fiber tissue burden. The latter usually requires special procedures.

  17. Allopurinol protects human glomerular endothelial cells from high glucose-induced reactive oxygen species generation, p53 overexpression and endothelial dysfunction.

    Science.gov (United States)

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2017-11-01

    Mitochondrial reactive oxygen species (ROS) overproduction in capillary endothelial cells is a prerequisite for the development of diabetic nephropathy. Inhibition of xanthine oxidase, another ROS generator, ameliorates experimental diabetic nephropathy. To test the hypothesis that the initial high glucose-induced ROS production by the mitochondria activates xanthine oxidase, which afterward remains as the major source of ROS, we cultured primary human glomerular endothelial cells (GEnC) under normal or high-glucose conditions, with or without the xanthine oxidase inhibitor allopurinol. ROS generation and nitric oxide synthase (NOS) activity were assessed by chemiluminescence or colorimetrically. Levels of intercellular adhesion molecule 1 (ICAM-1), p53 and phosphorylated p53 (p-p53) were assessed by western blotting. Allopurinol prevented high glucose-induced ROS generation indicating that xanthine oxidase is the major source of ROS. Allopurinol protected GEnC from endothelial dysfunction since it prevented the high glucose-induced decrease in NOS activity and increase in ICAM-1 expression. Allopurinol reduced p53 and p-p53 levels induced by high glucose suggesting an axis of xanthine oxidase-derived ROS, DNA damage, p53 stabilization and endothelial dysfunction that may contribute to the pathogenesis of diabetic nephropathy. Allopurinol protects GEnC from high glucose-induced ROS generation, p53 overexpression and endothelial dysfunction. These data provide a pathogenetic mechanism that supports the results of experimental and clinical studies about the beneficial effect of xanthine oxidase inhibitors on the development of diabetic nephropathy.

  18. Green tea polyphenol induces significant cell death in human lung ...

    African Journals Online (AJOL)

    Green tea polyphenol induces significant cell death in human lung cancer cells. Jie Huang, Fa-jiu Li, Shi Chen, Yi Shi, Xiao-jiang Wang, Chuan-hai Wang, Qing- ..... method for the determination of green and black tea polyphenols in biomatrices by high-performance liquid chromatography with coulometric array detection.

  19. Lipidomic characterization and localization of phospholipids in the human lung.

    Science.gov (United States)

    Zemski Berry, Karin A; Murphy, Robert C; Kosmider, Beata; Mason, Robert J

    2017-05-01

    Lipids play a central role in lung physiology and pathology; however, a comprehensive lipidomic characterization of human pulmonary cells relevant to disease has not been performed. The cells involved in lung host defense, including alveolar macrophages (AMs), bronchial epithelial cells (BECs), and alveolar type II cells (ATIIs), were isolated from human subjects and lipidomic analysis by LC-MS and LC-MS/MS was performed. Additionally, pieces of lung tissue from the same donors were analyzed by MALDI imaging MS in order to determine lipid localization in the tissue. The unique distribution of phospholipids in ATIIs, BECs, and AMs from human subjects was accomplished by subjecting the large number of identified phospholipid molecular species to univariant statistical analysis. Specific MALDI images were generated based on the univariant statistical analysis data to reveal the location of specific cell types within the human lung slice. While the complex composition and function of the lipidome in various disease states is currently poorly understood, this method could be useful for the characterization of lipid alterations in pulmonary disease and may aid in a better understanding of disease pathogenesis. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  20. Tangeretin sensitises human lung cancer cells to TRAIL- induced ...

    African Journals Online (AJOL)

    necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. (H1299 and H1975). Methods: ... Western blotting was performed to assess the expression of death receptors, apoptosis pathway proteins, JNK and ERK1/2. ...... upregulation in hepatocellular carcinoma cells. World J.

  1. Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells

    OpenAIRE

    Cossu, Annalisa

    2012-01-01

    Cardiovascular (CV) benefits of Natural Antioxidant (NA) supplementation are contradictory. Endothelial Cells (EC) are pivotal player on CV diseases onset/progression and thus represent a good model to study the NA impact on vascular pathophysiology. We show that two NA, coumaric acid and resveratrol, affect intracellular ROS levels and cell physiology in human EC. While at lower doses both compounds were antioxidant, at mildly high doses they became pro-oxidant, eliciting cell death by apopt...

  2. Characterization of Influenza Virus-Induced Leukocyte Adherence to Human Umbilical Vein Endothelial Cell Monolayers

    Science.gov (United States)

    1993-07-01

    with other viruses. HL-60 cell adherence to endothelial cell virus type A, which did not infect human venous or bovine monolayers was modulated by...LEUCOCYTE ADHERENC:E TO [NDOTIIELIL (FS1% A. B reawsd on parainfluenza virus-infected airway epithelial Poiy-iiysine Codled IPLC) Wells PLC.Wells cells...an antibody against ICAN1- I has no significant effect PLC Wells Virus on parainfluenza -induced neutrophil adherence (58). In 25 *HSV-intected HUVEC

  3. Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds

    OpenAIRE

    Matschegewski Claudia; Matthies Jörn-Bo; Grabow Niels; Schmitz Klaus-Peter

    2016-01-01

    The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide) PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926). Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation...

  4. CXCL12 enhances angiogenesis through CXCR7 activation in human umbilical vein endothelial cells

    OpenAIRE

    ZHANG Min; Qiu, Lisha; Zhang, Yanyan; Xu, Dongsheng; Zheng, Jialin C.; Jiang, Li

    2017-01-01

    Angiogenesis is the process by which new vessels form from existing vascular networks. Human umbilical vein endothelial cells (HUVECs) may contribute to the study of vascular repair and angiogenesis. The chemokine CXCL12 regulates multiple cell functions, including angiogenesis, mainly through its receptor CXCR4. In contrast to CXCL12/CXCR4, few studies have described roles for CXCR7 in vascular biology, and the downstream mechanism of CXCR7 in angiogenesis remains unclear. The results of the...

  5. Lung Volume Reduction Surgery and Improvement of Endothelial Function and Blood Pressure in Patients with Chronic Obstructive Pulmonary Disease. A Randomized Controlled Trial.

    Science.gov (United States)

    Clarenbach, Christian F; Sievi, Noriane A; Brock, Matthias; Schneiter, Didier; Weder, Walter; Kohler, Malcolm

    2015-08-01

    Cardiovascular disease is a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD). Preliminary studies have shown that both airflow obstruction and systemic inflammation may contribute to endothelial dysfunction in COPD. Lung volume reduction surgery (LVRS) is a treatment option in selected patients with COPD with emphysema that improves breathing mechanics and lung function. To determine the effect of LVRS on endothelial function and systemic inflammation. We conducted a randomized controlled trial in 30 patients scheduled for LVRS. In the intervention group, immediate LVRS was performed after baseline evaluation followed by reassessment 3 months later. In the control group, reassessment followed 3 months after baseline evaluation, and thereafter LVRS was performed. The primary outcome measures were the treatment effect on endothelial function and systemic inflammation. In the LVRS group 14 patients completed the trial and 13 in the control group. LVRS led to a relative reduction in mean (SD) residual volume/total lung capacity of -12% (12%) and an increase in FEV1 of 29% (27%). Flow-mediated dilatation of the brachial artery increased in the intervention group as compared with the control group (+2.9%; 95% confidence interval, +2.1 to +3.6%; P < 0.001), whereas there was no significant change in systemic inflammation. A significant treatment effect on mean blood pressure was observed (-9.0 mm Hg; 95% confidence interval, -17.5 to -0.5; P = 0.039). Endothelial function and blood pressure are improved 3 months after LVRS in patients with severe COPD and emphysema. LVRS may therefore have beneficial effects on cardiovascular outcomes. Clinical trial registered with www.clinicaltrials.gov (NCT 01020344).

  6. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  7. Characterization of ionizing radiation-induced unfolded protein response in human vascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun Ju; Lee, Yoon Jin; Kang, Seong Man [Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2013-04-15

    Misfolded or unfolded proteins within the endoplasmic reticulum (ER stress), viral infection, or amino acid deprivation induce eukaryotic translation initiation factor 2α phosphorylation (eIF2α) in eukaryotic cells, repressing global protein synthesis coincident with preferential translation of activating transcription factor 4 (ATF4). ATF4 is a transcriptional activator of genes involved in amino acid metabolism, cellular redox homeostasis, and regulation of apoptosis. When the eIF2α/ATF4 pathway is initiated by ER stress, the pathway is referred toas the unfolded protein response (UPR). In addition to DNA, proteins may be initial and important targets of ionizing radiation (IR), and the damaged protein can trigger ER stress pathway. Recent investigations suggested that IR induces ER stress followed by UPR in various cell types including intestinal epithelial cells. We conducted this study to determine whether IR can activate UPR in human vascular endothelial cells. Our data have shown that IR increased PERK-dependent eIF2α phosphorylation accompanied by induction in ATF4 protein levels in human vascular endothelial cells without alterations in expressions of XBP-1s and GRP78. Based on these data, we suggest that IR selectively activates PERK branch of unfolded protein response in human vascular endothelial cells.

  8. Human Haemato-Endothelial Precursors: Cord Blood CD34+ Cells Produce Haemogenic Endothelium

    Science.gov (United States)

    Pelosi, Elvira; Castelli, Germana; Martin-Padura, Ines; Bordoni, Veronica; Santoro, Simona; Conigliaro, Alice; Cerio, Anna Maria; De Santis Puzzonia, Marco; Marighetti, Paola; Biffoni, Mauro; Alonzi, Tonino; Amicone, Laura; Alcalay, Myriam; Bertolini, Francesco; Testa, Ugo; Tripodi, Marco

    2012-01-01

    Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144−), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45−) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level. PMID:23226561

  9. Human haemato-endothelial precursors: cord blood CD34+ cells produce haemogenic endothelium.

    Directory of Open Access Journals (Sweden)

    Elvira Pelosi

    Full Text Available Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-, triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45- capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.

  10. Detection of histidine decarboxylase mRNA in human vascular smooth muscle and endothelial cells.

    Science.gov (United States)

    Tippens, A S; Gruetter, C A

    2004-06-01

    The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA. HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively. Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons. These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

  11. Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model.

    Science.gov (United States)

    Lokmic, Zerina; Mitchell, Geraldine M; Koh Wee Chong, Nicholas; Bastiaanse, Jacqueline; Gerrand, Yi-Wen; Zeng, Yiping; Williams, Elizabeth D; Penington, Anthony J

    2014-01-01

    Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34(Neg)CD31(Pos) LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34(Neg)CD31(Pos) lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P lymphatic malformations.

  12. Magnetic Resonance Tracking of Endothelial Progenitor Cells Labeled with Alkyl-Polyethylenimine 2 kDa/Superparamagnetic Iron Oxide in a Mouse Lung Carcinoma Xenograft Model

    Directory of Open Access Journals (Sweden)

    Cong Chen

    2014-11-01

    Full Text Available The potential of using endothelial progenitor cells (EPCs in novel anticancer therapy and the repair of vascular injury has been increasingly recognized. In the present study, EPCs were labeled with N-alkyl-polyethylenimine 2 kDa (PEI2k-stabilized superparamagnetic iron oxide (SPIO to facilitate magnetic resonance imaging (MRI of EPCs in a mouse lung carcinoma xenograft model. EPCs derived from human peripheral blood were labeled with alkyl-PEI2k/SPIO. The viability and activity of labeled cells were evaluated using proliferation, migration, and tubulogenesis assays. Alkyl-PEI2k/SPIO-labeled EPCs were injected intravenously (group 1 or mixed and injected together with A549 cells subcutaneously (group 2 into groups of six mice with severe combined immunodeficiency. The labeling efficiency with alkyl-PEI2k/SPIO at 7 mg Fe/mL concentration was approximately 100%. Quantitative analysis of cellular iron was 6.062 ± 0.050 pg/cell. No significant effects on EPC proliferation, migration, or tubulogenesis were seen after labeling. Seventesla micro-MRI showed the presence of schistic or linear hypointense regions at the tumor margins starting from days 7 to 8 after EPC administration. This gradually extended into the inner tumor layers in group 1. In group 2, tumor growth was accompanied by dispersion of low-signal intensity regions inside the tumor. Iron-positive cells identified by Prussian blue dye were seen at the sites identified using MRI. Human CD31-positive cells and mouse CD31-positive cells were present in both groups. Labeling EPCs with alkyl-PEI2k/SPIO allows noninvasive magnetic resonance investigation of EPC involvement in tumor neovasculature and is associated with excellent biocompatibility and MRI sensitivity.

  13. Endothelial progenitor cells from human fetal aorta cure diabetic foot in a rat model.

    Science.gov (United States)

    Zhao, Wan-Ni; Xu, Shi-Qing; Liang, Jian-Feng; Peng, Liang; Liu, Hong-Lin; Wang, Zai; Fang, Qing; Wang, Meng; Yin, Wei-Qin; Zhang, Wen-Jian; Lou, Jin-Ning

    2016-12-01

    Recent evidence has suggested that circulating endothelial progenitor cells (EPCs) can repair the arterial endothelium during vascular injury. However, a reliable source of human EPCs is needed for therapeutic applications. In this study, we isolated human fetal aorta (HFA)-derived EPCs and analyzed the capacity of EPCs to differentiate into endothelial cells. In addition, because microvascular dysfunction is considered to be the major cause of diabetic foot (DF), we investigated whether transplantation of HFA-derived EPCs could treat DF in a rat model. EPCs were isolated from clinically aborted fetal aorta. RT-PCR, fluorescence-activated cell sorting, immunofluorescence, and an enzyme-linked immunosorbent assay were used to examine the expressions of CD133, CD34, CD31, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), von Willebrand Factor (vWF), and Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1). Morphology and Dil-uptake were used to assess function of the EPCs. We then established a DF model by injecting microcarriers into the hind-limb arteries of Goto-Kakizaki rats and then transplanting the cultured EPCs into the ischemic hind limbs. Thermal infrared imaging, oxygen saturation apparatus, and laser Doppler perfusion imaging were used to monitor the progression of the disease. Immunohistochemistry was performed to examine the microvascular tissue formed by HFA-derived EPCs. We found that CD133, CD34, and VEGFR2 were expressed by HFA-derived EPCs. After VEGF induction, CD133 expression was significantly decreased, but expression levels of vWF and ELAM-1 were markedly increased. Furthermore, tube formation and Dil-uptake were improved after VEGF induction. These observations suggest that EPCs could differentiate into endothelial cells. In the DF model, temperature, blood flow, and oxygen saturation were reduced but recovered to a nearly normal level following injection of the EPCs in the hind limb. Ischemic symptoms also improved. Injected EPCs were

  14. Isoflurane Protects Against Human Endothelial Cell Apoptosis by Inducing Sphingosine Kinase-1 via ERK MAPK

    Directory of Open Access Journals (Sweden)

    H. Thomas Lee

    2012-01-01

    Full Text Available Endothelial dysfunction is a major clinical problem affecting virtually every patient requiring critical care. Volatile anesthetics are frequently used during the perioperative period and protect the heart and kidney against ischemia and reperfusion injury. We aimed to determine whether isoflurane, the most commonly used volatile anesthetic in the USA, protects against endothelial apoptosis and necrosis and the mechanisms involved in this protection. Human endothelial EA.hy926 cells were pretreated with isoflurane or carrier gas (95% room air + 5% CO2 then subjected to apoptosis with tumor necrosis factor-α or to necrosis with hydrogen peroxide. DNA laddering and in situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL staining determined EA.hy926 cell apoptosis and percent LDH released determined necrosis. We also determined whether isoflurane modulates the expression and activity of sphingosine kinase-1 (SK1 and induces the phosphorylation of extracellular signal regulated kinase (ERK MAPK as both enzymes are known to protect against cell death. Isoflurane pretreatment significantly decreased apoptosis in EA.hy926 cells as evidenced by reduced TUNEL staining and DNA laddering without affecting necrosis. Mechanistically, isoflurane induces the phosphorylation of ERK MAPK and increased SK1 expression and activity in EA.hy926 cells. Finally, selective blockade of SK1 (with SKI-II or S1P1 receptor (with W146 abolished the anti-apoptotic effects of isoflurane. Taken together, we demonstrate that isoflurane, in addition to its potent analgesic and anesthetic properties, protects against endothelial apoptosis most likely via SK1 and ERK MAPK activation. Our findings have significant clinical implication for protection of endothelial cells during the perioperative period and patients requiring critical care.

  15. Impaired Endothelial Regeneration Through Human Parvovirus B19-Infected Circulating Angiogenic Cells in Patients With Cardiomyopathy.

    Science.gov (United States)

    Schmidt-Lucke, Caroline; Zobel, Thomas; Schrepfer, Sonja; Kuhl, Uwe; Wang, Dong; Klingel, Karin; Becher, Peter Moritz; Fechner, Henry; Pozzuto, Tanja; Van Linthout, Sophie; Lassner, Dirk; Spillmann, Frank; Escher, Felicitas; Holinski, Sebastian; Volk, Hans-Dieter; Schultheiss, Heinz-Peter; Tschope, Carsten

    2015-10-01

    Human parvovirus B19 (B19V) is a common pathogen in microvascular disease and cardiomyopathy, owing to infection of endothelial cells. B19V replication, however, is almost restricted to erythroid progenitor cells (ErPCs). Endothelial regeneration attributable to bone marrow-derived circulating angiogenic cells (CACs) is a prerequisite for organ function. Because of many similarities of ErPCs and CACs, we hypothesized that B19V is a perpetrator of impaired endogenous endothelial regeneration. B19V DNA and messenger RNA from endomyocardial biopsy specimens, bone marrow specimens, and circulating progenitor cells were quantified by polymerase chain reaction analysis. The highest B19V DNA concentrations were found in CD34(+)KDR(+) cells from 17 patients with chronic B19V-associated cardiomyopathy. B19V replication intermediates could be detected in nearly half of the patients. Furthermore, chronic B19V infection was associated with impaired endothelial regenerative capacity. B19V infection of CACs in vitro resulted in expression of transcripts encoding B19V proteins. The capsid protein VP1 was identified as a novel inducer of apoptosis, as were nonstructural proteins. Inhibition studies identified so-called death receptor signaling with activation of caspase-8 and caspase-10 to be responsible for apoptosis induction. B19V causally impaired endothelial regeneration with spreading of B19V in CACs in an animal model in vivo. We thus conclude that B19V infection and damage to CACs result in dysfunctional endogenous vascular repair, supporting the emergence of primary bone marrow disease with secondary end-organ damage. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity.

    Science.gov (United States)

    Hughes-Large, Jennifer M; Pang, Dominic K T; Robson, Debra L; Chan, Pak; Toma, Jelena; Borradaile, Nica M

    2014-12-01

    Niacin (nicotinic acid) as a monotherapy can reduce vascular disease risk, but its mechanism of action remains controversial, and may not be dependent on systemic lipid modifying effects. Niacin has recently been shown to improve endothelial function and vascular regeneration, independent of correcting dyslipidemia, in rodent models of vascular injury and metabolic disease. As a potential biosynthetic precursor for NAD(+), niacin could elicit these vascular benefits through NAD(+)-dependent, sirtuin (SIRT) mediated responses. Alternatively, niacin may act through its receptor, GPR109A, to promote endothelial function, though endothelial cells are not known to express this receptor. We hypothesized that niacin directly improves endothelial cell function during exposure to lipotoxic conditions and sought to determine the potential mechanism(s) involved. Angiogenic function in excess palmitate was assessed by tube formation following treatment of human microvascular endothelial cells (HMVEC) with either a relatively low concentration of niacin (10 μM), or nicotinamide mononucleotide (NMN) (1 μM), a direct NAD(+) precursor. Although both niacin and NMN improved HMVEC tube formation during palmitate overload, only NMN increased cellular NAD(+) and SIRT1 activity. We further observed that HMVEC express GRP109A. Activation of this receptor with either acifran or MK-1903 recapitulated niacin-induced improvements in HMVEC tube formation, while GPR109A siRNA diminished the effect of niacin. Niacin, at a low concentration, improves HMVEC angiogenic function under lipotoxic conditions, likely independent of NAD(+) biosynthesis and SIRT1 activation, but rather through niacin receptor activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation.

    Science.gov (United States)

    Lee, Yu-Hui; Shynlova, Oksana; Lye, Stephen J

    2015-03-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte-endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), Pcytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution.

  18. Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation

    Science.gov (United States)

    Lee, Yu-Hui; Shynlova, Oksana; Lye, Stephen J

    2015-01-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte–endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), Pstretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution

  19. The use of adipose mesenchymal stem cells and human umbilical vascular endothelial cells on a fibrin matrix for endothelialized skin substitute.

    Science.gov (United States)

    Sánchez-Muñoz, Isabel; Granados, Rosario; Holguín Holgado, Purificación; García-Vela, José Antonio; Casares, Celia; Casares, Miguel

    2015-01-01

    In recent years, the reconstruction of human skin by tissue engineering represents a clinical challenge and has offered a therapeutic alternative. Avascular engineered skin equivalents have been available for several years and used to treat wounds due to burns, nonhealing ulcers, and surgical excisions. They are constituted by different types of cultured cells included in a three-dimensional structure that permits cellular proliferation to create tissue substitutes. The major drawback of these artificial skin substitutes is their lack of blood supply, since the endurance and cell proliferation of the substitute depend on an adequate oxygen and nutrient supply and on toxin removal. These functions are served by the vascular system. We have produced a new model of endothelialized skin substitute that promotes the formation of capillary-like structures by seeding human umbilical vein endothelial cells (HUVECs) with dermal fibroblasts and human adipose-derived mesenchymal stem cells (hADMSCs) in a fibrin matrix. Dermal fibroblasts and hADMSCs produce extracellular matrix that stimulates cellular growth and proliferation. hADMSCs secrete significant quantities of angiogenic and antiapoptotic factors (vascular endothelial growth factor and hepatocyte growth factor), which induce in vitro differentiation of these cells into endothelial cells promoting angiogenesis and participating in tissue repair and skin regeneration processes. We obtained the artificial skin substitute with similar structure to native skin, including dermis and epidermis. We demonstrated that endothelial cells (CD31 and von Willebrand factor positive) proliferated and organized themselves into capillary-like structures within the fibrin matrix. The epidermis showed a complete epithelization by squamous cells (AE1/AE3 cytokeratin positive) with intracytoplasmic keratohyalin granules, hyperkeratosis, and parakeratosis. We have established a novel artificial skin substitute that facilitates the formation

  20. Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.

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    Saem Mul Park

    Full Text Available The lymphatic sinuses in human lymph nodes (LNs are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs. A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs; in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

  1. Retrograde flow and shear rate acutely impair endothelial function in humans.

    Science.gov (United States)

    Thijssen, Dick H J; Dawson, Ellen A; Tinken, Toni M; Cable, N Timothy; Green, Daniel J

    2009-06-01

    Changes in arterial shear stress induce functional and structural vasculature adaptations. Recent studies indicate that substantial retrograde flow and shear can occur through human conduit arteries. In animals, retrograde shear is associated with atherogenic effects. The aim of this study was to examine the impact of incremental levels of retrograde shear on endothelial function in vivo. On 3 separate days, we examined bilateral brachial artery flow-mediated dilation, an index of NO-mediated endothelial function, in healthy men (24+/-3 years) before and after a 30-minute intervention consisting of cuff inflation to 25, 50, or 75 mm Hg. Cuff inflations resulted in "dose"-dependent increases in retrograde shear rate, compared with the noncuffed arm, within subjects (P<0.001). Flow-mediated dilation in the cuffed arm did not change in response to the 25-mm Hg stimulus but decreased significantly after both the 50- and 75-mm Hg interventions (P<0.05). The decrease in flow-mediated dilation after the 75-mm Hg intervention was significantly larger than that observed after a 50-mm Hg intervention (P=0.03). In the noncuffed arm, no changes in shear rate or flow-mediated dilation were observed. These results demonstrate that an increase in retrograde shear rate induces a dose-dependent attenuation of endothelial function in humans. This finding contributes to our understanding regarding the possible detrimental effects of retrograde shear rate in vivo.

  2. Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells

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    A. Van Der Meeren

    1997-01-01

    Full Text Available Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of 60Co-irradiated human umbilical vein endothelial cells (HUVEC have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL-1α, IL-1β and tumor necrosis factor (TNF-α remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.

  3. Bioinformatics Analyses of the Role of Vascular Endothelial Growth Factor in Patients with Non-Small Cell Lung Cancer.

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    Ying Wang

    Full Text Available This study was aimed to identify the expression pattern of vascular endothelial growth factor (VEGF in non-small cell lung cancer (NSCLC and to explore its potential correlation with the progression of NSCLC.Gene expression profile GSE39345 was downloaded from the Gene Expression Omnibus database. Twenty healthy controls and 32 NSCLC samples before chemotherapy were analyzed to identify the differentially expressed genes (DEGs. Then pathway enrichment analysis of the DEGs was performed and protein-protein interaction networks were constructed. Particularly, VEGF genes and the VEGF signaling pathway were analyzed. The sub-network was constructed followed by functional enrichment analysis.Total 1666 up-regulated and 1542 down-regulated DEGs were identified. The down-regulated DEGs were mainly enriched in the pathways associated with cancer. VEGFA and VEGFB were found to be the initiating factor of VEGF signaling pathway. In addition, in the epidermal growth factor receptor (EGFR, VEGFA and VEGFB associated sub-network, kinase insert domain receptor (KDR, fibronectin 1 (FN1, transforming growth factor beta induced (TGFBI and proliferating cell nuclear antigen (PCNA were found to interact with at least two of the three hub genes. The DEGs in this sub-network were mainly enriched in Gene Ontology terms related to cell proliferation.EGFR, KDR, FN1, TGFBI and PCNA may interact with VEGFA to play important roles in NSCLC tumorigenesis. These genes and corresponding proteins may have the potential to be used as the targets for either diagnosis or treatment of patients with NSCLC.

  4. The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells

    OpenAIRE

    Ge, Gaoyuan; WU, HENGFANG; Xiong,Fei; Zhang, Yu; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Gu, Chunrong; Gu, Ning; Chen, Xiangjian; Yang, Di

    2013-01-01

    One major obstacle for successful application of nanoparticles in medicine is its potential nanotoxicity on the environment and human health. In this study, we evaluated the cytotoxicity effect of dimercaptosuccinic acid-coated iron oxide (DMSA-Fe2O3) using cultured human aortic endothelial cells (HAECs). Our results showed that DMSA-Fe2O3 in the culture medium could be absorbed into HAECs, and dispersed in the cytoplasm. The cytotoxicity effect of DMSA-Fe2O3 on HAECs was dose-dependent, and ...

  5. Expression of ADAMs ("a disintegrin and metalloprotease") in the human lung

    NARCIS (Netherlands)

    Dijkstra, Antoon; Postma, Dirkje S.; Noordhoek, Jacobien A.; Lodewijk, Monique E.; Kauffman, Henk F.; ten Hacken, Nick H. T.; Timens, Wim

    In view of the associations of "a disintegrin and metalloprotease" (ADAM) with respiratory diseases, we assessed the expression of various ADAMs in human lung tissue. Lung tissue was obtained from nine individuals who underwent surgery for lung cancer or underwent lung transplantation for emphysema.

  6. Tumor Associated Neutrophils in Human Lung Cancer

    Science.gov (United States)

    2016-10-01

    isolated from the samepatientwithNSCLC.Tcellproliferation in responsetoCD3/CD28wasperformedasdescribed inMaterials andMethods. Cell proliferationwas...PMNswere isolated as described inMaterials andMethods and then added to allogeneic MLR in the presence of neutralizing mouse anti-human LOX-1 antibody (10 mg

  7. SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells.

    Science.gov (United States)

    Zhang, Lianghui; Jambusaria, Ankit; Hong, Zhigang; Marsboom, Glenn; Toth, Peter T; Herbert, Brittney-Shea; Malik, Asrar B; Rehman, Jalees

    2017-06-20

    The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. CD34+ progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34+ cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34+ progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. Dedifferentiation of fibroblasts to intermediate CD34+ progenitors gives rise to endothelial cells and

  8. Thalidomide inhibits inflammatory and angiogenic activation of human intestinal microvascular endothelial cells (HIMEC)

    Science.gov (United States)

    Stein, Daniel J.; Nelson, Victoria M.; Otterson, Mary F.; Shaker, Reza; Binion, David G.

    2010-01-01

    The glutamic acid derivative thalidomide is a transcriptional inhibitor of TNF-α but is also known to affect human blood vessels, which may underlie its teratogenicity. Thalidomide has been used in the treatment of refractory Crohn's disease (CD), but the therapeutic mechanism is not defined. We examined the effect of thalidomide on primary cultures of human intestinal microvascular endothelial cells (HIMEC), the relevant endothelial cell population in inflammatory bowel disease (IBD), to determine its effect on endothelial activation, leukocyte interaction, and VEGF-induced angiogenesis. HIMEC cultures were pretreated with thalidomide before activation with either TNF-α/LPS or VEGF. A low-shear-stress flow adhesion assay with either U-937 or whole blood was used to assess HIMEC activation following TNF-α/LPS, and a Wright's stain identified adherent leukocytes. Expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) was assessed using radioimmunoassay. Effects of thalidomide on NF-κB activation, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression in TNF-α/LPS-activated HIMEC were determined by RT-PCR and Western blotting. Thalidomide blocked adhesion of both U-937 and whole blood leukocytes by 50% in HIMEC, inhibiting binding of all classes of leukocytes. Thalidomide also blocked NF-κB and cell adhesion molecule expression in HIMEC. In marked contrast, thalidomide did not affect either iNOS or COX-2 expression, two key molecules that play a role in the downregulation of HIMEC activation. VEGF-induced HIMEC transmigration, growth, proliferation, tube formation, and Akt phosphorylation were significantly inhibited by thalidomide. In summary, thalidomide exerted a potent effect on HIMEC growth and activation, suggesting that it may also function via an endothelial mechanism in the treatment of CD. PMID:19926820

  9. Glucosamine exposure reduces proteoglycan synthesis in primary human endothelial cells in vitro

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    Trine M. Reine

    2016-09-01

    Full Text Available Purpose: Glucosamine (GlcN supplements are promoted for medical reasons, for example, for patients with arthritis and other joint-related diseases. Oral intake of GlcN is followed by uptake in the intestine, transport in the circulation and thereafter delivery to chondrocytes. Here, it is postulated to have an effect on synthesis and turnover of extracellular matrix constituents expressed by these cells. Following uptake in the intestine, serum levels are transiently increased, and the endothelium is exposed to increased levels of GlcN. We investigated the possible effects of GlcN on synthesis of proteoglycans (PGs, an important matrix component, in primary human endothelial cells. Methods: Primary human endothelial cells were cultured in vitro in medium with 5 mM glucose and 0–10 mM GlcN. PGs were recovered and analysed by western blotting, or by SDS-PAGE, gel chromatography or ion-exchange chromatography of 35S-PGs after 35S-sulphate labelling of the cells. Results: The synthesis and secretion of 35S-PGs from cultured endothelial cells were reduced in a dose- and time-dependent manner after exposure to GlcN. PGs are substituted with sulphated glycosaminoglycan (GAG chains, vital for PG function. The reduction in 35S-PGs was not related to an effect on GAG chain length, number or sulphation, but rather to the total expression of PGs. Conclusion: Exposure of endothelial cells to GlcN leads to a general decrease in 35S-PG synthesis. These results suggest that exposure to high levels of GlcN can lead to decreased matrix synthesis, contrary to what has been claimed by supporters of such supplements.

  10. Aging-related changes in oxidative stress response of human endothelial cells.

    Science.gov (United States)

    Conti, Valeria; Corbi, Graziamaria; Simeon, Vittorio; Russomanno, Giusy; Manzo, Valentina; Ferrara, Nicola; Filippelli, Amelia

    2015-08-01

    Oxidative stress is strongly associated with aging and age-related diseases and plays a crucial role in endothelial dysfunction development. To better understand the molecular mechanisms of aging and stress response in humans, we examined changes to young and older human endothelial cells over time (72, 96 and 120 h), before and after H2O2-induced stress. We measured the expression of the deacetylase Sirtuin 1 (Sirt1) and its transcriptional target Forkhead box O3a (Foxo3a); TBARS, a well-known marker of overall oxidative stress, and catalase activity as index of antioxidation. Moreover, we quantified levels of cellular senescence by senescence-associated β galactosidase (SA-βgal) assay. Under oxidative stress induction older cells showed a progressive decrease of Sirt1 and Foxo3a expression, persistently high TBARS levels with high, but ineffective Cat activity to counteract such levels. In addition cellular senescence drastically increased in older cells compared with Young cells both in presence and in the absence of oxidative stress. By following the cell behavior during the time course, we can hypothesize that while in young cells an oxidative stress induction stimulated an adequate response through activation of molecular factor crucial to counteract oxidative stress, the older cells are not able to adequately adapt themselves to external stress stimuli. During their life, endothelial cells impair the ability to defend themselves from oxidative stress stimuli. This dysfunction involves the pathway of Sirt1 a critical regulator of oxidative stress response and cellular lifespan, underlining its crucial role in endothelial homeostasis control during aging and age-associated diseases.

  11. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-11-04

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  12. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Science.gov (United States)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  13. Single-dose rosuvastatin ameliorates lung ischemia-reperfusion injury via upregulation of endothelial nitric oxide synthase and inhibition of macrophage infiltration in rats with pulmonary hypertension.

    Science.gov (United States)

    Matsuo, Satoshi; Saiki, Yuriko; Adachi, Osamu; Kawamoto, Shunsuke; Fukushige, Shinichi; Horii, Akira; Saiki, Yoshikatsu

    2015-03-01

    Lung ischemia-reperfusion (IR) injury during cardiopulmonary surgery is associated with postoperative morbidity and mortality, particularly in patients with pulmonary hypertension (PH). Using a rat model for monocrotaline-induced PH, we investigated the protective effect of rosuvastatin against IR injury in lungs affected by PH and attempted to elucidate its mechanism of action. Male Sprague-Dawley monocrotaline-treated rats were divided into 4 groups (n = 8-9): sham, control + IR, statin + IR, and statin + mevalonolactone + IR. Lung ischemia was induced by left pulmonary artery occlusion (1 hour), followed by reperfusion (4 hours). Rosuvastatin (2 mg/kg) was injected 18 hours before reperfusion and mevalonolactone (1 mg/kg) was injected immediately before reperfusion. The arterial oxygen tension/inspired oxygen fraction ratio was used as a measure of lung oxygenation. Left lung tissue was analyzed for the wet-to-dry lung weight ratio and protein expression of endothelial nitric oxide synthase (eNOS) and phospho-eNOS. Macrophage recruitment was assessed by CD68 immunostaining. Our results showed that rosuvastatin decreased IR lung injury (control + IR vs statin + IR) in terms of the arterial oxygen tension/inspired oxygen fraction ratio (272 ± 43 vs 442 ± 13), wet-to-dry ratio (5.7 ± 0.7 vs 4.8 ± 0.6), and macrophage infiltration (8.0 ± 0.6/field vs 4.0 ± 0.5/field) (P < .05 for all). eNOS and phospho-eNOS were downregulated by IR, which was blocked by rosuvastatin. Effects of rosuvastatin were blunted by mevalonolactone. Single-dose rosuvastatin decreased IR injury in lungs affected by PH via 2 anti-inflammatory mechanisms: preserving eNOS function and inhibiting macrophage infiltration. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  14. Pathogen sensing pathways in human embryonic stem cell derived-endothelial cells: role of NOD1 receptors.

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    Daniel M Reed

    Full Text Available Human embryonic stem cell-derived endothelial cells (hESC-EC, as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR Toll-like receptor (TLR-4 and nucleotide-binding oligomerisation domain-containing protein (NOD-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC. HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC, and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

  15. Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

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    Jorge S Burns

    Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1

  16. [Human lung topography in the early fetal period of ontogenesis].

    Science.gov (United States)

    Zheleznov, L M; Shcherbakov, S M

    2012-01-01

    Lung holotopy, skeletotopy and syntopy were studied in 70 human fetuses at developmental weeks 16-24 with N. I. Pirogov method, macro-microscopical preparation and using histotopographical sections in three imutually perpendicular planes. It was found that during weeks 16-18, the apex of the left lung was located posteriorly at the level of I intercostal space, at weeks 22-24--at the level of lower surface of I rib. At the right side, the apex was located at the level of upper surface of I rib during the whole period. The lower margin of the right lung was located at the level of IV rib during the whole period, while that of the left lung was detected at the level of III rib only during the beginning of the period. In the early fetal period, the projection of the root of the right lung extended from the lower margin of T(III) vertebral body toT(VI), while that one of the left lung was located at the level of the upper margins of T(IV)-T(VII) vertebral bodies. In the late period, these projections were found at the level of T(IV) (upper vertebral margin)--T(VII) (lower vertebral margin), and T(IV) (lower vertebral margin)--T(VIII) (upper vertebral margin) respectively. Intraorgan bronchi and pulmonary vessels were most clearly visualized in horizontal sections at T(III) -T(IX) levels. The results obtained should be taken into account when carrying out of diagnostic ultrasound and magnetic resonance studies of the fetus and surgical interventions on fetuses.

  17. Loss of CD34 expression in aging human choriocapillaris endothelial cells.

    Directory of Open Access Journals (Sweden)

    Elliott H Sohn

    Full Text Available Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD. In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40, age-matched controls (> age 60 without AMD, or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization. Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6. Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse or a trend toward decreased CD34 (human in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

  18. Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ana-María Zaske

    2013-01-01

    Full Text Available Although atomic force microscopy (AFM has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

  19. Effects of drospirenone on adhesion molecule expression and monocyte adherence in human endothelial cells.

    Science.gov (United States)

    Ito, Fumitake; Mori, Taisuke; Takaoka, Osamu; Tanaka, Yukiko; Koshiba, Akemi; Tatsumi, Hiroshi; Iwasa, Koichi; Kitawaki, Jo

    2016-06-01

    A major concern in hormone replacement therapy is the associated increased risk of cardiovascular diseases. A progestogen without the unfavorable effects on cardiovascular disease should be explored. Monocyte adhesion to endothelial cells is an important initial event in atherosclerosis. In this study, the effects of the alternative progestogen drospirenone (DRSP) on monocyte adhesion in human umbilical venous endothelial cells (HUVECs) were examined. In HUVECs treated with estrogens and progestogens, including DRSP and medroxyprogesterone acetate (MPA), the expression of the adhesion molecules E-selectin, P-selectin, ICAM-1, and VCAM-1 were examined by real-time PCR and using an enzyme-linked immunosorbent assay. A flow chamber system was used to investigate the effects of DRSP on U937 monocytoid cell adherence to HUVEC monolayers. All experimental data were compared using one-way Analysis of Variance. Upregulation of adhesion molecule mRNA or protein was not seen in HUVECs treated with DRSP alone or with 17β-estradiol+DRSP. DRSP alone, 17β-estradiol+DRSP or ethinylestradiol+DRSP did not increase the number of adherent monocytoid cells to HUVECs in the flow chamber system. However, MPA significantly enhanced the monocytoid cell adherence (Padhesion molecules or monocytoid cell adherence to endothelial cells, indicating that DRSP could reduce the risk of atherogenesis caused by MPA. These results suggest that DRSP may be an alternative to MPA in hormone replacement therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.

  20. Commercial single-walled carbon nanotubes effects in fibrinolysis of human umbilical vein endothelial cells.

    Science.gov (United States)

    Rodríguez-Yáñez, Yury; Bahena-Uribe, Daniel; Chávez-Munguía, Bibiana; López-Marure, Rebeca; González-Monroy, Stuart; Cisneros, Bulmaro; Albores, Arnulfo

    2015-08-01

    Recent studies have demonstrated that carbon nanotubes (CNTs) induce platelet aggregation, endothelial dysfunction and vascular thrombosis. However, there is little information on the effects of CNTs on fibrinolysis. We investigated the role of pristine-commercial single-walled carbon nanotubes (SWCNTs) with fibrinolysis and their contribution to the induction of pro-thrombotic processes in human vein endothelial cells (HUVEC). SWCNTs alone produced concentration-dependent oxidation, as measured by a dithiothreitol oxidation assay. Internalized SWCNTs were located in HUVEC treated with 25 μg/ml using transmission electron microscopy, whereas treatment with 50 μg/ml compromised cell viability, and oxidative stress increased significantly at 5 μg/ml. The study showed that in HUVEC treated with 25 μg SWCNT/ml, fibrinolysis-related gene expression and protein levels had increased by 3-12 h after treatment (serpine-1: 13-fold; PLAT: 11-fold and PLAU: 2-fold), but only the PAI-1 protein was increased (1.5-fold), whereas tissue and urokinase plasminogen activator proteins (tPA and uPA, respectively) tended to decrease. In summary, pristine SWCNTs treatment resulted in evident HUVEC damage caused by cell fiber contact, internalization, and oxidative stress due to contaminant metals. The generation of endothelial dysfunction, as shown by the altered expression of genes and proteins involved in fibrinolysis, suggest that SWCNTs display pro-thrombotic effects. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Implanted neonatal human dermal fibroblasts influence the recruitment of endothelial cells in mice.

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    Guerreiro, Susana G; Brochhausen, Christoph; Negrão, Rita; Barbosa, Mário A; Unger, Ronald E; Kirkpatrick, C James; Soares, Raquel; Granja, Pedro L

    2012-01-01

    The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The vessels exhibited higher levels of hemoglobin, compared with the control matrix, implanted without fibroblasts, in which no vessel formation could be observed. No significant differences were detected in systemic inflammation. The presence of vessels originated from the host vasculature suggested that host vascular response was involved, which constitutes a fundamental aspect in the process of neovascularization. Fibroblasts implanted within matrigel increased the presence of endothelial cells with positive staining for CD31 and for CD34 and the production of collagen influencing the angiogenic process and promoting the formation of microvessels. New strategies in tissue engineering could be delineated with improved angiogenesis using neonatal fibroblasts.

  2. Reactive oxygen species modulate the barrier function of the human glomerular endothelial glycocalyx.

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    Anurag Singh

    Full Text Available Reactive oxygen species (ROS play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S(35 and (3H-glucosamine. GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.

  3. Mechanism of the toxicity induced by natural humic acid on human vascular endothelial cells.

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    Kihara, Yusuke; Yustiawati; Tanaka, Masato; Gumiri, Sulmin; Ardianor; Hosokawa, Toshiyuki; Tanaka, Shunitz; Saito, Takeshi; Kurasaki, Masaaki

    2014-08-01

    Humic acid (HA), a group of high-molecular weight organic compounds characterized by an ability to bind heavy metals, is normally found in natural water. Although the impairment of vascular endothelial cells in the presence of humic substances has been reported to be involved in some diseases, the mechanisms responsible for this involvement remain unclear. In this study, we examined the cytotoxicity of HA obtained from peatland in Central Kalimantan, Indonesia, to human vascular endothelial cells, as well as the mechanisms behind these effects. It was found that 50 mg/L HA showed cytotoxicity, which we considered to be mediated by apoptosis through the mitochondrial pathway because of an increase in the expression of caspases 6 and 9 in response to HA administration. In addition, this cytotoxicity was enhanced when cells in this experimental system were exposed to oxidative stress, while it was decreased by the addition of vitamin C. Thus, we conclude that the apoptosis induced by HA depends upon oxidative stress. Furthermore, an iron chelator, DFO, showed a tendency to decrease HA-induced cytotoxicity, suggesting that iron may potentially mediate HA-induced oxidative stress. In conclusion, long-term consumption of HA-rich water obtained from our study area may cause damage to endothelial cells and subsequent chronic health problems. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  4. A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

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    Laura Giusti

    2017-01-01

    Full Text Available Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs, bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG, on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation.

  5. The influence of statins on the free intracellular calcium concentration in human umbilical vein endothelial cells

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    Figulla Hans R

    2004-05-01

    Full Text Available Abstract Background Statins are cholesterol-lowering drugs that are widely used to reduce the risk of cardiac infarction. Their beneficial clinical effects, however, are not restricted to their influence on cholesterol production. As several studies have shown that they have a potency of relaxing blood vessels. Methods We measured the effects of statins on the intracellular free calcium concentration ([Ca2+]i in human umbilical vein endothelial cells (HUVEC after acute application and 24-h-preincubation of statins. Results Incubation of the cells for 24 h with cerivastatin or fluvastatin significantly increased the resting [Ca2+]i. For cerivastatin this effect manifested at a concentration of 1 μM. Increase of resting [Ca2+]i in the presence of cerivastatin also occurred when the nitric oxide synthase was inhibited. Transient Ca2+ release induced by histamine was not affected. Conclusions The increase of resting [Ca2+]i after incubation with cerivastatin or fluvastatin may provide an explanation for the direct effects of statins on the endothelial-dependent vasodilatation and restoration of endothelial activity in vivo.

  6. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

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    Grechowa, Irina; Horke, Sven; Wallrath, Anja; Vahl, Christian-Friedrich; Dorweiler, Bernhard

    2017-09-01

    Human neutrophil elastase impacts on atherosclerotic plaque stability by inducing apoptosis in endothelial cells. Our aim was to investigate the proapoptotic mechanism of elastase on endothelial cells and to evaluate the presence of elastase in human plaque material. Human endothelial cells were treated with purified human neutrophil elastase. Apoptosis was assayed by capsase-3/7 activation, TUNEL, and sub-G 1 assay. Activation of unfolded protein response (UPR) effector molecules binding Ig protein, soluble X-binding protein-1, protein kinase RNA-like ER kinase (PERK), and C/EBP-homologous protein (CHOP) was analyzed by RT-PCR, immunocytochemistry, and Western blot. Genetic silencing of CHOP was achieved by small interfering RNA. Elastase induces autophagic-apoptotic forms of endothelial cell death in a time- and dose-dependent manner, in conjunction with a significant increase in phosphorylation/expression of the canonical UPR-activation markers PERK and CHOP. By using CHOP knockdown, we identified CHOP as a key mediator of elastase-induced endothelial cell death. Immunohistochemical analysis of human rupture-prone plaque specimens confirmed the presence of elastase and colocalization with apoptosis. We have demonstrated for the first time that the PERK-CHOP branch of the UPR is causally involved in elastase-induced apoptosis of endothelial cells. Ex vivo analysis of human rupture-prone plaques confirmed the presence of elastase and its colocalization with markers of apoptosis. This novel role of elastase underlines the potential of combined targeting of elastase and endoplasmic reticulum stress in the prevention of plaque progression and cardiovascular events.-Grechowa, I., Horke, S., Wallrath, A., Vahl, C.-F., Dorweiler, B. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response. © FASEB.

  7. Influence of lung CT changes in chronic obstructive pulmonary disease (COPD on the human lung microbiome.

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    Marion Engel

    Full Text Available Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited.Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons.We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group (PC1, Padj = 0.002. Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype.Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system.

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

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

    2015-07-03

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

  9. AXL-Mediated Productive Infection of Human Endothelial Cells by Zika Virus.

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    Liu, Shufeng; DeLalio, Leon J; Isakson, Brant E; Wang, Tony T

    2016-11-11

    The mosquito-borne Zika virus (ZIKV) is now recognized as a blood-borne pathogen, raising an important question about how the virus gets into human bloodstream. The imminent threat of the ZIKV epidemic to the global blood supply also demands novel therapeutics to stop virus transmission though transfusion. We intend to characterize ZIKV tropism for human endothelial cells (ECs) and provide potential targets for intervention. We conducted immunostaining, plaque assay, and quantitative reverse transcription-polymerase chain reaction of ZIKV RNA to evaluate the possible infection of ECs by ZIKV. Both the African and the South American ZIKV strains readily infect human umbilical vein endothelial cells and human ECs derived from aortic and coronary artery, as well as the saphenous vein. Infected ECs released infectious progeny virus. Compared with the African strains, South American ZIKV isolates replicate faster in ECs and are partially cytopathic, suggesting enhanced virulence of these isolates. Flow cytometric analyses showed that the susceptibility of ECs positively correlated with the cell surface levels of tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase. Gain- and loss-of-function studies further revealed that AXL is required for ZIKV entry at a postbinding step. Finally, small-molecule inhibitors of the AXL kinase significantly reduced ZIKA infection of ECs. We identified EC as a key cell type for ZIKV infection. These data support the view of hematogenous dissemination of ZIKV and implicate AXL as a new target for antiviral therapy. © 2016 American Heart Association, Inc.

  10. Suitability of endogenous reference genes for gene expression studies with human intraocular endothelial cells

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    Wei Ruoxin

    2013-02-01

    Full Text Available Abstract Background The use of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR has become widely applied as a method to measure transcript abundance. In order to be reflective of biological processes during health and disease this method is dependent on normalisation of data against stable endogenous controls. However, these genes can vary in their stability in different cell types. The importance of reference gene validation for a particular cell type is now well recognised and is an important step in any gene expression study. Results Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms. Conclusions We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.

  11. Oxidation of 2-cys peroxiredoxins in human endothelial cells by hydrogen peroxide, hypochlorous acid, and chloramines.

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    Stacey, Melissa M; Vissers, Margreet C; Winterbourn, Christine C

    2012-08-01

    Reactive oxygen species released from neutrophils during vascular inflammation could contribute to endothelial dysfunction seen in diseases such as atherosclerosis. Activated neutrophils generate hydrogen peroxide (H(2)O(2)) and hypochlorous acid (HOCl), as well as chloramines that are formed when HOCl reacts with amino compounds. These oxidants preferentially target thiol groups and thiol-containing proteins. The peroxiredoxins (Prxs) are thiol proteins that have high reactivity with H(2)O(2) and may also be sensitive to HOCl and chloramines. We have investigated human umbilical vein endothelial cells and shown that their cytoplasmic (Prx1 and Prx2) and mitochondrial (Prx3) Prxs are oxidized when they are exposed to H(2)O(2), HOCl, or cell-permeable chloramines. H(2)O(2) converted the Prxs to hyperoxidized, inactive forms, with little accumulation of disulfide-linked dimers. The oxidized Prxs were reduced over hours, presumably due to the action of endothelial sulfiredoxin. In contrast to the hyperoxidation seen with H(2)O(2), HOCl and the chloramine derivatives of glycine and ammonia converted the Prxs to disulfide-linked dimers and dimerization was reversed within 10-30 min of oxidant removal. HOCl treatment caused thioredoxin reductase (TrxR) inhibition with no reversal of dimerization. The cytotoxicity of ammonia chloramine was increased when cells were pretreated with H(2)O(2) to hyperoxidize the Prxs, or when the chloramine was added in the presence of the TrxR inhibitor, auranofin. We describe the novel observation that exposure of nucleated cells to inflammatory oxidants results in the accumulation of Prxs in the dimeric form. Endothelial cell Prxs are sensitive targets for neutrophil-derived oxidants and may protect against their damaging effects.

  12. Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

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    Asmat, Tauseef M; Tenenbaum, Tobias; Jonsson, Ann-Beth; Schwerk, Christian; Schroten, Horst

    2014-01-01

    The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

  13. Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

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    Tauseef M Asmat

    Full Text Available The pili and outer membrane proteins of Neisseria meningitidis (meningococci facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

  14. Pollutant particles enhanced H2O2 production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells.

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    Li, Zhuowei; Hyseni, Xhevahire; Carter, Jacqueline D; Soukup, Joleen M; Dailey, Lisa A; Huang, Yuh-Chin T

    2006-08-01

    Particulate matter (PM) induces oxidative stress and cardiovascular adverse health effects, but the mechanistic link between the two is unclear. We hypothesized that PM enhanced oxidative stress in vascular endothelial cells and investigated the enzymatic sources of reactive oxygen species and their effects on mitogen-activated protein kinase (MAPK) activation and vasoconstriction. We measured the production of extracellular H2O2, activation of extracellular signal-regulated kinases1/2 (ERK1/2) and p38 MAPKs in human pulmonary artery endothelial cells (HPAEC) treated with urban particles (UP; SRM1648), and assessed the effects of H2O2 on vasoconstriction in pulmonary artery ring and isolated perfused lung. Within minutes after UP treatment, HPAEC increased H2O2 production that could be inhibited by diphenyleneiodonium (DPI), apocynin (APO), and sodium azide (NaN3). The water-soluble fraction of UP as well as its two transition metal components, Cu and V, also stimulated H2O2 production. NaN3 inhibited H2O2 production stimulated by Cu and V, whereas DPI and APO inhibited only Cu-stimulated H2O2 production. Inhibitors of other H2O2-producing enzymes, including Nomega-methyl-L-argnine, indomethacin, allopurinol, cimetidine, rotenone, and antimycin, had no effects. DPI but not NaN3 attenuated UP-induced pulmonary vasoconstriction and phosphorylation of ERK1/2 and p38 MAPKs. Knockdown of p47phox gene expression by small interfering RNA attenuated UP-induced H2O2 production and phosphorylation of ERK1/2 and p38 MAPKs. Intravascular administration of H2O2 generated by glucose oxidase increased pulmonary artery pressure. We conclude that UP induce oxidative stress in vascular endothelial cells by activating NAD(P)H oxidase and the mitochondria. The endothelial oxidative stress may be an important mechanism for PM-induced acute cardiovascular health effects.

  15. Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

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    Tianying Bian

    2017-01-01

    Full Text Available The aim of this study was to investigate the role of human β-defensin 3 (hBD3 in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs triggered by tumor necrosis factor- (TNF- α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage migration inhibitory factor (MIF in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK in the mitogen-activated protein kinase (MAPK pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.

  16. Sex-specific differences in hyperoxic lung injury in mice: Implications for acute and chronic lung disease in humans

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    Lingappan, Krithika, E-mail: lingappa@bcm.edu [Department of Pediatrics, Section of Neonatology, Texas Children' s Hospital, Baylor College of Medicine, 1102 Bates Avenue, MC: FC530.01, Houston, TX 77030 (United States); Jiang, Weiwu; Wang, Lihua; Couroucli, Xanthi I. [Department of Pediatrics, Section of Neonatology, Texas Children' s Hospital, Baylor College of Medicine, 1102 Bates Avenue, MC: FC530.01, Houston, TX 77030 (United States); Barrios, Roberto [Department of Pathology and Genomic Medicine, The Methodist Hospital Physician Organization, 6565 Fannin Street, Suite M227, Houston, TX 77030 (United States); Moorthy, Bhagavatula [Department of Pediatrics, Section of Neonatology, Texas Children' s Hospital, Baylor College of Medicine, 1102 Bates Avenue, MC: FC530.01, Houston, TX 77030 (United States)

    2013-10-15

    Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72 h of hyperoxia (FiO{sub 2} > 0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F{sub 2} alpha (8-iso-PGF 2α) (LC–MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. Cytochrome P450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F > M) and VEGF (M > F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans. - Highlights: • Male mice were more susceptible to hyperoxic lung injury than females. • Sex differences in inflammatory markers were observed. • CYP1A expression was higher in females after hyperoxia exposure.

  17. Distinct and overlapping roles of Nipah virus P gene products in modulating the human endothelial cell antiviral response.

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    Michael K Lo

    Full Text Available Nipah virus (NiV is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis in up to 75% of infected humans. Like other paramyxoviruses, NiV employs co-transcriptional mRNA editing during transcription of the phosphoprotein (P gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternative reading frame. There is evidence from both in vitro and in vivo studies to show that the P gene products play a role in NiV pathogenesis. We have developed a reverse genetic system to dissect the individual roles of the NiV P gene products in limiting the antiviral response in primary human microvascular lung endothelial cells, which represent important targets in human NiV infection. By characterizing growth curves and early antiviral responses against a number of recombinant NiVs with genetic modifications altering expression of the proteins encoded by the P gene, we observed that multiple elements encoded by the P gene have both distinct and overlapping roles in modulating virus replication as well as in limiting expression of antiviral mediators such as IFN-β, CXCL10, and CCL5. Our findings corroborate observations from in vivo hamster infection studies, and provide molecular insights into the attenuation and the histopathology observed in hamsters infected with C, V, and W-deficient NiVs. The results of this study also provide an opportunity to verify the results of earlier artificial plasmid expression studies in the context of authentic viral infection.

  18. Distinct and overlapping roles of Nipah virus P gene products in modulating the human endothelial cell antiviral response.

    Science.gov (United States)

    Lo, Michael K; Peeples, Mark E; Bellini, William J; Nichol, Stuart T; Rota, Paul A; Spiropoulou, Christina F

    2012-01-01

    Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis in up to 75% of infected humans. Like other paramyxoviruses, NiV employs co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternative reading frame. There is evidence from both in vitro and in vivo studies to show that the P gene products play a role in NiV pathogenesis. We have developed a reverse genetic system to dissect the individual roles of the NiV P gene products in limiting the antiviral response in primary human microvascular lung endothelial cells, which represent important targets in human NiV infection. By characterizing growth curves and early antiviral responses against a number of recombinant NiVs with genetic modifications altering expression of the proteins encoded by the P gene, we observed that multiple elements encoded by the P gene have both distinct and overlapping roles in modulating virus replication as well as in limiting expression of antiviral mediators such as IFN-β, CXCL10, and CCL5. Our findings corroborate observations from in vivo hamster infection studies, and provide molecular insights into the attenuation and the histopathology observed in hamsters infected with C, V, and W-deficient NiVs. The results of this study also provide an opportunity to verify the results of earlier artificial plasmid expression studies in the context of authentic viral infection.

  19. Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness.

    Science.gov (United States)

    Ding, Vanessa; Chin, Angela; Peh, Gary; Mehta, Jodhbir S; Choo, Andre

    2014-01-01

    Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good 'cobblestone-like' morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.

  20. Lipoprotein lipase and endothelial lipase in human testis and in germ cell neoplasms

    DEFF Research Database (Denmark)

    Nielsen, J E; Lindegaard, M L; Friis-Hansen, L

    2009-01-01

    The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL m......RNA and protein were found in all germ cells as well as in Sertoli and Leydig cells. EL mRNA was abundant in pre-invasive carcinoma in situ (CIS) cells and GCTs, and EL protein was present in the cytoplasm of these cells. LPL mRNA was also relatively abundant in germ cells, Sertoli cells, CIS cells and GCTs...

  1. Vascular endothelial growth factor -634G/C and vascular endothelial growth factor -2578C/A polymorphisms and lung cancer risk: a case-control study and meta-analysis.

    Science.gov (United States)

    Deng, Zai-Chun; Cao, Chao; Yu, Yi-Ming; Ma, Hong-Ying; Ye, Meng

    2014-03-01

    Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis in the process of tumor growth and metastasis. In present study, we conducted a case-control study and meta-analysis to evaluate the genetic effects of VEGF -634G/C and VEGF -2578C/A polymorphisms and risk of lung cancer. A total of 175 subjects were recruited for case-control study and seven studies were included in the meta-analysis. Our case-control study showed that VEGF -634G/C polymorphism had no association with lung cancer risk (CC vs. GG: OR = 0.88, 95% CI = 0.37-2.11), whereas there was an association between VEGF -2578CC genotype and decrease in lung cancer risk (CC vs. OR = 0.52, 95% CI = 0.28-0.96). A meta-analysis was further performed and statistically similar results were obtained (CC vs. GG: OR = 0.91, 95% CI = 0.60-1.39 for VEGF -634; CC vs. AA: OR = 0.53, 95% CI = 0.32-0.89 for VEGF -2578). Our study showed that the variant genotypes of the VEGF -2578C/A polymorphism, but not the VEGF -634G/C polymorphism, was associated with lung cancer risk. More studies are needed to detect VEGF -634G/C and VEGF -2578 polymorphisms and their association with lung cancer in different ethnic populations incorporated with environmental exposures.

  2. Uterine Vasculature Remodeling in Human Pregnancy Involves Functional Macrochimerism by Endothelial Colony Forming Cells of Fetal Origin

    Science.gov (United States)

    Sipos, Peter I; Rens, Willem; Schlecht, HÉlène; Fan, Xiaohu; Wareing, Mark; Hayward, Christina; Hubel, Carl A; Bourque, Stephane; Baker, Philip N; Davidge, Sandra T; Sibley, Colin P; Crocker, Ian P

    2013-01-01

    The potency of adult-derived circulating progenitor endothelial colony forming cells (ECFCs) is drastically surpassed by their fetal counterparts. Human pregnancy is associated with robust intensification of blood flow and vascular expansion in the uterus, crucial for placental perfusion and fetal supply. Here, we investigate whether fetal ECFCs transmigrate to maternal bloodstream and home to locations of maternal vasculogenesis, primarily the pregnant uterus. In the first instance, endothelial-like cells, originating from mouse fetuses expressing paternal eGFP, were identified within uterine endothelia. Subsequently, LacZ or enhanced green fluorescent protein (eGFP)-labeled human fetal ECFCs, transplanted into immunodeficient (NOD/SCID) fetuses on D15.5 pregnancy, showed similar integration into the mouse uterus by term. Mature endothelial controls (human umbilical vein endothelial cells), similarly introduced, were unequivocally absent. In humans, SRY was detected in 6 of 12 myometrial microvessels obtained from women delivering male babies. The copy number was calculated at 175 [IQR 149–471] fetal cells per millimeter square endothelium, constituting 12.5% of maternal vessel lumina. Cross-sections of similar human vessels, hybridized for Y-chromosome, positively identified endothelial-associated fetal cells. It appears that through ECFC donation, fetuses assist maternal uterine vascular expansion in pregnancy, potentiating placental perfusion and consequently their own fetal supply. In addition to fetal growth, this cellular mechanism holds implications for materno-fetal immune interactions and long-term maternal vascular health. PMID:23554274

  3. Human very Small Embryonic-like Cells Support Vascular Maturation and Therapeutic Revascularization Induced by Endothelial Progenitor Cells.

    Science.gov (United States)

    Guerin, Coralie L; Rossi, Elisa; Saubamea, Bruno; Cras, Audrey; Mignon, Virginie; Silvestre, Jean-Sébastien; Smadja, David M

    2017-08-01

    Very small embryonic-like stem cells (VSELs) are major pluripotent stem cells defined as cells of small size being Lineage- negative, CD133-positive, and CD45-negative. We previously described that human bone marrow VSELs were able to differentiate into endothelial cells and promoted post-ischemic revascularization in mice with surgically induced critical limb ischemia. In the present work, we isolated bone marrow VSELs from patients with critical limb ischemia and studied their ability to support endothelial progenitor cells therapeutic capacity and revascularization potential. Sorted bone marrow VSELs cultured in angiogenic media were co-injected with endothelial progenitor cells and have been show to trigger post-ischemic revascularization in immunodeficient mice, and support vessel formation in vivo in Matrigel implants better than human bone marrow mesenchymal stem cells. In conclusion, VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial and perivascular lineage in humans. VSELs are the first real vasculogenic stem cells able to differentiate in endothelial and perivascular lineage in human adult described from now. Thus, because VSELs presence have been proposed in adult tissues, we think that VSELs are CD45 negative stem cells able to give rise to vascular regeneration in human tissues and vessels.

  4. Calculation of hygroscopic particle deposition in the human lung.

    Science.gov (United States)

    Winkler-Heil, Renate; Ferron, George; Hofmann, Werner

    2014-02-01

    Inhaled hygroscopic aerosols will absorb water vapor from the warm and humid air of the human lung, thus growing in size and consequently changing their deposition properties. The objectives of the present study are to study the effect of a stochastic lung structure on individual particle growth and related deposition patterns and to predict local deposition patterns for different hygroscopic aerosols. The hygroscopic particle growth model proposed by Ferron et al. has been implemented into the stochastic asymmetric lung deposition model IDEAL. Deposition patterns were calculated for sodium chloride (NaCl), cobalt chloride (CoCl2 · 6H2O), and zinc sulfate (ZnSO4 · 7H2O) aerosols, representing high, medium and low hygroscopic growth factors. Hygroscopic growth decreases deposition of submicron particles compared to hydrophobic particles with equivalent diameters due to a less efficient diffusion mechanism, while the more efficient impaction and sedimentation mechanisms increase total deposition for micron-sized particles. Due to the variability and asymmetry of the human airway system, individual trajectories of inhaled particles are associated with individual growth factors, thereby enhancing the variability of the resulting deposition patterns. Comparisons of model predictions with several experimental data for ultrafine and micrometer-sized particles indicate good agreement, considering intersubject variations of morphometric parameters as well as differences between experimental conditions and modeling assumptions.

  5. Key role of the endothelial TGF-β/ALK1/endoglin signaling pathway in humans and rodents pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Benoît Gore

    Full Text Available Mutations affecting transforming growth factor-beta (TGF-β superfamily receptors, activin receptor-like kinase (ALK-1, and endoglin (ENG occur in patients with pulmonary arterial hypertension (PAH. To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs and pulmonary endothelial cells (PECs from 14 patients with idiopathic PAH (iPAH and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV systolic pressure (RVSP, RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (Eng+/- and wild-type (Eng+/+ mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, Eng+/- mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.

  6. Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

    Science.gov (United States)

    Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

    2017-11-01

    The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

  7. Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhong-Xiu-Zi [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Huang, Da-Yong [Department of Oncology, The Second Clinical Hospital, Harbin Medical University, Harbin (China); Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Zheng, Jin-Hua, E-mail: jhzhenghrbmu@yahoo.cn [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China)

    2010-09-10

    Research highlights: {yields} It has been shown that scutellarin exhibits a variety of pharmacological actions, including anti-oxidative, anti-inflammatory, vasodilator as well as cardiovascular and cerebrovascular ischemia protective effects, indicating beneficial vascular effects of scutellarin. Therefore, it is speculated that scutellarin may be able to stimulate angiogenesis, which could be beneficial in the treatment of ischemic disease, wound healing and tissue regeneration. {yields} The purpose of the present study was to elucidate the direct angiogenic actions of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. {yields} Our results showed that scutellarin to directly induce in vitro angiogenesis, which is closely correlated with upregulated MMP-2 expression, suggesting a potential for increasing angiogenesis. -- Abstract: Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin's effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin's angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly

  8. The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells.

    Science.gov (United States)

    Ge, Gaoyuan; Wu, Hengfang; Xiong, Fei; Zhang, Yu; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Gu, Chunrong; Gu, Ning; Chen, Xiangjian; Yang, Di

    2013-05-07

    One major obstacle for successful application of nanoparticles in medicine is its potential nanotoxicity on the environment and human health. In this study, we evaluated the cytotoxicity effect of dimercaptosuccinic acid-coated iron oxide (DMSA-Fe2O3) using cultured human aortic endothelial cells (HAECs). Our results showed that DMSA-Fe2O3 in the culture medium could be absorbed into HAECs, and dispersed in the cytoplasm. The cytotoxicity effect of DMSA-Fe2O3 on HAECs was dose-dependent, and the concentrations no more than 0.02 mg/ml had little toxic effect which were revealed by tetrazolium dye assay. Meanwhile, the cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without DMSA-Fe2O3). However, the endocrine function for endothelin-1 and prostacyclin I-2, as well as the urea transporter function, was altered even without obvious evidence of cell injury in this context. We also showed by real-time PCR analysis that DMSA-Fe2O3 exposure resulted in differential effects on the expressions of pro- and anti-apoptosis genes of HAECs. Meanwhile, it was noted that DMSA-Fe2O3 exposure could activate the expression of genes related to oxidative stress and adhesion molecules, which suggested that inflammatory response might be evoked. Moreover, we demonstrated by in vitro endothelial tube formation that even a small amount of DMSA-Fe2O3 (0.01 and 0.02 mg/ml) could inhibit angiogenesis by the HAECs. Altogether, these results indicate that DMSA-Fe2O3 have some cytotoxicity that may cause side effects on normal endothelial cells.

  9. Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro

    Science.gov (United States)

    2013-01-01

    Background Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein endothelial cells (HUVEC) in vitro. Methods Cell viability after prolonged treatment with papain was investigated by life cell staining and lactate dehydrogenase release assay. Angiogenic activation was assessed by ELISA against phosphorylated proteins AKT, MEK1/2, ERK1/2, SAPK/JNK and p38-MAPK. Growth inhibition was determined by means of an MTT-assay and cell migration by means of a scratch assay. Capability to form a capillary network was investigated using a tube formation assay. Results Papain did not induce proteolysis or cell detachment of HUVEC in a concentration range between 0 and 25 μg/mL. Four hours treatment with 10 μg/mL papain resulted in a reduced susceptibility of endothelial cells to activation by VEGF as determined by phosphorylation levels of Akt, MEK1/2, SAPK/JNK. Papain exerted a distinct inhibitory effect on cell growth, cell migration and tube formation with inhibition of tube formation detectable at concentrations as low as 1 μg/mL. Bromelain and ficin displayed similar effects with regard to cell growth and tube formation. Conclusion Papain showed a strong anti-angiogenic effect in VEGF activated HUVEC. This effect may be due to interference with AKT, MEK1/2 and SAPK/JNK phosphorylation. Two other plant derived cysteine proteases displayed similar inhibition of HUVEC cell growth and tube formation. These findings indicate that plant proteolytic enzymes may have potential as preventive and therapeutic agents against angiogenesis related human diseases

  10. Engineered Microvasculature in PDMS Networks Using Endothelial Cells Derived from Human Induced Pluripotent Stem Cells

    Science.gov (United States)

    Sivarapatna, Amogh; Ghaedi, Mahboobe; Xiao, Yang; Han, Edward; Aryal, Binod; Zhou, Jing; Fernandez-Hernando, Carlos; Qyang, Yibing; Hirschi, Karen K.

    2017-01-01

    In this study, we used a polydimethylsiloxane (PDMS)-based platform for the generation of intact, perfusion-competent microvascular networks in vitro. COMSOL Multiphysics, a finite-element analysis and simulation software package, was used to obtain simulated velocity, pressure, and shear stress profiles. Transgene-free human induced pluripotent stem cells (hiPSCs) were differentiated into partially arterialized endothelial cells (hiPSC-ECs) in 5 d under completely chemically defined conditions, using the small molecule glycogen synthase kinase 3β inhibitor CHIR99021 and were thoroughly characterized for functionality and arterial-like marker expression. These cells, along with primary human umbilical vein endothelial cells (HUVECs), were seeded in the PDMS system to generate microvascular networks that were subjected to shear stress. Engineered microvessels had patent lumens and expressed VE-cadherin along their periphery. Shear stress caused by flowing medium increased the secretion of nitric oxide and caused endothelial cells s to align and to redistribute actin filaments parallel to the direction of the laminar flow. Shear stress also caused significant increases in gene expression for arterial markers Notch1 and EphrinB2 as well as antithrombotic markers Kruppel-like factor 2 (KLF-2)/4. These changes in response to shear stress in the microvascular platform were observed in hiPSC-EC microvessels but not in microvessels that were derived from HUVECs, which indicated that hiPSC-ECs may be more plastic in modulating their phenotype under flow than are HUVECs. Taken together, we demonstrate the feasibly of generating intact, engineered microvessels in vitro, which replicate some of the key biological features of native microvessels. PMID:28901188

  11. Human endothelial and platelet septin SEPT11: cloning of novel variants and characterisation of interaction partners.

    Science.gov (United States)

    Bartsch, Ingrid; Bläser, Susanne; Röseler, Sabrina; Sandrock, Kirstin; Busse, Anja; Huber, Michael; Rempp, Hansjörg; Lieber, Mareike; Horn, Julia; Brendle, Cornelia; Zieger, Barbara

    2010-12-01

    Septins are cytoskeletal GTPases forming heteropolymeric complexes involved in processes characterised by active membrane movement such as cytokinesis, vesicle trafficking, and exocytosis. Septins are expressed in non-mitotic cells such as neurons and platelets. SEPT11 belongs to the SEPT6 group and was identified as interaction partner of SEPT5. We cloned and characterised novel SEPT11 variants and investigated interaction partners of SEPT11 in platelets and human umbilical vein endothelial cells. An endothelial cell library was used for cloning novel SEPT11 variants. Using Northern analysis the different SEPT11 transcripts were illustrated. Interaction studies were performed using yeast two-hybrid system, precipitation, FRET, and immunofluorescence microscopy. We demonstrate that SEPT11 partners with SEPT2, SEPT4 and SEPT7 using yeast two-hybrid system and precipitation. The interaction of SEPT11 with SEPT7 is also demonstrated by FRET. In addition to the known SEPT11 transcript (SEPT11_v1) we identified a novel SEPT11 variant (SEPT11_v2) as interaction partner of SEPT4 and SEPT7. Library screening of an endothelial cell library also revealed the presence of this novel SEPT11_v2 transcript. In addition, a third SEPT11 variant (SEPT11_v3) was identified. Expression of SEPT11_v1 and of SEPT11_v2 and SEPT11_v3 in human brain regions was investigated by Northern analysis. Further interaction partners of SEPT11 are characterised using immunofluorescence. Co-localisation of SEPT2, SEPT4, SEPT7 and SEPT11 with tubulin and transferrin receptor (endocytotic marker) is demonstrated. In addition, co-localisation of SEPT4 and SEPT11 with the vesicle-associated protein synaptobrevin 1 (VAMP1), but not clearly with actin, was shown. Only SEPT2 and SEPT7 definitely co-localised with actin, but not clearly with VAMP1.

  12. Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Warke, Rajas V.; Xhaja, Kris; Martin, Katherine J.; Fournier, Marcia F.; Shaw, Sunil K.; Brizuela, Nathaly; de Bosch, Norma; Lapointe, David; Ennis, Francis A.; Rothman, Alan L.; Bosch, Irene

    2003-01-01

    Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate. To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays. DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1. Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection. Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways. These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients. Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection. These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance. PMID:14557666

  13. Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/bioglass nanofibrous scaffolds.

    Science.gov (United States)

    Shamosi, Atefeh; Mehrabani, Davood; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Siavashi, Vahid; Ghanbari, Hossein; Sharifi, Esmaeel; Roozafzoon, Reza; Ai, Jafar

    2017-02-01

    The capacity of gelatin/chitosan/bioactive glass nanopowders (GEL/CS/BGNPs) scaffolds was investigated for increasing human endometrial stem cells (hEnSCs) differentiation into the endothelial cells in the presence of angiogenic factors. GEL/CS nanofibrous scaffold with different contents of BGNPs were fabricated and assessed. Expression of endothelial markers (CD31, vascular endothelial cadherin (VE-cadherin), and KDR) in differentiated cells was evaluated. Results showed the diameter of nanofiber increases with decreasing the BG content in GEL/CS scaffolds. Moreover, in vitro study indicated that the GEL/CS/BGNPs scaffold with 1.5% BGNPs content provided a suitable three-dimensional structure for endothelial cells differentiation. Thus, the GEL/CS/BGNPs scaffold can be recommended for blood vessels repair.

  14. Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

    Science.gov (United States)

    Kim, Ha Ryong; Cho, Han Soo; Shin, Da Young; Chung, Kyu Hyuck

    2017-02-01

    In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Endosulfan inducing apoptosis and necroptosis through activation RIPK signaling pathway in human umbilical vascular endothelial cells.

    Science.gov (United States)

    Zhang, Lianshuang; Wei, Jialiu; Ren, Lihua; Zhang, Jin; Yang, Man; Jing, Li; Wang, Ji; Sun, Zhiwei; Zhou, Xianqing

    2017-01-01

    Endosulfan, an organochlorine pesticide, was found in human blood, and its possible cardiovascular toxicity has been suggested. However, the mechanism about endothelial cell injuries induced by endosulfan has remained unknown. In the present study, human umbilical vein endothelial cells (HUVECs) were chosen to explore the toxicity mechanism and were treated with 0, 1, 6, and 12 μg/mL-1 endosulfan for 24 h, respectively. The results showed that exposure to endosulfan could inhibit the cell viability, increase the release of lactate dehydrogenase (LDH), damage the ultrastructure, and lead to apoptosis and necroptosis in HUVECs. Furthermore, endosulfan upregulated the expressions of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like (MLKL), caspase 8, and caspase 3, which means the activation of RIPK1 pathways. In addition, endosulfan promoted the increases of ROS, IL-1α, and IL-33 levels while antioxidant N-acetyl-L-cysteine (NAC) effectively attenuated the cytotoxicity from endosulfan. Taken together, these results have demonstrated that endosulfan induces the apoptosis and necroptosis of HUVECs, where the RIPK pathway plays a pro-necroptotic role and NAC plays an anti-necroptotic role. Our results may contribute to understanding cellular mechanisms for endosulfan-induced cardiovascular toxicity.

  16. Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells.

    Science.gov (United States)

    Bidarra, Sílvia J; Barrias, Cristina C; Barbosa, Mário A; Soares, Raquel; Amédée, Joelle; Granja, Pedro L

    2011-11-01

    The purpose of this work was to investigate if a coculture system of human mesenchymal stem cells (hMSC) with endothelial cells (human umbilical vein endothelial cells, HUVEC) could modulate the phenotype and proliferation of harvested MSCs. In addition to previous investigations on the crosstalk between these two cell types, in the present work different relative cell ratios were analyzed for long, therapeutically relevant, culture periods. Moreover, MSCs osteogenic commitment was assessed in a non-osteogenic medium and in the presence of HUVECs through magnetic cell separation, cell quantification by flow cytometry, morphology by fluorescent microscopy, metabolic activity and gene expression of osteogenic markers. Collectively, the present findings demonstrate that, by coculturing MSCs with HUVECs, there was not only the promotion of osteogenic differentiation (and its enhancement, depending on the relative cell ratios used), but also a significant increase on MSCs proliferation. This augmentation in cell proliferation occurred independently of relative cell ratios, but was favored by higher relative amounts of HUVECs. Taken together, this data suggests that HUVECs not only modulate MSC phenotype but also their proliferation rate. Therefore, a coculture system of MSCs and HUVECs can a have a broad impact on bone tissue engineering approaches. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Mishima Satoshi

    2009-11-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ, bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs. Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

  18. Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

    Directory of Open Access Journals (Sweden)

    Lehtonen Siri

    2010-05-01

    Full Text Available Abstract Background Claudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation. Methods 47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4. Results Claudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied. Conclusion Claudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.

  19. In vitro anti-angiogenic properties of LGD1069, a selective retinoid X-receptor agonist through down-regulating Runx2 expression on Human endothelial cells

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    Lu Hong

    2011-06-01

    Full Text Available Abstract Background LGD1069 (Targretin® is a selective retinoid X receptor (RXR ligand, which is used in patients for cutaneous T-cell lymphoma. Our published study reported that LGD1069 inhibited tumor-induced angiogenesis in non-small cell lung cancer. In present study, we found that LGD1069 suppressed the proliferation, adhesion, invasion and migration of endothelial cells directly, and affected the expression of vegf and some matrix genes. Methods Human umbilical vein endothelial cells (HUVECs were used for in vitro study. MTT assay and Sulforhodamine B assay were used for cell viability assay; the tube formation assay was used to investigate the effect of LGD1069 on angiogenesis in vitro. In vitro adhesion, migration and invasion of HUVEC cells were analyzed by Matrigel adhesion, migration and invasion assay. Gene expressions were measured by RT-PCR and Western blot analysis. Results Our data showed here that LGD1069 inhibited the activation of TGF-β/Smad pathway significantly. Furthermore, it was demonstrated that expression of Runx2 was suppressed pronouncedly during incubation with LGD1069. Runx2 is a DNA-binding transcription factor which plays a master role in tumor-induced angiogenesis and cancer cells metastasis by interaction with the TGF-β/Smad pathway of transcriptional modulators. Conclusions Our results suggested that LGD1069 may impair angiogenic and metastatic potential induced by tumor cells through suppressing expression of Runx2 directly on human endothelial cells, which may point out new pathway through which LGD1069 display anti-angiogenic properties, and provide new molecular evidence to support LGD1069 as a potent anti-metastatic agent in cancer therapy.

  20. Active trafficking of alpha 1 antitrypsin across the lung endothelium.

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    Angelia D Lockett

    Full Text Available The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.

  1. Effect of peritoneal dialysis fluid containing osmo-metabolic agents on human endothelial cells

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    Bonomini M

    2016-11-01

    Full Text Available Mario Bonomini,1,2 Sara Di Silvestre,3,4 Pamela Di Tomo,3,4 Natalia Di Pietro,2,4 Domitilla Mandatori,3,4 Lorenzo Di Liberato,1 Vittorio Sirolli,1,2 Francesco Chiarelli,2,4 Cesare Indiveri,5 Assunta Pandolfi,3,4 Arduino Arduini6 1Unit of Nephrology and Dialysis, 2Department of Medicine and Aging Sciences, 3Department of Medical, Oral and Biotechnological Sciences, 4Aging Research Center and Translational Medicine, CeSI-MeT, University “G. d’Annunzio”, Chieti-Pescara, 5Department DiBEST (Biologia, Ecologia, Scienze della Terra, Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende CS, Italy; 6CoreQuest, Manno, Switzerland Background: The use of glucose as the only osmotic agent in peritoneal dialysis (PD solutions (PDSs is believed to exert local (peritoneal and systemic detrimental actions, particularly in diabetic PD patients. To improve peritoneal biocompatibility, we have developed more biocompatible PDSs containing xylitol and carnitine along with significantly less amounts of glucose and have tested them in cultured Human Vein Endothelial Cells (HUVECs obtained from the umbilical cords of healthy (C and gestational diabetic (GD mothers. Methods: Primary C- and GD-HUVECs were treated for 72 hours with our PDSs (xylitol 0.7% and 1.5%, whereas carnitine and glucose were fixed at 0.02% and 0.5%, respectively and two glucose-based PDSs (glucose 1.36% or 2.27%. We examined their effects on endothelial cell proliferation (cell count, viability (3-(4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium bromide assay, intracellular nitro-oxidative stress (peroxynitrite levels, Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 membrane exposure (flow cytometry, and HUVEC-monocyte interactions (U937 adhesion assay. Results: Compared to glucose-based PDSs, our in vitro studies demonstrated that the tested PDSs did not change the proliferative potential both in C- and GD-HUVECs. Moreover, our

  2. Human Immunodeficiency Virus Infection and Host Defense in the Lungs.

    Science.gov (United States)

    Charles, Tysheena P; Shellito, Judd E

    2016-04-01

    Immunosuppression associated with human immunodeficiency virus (HIV) infection impacts all components of host defense against pulmonary infection. Cells within the lung have altered immune function and are important reservoirs for HIV infection. The host immune response to infected lung cells further compromises responses to a secondary pathogenic insult. In the upper respiratory tract, mucociliary function is impaired and there are decreased levels of salivary immunoglobulin A. Host defenses in the lower respiratory tract are controlled by alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes. As HIV infection progresses, lung CD4 T cells are reduced in number causing a lack of activation signals from CD4 T cells and impaired defense by macrophages. CD8 T cells, on the other hand, are increased in number and cause lymphocytic alveolitis. Specific antibody responses by B-lymphocytes are decreased and opsonization of microorganisms is impaired. These observed defects in host defense of the respiratory tract explain the susceptibility of HIV-infected persons for oropharyngeal candidiasis, bacterial pneumonia, Pneumocystis pneumonia, and other opportunistic infections. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  3. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Medicine, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Epperly, Michael W. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Radiation Oncology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Basse, Per H. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wang, Hong [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Biostatistics, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wang, Xinhui [Harvard Medical School, Harvard University, 25 Shattuck Street, Boston, MA 02115 (United States); Proia, David A. [Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421 (United States); Greenberger, Joel S. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Radiation Oncology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Socinski, Mark A.; Levina, Vera, E-mail: levinav@upmc.edu [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Medicine, The University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-05-22

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

  4. Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

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    Sato, Hiromi

    2017-01-01

    Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways

  5. Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Hiromi Sato

    2017-07-01

    Full Text Available Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1 extracellular matrix, 2 intercellular adhesion molecules and cell surface receptors, 3 intracellular proteins, 4 cell-cell junction proteins, and 5 a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or

  6. Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells

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    W-J. Wang

    2017-10-01

    Full Text Available Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA using human umbilical vein endothelial cells (HUVEC. HUVECs (5×106 cells/mL were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS and incubated with RA-supplemented hemodialysis fluid (HDF. Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.

  7. Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells.

    Science.gov (United States)

    Wang, W-J; Cheng, M-H; Lin, J-H; Weng, C-S

    2017-10-19

    Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA) using human umbilical vein endothelial cells (HUVEC). HUVECs (5×106 cells/mL) were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS) and incubated with RA-supplemented hemodialysis fluid (HDF). Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB) were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.

  8. Human neutrophil cytoskeletal dynamics and contractility actively contribute to trans-endothelial migration.

    Science.gov (United States)

    Stroka, Kimberly M; Hayenga, Heather N; Aranda-Espinoza, Helim

    2013-01-01

    Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.

  9. Human neutrophil cytoskeletal dynamics and contractility actively contribute to trans-endothelial migration.

    Directory of Open Access Journals (Sweden)

    Kimberly M Stroka

    Full Text Available Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.

  10. The inhibitory effect of simvastatin and aspirin on histamine responsiveness in human vascular endothelial cells.

    Science.gov (United States)

    Absi, Mais; Bruce, Jason I; Ward, Donald T

    2014-04-01

    Statins and aspirin deliver well-established cardiovascular benefits resulting in their increased use as combined polypills to decrease risk of stroke and heart disease. However, the direct endothelial effect of combined statin/aspirin cotreatment remains unclear. Histamine is an inflammatory mediator that increases vascular permeability, and so we examined the effect of treating human umbilical vein endothelial cells (HUVECs) for 24 h with 1 μM simvastatin and 100 μM aspirin on histamine responsiveness. Subsequent histamine (1 μM) challenge increased intracellular calcium (Ca(2+)i) concentration, an effect that was significantly inhibited by combined simvastatin/aspirin pretreatment but not when then the compounds were given separately, even at 10-fold higher concentrations. In contrast, the Ca(2+)i mobilization response to ATP challenge (10 μM) was not inhibited by combined simvastatin/aspirin pretreatment. The H1 receptor antagonist pyrilamine significantly inhibited both histamine-induced Ca(2+)i mobilization and extracellular signal-regulated kinase (ERK) activation, whereas ranitidine (H2 receptor antagonist) was without effect. However, combined simvastatin/aspirin pretreatment failed to decrease H1 receptor protein expression ruling out receptor downregulation as the mechanism of action. Histamine-induced ERK activation was also inhibited by atorvastatin pretreatment, while simvastatin further inhibited histamine-induced vascular endothelial cadherin phosphorylation as well as altered HUVEC morphology and inhibited actin polymerization. Therefore, in addition to the known therapeutic benefits of statins and aspirin, here we provide initial cellular evidence that combined statin/aspirin treatment inhibits histamine responsiveness in HUVECs.

  11. Gene Expression Analysis to Assess the Relevance of Rodent Models to Human Lung Injury.

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    Sweeney, Timothy E; Lofgren, Shane; Khatri, Purvesh; Rogers, Angela J

    2017-08-01

    The relevance of animal models to human diseases is an area of intense scientific debate. The degree to which mouse models of lung injury recapitulate human lung injury has never been assessed. Integrating data from both human and animal expression studies allows for increased statistical power and identification of conserved differential gene expression across organisms and conditions. We sought comprehensive integration of gene expression data in experimental acute lung injury (ALI) in rodents compared with humans. We performed two separate gene expression multicohort analyses to determine differential gene expression in experimental animal and human lung injury. We used correlational and pathway analyses combined with external in vitro gene expression data to identify both potential drivers of underlying inflammation and therapeutic drug candidates. We identified 21 animal lung tissue datasets and three human lung injury bronchoalveolar lavage datasets. We show that the metasignatures of animal and human experimental ALI are significantly correlated despite these widely varying experimental conditions. The gene expression changes among mice and rats across diverse injury models (ozone, ventilator-induced lung injury, LPS) are significantly correlated with human models of lung injury (Pearson r = 0.33-0.45, P human lung injury. Predicted therapeutic targets, peptide ligand signatures, and pathway analyses are also all highly overlapping. Gene expression changes are similar in animal and human experimental ALI, and provide several physiologic and therapeutic insights to the disease.

  12. Transendothelial migration of human umbilical mesenchymal stem cells across uterine endothelial monolayers: Junctional dynamics and putative mechanisms.

    Science.gov (United States)

    Ebrahim, Neven A; Leach, Lopa

    2016-12-01

    During pregnancy, fetal stem cells can transfer to the maternal circulation and participate in tissue repair. How they transmigrate across maternal endothelial barriers and whether they can subsequently influence maternal endothelial integrity is not known. Mesenchymal stem cells (WJ-MSC) were isolated from Wharton's jelly and their interactions with human uterine microvascular endothelial cell (HUtMEC) monolayers, junctional occupancy and expression/phosphorylation of vascular endothelial (VE)- cadherin and vascular endothelial growth factor (VEGF-A) secretion was studied over 48h by real time, confocal microscopy, immunoblotting and ELISA. WJ-MSC displayed exploratory behaviour with interrogation of paracellular openings and spreading into the resultant increased gaps followed by closing of the endothelium over the WJ-MSC. 62% of added cells crossed within 22h to sub-endothelial niches. There was a concomitant loss of junctional VE-cadherin in HUtMEC followed by a full return and increased VE-cadherin expression after 22h. During early hours, VE-cadherin showed a transient phosphorylation at Tyrosine (Tyr)-685 when VEGF-A secretion were high. From 16 to 22h, there was increased de-phosphorylation of Tyr-731. Anti-VEGF-A blocked Tyr-685 phosphorylation but not the decrease in P-Tyr731; this partially inhibited WJ-MSC transmigration. Fetal WJ-MSC can traverse uterine endothelial monolayers by mediating a non-destructive paracellular pathway. They can promote junctional stability of uterine endothelium from the sub-endothelial niche. Mechanistically, WJ-MSC induces VEGF-dependent phosphorylation events linked with paracellular permeability and VEGF-independent de-phosphorylation events associated with leukocyte extravasation. Our data also allows consideration of a possible role of fetal MSC in mature functioning of the uterine vasculature needed for optimal utero-placental perfusion. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

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

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    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. Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides.

    Science.gov (United States)

    Bode, Lars; Kunz, Clemens; Muhly-Reinholz, Marion; Mayer, Konstantin; Seeger, Werner; Rudloff, Silvia

    2004-12-01

    Excessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. The initial step in leukocyte extravasation is mediated by selectins and oligosaccharides on their glycoconjugate ligands. Human milk is a rich source of lactose-derived oligosaccharides that are partly absorbed in the intestine and excreted with the urine. As these components contain binding determinants for the selectins we investigated whether human milk oligosaccharides are able to affect leukocyte rolling and adhesion to endothelial cells under dynamic conditions. Therefore, monocytes, lymphocytes, or neutrophils isolated from human peripheral blood were passed over TNF-alpha-activated HUVEC under shear stress. The influence of different oligosaccharide fractions was determined by video-microscopy and compared with the effects of various individual oligosaccharides. Within a physiological range (12.5 - 125 microg/ml) the acidic fraction significantly inhibited leukocyte rolling and adhesion (up to 24.0% and 52.8%, respectively) in a concentration-dependent manner. These effects were even more pronounced than those achieved by soluble sialyl-Lewis x, a physiological binding determinant for selectins. Several active components within the oligosaccharide fraction of human milk were identified, e.g. 3'-sialyl-lactose and 3'-sialyl-3-fucosyl-lactose. These results indicate that specific oligosaccharides in human milk may serve as anti-inflammatory components and might therefore contribute to the lower incidence of inflammatory diseases in human milk-fed infants.

  15. Regulation of thrombomodulin expression and release in human aortic endothelial cells by cyclic strain.

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    Fiona A Martin

    Full Text Available 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.This study employed human aortic endothelial cells (HAECs to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs, and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs, on TM expression and release. Time-, dose- and frequency-dependency studies were performed.Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0-7.5% and frequency (0.5-2.0 Hz dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2, receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml and ox-LDL (10-50 µg/ml appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001 nor rhomboids (3,4-dichloroisocoumarin had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM

  16. The Effect of Shiga Toxin on Weibel-Palade Bodies in Primary Human Endothelial Cells

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    Joyce Geelen

    2014-07-01

    Full Text Available Background/Aims: Diarrhea-associated hemolytic uremic syndrome is associated with the presence of Shiga toxin (Stx1, Stx2 and several variants in the circulation. The aim of this study is to examine the possible triggering effect of Stx1 on the exocytosis of Weibel-Palade bodies (WPbs. Methods: Cultured human umbilical venous endothelial cells (HUVECs and glomerular microvascular endothelial cells (GMVECs were stimulated by thrombin and Stx1 in both static and flowing conditions. The amount of secreted von Willebrand factor (VWF in the supernatant as well as the remaining intracellular fraction was determined. Results: In HUVECs and in 2 out of 4 GMVECs, the stimulation of Stx1 in flow at 1 dyne/cm2 resulted in a decrease of intracellular VWF. This is contrary to the results of Stx1 applied in static conditions. At a higher flow rate of 5 dyne/cm2, no effect in GMVECs was observed. Conclusion: Stx1 can contribute, via an effect on WPbs, to the exocytosis of WPbs in flow conditions in HUVECs and probably in GMVECs. This results in the release of VWF, suggesting an initiating role of the coagulation system in the pathogenesis.

  17. LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells.

    Science.gov (United States)

    Figarola, James L; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay; Singhal, Sharad S

    2014-05-01

    Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.

  18. Chronic nitric oxide deprivation induces an adaptive antioxidant status in human endothelial cells.

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    Cattaneo, Maria Grazia; Cappellini, Elisa; Ragni, Maurizio; Tacchini, Lorenza; Scaccabarozzi, Diletta; Nisoli, Enzo; Vicentini, Lucia Maria

    2013-11-01

    In a previous work, we showed an increased cell motility due to the accumulation and transcriptional activation of the Hypoxia Inducible Factor-1α (HIF-1α) and a reduced mitochondrial energy production in an in vitro model of endothelial dysfunction (ED) represented by human endothelial cells (ECs) chronically deprived of nitric oxide (NO) by L-NAME treatment. In the present study, in the attempt to unravel the pathway(s) linking NO deficiency to HIF-1α accumulation and activation, we focused our attention on Reactive Oxygen Species (ROS). We found that ROS were partially involved in HIF-1α stabilization, but not in the pro-migratory phenotype. Regarding mitochondrial dysfunction, it did not require neither ROS generation nor HIF-1α activity, and was not due to autophagy. Very interestingly, while acute treatment with L-NAME induced a transient increase in ROS formation, chronic NO deprivation by long term L-NAME exposure drastically reduced cellular ROS content giving rise to an antioxidant environment characterized by an increase in superoxide dismutase-2 (SOD-2) expression and activity, and by nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2). These results might have important implications for our understanding of the consequences of NO deprivation in endothelium behavior and in the onset of cardiovascular diseases. © 2013.

  19. On the Normal Force Mechanotransduction of Human Umbilical Vein Endothelial Cells

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    Vahabikashi, Amir; Wang, Qiuyun; Wilson, James; Wu, Qianhong; Vucbmss Team

    2016-11-01

    In this paper, we report a cellular biomechanics study to examine the normal force mechanotransduction of Human Umbilical Vein Endothelial Cells (HUVECs) with their implications on hypertension. Endothelial cells sense mechanical forces and adjust their structure and function accordingly. The mechanotransduction of normal forces plays a vital role in hypertension due to the higher pressure buildup inside blood vessels. Herein, HUVECs were cultured to full confluency and then exposed to different mechanical loadings using a novel microfluidic flow chamber. One various pressure levels while keeps the shear stress constant inside the flow chamber. Three groups of cells were examined, the control group (neither shear nor normal stresses), the normal pressure group (10 dyne/cm2 of shear stress and 95 mmHg of pressure), and the hypertensive group (10 dyne/cm2 of shear stress and 142 mmHg of pressure). Cellular response characterized by RT-PCR method indicates that, COX-2 expressed under normal pressure but not high pressure; Mn-SOD expressed under both normal and high pressure while this response was stronger for normal pressure; FOS and e-NOS did not respond under any condition. The differential behavior of COX-2 and Mn-SOD in response to changes in pressure, is instrumental for better understanding the pathogenesis of hypertensive cardiovascular diseases. This research was supported by the National Science Foundation under Award #1511096.

  20. Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells

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    I-Rang Lim

    2017-01-01

    Full Text Available Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-ylethylcarbamoyl-2-(benzo[d][1,3]dioxol-5-yloxyacetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 h of treatment, KCH-1521 changed the cell morphology of human umbilical vein endothelial cells (HUVECs and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via focal adhesion kinase (FAK, kinase B (AKT, and extracellular signal-regulated kinase (ERK pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

  1. Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC Damages.

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    Yumei Zhang

    Full Text Available Here, we studied the underlying mechanism of aldosterone (Aldo-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L inhibited human umbilical vein endothelial cells (HUVEC survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18 production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P, an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS inhibitor PDMP or the ceramide (C6 potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1 is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.

  2. Turkish propolis protects human endothelial cells in vitro from homocysteine-induced apoptosis.

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    Darendelioglu, Ekrem; Aykutoglu, Gurkan; Tartik, Musa; Baydas, Giyasettin

    2016-05-01

    Chronic cardiovascular and neurodegenerative complications induced by hyperhomocysteinemia have been most relatively associated with endothelial cell injury. Elevated homocysteine (Hcy) generates reactive oxygen species (ROS) accompanying with oxidative stress which is hallmarks of the molecular mechanisms responsible for cardiovascular disease. Propolis is a natural product, obtained by honeybee from various oils, pollens, special resins and wax materials, conventionally used with the purpose of treatment by folks Propolis has various biological activities and powerful antioxidant capacity. The flavonoids and phenolic acids, most bioactive components of propolis, have superior antioxidant ability to defend cell from free radicals. This study was designed to examine the protective effects of Turkish propolis (from east of country) on Hcy induced ROS production and apoptosis in human vascular endothelial cells (HUVECs). According to results, co-treatment of HUVECs with propolis decreased Hcy-induced ROS overproduction and lipid peroxidation (LPO) levels. Furthermore, overproductions of Bax, caspase-9 and caspase-3 protein, elevation of cytochrome c release in Hcy-treated HUVECs were significantly reduced by propolis. It was concluded that propolis has cytoprotective ability against cytotoxic effects of high Hcy in HUVECs. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. Crocetin Inhibits Lipopolysaccharide-Induced Inflammatory Response in Human Umbilical Vein Endothelial Cells

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    Lei Song

    2016-11-01

    Full Text Available Background/Aim: Crocetin is a readily bioavailable and bioactive compound extracted from Saffron. Previous studies indicated its various biomedical properties including antioxidant and anti-coagulation potencies. However, its effect on inflammation, notably within the cardiovascular system, has not been investigated yet. In the present study, we utilized human umbilical vein endothelial cell (HUVEC to elucidate the effect of Crocetin on vascular inflammation. Methods: Cell viability and toxicity were evaluated by MTT and Lactate dehydrogenase (LDH assay, respectively. Pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1 and Interleukin-8 (IL-8 expressions were determined by RT-PCR and ELISA. With fluorescence labeled U937 cells, we examined immune cell adhesion to the inflamed HUVEC in vitro, which was further confirmed by the H&E staining in the murine subcutaneous endothelium in vivo. Results: Upon Lipopolysaccharide (LPS-induced inflammatory response in HUVECs, Crocetin ameliorated cell cytotoxicity, suppressed MCP-1 and IL-8 expressions through blocking NF-κB p65 signaling transduction. Moreover, Crocetin inhibited immune cells adhesion and infiltration to inflamed endothelium, which is a key step in inflammatory vascular injury. Conclusions: These findings suggest that Crocetin, a natural herb extract, is a potent suppressor of vascular endothelial inflammation.

  4. Anti-Inflammatory effect of Buddleja officinalis on vascular inflammation in human umbilical vein endothelial cells.

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    Lee, Yun Jung; Moon, Mi Kyoung; Hwang, Sun Mi; Yoon, Jung Joo; Lee, So Min; Seo, Kwan Soo; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

    2010-01-01

    Vascular inflammation process has been suggested to be an important risk factor in the initiation and development of atherosclerosis. In this study, we investigated whether and by what mechanisms an aqueous extract of Buddleja officinalis (ABO) inhibited the expressions of cellular adhesion molecules, which are relevant to inflammation and atherosclerosis. Pretreatment of human umbilical vein endothelial cells (HUVEC) with ABO (1-10 microg/ml) for 18 hours dose-dependently inhibited TNF-alpha-induced adhesion U937 monocytic cells, as well as mRNA and protein expressions of vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1). Pretreatment with ABO also blocked TNF-alpha-induced ROS formation. Nuclear factor-kappa B (NF-kappaB) is required in the transcription of these adhesion molecule genes. Western blot analysis revealed that ABO inhibits the translocation of the p65 subunit of NF-kappaB to the nucleus. ABO inhibited the TNF-alpha-induced degradation of IkappaB-alpha, an inhibitor of NF-kappaB, by inhibiting the phosphorylation of IkappaB-alpha in HUVEC. Taken together, ABO could reduce cytokine-induced endothelial adhesiveness throughout down-regulating intracellular ROS production, NF-kappaB, and adhesion molecule expression in HUVEC, suggesting that the natural herb Buddleja officinalis may have potential implications in atherosclerosis.

  5. Buddleja officinalis inhibits high glucose-induced matrix metalloproteinase activity in human umbilical vein endothelial cells.

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    Lee, Yun Jung; Kang, Dae Gill; Kim, Jin Sook; Lee, Ho Sub

    2008-12-01

    The aim of the present investigation was to investigate whether an aqueous extract of Buddleja officinalis (ABO), a traditional Korean herbal medicine, suppresses the endothelial extracellular matrix degradation under high glucose condition. The incubation with high concentration of glucose (25 mM) increased significantly matrix metalloproteinase (MMP)-2/-9 expressions and activities in primary cultured human umbilical vein endothelial cells (HUVEC). Pretreatment with ABO decreased high glucose-induced increase of MMP-2/-9 activities in a dose-dependent manner. Real time qRT-PCR revealed that high glucose-induced MMP-2/-9 mRNA expression levels were attenuated by pretreatment with ABO. High glucose-induced MCP-1 and IL-8 mRNA expression levels also decreased by ABO. ABO decreased high glucose-induced hydrogen peroxide production, oxidative stress marker. These results provide new insights into the pathophysiological mechanisms for anti-inflammatory properties of ABO in vascular diseases associated with diabetes mellitus. (c) 2008 John Wiley & Sons, Ltd.

  6. Colocalization of serum amyloid a with microtubules in human coronary artery endothelial cells.

    Science.gov (United States)

    Lakota, Katja; Resnik, Nataša; Mrak-Poljšak, Katjuša; Sodin-Šemrl, Snežna; Veranič, Peter

    2011-01-01

    Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO₆. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

  7. Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

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    Katja Lakota

    2011-01-01

    Full Text Available Serum amyloid A (SAA acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

  8. Triamcinolone up-regulates GLUT 1 and GLUT 3 expression in cultured human placental endothelial cells.

    Science.gov (United States)

    Kipmen-Korgun, Dijle; Ozmen, Asli; Unek, Gozde; Simsek, Mehmet; Demir, Ramazan; Korgun, Emin Turkay

    2012-01-01

    The placenta is a glucocorticoid target organ, and glucocorticoids (GCs) are essential for the development and maturation of fetal organs. They are widely used for treatment of a variety of diseases during pregnancy. In various tissues, GCs have regulated by glucose transport systems; however, their effects on glucose transporters in the human placental endothelial cells (HPECs) are unknown. In the present study, HPECs were cultured 24 h in the presence or absence of 0.5, 5 and 50 µmol · l(-1) of synthetic GC triamcinolone (TA). The glucose carrier proteins GLUT 1, GLUT 3 and GC receptor (GR) were detected in the HPECs. We showed increased expression of GLUT 1 and GLUT 3 proteins and messenger RNA (mRNA) levels (p GLUT 1 and GLUT 3 expression through GR. Excessive exposure to GCs causes maternal and fetal hypoglycemia and diminished fetal growth. We speculate that to compensate for fetal hypoglycemia and diminished fetal growth, the expression of placental endothelial glucose transporters might be increased. Copyright © 2011 John Wiley & Sons, Ltd.

  9. Interaction of Tissue Engineering Substrates with Serum Proteins and Its Influence on Human Primary Endothelial Cells.

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    Mohan, Tamilselvan; Niegelhell, Katrin; Nagaraj, Chandran; Reishofer, David; Spirk, Stefan; Olschewski, Andrea; Stana Kleinschek, Karin; Kargl, Rupert

    2017-02-13

    Polymer-based biomaterials particularly polycaprolactone (PCL) are one of the most promising substrates for tissue engineering. The surface chemistry of these materials plays a major role since it governs protein adsorption, cell adhesion, viability, degradation, and biocompatibility in the first place. This study correlates the interaction of the most abundant serum proteins (albumin, immunoglobulins, fibrinogen) with the surface properties of PCL and its influence on the morphology and metabolic activity of primary human arterial endothelial cells that are seeded on the materials. Prior to that, thin films of PCL are manufactured by spin-coating and characterized in detail. A quartz crystal microbalance with dissipation (QCM-D), a multiparameter surface plasmon resonance spectroscopy instrument (MP-SPR), wettability data, and atomic force microscopy are combined to elucidate the pH-dependent protein adsorption on the PCL substrates. Primary endothelial cells are cultured on the protein modified polymer, and conclusions are drawn on the significant impact of type and form of proteins coatings on cell morphology and metabolic activity.

  10. Knockdown of ezrin suppresses the migration and angiogenesis of human umbilical vein endothelial cells in vitro.

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    Zhao, Liang-ping; Huang, Lei; Tian, Xun; Liang, Feng-qi; Wei, Jun-cheng; Zhang, Xian; Li, Sha; Zhang, Qing-hua

    2016-04-01

    Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.

  11. Cancer-associated loss of TARSH gene expression in human primary lung cancer.

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    Terauchi, Kunihiko; Shimada, Junichi; Uekawa, Natsuko; Yaoi, Takeshi; Maruyama, Mitsuo; Fushiki, Shinji

    2006-01-01

    We have previously identified mouse Tarsh as one of the cellular senescence-related genes and showed the loss of expression of TARSH mRNA in four human lung cancer cell lines. TARSH is a presumptive signal transduction molecule interacting with NESH, which is implicated to have some roles in lung cancer metastasis. The amplification of complete ORF-encoding TARSH cDNA was done with reverse transcription-PCR. Northern blotting was carried out using TARSH cDNA probes. To clarify the relationship between TARSH and lung cancer, we quantified TARSH mRNA expression in 15 human lung cancer cell lines and 32 primary non-small cell lung cancers. We first determined the complete ORF-encoding cDNA sequence which is expressed in the human lung. On the Northern hybridization analysis, TARSH was strongly expressed in the human lung. The expression of TARSH mRNA is remarkably downregulated in all the lung cancer cell lines examined. Furthermore, TARSH expression was significantly low in all of the tumor specimens when compared to the expression in corresponding non-neoplastic lung tissue specimens. The cancer-associated transcriptional inactivation of TARSH suggests that TARSH could be used as a biomarker for lung cancer development as well as a molecular adjunct for lung carcinogenesis in human.

  12. IMP3 Predicts Invasion and Prognosis in Human Lung Adenocarcinoma.

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    Yan, Jinhai; Wei, Qingzhu; Jian, Wenjing; Qiu, Bo; Wen, Jing; Liu, Jianghuan; Fu, Bo; Zhou, Xinhua; Zhao, Tong

    2016-02-01

    Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with several aggressive and advanced cancers. Whether IMP3 can predict invasion, and prognosis in patients with human lung adenocarcinoma (LAC) remains unclear. Ninety-five LAC and 75 non-tumor lung tissue samples were included in a tissue microarray. IMP3 expression was assessed by immunohistochemical examination. Correlation between IMP3 expression levels, clinicopathological characteristics, and overall prognosis was evaluated. In a separate in vitro study, RNA interference method was applied for knockdown of IMP3 gene in human LAC cell lines. Invasive potential of LAC cells was then evaluated by transwell migration assay. IMP3 immunoreactivity was observed in 39 out of 95 (41.1 %) LAC patients, but not in non-tumor lung tissues. IMP3 expression levels were closely associated with histological grade (P = 0.037), TNM stage (P = 0.034), and lymph node metastasis (P = 0.011). Patients presenting with positive IMP3 expression (P = 0.000), an advanced TNM stage (P = 0.000), and lymph node metastasis (P = 0.001) had a worse overall survival, compared to those lacking these characteristics. Both IMP3 expression (hazard ratio [HR], 2.310; 95 % confidence interval [CI] 1.192-4.476; P = 0.013) and TNM stage (HR 2.338; 95 % CI 1.393-3.925; P = 0.001) were independent predictors of poor prognosis. The invasive potential of LAC cells was significantly inhibited by IMP3 knockdown. IMP3 appears to play an important role in tumor invasion in patients with LAC and may serve as a useful prognostic biomarker in these patients.

  13. Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice

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    Yu-Qin Chen

    2015-01-01

    Full Text Available Objective: This work was aimed at studying the inhibitory activity of metformin combined with the commonly used chemotherapy drug cisplatin in human lung cancer xenografts in nude mice. We also examined the combined effects of these drugs on the molecular expression of survivin, matrix metalloproteinase-2 (MMP-2, vascular endothelial growth factor-C (VEGF-C, and vascular endothelial growth factorreceptor-3 (VEGFR-3 to determine the mechanism of action and to explore the potential applications of the new effective drug therapy in lung cancer. Materials and Methods: The nude mice model of lung cancer xenografts was established, and mice were randomly divided into the metformin group, the cisplatin group, the metformin + cisplatin group, and the control group. The animals were killed 42 days after drug administration, and the tumor tissues were then sampled to detect the messenger ribonucleic acid (mRNA and protein expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR. Results: The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the cisplatin group and the combined treatment group were lower than that in the control group (P < 0.05. In the metformin group, the expression of MMP-2 protein and mRNA was lower than that in the control group (P < 0.05. The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the combined treatment group were lower than that in the cisplatin group and the metformin group (P < 0.05. Conclusions: Metformin inhibited the expression of MMP-2, cisplatin and the combined treatment inhibited the expression of survivin, MMP-2, VEGF-C, and VEGFR-3, and the combined treatment of metformin with cisplatin resulted in enhanced anti-tumor efficacy.

  14. Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice.

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    Chen, Yu-Qin; Chen, Gang

    2015-01-01

    This work was aimed at studying the inhibitory activity of metformin combined with the commonly used chemotherapy drug cisplatin in human lung cancer xenografts in nude mice. We also examined the combined effects of these drugs on the molecular expression of survivin, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor-C (VEGF-C), and vascular endothelial growth factor receptor-3 (VEGFR-3) to determine the mechanism of action and to explore the potential applications of the new effective drug therapy in lung cancer. The nude mice model of lung cancer xenografts was established, and mice were randomly divided into the metformin group, the cisplatin group, the metformin + cisplatin group, and the control group. The animals were killed 42 days after drug administration, and the tumor tissues were then sampled to detect the messenger ribonucleic acid (mRNA) and protein expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the cisplatin group and the combined treatment group were lower than that in the control group (P metformin group, the expression of MMP-2 protein and mRNA was lower than that in the control group (P metformin group (P Metformin inhibited the expression of MMP-2, cisplatin and the combined treatment inhibited the expression of survivin, MMP-2, VEGF-C, and VEGFR-3, and the combined treatment of metformin with cisplatin resulted in enhanced anti-tumor efficacy.

  15. Experimental acute lung injury induces multi-organ epigenetic modifications in key angiogenic genes implicated in sepsis-associated endothelial dysfunction.

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    Bomsztyk, Karol; Mar, Daniel; An, Dowon; Sharifian, Roya; Mikula, Michal; Gharib, Sina A; Altemeier, William A; Liles, W Conrad; Denisenko, Oleg

    2015-05-11

    The Tie2/angiopoietin (Tie2/Ang) and vascular endothelial growth factor receptor-ligand systems (VEGFR/VEGF) are recognized to play important roles in the regulation of microvascular endothelial function. Downregulation of these genes during sepsis has been implicated in the pathogenesis of sepsis-related microvascular leak and multiple organ dysfunction syndrome. Mechanisms responsible for dysregulation of angiogenic genes in sepsis are poorly defined. Western blot, reverse transcription-polymerase chain reaction, and multiplex chromatin immunoprecipitation platform (Matrix ChIP) were used to investigate serum albumin leak, changes in gene expression, and associated epigenetic alterations in a murine model of acute lung injury-induced sepsis (ALI-sepsis). Experimental ALI-sepsis induced microvascular leak and downregulation of expression of Angpt1 (Ang1), Tek (Tie2), and Kdr (Vegfr2 or Flk-1) genes in the lung, kidney, and liver. These changes correlate with a decrease in RNA polymerase II density at these genes, and the greatest response was observed in the lung. ALI-sepsis reduced levels of transcription-permissive histone H3 lysine acetylation (H3KAc) at these loci in all examined tissues. Decreases in permissive H3K4m3 and H3Km2 marks were detected only in the lung. In contrast, only minimal alterations in transcription-repressive histone modifications (H3K27m3, H3K9m2, H3K9m3, and H4K20m3) were observed in all tissues. Our results demonstrate that decreases in transcription-permissive, but not increases in transcription-repressive, histone modifications at Angpt1, Tek, and Kdr are a systemic, rather than a lung-restricted, response, involving key end-organs in experimental ALI-sepsis. Given that ventilator-associated pneumonia is a major cause of sepsis in critically ill patients, elucidation of mechanisms mediating epigenetic alterations during sepsis provides fundamental new insights into the pathogenesis of sepsis-induced microvascular leak and subsequent

  16. A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.

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    Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere

    2014-08-27

    Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.

  17. Measuring endothelial glycocalyx dimensions in humans: a potential novel tool to monitor vascular vulnerability.

    Science.gov (United States)

    Nieuwdorp, Max; Meuwese, Marijn C; Mooij, Hans L; Ince, Can; Broekhuizen, Lysette N; Kastelein, John J P; Stroes, Erik S G; Vink, Hans

    2008-03-01

    The endothelial glycocalyx is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults. The purpose of this study was to translate an established experimental method of estimating capillary glycocalyx dimension into a clinically useful tool and to assess its reproducibility in humans. We first evaluated by intravital microscopy the relation between the distance between the endothelium and erythrocytes, as a measure of glycocalyx thickness, and the transient widening of the erythrocyte column on glycocalyx compression by passing leukocytes in hamster cremaster muscle capillaries. We subsequently assessed sublingual microvascular glycocalyx thickness in 24 healthy men using orthogonal polarization spectral imaging. In parallel, systemic glycocalyx volume (using a previously published tracer dilution technique) as well as cardiovascular risk profiles were assessed. Estimates of microvascular glycocalyx dimension from the transient erythrocyte widening correlated well with the size of the erythrocyte-endothelium gap (r = 0.63). Measurements in humans were reproducible (0.58 +/- 0.16 and 0.53 +/- 0.15 microm, coefficient of variance 15 +/- 5%). In univariate analysis, microvascular glycocalyx thickness significantly correlated with systemic glycocalyx volume (r = 0.45), fasting plasma glucose (r = 0.43), and high-density lipoprotein-cholesterol (r = 0.40) and correlated negatively with low-density lipoprotein-cholesterol (r = -0.41) as well as body mass index (r = -0.45) (all P < 0.05). In conclusion, the dimension of the endothelial glycocalyx can be measured reproducibly in humans and is related to cardiovascular risk factors. It remains to be tested whether glycocalyx dimension can be used as an early marker of vascular damage and whether therapies aimed at glycocalyx repair can protect the vasculature against pathogenic challenges.

  18. Visfatin as a novel mediator released by inflamed human endothelial cells.

    Science.gov (United States)

    Romacho, Tania; Villalobos, Laura A; Cercas, Elena; Carraro, Raffaele; Sánchez-Ferrer, Carlos F; Peiró, Concepción

    2013-01-01

    Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC). However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL)-1β (1 to 10 ng/mL), tumor necrosis factor-α (1 to 10 ng/mL) or angiotensin II (10 pmol/L to 1 μmol/L) markedly enhanced intracellular visfatin content. Using IL-1β (10 ng/mL; 18 h), it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose) polymerase-1 activation. Moreover, IL-1β modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1β-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1β promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation.

  19. Visfatin as a novel mediator released by inflamed human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Tania Romacho

    Full Text Available BACKGROUND: Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. METHODS AND RESULTS: Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC. However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL-1β (1 to 10 ng/mL, tumor necrosis factor-α (1 to 10 ng/mL or angiotensin II (10 pmol/L to 1 μmol/L markedly enhanced intracellular visfatin content. Using IL-1β (10 ng/mL; 18 h, it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose polymerase-1 activation. Moreover, IL-1β modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1β-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1β promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. CONCLUSIONS: Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation.

  20. Contribution of Endothelial-to-Mesenchymal Transition to the Pathogenesis of Human Cerebral and Orbital Cavernous Malformations.

    Science.gov (United States)

    Takada, Shigeki; Hojo, Masato; Tanigaki, Kenji; Miyamoto, Susumu

    2017-07-01

    The analysis of gene-targeted mouse mutants has demonstrated that endothelial-to-mesenchymal transition (EndMT) is crucial to the onset and progression of cerebral cavernous malformations (CMs). It has also been shown that Notch and ephrin/Eph signaling are involved in EndMT. However, their roles in the pathogenesis of human intracranial CMs remain unclear. To elucidate the contribution of EndMT, the Notch pathway, and ephrin-B2/EphB4 signaling to the pathogenesis of human intracranial CMs. Eight human intracranial CMs (5 cerebral and 3 orbital CMs) were immunohistochemically investigated. CD31 (an endothelial marker) and EndMT markers, such as α-smooth muscle actin (a mesenchymal marker) and CD44 (a mesenchymal stem cell marker), were expressed in the endothelial layer of vascular sinusoids in all cases, suggesting that endothelial cells (ECs) have acquired mesenchymal and stem-cell-like characteristics and undergone EndMT in all cerebral and orbital CMs. EndMT was observed in about 70% and 35% of ECs in cerebral and orbital CMs, respectively. In all cases, Notch3 was expressed in the endothelial layer, indicating that ECs of vascular sinusoids have acquired mesenchymal features. In all cases, both ephrin-B2 and EphB4 were detected in the endothelial layer, suggesting that ECs of vascular sinusoids are immature or malformed cells and have both arterial and venous characteristics. EndMT plays a critical role in the pathogenesis of human cerebral and orbital CMs. Modulating EndMT is expected to be a new therapeutic strategy for cerebral and orbital CMs.

  1. Docosahexaenoic acid inhibits the adhesion of flowing neutrophils to cytokine stimulated human umbilical vein endothelial cells.

    Science.gov (United States)

    Yates, Clara M; Tull, Samantha P; Madden, Jackie; Calder, Philip C; Grimble, Robert F; Nash, Gerard B; Rainger, G Ed

    2011-07-01

    The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

  2. Resistance of human brain microvascular endothelial cells in culture to methylmercury: cell-density-dependent defense mechanisms.

    Science.gov (United States)

    Hirooka, Takashi; Fujiwara, Yasuyuki; Shinkai, Yasuhiro; Yamamoto, Chika; Yasutake, Akira; Satoh, Masahiko; Eto, Komyo; Kaji, Toshiyuki

    2010-06-01

    Vascular toxicity is important for understanding the neurotoxicity of methylmercury, because microvessels strongly influence the construction of microenvironment around neurons. Previously, we found that low density-human brain microvascular pericytes are markedly susceptible to methylmercury cytotoxicity due to high expression levels of the L-type amino acid transporter 1 (LAT-1) that transports methylmercury into the cells. Although LAT-1 can be, in general, highly expressed in sparse cells that require amino acids for growth, we found that human brain microvascular endothelial cells, regardless of cell density, were resistant to methylmercury cytotoxicity. To investigate the mechanisms underlying this resistance, we exposed the endothelial cells at low and high cell densities to methylmercury and determined the extent of nonspecific cell damage, intracellular accumulation of methylmercury, expression of LAT-1 and LAT-2 mRNAs, and intracellular expression of reduced glutathione and metallothionein. These experiments indicate that sparse endothelial cells intracellularly accumulate more methylmercury via the highly expressed LAT-1, but are resistant to methylmercury cytotoxicity by higher expression of the protective sulfhydryl peptides, namely, reduced glutathione and metallothionein. It is suggested that both nonspecific and functional damage is caused in pericytes, whereas functional abnormalities rather than nonspecific damage may occur to a greater extent in the endothelial cells in the brain microvessels exposed to methylmercury. The previous and present data also suggest that methylmercury exhibits toxicity in endothelial cells in a manner different from that in pericytes in the brain microvessels.

  3. Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

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    Fatemeh Kazemi-Lomedasht

    2014-12-01

    Full Text Available Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

  4. Microbial volatile communication in human organotypic lung models.

    Science.gov (United States)

    Barkal, Layla J; Procknow, Clare L; Álvarez-García, Yasmín R; Niu, Mengyao; Jiménez-Torres, José A; Brockman-Schneider, Rebecca A; Gern, James E; Denlinger, Loren C; Theberge, Ashleigh B; Keller, Nancy P; Berthier, Erwin; Beebe, David J

    2017-11-24

    We inhale respiratory pathogens continuously, and the subsequent signaling events between host and microbe are complex, ultimately resulting in clearance of the microbe, stable colonization of the host, or active disease. Traditional in vitro methods are ill-equipped to study these critical events in the context of the lung microenvironment. Here we introduce a microscale organotypic model of the human bronchiole for studying pulmonary infection. By leveraging microscale techniques, the model is designed to approximate the structure of the human bronchiole, containing airway, vascular, and extracellular matrix compartments. To complement direct infection of the organotypic bronchiole, we present a clickable extension that facilitates volatile compound communication between microbial populations and the host model. Using Aspergillus fumigatus, a respiratory pathogen, we characterize the inflammatory response of the organotypic bronchiole to infection. Finally, we demonstrate multikingdom, volatile-mediated communication between the organotypic bronchiole and cultures of Aspergillus fumigatus and Pseudomonas aeruginosa.

  5. Cellular morphometry of the bronchi of human and dog lungs

    Energy Technology Data Exchange (ETDEWEB)

    Robbins, E.S.

    1992-09-01

    Quantitative data of the human bronchial epithelial cells at possible risk for malignant transformation in lung cancer is crucial for accurate radon dosimetry and risk analysis. The locations and other parameters of the nuclei which may be damaged by [alpha] particles must be determined and compared in different airway generations, among smokers, non-smokers and ex-smokers, between men and women and in people of different ages. This proposal includes extended morphometric studies on electron micrographs of human epithelium of defined airway generations and in parallel on electron micrographs of the dog bronchial lining. The second part of this proposal describes studies to quantitate the cycling bronchial epithelial population(s) using proliferation markers and immunocytochemistry on frozen and paraffin sections and similar labeling of isolated bronchial epithelial cells sorted flow cytometry.

  6. The Role of Serotonin Transporter in Human Lung Development and in Neonatal Lung Disorders

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    E. C. C. Castro

    2017-01-01

    Full Text Available Introduction. Failure of the vascular pulmonary remodeling at birth often manifests as pulmonary hypertension (PHT and is associated with a variety of neonatal lung disorders including a uniformly fatal developmental disorder known as alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV. Serum serotonin regulation has been linked to pulmonary vascular function and disease, and serotonin transporter (SERT is thought to be one of the key regulators in these processes. We sought to find evidence of a role that SERT plays in the neonatal respiratory adaptation process and in the pathomechanism of ACD/MPV. Methods. We used histology and immunohistochemistry to determine the timetable of SERT protein expression in normal human fetal and postnatal lungs and in cases of newborn and childhood PHT of varied etiology. In addition, we tested for a SERT gene promoter defect in ACD/MPV patients. Results. We found that SERT protein expression begins at 30 weeks of gestation, increases to term, and stays high postnatally. ACD/MPV patients had diminished SERT expression without SERT promoter alteration. Conclusion. We concluded that SERT/serotonin pathway is crucial in the process of pulmonary vascular remodeling/adaptation at birth and plays a key role in the pathobiology of ACD/MPV.

  7. A SCID mouse-human lung xenograft model of varicella-zoster virus infection.

    Science.gov (United States)

    Wang, Wei; Pan, Dequan; Fu, Wenkun; Cai, Linli; Ye, Jianghui; Liu, Jian; Liu, Che; Huang, Xiumin; Lin, Yanzhen; Xia, Ningshao; Cheng, Tong; Zhu, Hua

    2017-10-01

    Varicella pneumonia is one of the most serious, potentially life-threatening complications of primary varicella-zoster virus (VZV) infection in adults and immunocompromised individuals. However, studies on the lung pathogenesis of VZV infection as well as development and testing of antivirals have long been hindered by limited access to clinical samples and a lack of suitable animal models. In this study, we report for the first time the use of human lung xenografts in SCID mice for investigating VZV infection. Human fetal lung tissues grafted under the kidney capsule of SCID mice rapidly grew and developed mature structures closely resembling normal human lung. Following infection, VZV replicated and spread efficiently in human lung xenografts, where the virus targeted both alveolar epithelial and mesenchymal cells, and resulted in formation of large viral lesions. VZV particles were readily detected in the nuclei and cytoplasm of infected lung cells by electron microscopy. Additionally, VZV infection resulted in a robust pro-inflammatory cytokine response in human lung xenografts. In conclusion, infecting human lung xenografts in SCID mice provides a useful, biological relevant tool for future mechanistic studies on VZV lung pathogenesis, and may potentially facilitate the evaluation of new antiviral therapies for VZV lung infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

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    Takamitsu Sasaki

    2007-12-01

    Full Text Available The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR and vascular endothelial growth factor receptor (VEGFR signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α and vascular endothelial growth factor (VEGF but were negative for EGFR, human epidermal growth factor receptor 2 (HER2, VEGFR. Double immunofluorescence staining revealed that tumorassociated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR, phosphorylated VEGFR (pVEGFR. Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01; this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001. AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, increased the level of apoptosis in both tumorassociated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.

  9. Bromodomain and extra-terminal protein mimic JQ1 decreases inflammation in human vascular endothelial cells: Implications for pulmonary arterial hypertension.

    Science.gov (United States)

    Mumby, Sharon; Gambaryan, Natalia; Meng, Chao; Perros, Frederic; Humbert, Marc; Wort, S John; Adcock, Ian M

    2017-01-01

    Nuclear factor kappa B (NF-kB)-mediated inflammatory gene expression and vascular endothelial cell proliferation/remodelling are implicated in the pathophysiology of the fatal disease, pulmonary arterial hypertension (PAH). Bromodomain and extra-terminal (BET) proteins are essential for the expression of a subset of NF-kB-induced inflammatory genes. BET mimics including JQ1+ prevent binding of BETs to acetylated histones and down-regulate the expression of selected genes. The effects of JQ1+ on the proliferation of primary human pulmonary microvascular endothelial cells (HPMECs) from healthy subjects were measured by bromodeoxyuridine (BrdU) incorporation. Cell cycle progression was assessed by flow cytometry; mRNA and protein levels of cyclin-dependent kinases (CDKs), inhibitors and cytokines were determined by reverse transcription-quantitative PCR (RT-qPCR), Western blotting or ELISA. Histone acetyltransferase (HAT) and deacetylase (HDAC) activities were determined in nuclear extracts from whole lung of PAH and control patients. JQ1+ significantly inhibited IL6 and IL8 (IL6 and CXCL8) mRNA and protein in HPMECs compared with its inactive enantiomer JQ1-. JQ1+ decreased NF-kB p65 recruitment to native IL6 and IL8 promoters. JQ1+ showed a concentration-dependent decrease in HPMEC proliferation compared with JQ1--treated cells. JQ1+ induced G1 cell cycle arrest by increasing the expression of the CDK inhibitors (CDKN) 1A (p21 cip ) and CDKN2D (p19 INK4D ) and decreasing that of CDK2, CDK4 and CDK6. JQ1+ also inhibited serum-stimulated migration of HPMECs. Finally, HAT activity was significantly increased in the lung of PAH patients. Inhibition of BETs in primary HPMECs decreases inflammation and remodelling. BET proteins could be a target for future therapies for PAH. © 2016 The Authors. Respirology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Respirology.

  10. Brain Tumor Tropism of Transplanted Human Neural Stem Cells Is Induced by Vascular Endothelial Growth Factor

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    Nils Ole Schmidt

    2005-06-01

    Full Text Available The transplantation of neural stem cells (NSCs offers a new potential therapeutic approach as a cell-based delivery system for gene therapy in brain tumors. This is based on the unique capacity of NSCs to migrate throughout the brain and to target invading tumor cells. However, the signals controlling the targeted migration of transplanted NSCs are poorly defined. We analyzed the in vitro and in vivo effects of angiogenic growth factors and protein extracts from surgical specimens of brain tumor patients on NSC migration. Here, we demonstrate that vascular endothelial growth factor (VEGF is able to induce a long-range attraction of transplanted human NSCs from distant sites in the adult brain. Our results indicate that tumorupregulated VEGF and angiogenic-activated microvasculature are relevant guidance signals for NSC tropism toward brain tumors.

  11. Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds

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    Matschegewski Claudia

    2016-09-01

    Full Text Available The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926. Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation of diminished cell spreading accompanied with reduced cell viability on nonwovens. Among those, highest biocompatibility was assessed for PLLA L214, although being generally low when compared to the planar control surface. Electrospinning was demonstrated as an innovative technique for the fabrication of advanced biomaterials aiming at guided cellular behavior as well as the design of novel implant platforms. A better understanding of cell–biomaterial interactions is desired to further improve implant development.

  12. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

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    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  13. Oxytocin inhibits ox-LDL-induced adhesion of monocytic THP-1 cells to human brain microvascular endothelial cells.

    Science.gov (United States)

    Liu, Shuyan; Pan, Shengying; Tan, Jing; Zhao, Weina; Liu, Fengguo

    2017-12-15

    The attachment of monocytes to human brain microvascular endothelial cells (HBMVEs) caused by oxidized low-density lipoprotein (ox-LDL) is associated with an early event and the pathological progression of cerebrovascular diseases. Oxytocin (OT) is a human peptide hormone that is traditionally used as a medication to facilitate childbirth. However, little information is available regarding the physiological function of OT in brain endothelial dysfunction. In the present study, our results indicate that the oxytocin receptor (OTR) was expressed in human brain microvascular endothelial cells (HBMVEs) and was upregulated in response to ox-LDL in a concentration-dependent manner. Notably, OT significantly suppressed ox-LDL-induced attachment of THP-1 monocytes to HBMVEs. Furthermore, we found that OT reduced the expression of adhesion molecules, such as VCAM-1 and E-selectin. Interestingly, it was shown that OT could restore ox-LDL-induced reduction of KLF4 in HBMVEs. Importantly, knockdown of KLF4 abolished the inhibitory effects of OT on ox-LDL-induced expressions of VCAM-1 and E-selectin as well as the adhesion of human monocytic THP-1 cells to endothelial HBMVEs. Mechanistically, we found that the stimulatory effects of OT on KLF4 expression are mediated by the MEK5/MEF2A pathway. Copyright © 2017. Published by Elsevier Inc.

  14. Characterization of heme oxygenase 1 (heat shock protein 32) induction by atrial natriuretic peptide in human endothelial cells

    NARCIS (Netherlands)

    Kiemer, Alexandra K.; Bildner, Nicole; Weber, Nina C.; Vollmar, Angelika M.

    2003-01-01

    Atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory and cytoprotective potential. The aim of this study was to characterize induction of heme oxygenase (HO)-1 by ANP in human umbilical vein endothelial cells (HUVEC). HUVEC were treated with ANP, 8-bromo-cyclic

  15. Transcriptome dysregulation by anthrax lethal toxin plays a key role in induction of human endothelial cell cytotoxicity

    CSIR Research Space (South Africa)

    Rolando, M

    2010-07-01

    Full Text Available The authors have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase-3 activation and the formation of thick actin cables in human endothelial cells. By DNA array analysis they show that LT has a major impact on the cell...

  16. Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

    NARCIS (Netherlands)

    Kusters, B.; Waal, R.M.W. de; Wesseling, P.; Verrijp, K.; Maass, C.N.; Heerschap, A.; Barentsz, J.O.; Sweep, C.G.J.; Ruiter, D.J.; Leenders, W.P.J.

    2003-01-01

    We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood

  17. Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy, E-mail: shivanna@bcm.edu

    2015-07-15

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

  18. Antiproliferative action of metformin in human lung cancer cell lines.

    Science.gov (United States)

    Ashinuma, Hironori; Takiguchi, Yuichi; Kitazono, Satoru; Kitazono-Saitoh, Miyako; Kitamura, Atsushi; Chiba, Tetsuhiro; Tada, Yuji; Kurosu, Katsushi; Sakaida, Emiko; Sekine, Ikuo; Tanabe, Nobuhiro; Iwama, Atsushi; Yokosuka, Osamu; Tatsumi, Koichiro

    2012-07-01

    The oral antidiabetic agent metformin has anticancer properties, probably via adenosine monophosphate-activated protein kinase activation. In the present study, growth inhibition was assessed by a clonogenic and by a cell survival assay, apoptosis induction was assessed by Hoechst staining and caspase activities and cell cycle alteration after exposure to metformin, and the interaction of metformin with cisplatin in vitro were elucidated in four human lung cancer cell lines representing squamous, adeno-, large cell and small cell carcinoma. Clonogenicity and cell proliferation were inhibited by metformin in all the cell lines examined. This inhibitory effect was not specific to cancer cells because it was also observed in a non-transformed human mesothelial cell line and in mouse fibroblast cell lines. Inhibition of clonogenicity was observed only when the cells were exposed to metformin for a long period, (10 days) and the surviving fraction, obtained after inhibiting proliferation by increasing the dose, reached a plateau at approximately 0.1-0.3, indicating the cytostatic characteristics of metformin. Metformin induced significant apoptosis only in the small cell carcinoma cell line. A tendency of cell cycle accumulation at the G0/G1 phase was observed in all four cell lines. Cisplatin, in a dose-dependent manner, severely antagonized the growth inhibitory effect of metformin, and even reversed the effect in three cell lines but not in the adenocarcinoma cell line. The present data obtained using various histological types of human lung cancer cell lines in vitro illustrate the cytostatic nature of metformin and its cytoprotective properties against cisplatin.

  19. Inhibition of normal human lung fibroblast growth by beryllium.

    Science.gov (United States)

    Lehnert, N M; Gary, R K; Marrone, B L; Lehnert, B E

    2001-03-07

    Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset ( approximately 1-6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO(4) (0.1-100 microM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be(2+)-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G(0)-G(1)/pre-S phase arrest. Western blot analyses indicated that the Be-induced G(0)-G(1)/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21(Waf-1,Cip1)). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

  20. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

    Science.gov (United States)

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

  1. Pulmonary venous hypertension and mechanical strain stimulate monocyte chemoattractant protein-1 release and structural remodelling of the lung in human and rodent chronic heart failure models.

    Science.gov (United States)

    Park, John E S; Lyon, Alexander R; Shao, Dongmin; Hector, Lauren R; Xu, Hua; O'Gara, Peter; Pinhu, Liao; Chambers, Rachel C; Wort, S John; Griffiths, Mark J D

    2014-12-01

    The burden of chronic heart failure (HF) is rising owing to an increased survivorship after myocardial infarction (MI). Pulmonary structural remodelling in patients with HF may protect against oedema while causing dyspnoea, the predominant symptom associated with HF. The cellular and molecular mechanisms underlying these processes in HF are poorly understood. We hypothesised that pulmonary venous hypertension (PVH) following MI provides a mechanical stimulus for structural remodelling of the lung via monocyte chemoattractant protein-1 (MCP-1). Human lung microvascular endothelial cells (HLMVEC) and Ea.Hy 926 cells exposed to cyclic mechanical strain (CMS) in vitro were analysed for MCP-1 expression and activation of signalling intermediates. HF was induced in Sprague-Dawley rats 16 weeks after MI; a cohort was rescued with AAV9.SERCA2a gene therapy to reduce PVH. HLMVEC and Ea.Hy 926 cells exposed to CMS upregulated MCP-1 gene expression and protein release in an extracellular-signal-regulated kinase (ERK) 1/2 dependent manner. Supernatants from these experiments stimulated fibroblast (human fetal lung fibroblast -1) and pulmonary artery smooth muscle cell proliferation and differentiation. Total lung collagen, a marker of structural remodelling, and MCP-1 gene expression were increased in the lungs of rats with post-MI HF. SERCA2a gene therapy that attenuated PVH after MI was associated with lower levels of lung collagen and MCP-1 gene expression in the lung. Mechanical strain associated with PVH may stimulate pulmonary structural remodelling through ERK 1/2 dependent induction of MCP-1. These findings provide insights into the pathophysiology of lung remodelling in HF and highlight novel, potential therapeutic targets. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  2. Isolation and Culture of Human Microvascular endothelium for comparison of the morphological and molecular characteristics of Microvascular endothelial cells under normal gravity against simulated micro gravity

    Directory of Open Access Journals (Sweden)

    Tholcopiyan L

    2010-01-01

    Full Text Available BACKGROUND: Vascular endothelial cells play a major role in wound healing and also in growth of the tumors. Angiogenesis can be a target for treating diseases that are due to either poor vascularisation or decreased blood supply as in stroke, ulcers, heart disease, etc or abnormal and increased vasculature like in tumours. Application of specific compounds that may inhibit or induce the creation of new blood vessels in the body may help in the treatment of such diseases (1. Ex vivo generation of blood vessels may offer an excellent alternative to the synthetic valves that are being currently used in cardiology. Micro gravity also referred to, as weightlessness is not essentially zero gravity but rather minimal gravity. According to cell type, micro gravity causes variety of changes in proliferation and differentiation of cells while also affecting the migration of cells and cellular functions (2, 3. Siamwala et al from AUKBC have already studied the effects of microgravity on the microvascular endothelial cells from bovine lung and macrovascular endothelial cells from the bovine pulmonary artery. It was observed that the proliferation and migration of macrovascular endothelial cells were increased in microgravity (4, 5. Nitric oxide production was also studied and observed that microgravity treatment did not change nitric oxide production by microvascular endothelial cells (4OBJECTIVE: Isolation and Comparison of culture characteristics of Human microvascular endothelium cultured conventionally and in novel nanomaterial scaffold and further study the morphological and molecular characteristics of microvascular endothelial cells under normal gravity against simulated micro gravityMATERIALS AND METHODS: The human Omentum samples were obtained using surgical procedures after informed consent. The microvascular endothelial cells were isolated following the protocol described by Scott et al (6.The isolated cells were seeded in two groups; Group I

  3. Silk biomaterials functionalized with recombinant domain V of human perlecan modulate endothelial cell and platelet interactions for vascular applications.

    Science.gov (United States)

    Rnjak-Kovacina, Jelena; Tang, Fengying; Whitelock, John M; Lord, Megan S

    2016-12-01

    Modulation of endothelial cell and platelet interactions is an essential feature of vascular materials. Silk biomaterials were functionalized with recombinantly expressed domain V of human perlecan, an essential vascular proteoglycan involved in vasculogenesis, angiogenesis and wound healing, using passive adsorption or covalent cross-linking via carbodiimide chemistry. The orientation of domain V on the surface of silk biomaterials was modulated by the immobilization technique and glycosaminoglycan chains played an essential role in the proteoglycan presentation on the material surface. Covalent immobilization supported improved integrin binding site presentation to endothelial cells compared to passive adsorption in the presence of glycosaminoglycan chains, but removal of glycosaminoglycan chains resulted in reduced integrin site availability and thus cell binding. Silk biomaterials covalently functionalized with domain V supported endothelial cell adhesion, spreading and proliferation and were anti-adhesive for platelets, making them promising surfaces for the development of the next-generation vascular grafts. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.

    Science.gov (United States)

    García-Morales, Verónica; Luaces-Regueira, María; Campos-Toimil, Manuel

    2017-12-01

    3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca2+ concentration ([Ca2+]c) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca2+]c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca2+, was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca2+]c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Effects of incretin agonists on endothelial nitric oxide synthase expression and nitric oxide synthesis in human coronary artery endothelial cells exposed to TNFα and glycated albumin.

    Science.gov (United States)

    Garczorz, Wojciech; Francuz, Tomasz; Siemianowicz, Krzysztof; Kosowska, Agnieszka; Kłych, Agnieszka; Aghdam, Mohammad Reza F; Jagoda, Krystyna

    2015-02-01

    There have been a number of beneficial effects of incretin agonists on the cardiovascular system. Glycated albumin (GA) and tumor necrosis factor (TNFα) may lead to endothelial dysfunction. Due to reports of cardioprotective effects of incretin agonist, we wanted to determine if GLP-1 and exendin-4 can reverse diminished production of nitric oxide (NO) after treatment with TNFα and GA. The objective of our experiment was to study the interaction between incretin agonists and proinflammatory substances like TNFα and GA on production of NO in HCAEC. Human vascular endothelial cells from the coronary artery (HCAEC) were used. The mRNA expression and protein level of endothelial nitric oxide synthase (eNOS) and inducible (iNOS) were quantified. NO production was measured in cells using DAF-FM/DA and flow cytometry. TNFα (10 ng/mL) decreased eNOS: mRNA by 90% and protein level by 31%. TNFα also decreased NO by 33%. GA (500 μg/mL) neither affected eNOS expression nor the protein level, but inhibited nearly all formation of NO in endothelium. GLP-1 (100 nM) and exendin-4 (1 and 10nM) decreased the amount of NO compared to control. Incubation of HCAEC with TNFα and incretin agonists did not change or moderately reduce the amount of NO compared to TNFα alone. TNFα and GA decrease production of NO in HCAEC, presumably by inducing reactive oxygen species or eNOS uncoupling. Incretin agonists in tested concentrations in the presence of l-arginine were not able to reverse this effect and instead led to a further reduction in NO production. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  6. Curcumin inhibits activation induced by urban particulate material or titanium dioxide nanoparticles in primary human endothelial cells.

    Science.gov (United States)

    Montiel-Dávalos, Angélica; Silva Sánchez, Guadalupe Jazmin; Huerta-García, Elizabeth; Rueda-Romero, Cristhiam; Soca Chafre, Giovanny; Mitre-Aguilar, Irma B; Alfaro-Moreno, Ernesto; Pedraza-Chaverri, José; López-Marure, Rebeca

    2017-01-01

    Curcumin has protective effects against toxic agents and shows preventive properties for various diseases. Particulate material with an aerodynamic diameter of ≤10 μm (PM10) and titanium dioxide nanoparticles (TiO2-NPs) induce endothelial dysfunction and activation. We explored whether curcumin is able to attenuate different events related to endothelial activation. This includes adhesion, expression of adhesion molecules and oxidative stress induced by PM10 and TiO2-NPs. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 10 and 100 μM curcumin for 1 h and then exposed to PM10 at 3 μg/cm2 or TiO2-NPs at 10 μg/cm2. Cell adhesion was evaluated by co-culture with U937 human myelomonocytic cells. Adhesion molecules expression was measured by flow cytometry after 3 or 24 h of exposure. Oxidative stress was determined by 2,7-dichlorodihydrofluorescein (H2DCF) oxidation. PM10 and TiO2-NPs induced the adhesion of U937 cells and the expression of E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1). The expression of E- and P-selectins matched the adhesion of monocytes to HUVEC after 3 h. In HUVEC treated with 1 or 10 μM curcumin, the expression of adhesion molecules and monocytes adhesion was significantly diminished. Curcumin also partially reduced the H2DCF oxidation induced by PM10 and TiO2-NPs. Our results suggest an anti-inflammatory and antioxidant role by curcumin attenuating the activation caused on endothelial cells by exposure to particles. Therefore, curcumin could be useful in the treatment of diseases where an inflammatory process and endothelial activation are involved.

  7. The influence of C-ions and X-rays on human umbilical vein endothelial cells

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    Alexander eHelm

    2016-01-01

    Full Text Available Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells (HUVEC as a model system to examine the effect of low and high linear energy transfer (LET radiation. Cells were exposed to 250 kV X-rays or C-ions with the energies of either 9.8 MeV/u (LET=170 keV/µm or 91 MeV/u (LET=28 keV/µm. Subculture of cells was performed regularly up to 49 days (~22 population doublings post-irradiation. Immediately after exposure cells were seeded for the colony forming assay. Additionally, at regular intervals mitochondrial membrane potential (JC-1 staining and cellular senescence (senescence associated β-galactosidase staining were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution mFISH technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay as observed in other cell systems. For 9.8 and 91 MeV/u C-ions relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time-points the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the mitochondrial membrane potential was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time-point investigated. Altogether our data indicate that C-ions were LET-dependently more effective in damaging endothelial cells shortly after exposure. Late damage to endothelial cells, however, was not found for the applied conditions and endpoints.

  8. Desflurane preconditioning induces oscillation of NF-κB in human umbilical vein endothelial cells.

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    Juan Yi

    Full Text Available BACKGROUND: Nuclear factor kappa B (NF-κB has been implicated in anesthetic preconditioning (APC induced protection against anoxia and reoxygenation (A/R injury. The authors hypothesized that desflurane preconditioning would induce NF-κB oscillation and prevent endothelial cells apoptosis. METHODS: A human umbilical vein endothelial cells (HUVECs A/R injury model was used. A 30 minute desflurane treatment was initiated before anoxia. NF-κB inhibitor BAY11-7082 was administered in some experiments before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT assay. The cellular superoxide dismutases (SOD activitiy were tested by water-soluble tetrazolium salt (WST-1 assay. NF-κB p65 subunit nuclear translocation was detected by immunofluorescence staining. Expression of inhibitor of NF-κB-α (IκBα, NF-κB p65 and cellular inhibitor of apoptosis 1 (c-IAP1, B-cell leukemia/lymphoma 2 (Bcl-2, cysteine containing aspartate specific protease 3 (caspases-3 and second mitochondrial-derived activator of caspase (SMAC/DIABLO were determined by western blot. RESULTS: Desflurane preconditioning caused phosphorylation and nuclear translocation of NF-κB before anoxia, on the contrary, induced the synthesis of IκBα and inhibition of NF-κB after reoxygenation. Desflurane preconditioning up-regulated the expression of c-IAP1 and Bcl-2, blocked the cleavage of caspase-3 and reduced SMAC release, and decreased the cell death of HUVECs after A/R. The protective effect was abolished by BAY11-7082 administered before desflurane. CONCLUSIONS: The results demonstrated that desflurane activated NF-κB during the preconditioning period and inhibited excessive activation of NF-κB in reperfusion. And the oscillation of NF-κB induced by desflurane preconditioning finally up-regulated antiapoptotic proteins expression and

  9. Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.

    Science.gov (United States)

    Stebbins, Matthew J; Wilson, Hannah K; Canfield, Scott G; Qian, Tongcheng; Palecek, Sean P; Shusta, Eric V

    2016-05-15

    The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Lung Cancer and Human Papilloma Viruses (HPVs: Examining the Molecular Evidence

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    Priya R. Prabhu

    2012-01-01

    Full Text Available Human papilloma virus (HPV, known to be an etiological agent for genital cancers, has been suggested also to be a possible contributory agent for lung cancer. Alternatively, lung cancer, formerly considered to be solely a smoker's disease, may now be more appropriately categorised into never smoker's and smoker's lung cancer. Through this paper we attempt to bring forth the current knowledge regarding mechanisms of HPV gaining access into the lung tissue, various strategies involved in HPV-associated tumorigenesis in lung tissue.

  11. Lung Cancer and Human Papilloma Viruses (HPVs): Examining the Molecular Evidence

    Science.gov (United States)

    Prabhu, Priya R.; Jayalekshmi, D.; Pillai, M. Radhakrishna

    2012-01-01

    Human papilloma virus (HPV), known to be an etiological agent for genital cancers, has been suggested also to be a possible contributory agent for lung cancer. Alternatively, lung cancer, formerly considered to be solely a smoker's disease, may now be more appropriately categorised into never smoker's and smoker's lung cancer. Through this paper we attempt to bring forth the current knowledge regarding mechanisms of HPV gaining access into the lung tissue, various strategies involved in HPV-associated tumorigenesis in lung tissue. PMID:22363346

  12. Sterols of Pneumocystis carinii hominis organisms isolated from human lungs

    DEFF Research Database (Denmark)

    Kaneshiro, E S; Amit, Z; Chandra, Jan Suresh

    1999-01-01

    The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting...... mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C(28) and C(29) 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently...... in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human...

  13. Racial differences in the responses to shear stress in human umbilical vein endothelial cells

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    Feairheller D

    2011-07-01

    Full Text Available Deborah L Feairheller1,4, Joon-Young Park2, Victor Rizzo3, Boa Kim2, Michael D Brown1,31Hypertension, Molecular and Applied Physiology Laboratory, 2Cardiovascular Genomics Laboratory, Department of Kinesiology, 3Cardiovascular Research Center, School of Medicine, Temple University, Philadelphia, PA, USA; 4Exercise and Metabolic Disease Research Laboratory, School of Nursing, University of California Los Angeles, Los Angeles, CA, USABackground: African American ethnicity is an independent risk factor for exaggerated oxidative stress, which is related to inflammation, hypertension, and cardiovascular disease. Recently, we reported that in vitro oxidative stress and inflammation levels differ between African American and Caucasian human umbilical vein endothelial cells (HUVECs, African American HUVECs having higher levels of both. However, it remains to be shown whether the cells would respond differently to external stimuli.Methods: We used a cone and plate viscometer to apply laminar shear stress (LSS as an aerobic exercise mimetic to compare the responses by race. HUVECs were exposed to static conditions (no LSS, low LSS (5 dyne/cm2, and moderate LSS (20 dyne/cm2.Results: It was found that African American HUVECs had higher levels of oxidative stress under static conditions, and when LSS was applied protein expression levels (NADPH oxidase NOX2, NOX4 and p47phox subunits, eNOS, SOD2, and catalase and biomarkers (NO, SOD, and total antioxidant capacity were modulated to similar levels between race.Conclusion: African American HUVECs may be more responsive to LSS stimulus indicating that aerobic exercise prescriptions may be valuable for this population since the potential exists for large in vivo improvements in oxidative stress levels along the endothelial layer in response to increased shear flow.Keywords: shear stress, African American, NADPH oxidase, HUVECs, oxidative stress

  14. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    Directory of Open Access Journals (Sweden)

    Masahito Yoshihara

    Full Text Available The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  15. Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells.

    Science.gov (United States)

    Dong, Lixue; Krewson, Elizabeth A; Yang, Li V

    2017-01-27

    Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

  16. PPARγ affects nitric oxide in human umbilical vein endothelial cells exposed to Porphyromonas gingivalis.

    Science.gov (United States)

    Li, Peng; Zhang, Dakun; Wan, Meng; Liu, Jianru

    2016-08-01

    Porphyromonas gingivalis induces nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs). Peroxisome proliferator-activated receptor (PPARγ) has an anti-inflammation function, and its involvement in this NO induction process requires elucidation. Here, we focused on PPARγ expression in HUVECs exposed to P. gingivalis, and investigated its effects on NO synthesis. HUVECs were time-dependently stimulated by P. gingivalis W83 for 0-24h. PPARγ expression was assessed at the mRNA and protein levels, and PPARγ activation was measured using dual-luciferase reporter assays. NO synthesis and NO synthase (NOS) expression in response to P. gingivalis were examined in HUVECs pretreated with representative PPARγ agonist (15-deoxy-Δ12,14-prostaglandin J2 10μM) or antagonist (GW9662 10μM). In addition, NO synthesis and NOS expression in the P. gingivalis infected and control groups were detected. The PPARγ mRNA level in HUVECs increased after exposure to P. gingivalis for 1h and its protein level increased at 2h. Luciferase-induced PPARγ increased in P. gingivalis-exposed HUVECs. NO synthesis in the infected group at 4h, and in the PPARγ-activated group at 8h, was higher than that in controls. Inducible NOS increased in the infected and PPARγ-activated groups at 4 and 8h. The total endothelial NOS (eNOS) and phospho-eNOS levels were lower in the infected group than controls, but did not change in the PPARγ-activated group. Activated PPARγ induces NO generation through the NOS pathway in HUVECs exposed to P. gingivalis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    Science.gov (United States)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  18. Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime

    Directory of Open Access Journals (Sweden)

    M. Mathy-Hartert

    1995-01-01

    Full Text Available We investigated the effects of the antibiotic ceftazidime (CAZ on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−. In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC were capable of taking up active MPO. In presence of H2O2 (10−4 M, this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC. Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2 was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon. So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

  19. Bradykinin preconditioning improves therapeutic potential of human endothelial progenitor cells in infarcted myocardium.

    Directory of Open Access Journals (Sweden)

    Zulong Sheng

    Full Text Available OBJECTIVES: Stem cell preconditioning (PC is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs with bradykinin (BK enhance cell survival, inhibit apoptosis and repair the infarcted myocardium. METHODS: The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging. RESULTS: In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively. CONCLUSIONS: The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used.

  20. Metformin modulates human leukocyte/endothelial cell interactions and proinflammatory cytokines in polycystic ovary syndrome patients.

    Science.gov (United States)

    Victor, Victor M; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Lopez-Domenech, Sandra; Escribano-López, Irene; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernandez-Mijares, Antonio

    2015-09-01

    We aim to assess the effect of metformin treatment on metabolic parameters, endothelial function and inflammatory markers in polycystic ovary syndrome (PCOS) subjects. The study population consisted of 40 reproductive-age women with PCOS, who underwent treatment with metformin during a 12-week period, and their corresponding matched controls (n = 44). We evaluated endocrinological parameters, adhesion molecules (vascular cell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecule 1 (ICAM-1) and E-selectin) and proinflammatory cytokines (interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα)) in serum. In addition, interactions between human umbilical vein endothelial cells and polymorphonuclear (PMN) cells were assessed by flow chamber microscopy. In addition, a group of type 2 diabetes patients who underwent treatment with metformin during a 12-week period was incorporated into the study. Metformin produced beneficial effects on PCOS patients by decreasing polymorphonuclear (PMN) rolling flux and adhesion. It also decreased levels of ICAM-1, E-selectin, IL-6 and ΤΝFα. In addition, metformin induced an improvement of endocrine and anthropometric parameters in PCOS subjects by reducing glucose, follicle-stimulating hormone (FSH) and androstendione, and by increasing dehydroepiandrosterone-sulfate (DHEA-S). Metformin also had beneficial effects in type 2 diabetic subjects by reducing body weight, waist circumference and PMN adhesion, and by increasing PMN rolling velocity. Our results highlight the modulating effect of metformin on leukocyte/endothelium interactions. These findings may explain the potential beneficial effect of metformin in reducing the risk of vascular events in PCOS patients and in insulin resistance conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Effects of fluorides on apoptosis and activation of human umbilical vein endothelial cells.

    Science.gov (United States)

    Szczepański, M; Kamianowska, M; Kamianowski, G

    2012-04-01

    To determine the effects of fluorides on endothelial functioning. We analyzed expressions of adhesion molecules, ICAM-1 and ICAM-3, and annexin V, on the surface of human umbilical vein endothelial cells (HUVECs) exposed to various concentrations of NaF and SnF(2) . We compared the effects of fluoride-induced changes with those obtained when stimulating HUVECs with TNF-α and verified whether N-acetyl cysteine (NAC), well-known antioxidant, can prevent both fluoride- and TNF-α-induced alterations. The expressions of annexin V and ICAM-1 increased significantly after adding NaF (5.0 or 7.5mM) or Sn(2) F (0.5 or 0.75mM) to the culture medium. Pre-incubating HUVECs with NAC prevented the effects induced by 5.0 mM of NaF and 0.5 mM of Sn(2) F. Only the highest concentration of NaF (7.5mM) triggered the expression of ICAM-3. The expressions of all three molecules increased significantly upon stimulating the cultures with TNF-α (20ng ml(-1) ); these changes were not reversed by pre-incubation with NAC. Fluorides induce oxidative stress, resulting in apoptosis and activation of HUVECs, manifested by an elevated expression of ICAM-1. The oxidative stress resulting from a stimulation by the highest NaF concentration triggers ICAM-3 expression on the HUVECs' surface. © 2011 John Wiley & Sons A/S.

  2. Simvastatin versus ezetimibe: pleiotropic and lipid-lowering effects on endothelial function in humans

    National Research Council Canada - National Science Library

    Landmesser, Ulf; Bahlmann, Ferdinand; Mueller, Maja; Spiekermann, Stephan; Kirchhoff, Nina; Schulz, Svenja; Manes, Costantina; Fischer, Dieter; de Groot, Kirsten; Fliser, Danilo; Fauler, Günter; März, Winfried; Drexler, Helmut

    2005-01-01

    .... We therefore tested the hypothesis that similar reductions in LDL cholesterol with simvastatin and ezetimibe, a novel cholesterol absorption inhibitor, result in different effects on endothelial function...

  3. Gene expression changes after ionizing radiation in endothelial cells derived from human endometrial cancer-preliminary outcomes.

    Science.gov (United States)

    Liu, Ting; Du, Xuelian; Sheng, Xiugui

    2014-06-01

    Accumulating evidence has demonstrated that death of microvascular endothelial cells plays a decisive role in the tumor response against radiotherapy. Nevertheless, radiation-induced gene alterations on cancer-associated endothelial cells of human endometrial carcinoma remain poorly understood. The purpose of this study was to elucidate the gene expression changes after X-ray radiation in human endometrial carcinoma vascular endothelial cells and to provide new targets for combined treatment of radiation and anti-angiogenesis in human endometrial carcinoma. Endometrial cancer-derived endothelial cells, which obtained before and 4 h after 400 cGy X-ray radiation from four endometrial carcinomas, were analyzed by gene expression profile. The selected meaningful genes from gene microarray experiments were validated by real-time quantitative PCR. Microarray analyses showed 49 significantly changed genes which were common to all the microarray experiments. There into, 14 genes were found to be in persistent up-regulation and 14 in persistent down-regulation 4 h after X-ray radiation when compared with the control group. These genes were involved in cell cycle and growth regulation, cell-apoptosis, chemokine, cell signaling, cellular stress response, angiogenesis, DNA synthesis and repair and cell adhesion. Eight randomly selected genes were validated by real-time PCR. The genes of cancer-derived endothelial cells regulated by X-ray radiation as well as their related signal pathways, which obtained from gene expression profiling data, were relevant to radiosensitivity of endometrial cancer. This study shows that the identified genes and their related signaling pathways are candidated biomarkers for radiation and anti-angiogenesis of human endometrial carcinoma.

  4. Prevention of Osmotic Injury to Human Umbilical Vein Endothelial Cells for Biopreservation: A First Step Toward Biobanking of Endothelial Cells for Vascular Tissue Engineering.

    Science.gov (United States)

    Niu, Dan; Zhao, Gang; Liu, Xiaoli; Zhou, Ping; Cao, Yunxia

    2016-03-01

    High-survival-rate cryopreservation of endothelial cells plays a critical role in vascular tissue engineering, while optimization of osmotic injuries is the first step toward successful cryopreservation. We designed a low-cost, easy-to-use, microfluidics-based microperfusion chamber to investigate the osmotic responses of human umbilical vein endothelial cells (HUVECs) at different temperatures, and then optimized the protocols for using cryoprotective agents (CPAs) to minimize osmotic injuries and improve processes before freezing and after thawing. The fundamental cryobiological parameters were measured using the microperfusion chamber, and then, the optimized protocols using these parameters were confirmed by survival evaluation and cell proliferation experiments. It was revealed for the first time that HUVECs have an unusually small permeability coefficient for Me2SO. Even at the concentrations well established for slow freezing of cells (1.5 M), one-step removal of CPAs for HUVECs might result in inevitable osmotic injuries, indicating that multiple-step removal is essential. Further experiments revealed that multistep removal of 1.5 M Me2SO at 25°C was the best protocol investigated, in good agreement with theory. These results should prove invaluable for optimization of cryopreservation protocols of HUVECs.

  5. Endothelial shear stress: a critical determinant of arterial remodeling and arterial stiffness in humans--a carotid 3.0-T MRI study.

    NARCIS (Netherlands)

    Duivenvoorden, R.; Bavel, E. van; Groot, E. de; Stroes, E.S.; Disselhorst, J.A.; Hutten, B.A.; Lameris, J.S.; Kastelein, J.J.; Nederveen, A.J.

    2010-01-01

    BACKGROUND: Low endothelial shear stress (ESS) elicits endothelial dysfunction. However, the relationship between ESS and arterial remodeling and arterial stiffness is unknown in humans. We developed a 3.0-T MRI protocol to evaluate the contribution of ESS to arterial remodeling and stiffness.

  6. Differential and specific labeling of epithelial and vascular endothelial cells of the rat lung by Lycopersicon esculentum and Griffonia simplicifolia I lectins.

    Science.gov (United States)

    Bankston, P W; Porter, G A; Milici, A J; Palade, G E

    1991-04-01

    In the rat lung, we found that the Lycopersicon esculentum (LEA) lectin specifically binds to the epithelium of bronchioles and alveoli whereas Griffonia simplicifolia I (GS-I) binds to the endothelium of alveolar capillaries. The differential binding affinity of these lectins was examined on semithin (approximately 0.5 microns) and thin (less than 0.1 (microns) frozen sections of rat lung lavaged to remove alveolar macrophages. On semithin frozen sections, LEA bound to epithelial cells lining bronchioles and the alveoli (type I, but not type II epithelial cells). On thin frozen sections, biotinylated Lycopersicon esculentum (bLEA)-streptavidin-gold conjugates were confined primarily to the luminal plasmalemma of type I cells. bGS-I-streptavidin-Texas Red was detected on the endothelial cells of alveolar capillaries and postcapillary venules but not on those of larger venules, veins or arterioles. By electron microscopy, GS-I-streptavidin-gold complexes were localized primarily to the luminal plasmalemma of thick and thin regions of the capillary endothelium. Neither lectin labeled type II alveolar cells, but both lectins labeled macrophages in the interstitia and in incompletely lavaged alveoli.

  7. Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells.

    Science.gov (United States)

    Ugusman, Azizah; Zakaria, Zaiton; Hui, Chua Kien; Nordin, Nor Anita Megat Mohd

    2010-07-01

    Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). HUVECS WERE DIVIDED INTO FOUR GROUPS: control, treatment with 180 microM hydrogen peroxide (H(2)O(2)), treatment with 150 microg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H(2)O(2) for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

  8. Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Azizah Ugusman

    2010-01-01

    Full Text Available OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs. METHODS: HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H2O2, treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

  9. Catechins inhibit vascular endothelial growth factor production and cyclooxygenase-2 expression in human dental pulp cells.

    Science.gov (United States)

    Nakanishi, T; Mukai, K; Hosokawa, Y; Takegawa, D; Matsuo, T

    2015-03-01

    To investigate the effect of catechins on vascular endothelial growth factor (VEGF) production and cyclooxygenase-2 (COX-2) expression in human dental pulp cells (HDPC) stimulated with bacteria-derived factors or pro-inflammatory cytokines. Morphologically fibroblastic cells established from explant cultures of healthy human dental pulp tissues were used as HDPC. HDPC pre-treated with catechins, epigallocatechin-3-gallate (EGCG) or epicatechin gallate (ECG), were exposed to lipopolysaccharide (LPS), peptidoglycan (PG), interlukin-1β (IL-1β) or tumour necrosis factor-α (TNF-α). VEGF production was examined by enzyme-linked immunosorbent assay, and COX-2 expression was assessed by immunoblot. EGCG and ECG significantly reduced LPS- or PG-mediated VEGF production in the HDPC in a dose-dependent manner. EGCG also prevented IL-1β-mediated VEGF production. Although TNF-α did not enhance VEGF production in the dental pulp cells, treatment of 20 μg mL(-1) of EGCG decreased the level of VEGF. In addition, the catechins attenuated COX-2 expression induced by LPS and IL-1β. The up-regulated VEGF and COX-2 expressions in the HDPC stimulated with these bacteria-derived factors or IL-1β were diminished by the treatment of EGCG and ECG. These findings suggest that the catechins may be beneficial as an anti-inflammatory tool of the treatment for pulpal inflammation. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  10. Epigenetic modulations in early endothelial cells and DNA hypermethylation in human skin after sulfur mustard exposure.

    Science.gov (United States)

    Steinritz, Dirk; Schmidt, Annette; Balszuweit, Frank; Thiermann, Horst; Simons, Thilo; Striepling, Enno; Bölck, Birgit; Bloch, Wilhelm

    2016-02-26

    Victims that were exposed to the chemical warfare agent sulfur mustard (SM) suffer from chronic dermal and ocular lesions, severe pulmonary problems and cancer development. It has been proposed that epigenetic perturbations might be involved in that process but this has not been investigated so far. In this study, we investigated epigenetic modulations in vitro using early endothelial cells (EEC) that were exposed to different SM concentrations (0.5, 1.0, 23.5 and 50μM). A comprehensive analysis of 78 genes related to epigenetic pathways (i.e., DNA-methylation and post-translational histone modifications) was performed. Moreover, we analyzed global DNA methylation in vitro in EEC after SM exposure as a maker for epigenetic modulations and in vivo using human skin samples that were obtained from a patient 1 year after an accidently exposure to pure SM. SM exposure resulted in a complex regulation pattern of epigenetic modulators which was accompanied by a global increase of DNA methylation in vitro. Examination of the SM exposed human skin samples also revealed a significant increase of global DNA methylation in vivo, underlining the biological relevance of our findings. Thus, we demonstrated for the first time that SM affects epigenetic pathways and causes epigenetic modulations both in vivo and in vitro. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2010-01-01

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

  12. Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S.; Shachter, Neil S.

    2003-12-01

    We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.

  13. Upregulation of P53 promoted G1 arrest and apoptosis in human umbilical cord vein endothelial cells from preeclampsia.

    Science.gov (United States)

    Gao, Qinqin; Zhu, Xiaolin; Chen, Jie; Mao, Caiping; Zhang, Lubo; Xu, Zhice

    2016-07-01

    Preeclampsia is a leading cause of maternal and perinatal morbidity and mortality. Current research has focused on endothelial dysfunction regarding pathogenesis of preeclampsia. However, very limited or no studies so far have been performed to assess possible damaged endothelial cell growth/development in the placenta-umbilical cord circulation system in human preeclampsia. We isolated and cultured human umbilical cord vein endothelial cells (HUVECs) from normal and preeclampsia pregnancies in vitro. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to measure cell growth and flow cytometric analysis to determine cell-cycle distribution. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was employed for cell apoptosis experiments. The study showed that the cell growth was significantly suppressed, accompanied by the increased G1 arrest and apoptosis in cultured HUVECs from preeclampsia pregnancies comparing with normotensive controls. Protein P53 was upregulated in the cultured HUVECs from preeclampsia pregnancies, which induced G1 arrest, followed by upregulating P21 expression, and downregulating cyclin E expression and CDK2-cyclin E complexes. On the other hand, upregulation of P53 also activated Bax gene and repressed Bcl-2 and BIRC5 genes, resulting in an increase of the Bax/Bcl-2 ratio and subsequently activating caspase cascade, ultimately led to an initiation of the apoptotic machinery. These results indicated that in preeclampsia, vascular endothelial cells could be damaged and cellular proliferation was depressed in human placenta-umbilical cord circulation, adding new information on endothelial cell injury for better understanding the pathogenesis of preeclampsia.

  14. Identification of novel vascular targets in lung cancer

    Science.gov (United States)

    Zhuang, X; Herbert, J M J; Lodhia, P; Bradford, J; Turner, A M; Newby, P M; Thickett, D; Naidu, U; Blakey, D; Barry, S; Cross, D A E; Bicknell, R

    2015-01-01

    Background: Lung cancer remains the leading cause of cancer-related death, largely owing to the lack of effective treatments. A tumour vascular targeting strategy presents an attractive alternative; however, the molecular signature of the vasculature in lung cancer is poorly explored. This work aimed to identify novel tumour vascular targets in lung cancer. Methods: Enzymatic digestion of fresh tissue followed by endothelial capture with Ulex lectin-coated magnetic beads was used to isolate the endothelium from fresh tumour specimens of lung cancer patients. Endothelial isolates from the healthy and tumour lung tissue were subjected to whole human genome expression profiling using microarray technology. Results: Bioinformatics analysis identified tumour endothelial expression of angiogenic factors, matrix metalloproteases and cell-surface transmembrane proteins. Predicted novel tumour vascular targets were verified by RNA-seq, quantitative real-time PCR analysis and immunohistochemistry. Further detailed expression profiling of STEAP1 on 82 lung cancer patients confirmed STEAP1 as a novel target in the tumour vasculature. Functional analysis of STEAP1 using siRNA silencing implicates a role in endothelial cell migration and tube formation. Conclusions: The identification of cell-surface tumour endothelial markers in lung is of interest in therapeutic antibody and vaccine development. PMID:25535734

  15. Engineering an endothelialized vascular graft: a rational approach to study design in a non-human primate model.

    Directory of Open Access Journals (Sweden)

    Deirdre E J Anderson

    Full Text Available After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data-correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.

  16. Time lapse phase contrast video microscopy of directed migration of human microvascular endothelial cells on matrigel

    NARCIS (Netherlands)

    Meade-Tollin, L. C.; van Noorden, C. J.

    2000-01-01

    Migration of microvascular endothelial cells is an early and critical step in angiogenesis. Formation of branching and polygonal cellular aggregates by endothelial cells on matrigel has often been considered to be an in vitro model for angiogenesis, although formation of lumens has not always been

  17. CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

    NARCIS (Netherlands)

    Siemerink, Martin J.; Klaassen, Ingeborg; Vogels, Ilse M. C.; Griffioen, Arjan W.; van Noorden, Cornelis J. F.; Schlingemann, Reinier O.

    2012-01-01

    The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend

  18. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    Science.gov (United States)

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  19. Nuclear IL-33 regulates soluble ST2 receptor and IL-6 expression in primary human arterial endothelial cells and is decreased in idiopathic pulmonary arterial hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Dongmin [Section of Vascular Biology, National Heart and Lung Institute, Imperial College London, London (United Kingdom); Perros, Frédéric [Faculté de Médecine, Université Paris-Sud, Paris, Clamart (France); Caramori, Gaetano [Dipartimento di Scienze Mediche, Sezione di Medicina Interna e Cardiorespiratoria, Centro Interdipartimentale per lo Studio delle Malattie Infiammatorie delle Vie Aeree e Patologie Fumo-Correlate, University of Ferrara, Ferrara (Italy); Meng, Chao [Section of Vascular Biology, National Heart and Lung Institute, Imperial College London, London (United Kingdom); Department of Geriatrics, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Dormuller, Peter [Faculté de Médecine, Université Paris-Sud, Paris, Clamart (France); Chou, Pai-Chien [Airways Disease, National Heart and Lung Institute (United Kingdom); Church, Colin [Scottish Pulmonary Vascular Unit, University of Glasgow (United Kingdom); Papi, Alberto; Casolari, Paolo [Dipartimento di Scienze Mediche, Sezione di Medicina Interna e Cardiorespiratoria, Centro Interdipartimentale per lo Studio delle Malattie Infiammatorie delle Vie Aeree e Patologie Fumo-Correlate, University of Ferrara, Ferrara (Italy); Welsh, David; Peacock, Andrew [Scottish Pulmonary Vascular Unit, University of Glasgow (United Kingdom); Humbert, Marc [Faculté de Médecine, Université Paris-Sud, Paris, Clamart (France); Adcock, Ian M. [Airways Disease, National Heart and Lung Institute (United Kingdom); Wort, Stephen J., E-mail: s.wort@imperial.ac.uk [Section of Vascular Biology, National Heart and Lung Institute, Imperial College London, London (United Kingdom)

    2014-08-15

    Highlights: • Nuclear IL-33 expression is reduced in vascular endothelial cells from PAH patients. • Knockdown of IL-33 leads to increased IL-6 and sST2 mRNA expression. • IL-33 binds homeobox motifs in target gene promoters and recruits repressor proteins. - Abstract: Idiopathic pulmonary arterial hypertension (IPAH) is an incurable condition leading to right ventricular failure and death and inflammation is postulated to be associated with vascular remodelling. Interleukin (IL)-33, a member of the “alarmin” family can either act on the membrane ST2 receptor or as a nuclear repressor, to regulate inflammation. We show, using immunohistochemistry, that IL-33 expression is nuclear in the vessels of healthy subjects whereas nuclear IL-33 is markedly diminished in the vessels of IPAH patients. This correlates with reduced IL-33 mRNA expression in their lung. In contrast, serum levels of IL-33 are unchanged in IPAH. However, the expression of the soluble form of ST2, sST2, is enhanced in the serum of IPAH patients. Knock-down of IL-33 in human endothelial cells (ECs) using siRNA is associated with selective modulation of inflammatory genes involved in vascular remodelling including IL-6. Additionally, IL-33 knock-down significantly increased sST2 release from ECs. Chromatin immunoprecipitation demonstrated that IL-33 bound multiple putative homeodomain protein binding motifs in the proximal and distal promoters of ST2 genes. IL-33 formed a complex with the histone methyltransferase SUV39H1, a transcriptional repressor. In conclusion, IL-33 regulates the expression of IL-6 and sST2, an endogenous IL-33 inhibitor, in primary human ECs and may play an important role in the pathogenesis of PAH through recruitment of transcriptional repressor proteins.

  20. Dimethylfumarate protects against TNF-α-induced secretion of inflammatory cytokines in human endothelial cells.

    Science.gov (United States)

    Gerhardt, Simon; König, Veronika; Doll, Monika; Hailemariam-Jahn, Tsige; Hrgovic, Igor; Zöller, Nadja; Kaufmann, Roland; Kippenberger, Stefan; Meissner, Markus

    2015-01-01

    Inflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression. We analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/- DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6. DMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42

  1. Analysis of biological effects in human endothelial cells after stimulated microgravity

    Science.gov (United States)

    Min, Zhang; Sun, Yeqing; Xu, Dan

    Space environment is characterized by strong radiation, ultra-high vacuum, weak magnetic field and microgravity. Among them, microgravity (10-4-10-6g) in space is different from gravity (1g) on earth, possibly causing visual disorders, muscle alterations, bone loss and dysfunction of cardiovascular systems. To study about microgravity environment, the most advanced rotary cell culture system (RCCS-1) was used to do stimulated microgravity (SMG) experiments in the ground. Up to now, most of studies focus on the biological effects under stimulated microgravity, but it is less known about the cellular response after stimulated microgravity. In the present study, we explored the subsequent effects of stimulated microgravity on human endothelial cells (HUVEC-C) after these cells were cultured on RCCS-1 for 48 hours. We co-cultured HUVEC-C cells with HillexⅡmicrocarriers in 60-mm culture dishes for 24h, followed by transferring them to RCCS-1 so that cells remain to be the state of SMG. In parallel, HUVEC-C cells were co-cultured with microcarriers in the ground condition. We found that stimulated microgravity induced cytoskeleton remodeling, cell cycle G2/M arrest and cellular senescence, consistent with previous reports. To study the subsequent effects of stimulated microgravity, we make cells detach from microcarriers and observed various effects including cell growth, cell adhesion, cytoskeleton, cell cycle, apoptosis and senescence. The results showed that those cells undergoing stimulated microgravity appeared obvious growth inhibition, a transition from the decrease in cell adhesion ability and cytoskeleton remodeling within 24h to induction of apoptosis and senescence-like phenotype in the later time with slight changes in cell cycle. Analysis of protein expression in western blot demonstrated that apoptosis-related protein PTEN was up-regulated on the time-dependent pattern after stimulated microgravity, indicating that PTEN-PI3K-Akt pathway might play an

  2. Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

    DEFF Research Database (Denmark)

    Nissen, Dan; Petersen, Lars Jelstrup; Nolte, H

    1998-01-01

    OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted...... responses were observed but data could be reproduced within individual donors. Monocyte chemoattractant protein-1, a potent basophil secretagogue, did not induce histamine release in lung tissue which indicated mast cells to be the histamine source. Substance P did not release histamine in the lung tissue....... CONCLUSIONS: The microdialysis technique allowed measurements of histamine release from mast cells in intact lung ex vivo. The method may prove useful since a number of experiments can be performed in a few hours in intact lung tissue without any dispersion or enzymatic treatment....

  3. Evoked corticospinal output to the human scalene muscles is altered by lung volume.

    Science.gov (United States)

    Hudson, Anna L; Taylor, Janet L; Anand, Ashima; Gandevia, Simon C; Butler, Jane E

    2012-03-15

    Increases in lung volume inhibit the inspiratory output from the medulla, but the effect of lung inflation on the voluntary control of breathing in humans is not known. We tested corticospinal excitability using transcranial magnetic stimulation (TMS) to evoke a response in the scalene muscles. TMS was delivered at rest at three different lung volumes between functional residual capacity (FRC) and total lung capacity (TLC) during incremental inspiratory and incremental expiratory manoeuvres. Motor evoked potentials (MEPs) in scalenes were ∼50% larger at a high lung volume (FRC+∼90% inspiratory capacity [IC]) compared to lower lung volumes (FRC and FRC+∼40% IC) in both inspiratory and expiratory manoeuvres (plung inflation on the automatic and voluntary control of breathing in humans. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells.

    Science.gov (United States)

    Li, Huige; Xia, Ning; Brausch, Isolde; Yao, Ying; Förstermann, Ulrich

    2004-09-01

    Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.

  5. Proteomic profiling of human retinal and choroidal endothelial cells reveals molecular heterogeneity related to tissue of origin.

    Science.gov (United States)

    Zamora, David O; Riviere, Michael; Choi, Dongseok; Pan, Yuzhen; Planck, Stephen R; Rosenbaum, James T; David, Larry L; Smith, Justine R

    2007-10-30

    The ocular vascular endothelium plays a key role in the development of several leading retinal causes of blindness in Western nations. Choroidal endothelial cells are integral to the subretinal neovascular lesions that characterize the exudative form of late age-related macular degeneration (AMD), and retinal endothelial cells participate in the initiation of diabetic retinopathy and posterior uveitis. Vascular endothelial cells at different sites exhibit considerable molecular diversity. This diversity has implications for understanding the pathogenesis of tissue-specific diseases and for the development of targeted therapies to treat these conditions. Previous work from our group has identified significant differences in the gene transcript profiles of human retinal and choroidal endothelial cells. Because the proteome ultimately determines the behavior of any given cell, however, it is critical to determine whether molecular differences exist at the level of protein expression. Retinal and choroidal endothelial cells were separately isolated from five sets of human eyes by enzymatic digestion with type II collagenase followed by anti-CD31 antibody-conjugated magnetic bead separation. Cells were washed to remove serum peptides in the culture medium, and lysed by sonication in buffer containing 2% sodium dodecyl sulfate. Protein was then precipitated with acetone. Retinal and choroidal endothelial samples from each donor were labeled with Cy3 and Cy5, respectively, mixed with a Cy2-labeled pooled protein sample to facilitate spot matching across gels, and separated by two-dimensional difference gel electrophoresis (2D-DIGE). Following a global normalization, differentially abundant protein spots that were visible in at least four of five donor gels were detected by the significance analysis of microarrays method, with false discovery rate set at 5%. Corresponding spots were excised from additional DIGE-labeled or Coomassie-stained 2D electrophoretic gels. Protein

  6. Lysophosphatidic acid enhances vascular endothelial growth factor-C expression in human prostate cancer PC-3 cells.

    Directory of Open Access Journals (Sweden)

    Chuan-En Lin

    Full Text Available Clinical evidence suggests that lymphangiogenesis and lymphatic metastasis are important processes during the progression of prostate cancer. Vascular endothelial growth factor (VEGF-C was shown to be a key regulator in these processes. Our previous studies demonstrated that lysophosphatidic acid (LPA, a low-molecular-weight lipid growth factor, enhances VEGF-C expression in human endothelial cells. We previously demonstrated that the LPA receptor plays an important role in lymphatic development in zebrafish embryos. However, the effects of LPA on VEGF-C expression in prostate cancer are not known. Herein, we demonstrate that LPA up-regulated VEGF-C expression in three different human prostate cancer cell lines. In PC-3 human prostate cancer cells, the enhancing effects of LPA were mediated through both LPA1 and LPA3. In addition, reactive oxygen species (ROS production and lens epithelium-derived growth factor (LEDGF expression were involved in LPA(1/3-dependent VEGF-C expression. Furthermore, autotaxin (ATX, an enzyme responsible for LPA synthesis, also participates in regulating VEGF-C expression. By interrupting LPA(1/3 of PC-3, conditioned medium (CM -induced human umbilical vein endothelial cell (HUVEC lymphatic markers expression was also blocked. In summary, we found that LPA enhances VEGF-C expression through activating LPA(1/3-, ROS-, and LEDGF-dependent pathways. These novel findings could potentially shed light on developing new strategies for preventing lymphatic metastasis of prostate cancer.

  7. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells.

    Science.gov (United States)

    Giacomelli, Elisa; Bellin, Milena; Sala, Luca; van Meer, Berend J; Tertoolen, Leon G J; Orlova, Valeria V; Mummery, Christine L

    2017-03-15

    Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs. © 2017. Published by The Company of Biologists Ltd.

  8. RNA-seq reveals novel transcriptome of genes and their isoforms in human pulmonary microvascular endothelial cells treated with thrombin.

    Directory of Open Access Journals (Sweden)

    Li Qin Zhang

    Full Text Available The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.

  9. RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin

    Science.gov (United States)

    Zhang, Li Qin; Cheranova, Dilyara; Gibson, Margaret; Ding, Shinghua; Heruth, Daniel P.; Fang, Deyu; Ye, Shui Qing

    2012-01-01

    The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91–94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets. PMID:22359579

  10. Protective Effect of Allium tuberosum Extract on Vascular Inflammation in Tumor Necrosis Factor-α-induced Human Vascular Endothelial Cells.

    Science.gov (United States)

    Hur, Haeng Jeon; Lee, Ae Sin

    2017-12-01

    Endothelial adhesion molecule expression induced by pro-inflammatory cytokine plays an important role in vascular endothelial cell injury, leading to vascular disease. Allium tuberosum (AT), which is used as a functional food, has a thrombolytic effect. It contains vitamin A, vitamin C, carbohydrate, calcium, iron, and phosphorus. There are many carotenes that turn into vitamin A in the body. Also, it helps blood circulation and stimulates metabolism. The purpose of the this study was to estimate the anti-inflammatory effects of the AT extract. Human vascular endothelial cells were pre-treated with 100 μg/mL AT extract for 30 minutes and subsequently co-treated with TNF-α (10 ng/mL) and AT extract (100 μg/mL) for 1, 4, and 6 hours. After treatment, the cells were lysed and used for quantitative reverse transcription PCR, Western blot analysis, and monocyte adhesion assay. We examined the effect of the AT extract on inflammatory gene expression in TNF-α-induced human umbilical vein endothelial cells (HUVECs). The extract reduced the expression levels of mRNA and protein of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in TNF-α-stimulated HUVECs. It also inhibited the TNF-α-induced phosphorylation of the NF-κB p65 subunit and degradation of IκBα. Furthermore, the AT extract prevented the increased adhesion capacity of monocyte to TNF-α-stimulated vascular endothelial cells by reducing ICAM-1 and VCAM-1 expression. The AT extract has preventive and anti-inflammatory effect against vascular disease and has potential for supporting prevention against the early process of atherosclerosis.

  11. Co-culture model consisting of human brain microvascular endothelial and peripheral blood mononuclear cells

    Science.gov (United States)

    Strazza, Marianne; Maubert, Monique E.; Pirrone, Vanessa; Wigdahl, Brian; Nonnemacher, Michael R.

    2016-01-01

    Background Numerous systems exist to model the blood-brain barrier (BBB) with the goal of understanding the regulation of passage into the central nervous system (CNS) and the potential impact of selected insults on BBB function. These models typically focus on the intrinsic cellular properties of the BBB, yet studies of peripheral cell migration are often excluded due to technical restraints. New Method This method allows for the study of in vitro cellular transmigration following exposure to any treatment of interest through optimization of co-culture conditions for the human brain microvascular endothelial cells (BMEC) cell line, hCMEC/D3, and primary human peripheral blood mononuclear cells (PBMCs). Results hCMEC/D3 cells form functionally confluent monolayers on collagen coated polytetrafluoroethylene (PTFE) transwell inserts, as assessed by microscopy and tracer molecule (FITC-dextran (FITC-D)) exclusion. Two components of complete hCMEC/D3 media, EBM-2 base-media and hydrocortisone (HC), were determined to be cytotoxic to PBMCs. By combining the remaining components of complete hCMEC/D3 media with complete PBMC media a resulting co-culture media was established for use in hCMEC/D3 – PBMC co-culture functional assays. Comparison with existing methods Through this method, issues of extensive differences in culture media conditions are resolved allowing for treatments and functional assays to be conducted on the two cell populations co-cultured simultaneously. Conclusion Described here is an in vitro co-culture model of the BBB, consisting of the hCMEC/D3 cell line and primary human PBMCs. The co-culture media will now allow for the study of exposure to potential insults to BBB function over prolonged time courses. PMID:27216631

  12. Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells.

    Science.gov (United States)

    Huang, Lan; Critser, Paul J; Grimes, Brenda R; Yoder, Mervin C

    2011-07-01

    A hierarchy of endothelial colony-forming cells (ECFC) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. ECFC has recently become an attractive target for new vascular regenerative therapies; however, in vitro expansion of ECFC typically depends on the presence of fetal bovine serum (FBS) or fetal calf serum (FCS) in the culture medium, which is not appropriate for its therapeutic application. To identify optimal conditions for in vitro expansion of ECFC, the effects of human endothelial serum-free medium (SFM) supplemented with six pro-angiogenic cytokines and human umbilical cord blood plasma (HCP) were investigated. The in vitro morphology, proliferation, surface antigen expression and in vivo vessel-forming ability were utilized for examining the effects of medium on ECFC. This novel formulation of endothelial cell culture medium allows us, for the first time, to isolate and expand human ECFC efficiently in vitro with a low concentration of HCP (1.5%) and without bovine serum additives. In this serum-reduced medium (SRM), human ECFC colony yields remained quantitatively similar to those cultured in a high concentration (10%) of bovine serum-supplemented medium. SRM-cultured ECFC displayed a robust clonal proliferative ability in vitro and human vessel-forming capacity in vivo. The present study provides a novel method for the expansion of human ECFC in vitro and will help to advance approaches for using the cells in human therapeutic trials.

  13. Evidence against a role for jaagsiekte sheep retrovirus in human lung cancer.

    Science.gov (United States)

    Miller, A Dusty; De Las Heras, Marcelo; Yu, Jingyou; Zhang, Fushun; Liu, Shan-Lu; Vaughan, Andrew E; Vaughan, Thomas L; Rosadio, Raul; Rocca, Stefano; Palmieri, Giuseppe; Goedert, James J; Fujimoto, Junya; Wistuba, Ignacio I

    2017-01-20

    Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats that can be transmitted by aerosols produced by infected animals. Virus entry into cells is initiated by binding of the viral envelope (Env) protein to a specific cell-surface receptor, Hyal2. Unlike almost all other retroviruses, the JSRV Env protein is also a potent oncoprotein and is responsible for lung cancer in animals. Of concern, Hyal2 is a functional receptor for JSRV in humans. We show here that JSRV is fully capable of infecting human cells, as measured by its reverse transcription and persistence in the DNA of cultured human cells. Several studies have indicated a role for JSRV in human lung cancer while other studies dispute these results. To further investigate the role of JSRV in human lung cancer, we used highly-specific mouse monoclonal antibodies and a rabbit polyclonal antiserum against JSRV Env to test for JSRV expression in human lung cancer. JSRV Env expression was undetectable in lung cancers from 128 human subjects, including 73 cases of bronchioalveolar carcinoma (BAC; currently reclassified as lung invasive adenocarcinoma with a predominant lepidic component), a lung cancer with histology similar to that found in JSRV-infected sheep. The BAC samples included 8 JSRV DNA-positive samples from subjects residing in Sardinia, Italy, where sheep farming is prevalent and JSRV is present. We also tested for neutralizing antibodies in sera from 138 Peruvians living in an area where sheep farming is prevalent and JSRV is present, 24 of whom were directly exposed to sheep, and found none. We conclude that while JSRV can infect human cells, JSRV plays little if any role in human lung cancer.

  14. Preeclampsia serum upregulates CD40/CD40L expression and induces apoptosis in human umbilical cord endothelial cells.

    Science.gov (United States)

    Wu, Chun-feng; Huang, Fu-dan; Sui, Ren-fang; Sun, Jing-xia

    2012-04-18

    The endothelial cell dysfunction observed in preeclampsia (PE) may be induced by CD40/CD40L signaling. This study investigated the role of CD40/CD40L in the pathogenesis of PE by comparing the effect of maternal serum obtained from healthy pregnant women and PE patients on HUVEC cell growth, apoptosis and CD40/CD40L expression. Maternal serum was obtained from 20 patients with PE (PE group) as well as 20 healthy pregnant women (control group). The human umbilical endothelial cell line, CRL1730, was cultured in the presence of maternal serum for 24, 48, and 72 h after which cell growth and apoptosis were assessed by MTT and flow cytometry analysis, respectively. CD40/CD40L expression was determined using flow cytometry and RT-PCR analyses. As compared to CRL1730 cells treated with control sera, those treated with PE sera had altered morphology, decreased cell growth, increased apoptosis and greater CD40/CD40L protein and mRNA expression. Stimulation of CD40/CD40L protein and mRNA expression by PE sera was greatest at 24 h. PE sera may induce endothelial cell damage possibly through increased CD40/CD40L expression in early-onset PE. Further studies are necessary to determine the factor(s) in PE sera responsible for the observed changes in endothelial cell viability.

  15. Preeclampsia serum upregulates CD40/CD40L expression and induces apoptosis in human umbilical cord endothelial cells

    Directory of Open Access Journals (Sweden)

    Wu Chun-feng

    2012-04-01

    Full Text Available Abstract Background The endothelial cell dysfunction observed in preeclampsia (PE may be induced by CD40/CD40L signaling. This study investigated the role of CD40/CD40L in the pathogenesis of PE by comparing the effect of maternal serum obtained from healthy pregnant women and PE patients on HUVEC cell growth, apoptosis and CD40/CD40L expression. Methods Maternal serum was obtained from 20 patients with PE (PE group as well as 20 healthy pregnant women (control group. The human umbilical endothelial cell line, CRL1730, was cultured in the presence of maternal serum for 24, 48, and 72 h after which cell growth and apoptosis were assessed by MTT and flow cytometry analysis, respectively. CD40/CD40L expression was determined using flow cytometry and RT-PCR analyses. Results As compared to CRL1730 cells treated with control sera, those treated with PE sera had altered morphology, decreased cell growth, increased apoptosis and greater CD40/CD40L protein and mRNA expression. Stimulation of CD40/CD40L protein and mRNA expression by PE sera was greatest at 24 h. Conclusions PE sera may induce endothelial cell damage possibly through increased CD40/CD40L expression in early-onset PE. Further studies are necessary to determine the factor(s in PE sera responsible for the observed changes in endothelial cell viability.

  16. Salidroside protects against homocysteine-induced injury in human umbilical vein endothelial cells via the regulation of endoplasmic reticulum stress.

    Science.gov (United States)

    Zhu, Lin; Jia, Fang; Wei, Jiang; Yu, Yang; Yu, Tianhong; Wang, Yanjun; Sun, Jianhui; Luo, Guanghua

    2017-02-01

    Previous studies showed that homocysteine (Hcy) could injure vascular endothelial cells via several mechanisms, including its promotion of oxidative stress pathway and endoplasmic reticulum stress (ER stress) pathway. Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. Emerging evidence conformed that SAL attenuated Hcy-induced endothelial dysfunction by reducing oxidative stress. However, its role in ER stress pathway remains unclarified. The purpose of this study was to explore the mechanism of the protective effect of SAL on Hcy-induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cell damage effects brought by Hcy in a dose-dependent manner. Functional studies on the HUVECs found that SAL rescued the endoplasmic reticulum stress induced by Hcy. The underlying mechanisms involve the inhibition of Hcy-induced activation of binding protein (Bip) and C/EBP homologous protein (CHOP), as well as the phosphorylation of protein kinase RNA-like ER kinase (PERK) or inositol-requiring enzyme 1 alpha (IRE1α). Taken together, these findings implicate that SAL could regulate ER stress pathway on the viability of endotheliocyte induced by Hcy in vitro. Our findings provide the first evidence that SAL plays an important role in endotheliocyte protection via suppressing ER stress pathway in HUVEC cells and that it may be a promising therapeutic target for atherosclerosis and cardiovascular disease. © 2016 John Wiley & Sons Ltd.

  17. miR-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting VEGF Signaling

    Science.gov (United States)

    Hassel, David; Cheng, Paul; White, Mark P.; Ivey, Kathryn N.; Kroll, Jens; Augustin, Hellmut G.; Katus, Hugo A.; Stainier, Didier Y.R.; Srivastava, Deepak

    2012-01-01

    Rationale Formation and remodeling of the vasculature during development and disease involves a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their post-transcriptional dose-titration by miRNAs is poorly understood. Objective To identify miRNAs that regulate angiogenesis. Methods and Results We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating pro-angiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth (ISV) in the trunk of zebrafish larvae, while overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells (HUVECs). We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters VEGF, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in HUVECs inhibited the angiogenic behavior of endothelial cells largely by antagonizing VEGF receptor-2 signaling. Conclusion Our study provides insights into how FLT1 and VEGF receptor-2 signaling is titrated in a miRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis. PMID:22955733

  18. Acute effects of hyperinsulinemia and hyperglycemia on vascular inflammatory biomarkers and endothelial function in overweight and obese humans

    Science.gov (United States)

    Perkins, Jennifer M.; Joy, Nino G.; Tate, Donna B.

    2015-01-01

    We investigated the separate and combined effects of hyperglycemia and hyperinsulinemia on markers of endothelial function, proinflammatory and proatherothrombotic responses in overweight/obese nondiabetic humans. Twenty-two individuals (13 F/9 M, BMI 30.1 ± 4.1 kg/m2) were studied during four randomized, single-blind protocols. The pancreatic clamp technique was combined with 4-h glucose clamps consisting of either 1) euinsulinemia-euglycemia, 2) euinsulinemia-hyperglycemia, 3) hyperinsulinemia-hyperglycemia, or 4) hyperinsulinemia-euglycemia. Insulin levels were higher (998 ± 66 vs. 194 ± 22 pmol/l) during hyperinsulinemia compared with euinsulinemia. Glucose levels were 11.1 mmol/l during hyperinsulinemia compared with 5.1 ± 0.1 mmol/l during euglycemia. VCAM, ICAM, P-selectin, E-selectin, IL-6, adiponectin, and PAI-1 responses were all increased (P hyperglycemia compared with other protocols. Hyperinsulinemia in the presence of hyperglycemia prevented the increase in proinflammatory and proatherothrombotic markers while also normalizing vascular endothelial function. We conclude that 4 h of moderate hyperglycemia can result in increases of proinflammatory markers (ICAM, VCAM, IL-6, E-selectin), platelet activation (P-selectin), reduced fibrinolytic balance (increased PAI-1), and disordered endothelial function in a group of obese and overweight individuals. Hyperinsulinemia prevents the actions of moderate hyperglycemia to reduce endothelial function and increase proinflammatory and proatherothrombotic markers. PMID:26015434

  19. Effect of silica nanoparticles with variable size and surface functionalization on human endothelial cell viability and angiogenic activity

    Science.gov (United States)

    Guarnieri, Daniela; Malvindi, Maria Ada; Belli, Valentina; Pompa, Pier Paolo; Netti, Paolo

    2014-02-01

    Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior effects of silica nanoparticles on primary endothelial cells. Here we investigated uptake, cytotoxicity and angiogenic properties of silica nanoparticle with positive and negative surface charge and sizes ranging from 25 to 115 nm in primary human umbilical vein endothelial cells. Dynamic light scattering measurements and nanoparticle tracking analysis were used to estimate the dispersion status of nanoparticles in cell culture media, which was a key aspect to understand the results of the in vitro cellular uptake experiments. Nanoparticles were taken up by primary endothelial cells in a size-dependent manner according to their degree of agglomeration occurring after transfer in cell culture media. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake, compared to negatively charged nanoparticles. However, this effect was contrasted by the tendency of particles to form agglomerates, leading to lower internalization efficiency. Silica nanoparticle uptake did not affect cell viability and cell membrane integrity. More interestingly, positively and negatively charged 25 nm nanoparticles did not influence capillary-like tube formation and angiogenic sprouting, compared to controls. Considering the increasing interest in nanomaterials for several biomedical applications, a careful study of nanoparticle-endothelial cells interactions is of high relevance to assess possible risks associated to silica nanoparticle exposure and their possible applications in nanomedicine as safe and effective nanocarriers for vascular transport of therapeutic agents.

  20. Zn(II released from zinc oxide nano/micro particles suppresses vasculogenesis in human endothelial colony-forming cells

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    Saeko Tada-Oikawa

    2015-01-01

    Full Text Available Zinc oxide (ZnO nanoparticles have been widely used in industry, cosmetics, and biomedicine. Recent studies suggested that these nanoparticles could have a major impact on the cardiovascular system. Endothelial progenitor cells (EPCs contribute to postnatal endothelial repair and regeneration. The present study dissected the effects of ZnO nanoparticles on vasculogenesis using human endothelial colony forming cells (ECFCs, which participate in post-natal vasculogenesis. Two types of ZnO particles were used (nano and micro, in addition to zinc chloride solutions with zinc ion concentrations equal to those in ZnO nanoparticles. Twenty-four-hour exposure induced cytotoxicity in a dose-dependent manner and increased ECFCs apoptosis in all groups. The exposure also reduced the functional capacity of ECFCs on Matrix gel to form tubules, compared with the control cells. These effects were associated with downregulation of expression of vascular endothelial growth factor receptor, VEGFR2 and CXC chemokine receptor, CXCR4. The results suggest that ZnO nanoparticles suppress vasculogenesis from ECFCs through downregulation of the expression of receptors related to vasculogenesis. These effects are based the concentration of released Zn(II.

  1. Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

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    Morihiko Maeda

    Full Text Available Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-β-galactosidase (SA-β-gal activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-β-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction.

  2. Ganoderma atrum polysaccharide ameliorates anoxia/reoxygenation-mediated oxidative stress and apoptosis in human umbilical vein endothelial cells.

    Science.gov (United States)

    Zhang, Yan-Song; Li, Wen-Juan; Zhang, Xian-Yi; Yan, Yu-Xin; Nie, Shao-Ping; Gong, De-Ming; Tang, Xiao-Fang; He, Ming; Xie, Ming-Yong

    2017-05-01

    Ganoderma atrum polysaccharide (PSG-1), a main polysaccharide from Ganoderma atrum, possesses potent antioxidant capacity and cardiovascular benefits. The aim of this study was to investigate the role of PSG-1 in oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs) under anoxia/reoxygenation (A/R) injury conditions. The results showed that exposure of HUVECs to A/R triggered cell death and apoptosis. Administration of PSG-1 significantly inhibited A/R-induced cell death and apoptosis in HUVECs. PSG-1-reduced A/R injury was mediated via mitochondrial apoptotic pathway, as evidenced by elevation of mitochondrial Bcl-2 protein and mitochondrial membrane potential, and attenuation of Bax translocation, cytochrome c release and caspases activation. Furthermore, PSG-1 enhanced the activities of superoxide dismutase, catalase and glutathione peroxidase and glutathione content, and concomitantly attenuated reactive oxygen species generation, lipid peroxidation and glutathione disulfide content. The antioxidant, N-acetyl-l-cysteine, significantly ameliorated all of these endothelial injuries caused by A/R, suggesting that antioxidant activities might play a key role in PSG-1-induced endothelial protection. Taken together, these findings suggested that PSG-1 could be as a promising adjuvant against endothelial dysfunction through ameliorating oxidative stress and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Angiotensin-converting enzyme inhibitors attenuate propofol-induced pro-oxidative and antifibrinolytic effect in human endothelial cells.

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    Wojewodzka-Zelezniakowicz, Marzena; Gromotowicz-Poplawska, Anna; Kisiel, Wioleta; Konarzewska, Emilia; Szemraj, Janusz; Ladny, Jerzy Robert; Chabielska, Ewa

    2017-01-01

    The aim of this study was to investigate the effects of plasma and tissue angiotensin-converting enzyme inhibitors (ACE-Is) against propofol-induced endothelial dysfunction and to elucidate the involved mechanisms in vitro. We examined the effects of propofol (50 μM), quinaprilat and enalaprilat (10-5 M) on fibrinolysis (t-PA, PAI-1, TAFI antigen levels), oxidative stress parameters (H2O2 and MDA antigen levels and SOD and NADPH oxidase mRNA levels) and nitric oxide bioavailability (NO2/NO3 concentration and NOS expression at the level of mRNA) in human umbilical vein endothelial cells (HUVECs). We found that both ACE-Is promoted similar endothelial fibrinolytic properties and decreased oxidative stress in vitro. Propofol alone increased the release of antifibrinolytic and pro-oxidative factors from the endothelium and increased mRNA iNOS expression. We also found that the incubation of HUVECs in the presence of propofol following ACE-Is pre-incubation caused weakness of the antifibrinolytic and pro-oxidative potential of propofol and this effect was similar after both ACE-Is. This observation suggests that the studied ACE-Is exerted protective effects against endothelial cell dysfunction caused by propofol, independently of hemodynamics.

  4. Effects of Citral on Lipopolysaccharide-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

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    Song, Yan; Zhao, Hongfeng; Liu, Jinyang; Fang, Chao; Miao, Renying

    2016-04-01

    Citral is an active compound of lemongrass oil which has been reported to have anti-inflammatory effects. In this study, we investigated the effects of citral on lipopolysaccharide (LPS)-induced inflammatory response in a rat model of peritonitis and human umbilical vein endothelial cells (HUVECs). LPS was intraperitoneally injected into rats to establish a peritonitis model. The HUVECs were treated with citral for 12 h before exposure to LPS. The levels of TNF-α and IL-8 were measured using ELISA. Western blotting was used to detect the expression of VCAM-1, ICAM-1, NF-κB, and PPAR-γ. The results showed that citral had a protective effect against LPS-induced peritonitis. Citral decreased the levels of WBCs and inflammatory cytokines TNF-α and IL-6. Citral also inhibited LPS-induced myeloperoxidase (MPO) activity in the peritoneal tissue. Treatment of HUVECs with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. LPS-induced VCAM-1 and ICAM-1 expression were also suppressed by citral. Meanwhile, we found that citral inhibited LPS-induced NF-κB activation in HUVECs. Furthermore, we found that citral activated PPAR-γ and the anti-inflammatory effects of citral can be reversed by PPAR-γ antagonist GW9662. In conclusion, citral inhibits LPS-induced inflammatory response via activating PPAR-γ which attenuates NF-κB activation and inflammatory mediator production.

  5. Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

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    Jing Wang

    2014-10-01

    Full Text Available AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2 on untransfected human corneal endothelial cells (HCECs in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01 and the length of F-actin, reduced the mean optical density (P<0.01 of the collagen type IV in extracellular matrix (ECM and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

  6. MDP Up-Regulates the Gene Expression of Type I Interferons in Human Aortic Endothelial Cells

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    Xiumei Xie

    2012-03-01

    Full Text Available Muramyldipeptide (MDP, the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2. Here, we obtained evidence that the treatment of human aortic endothelial cells (HAECs with MDP up-regulated the gene expression of type I interferons in a dose- and time-dependent manner. MDP also up-regulated the expression of the receptor NOD2, suggesting that MDP may induce a positive feedback response. The up-regulation of interferons was not dependent on the TNFa signaling, as HAECs did not express TNFa with the stimulation of MDP, and TNFa neutralizing antibody did not decrease the induction of IFNs induced by MDP. RT-PCR results showed that HAECs expressed the gene transcripts of interferon regulatory factor (IRF 1, 2, 3, 9. The western blot results showed that MDP induced the phosphorylation of IRF3. These results suggested that MDP induced the up-regulation of gene transcript of interferons through the activation of IRF3 signaling pathway. Meanwhile, MDP induced the gene expression of pro-inflammatory cytokines, including IL-1ß, IL-8, and MCP-1. Taken together, these results suggested that HAECs may play roles in the anti-infection immune response and in the induction of innate immunity.

  7. Dextromethorphan attenuates LPS-induced adhesion molecule expression in human endothelial cells.

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    Jiang, Shinn-Jong; Hsu, Sheng-Yao; Deng, Chuan-Rou; Huang, Huey-Chun; Liu, Shu-Lin; Shi, Guey-Yueh; Wu, Hua-Lin

    2013-02-01

    This study examines the effect of Dextromethorphan (d-3-methoxy-17-methylmorphinan; DXM), a commonly used cough-suppressing drug, on the expression of VCAM-1 and ICAM-1 in human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide (LPS). The effect of DXM on expression of cell adhesion molecules induced by LPS was evaluated by monocyte bindings in vitro and ex vivo and transmigration assays. The signaling pathways involved in the inflammation inhibitory effect of DXM were analyzed by Western blot and immunofluorescent stain. Pretreatment of HUVECs with DXM inhibited LPS-induced adhesion of THP-1 cells in vitro and ex vivo, and reduced transendothelial migration of these cells. Furthermore, treatment of HUVECs with DXM can significantly decrease LPS-induced expression of ICAM-1 and VCAM-1. DXM abrogated LPS-induced phosphorylation of ERK and Akt. The translocation of early growth response gene-1 (Egr-1), a downstream transcription factor involved in the mitogen-activated kinase (MEK)-ERK signaling pathway, was suppressed by DXM treatment. Furthermore, DXM inhibited LPS-induced IκBα degradation and nuclear translocation of p65. Dextromethorphan inhibits the adhesive capacity of HUVECs by reducing the LPS-induced ICAM-1 and VCAM-1 expression via the suppression of the ERK, Akt, and NF-κB signaling pathways. Thus, DXM is a potential anti-inflammatory therapeutic that may modulate atherogenesis. © 2012 John Wiley & Sons Ltd.

  8. Influence of nanoporous poly(ether imide) particle extracts on human aortic endothelial cells (HAECs).

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    Kumar, Reddi K; Basu, Sayantani; Lemke, Horst-Dieter; Jankowski, Joachim; Kratz, Karl; Lendlein, Andreas; Tetali, Sarada D

    2016-01-01

    Accumulated uremic toxins like indoxyl sulphate, hippuric acid and p-cresyl sulphates in renal failure patients stimulate proinflammatory effects, and consequently kidney and cardiovascular diseases. Low clearance rate of these uremic toxins from the blood of uremic patients by conventional techniques like hemodialysis is due to their strong covalent albumin binding (greater than 95%) and hydrophobic nature, which led to alternatives like usage of hydrophobic adsorber's in removing these toxins from the plasma of kidney patients. Polymers like polyethylene, polyurethane, polymethylmethacrylate, cellophane and polytetrafluoroethylene were already in use as substitutes for metal devices as dialysis membranes. Among new synthetic polymers, one such ideal adsorber material are highly porous microparticles of poly(ether imide) (PEI) with diameters in the range from 50-180μm and a porosity around 88±2% prepared by a spraying and coagulation process.It is essential to make sure that these synthetic polymers should not evoke any inflammatory or apoptotic response during dialysis. Therefore in our study we evaluated in vitro effect of PEI microparticle extracts in human aortic endothelial cells (HEACs) concerning toxicity, inflammation and apoptosis. No cell toxicity was observed when HAECs were treated with PEI extracts and inflammatory/apoptotic markers were not upregulated in presence of PEI extracts. Our results ensure biocompatibility of PEI particles and further hemocompatibility of particles will be tested.

  9. Low molecular weight heparin and aspirin exacerbate human endometrial endothelial cell responses to antiphospholipid antibodies.

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    Quao, Zola Chihombori; Tong, Mancy; Bryce, Elena; Guller, Seth; Chamley, Lawrence W; Abrahams, Vikki M

    2018-01-01

    Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications despite treatment with low molecular weight heparin (LMWH) or aspirin (ASA). aPL recognizing beta2 glycoprotein I can target the uterine endothelium, however, little is known about its response to aPL. This study characterized the effect of aPL on human endometrial endothelial cells (HEECs), and the influence of LMWH and ASA. HEECs were exposed to aPL or control IgG, with or without low-dose LMWH and ASA, alone or in combination. Chemokine and angiogenic factor secretion were measured by ELISA. A tube formation assay was used to measure angiogenesis. aPL increased HEEC secretion of pro-angiogenic VEGF and PlGF; increased anti-angiogenic sFlt-1; inhibited basal secretion of the chemokines MCP-1, G-CSF, and GRO-α; and impaired angiogenesis. LMWH and ASA, alone and in combination, exacerbated the aPL-induced changes in the HEEC angiogenic factor and chemokine profile. There was no reversal of the aPL inhibition of HEEC angiogenesis by either single or combination therapy. By aPL inhibiting HEEC chemokine secretion and promoting sFlt-1 release, the uterine endothelium may contribute to impaired placentation and vascular transformation. LMWH and ASA may further contribute to endothelium dysfunction in women with obstetric APS. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Piperine Decreases Binding of Drugs to Human Plasma and Increases Uptake by Brain Microvascular Endothelial Cells.

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    Dubey, Raghvendra K; Leeners, Brigitte; Imthurn, Bruno; Merki-Feld, Gabriele Susanne; Rosselli, Marinella

    2017-09-26

    We previously reported that piperine, an active alkaloidal principal of black and long peppers, enhances drug bioavailability by inhibiting drug metabolism. Another mechanism influencing drug availability/uptake is its free fraction. Since piperine is highly lipophilic, we hypothesize that it could also interact with drugs through binding displacement and influence their bioavailability. Accordingly, using equilibrium dialysis, we investigated whether piperine alters the binding of model drug ligands, that is flunitrazepam, diazepam, warfarin, salicylic acid, propranolol, lidocaine, and disopyramide to human plasma (n = 4). Since alterations in binding influence drug disposition, we also studied the effects of piperine on the uptake of plasma bound (3) H-propranolol and (14) C-warfarin by cultured bovine brain microvascular endothelial cells (BMECs). Piperine (1-1000 μM) increased the free fraction (fu) of both albumin and alpha-acid glycoprotein bound drugs in a concentration-dependent manner (p piperine (10 μM) increased the uptake of (3) H-propranolol and (14) C-warfarin by BMECs (p piperine displaces plasma bound drugs from both albumin and alpha-acid glycoprotein and facilitates drug uptake across biological membranes (e.g. BMEC). Moreover, it is feasible that piperine may similarly facilitate the transport of drugs into tissues, in vivo, and alter both pharmacokinetics and pharmacodynamics of administered drugs. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  11. Infectomic Analysis of Gene Expression Profiles of Human Brain Microvascular Endothelial Cells Infected with Cryptococcus neoformans

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    Ambrose Jong

    2008-01-01

    Full Text Available In order to dissect the pathogenesis of Cryptococcus neoformans meningoencephalitis, a genomic survey of the changes in gene expression of human brain microvascular endothelial cells infected by C. neoformans was carried out in a time-course study. Principal component analysis (PCA revealed sigificant fluctuations in the expression levels of different groups of genes during the pathogen-host interaction. Self-organizing map (SOM analysis revealed that most genes were up- or downregulated 2 folds or more at least at one time point during the pathogen-host engagement. The microarray data were validated by Western blot analysis of a group of genes, including β-actin, Bcl-x, CD47, Bax, Bad, and Bcl-2. Hierarchical cluster profile showed that 61 out of 66 listed interferon genes were changed at least at one time point. Similarly, the active responses in expression of MHC genes were detected at all stages of the interaction. Taken together, our infectomic approaches suggest that the host cells significantly change the gene profiles and also actively participate in immunoregulations of the central nervous system (CNS during C. neoformans infection.

  12. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

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    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Influence of heavy ions on cell survival, cytogenetic damage and mitochondrial function of human endothelial cells

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    Ritter, Sylvia; Helm, Alexander; Lee, Ryonfa; Pollet, Dieter; Durante, Marco

    There is increasing evidence that there is an elevated risk of cardiovascular disease among atomic bomb survivors and radiotherapy patients, typically developing with a long latency. However, essentially no information is available on the potential cardiovascular risks associated with space radiation, in particular heavy ions. To address this issue, we have chosen human umbilical vein endothelial cells (HUVEC) as a model system. Cells at an early passage number were irradiated with 0.1 to 4 Gy of either 9.8 MeV/u C-ions (LET=170 keV/µm), 91 MeV/u C-ions (LET=29 keV/µm) or 250 kV X-rays. Cells were regularly subcultured up to 40 days (20 population doublings) post-irradiation. Immediately after exposure cell inactivation was deter-mined by the colony forming assay. Furthermore, at selected time-points cytogenetic damage (formation of micronuclei in binucleated cells) and the mitochondrial membrane potential ΨM (flow cytometric analysis following JC-1 staining) were assessed. Measurement of the directly induced radiation damage showed that 9.8 MeV/u and 91 MeV/u C-ions were more effective than X-rays (i.e. about 3 and 2 times, respectively) with respect to cell inactivation or the in-duction of cytogenetic damage. At the subsequent days in the irradiated cultures the number of cells with micronuclei declined to the control level (3-5Altogether our data indicate that under the applied radiation conditions the integrity of mitochondria which play a significant role in the regulation of cardiovascular cell function is not impaired. With respect to directly induced genetic damage C-ions are more effective than X-rays as observed in other cell systems. If the effectiveness of charged particles for the occurrence of late chromosomal damage in endothelial cells is higher than that of sparsely ionizing radiation needs further clarification. The data obtained up to now indicate that sophisticated cytogenetic techniques have to be applied in order to draw any firm

  14. Facilitative interaction between angiotensin II and oxidised LDL in cultured human coronary artery endothelial cells

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    Jawahar L Mehta

    2001-03-01

    Full Text Available Background Several studies have shown that angiotensin II (Ang II and oxidised low-density lipoprotein (ox-LDL are critical factors in atherosclerosis. In this study, we examined the molecular basis of mutually facilitative interactions between Ang II and ox-LDL in human coronary artery endothelial cells (HCAECs.Methods and results We observed that incubation of cultured HCAECs with Ang II (10-12 to 10-6 M for 24 hours caused a concentration-dependent increase in the expression of mRNA and protein of a specialised receptor for ox-LDL (LOX-1. These effects of Ang II were completely blocked by pretreatment of HCAECs with candesartan (10-6 M, a specific AT1-receptor blocker, but not by PD 123319 (10-6 M, a specific AT2-receptor blocker. On the other hand, incubation of HCAECs with ox-LDL (10 and 40 µg/ml for 24 hours progressively upregulated AT1-, but not AT 2-, receptor mRNA and protein. Pretreatment of cells with the anti-oxidant alpha-tocopherol (1—5 x 10-6 M inhibited the upregulation of AT1-receptor expression induced by ox-LDL (p<0.05. To determine the significance of expression of AT1-receptors and LOX-1, we measured cell injury in response to Ang II and ox-LDL. Incubation of cells with both ox-LDL and Ang II synergistically increased cell injury, measured as cell viability and LDH release, compared with either ox-LDL or Ang II alone (both p<0.05. Alpha-tocopherol, as well as candesartan, attenuated cell injury in response to Ang II and ox-LDL (both p<0.05.Conclusions These observations show that Ang II upregulates a novel endothelial receptor for ox-LDL (LOX-1 gene expression and ox-LDL in turn upregulates Ang II AT 1receptor gene expression. This interaction between Ang II and ox-LDL further augments cell injury in HCAECs. These findings provide basis for the use of AT1-receptor blockers and anti-oxidants in designing therapy for atherosclerosis and myocardial ischaemia.

  15. Neurotrophins expression is decreased in lungs of human infants with congenital diaphragmatic hernia

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    O'Hanlon LD

    2014-02-01

    Full Text Available Lynn D O'Hanlon, Sherry M Mabry, Ikechukwu I EkekezieChildren's Mercy Hospitals/University of Missouri-Kansas City School of Medicine, Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Kansas City, MO, USAObjectives: To evaluate neurotrophin (NT (nerve growth factor [NGF], NT-3, and brain-derived neurotrophic factor [BDNF] expression in autopsy lung tissues of human congenital diaphragmatic hernia (CDH infants versus that of infants that expired with: 1 "normal" lungs (controls; 2 chronic lung disease (CLD; and 3 pulmonary hypertension (PPHN.Hypothesis: NT expression will be significantly altered in CDH lung tissue compared with normal lung tissue and other neonatal lung diseases.Study design: Immunohistochemical studies for NT proteins NGF, BDNF, and NT-3 were applied to human autopsy neonatal lung tissue samples.Subject selection: The samples included a control group of 18 samples ranging from 23-week gestational age to term, a CDH group of 15 samples, a PPHN group of six samples, and a CLD group of 12 samples.Methodology: The tissue samples were studied, and four representative slide fields of alveoli/saccules and four of bronchioles were recorded from each sample. These slide fields were then graded (from 0 to 3 by three blinded observers for intensity of staining.Results: BDNF, NGF, and NT-3 immunostaining intensity scores were significantly decreased in the CDH lung tissue (n=15 compared with normal neonatal lung tissue (n=18 (P<0.001. The other neonatal pulmonary diseases that were studied, CLD and PPHN, were much less likely to be affected and were much more variable in their neurotrophin expression.Conclusion: NT expression is decreased in CDH lungs. The decreased expression of NT in CDH lung tissue may suggest they contribute to the abnormality in this condition.Keywords: nerve growth factor, NGF, brain-derived neurotrophic factor, BDNF, neurotrophin-3, NT-3, chronic lung disease, persistent pulmonary hypertension, lung

  16. Circulating Endothelial-Derived Activated Microparticle: A Useful Biomarker for Predicting One-Year Mortality in Patients with Advanced Non-Small Cell Lung Cancer

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    Chin-Chou Wang

    2014-01-01

    Full Text Available Background. This study tested the hypothesis that circulating microparticles (MPs are useful biomarkers for predicting one-year mortality in patients with end-stage non-small cell lung cancer (ES-NSCLC. Methods and Results. One hundred seven patients were prospectively enrolled into the study between April 2011 and February 2012, and each patient received regular follow-up after enrollment. Levels of four MPs in circulation, (1 platelet-derived activated MPs (PDAc-MPs, (2 platelet-derived apoptotic MPs (PDAp-MPs, (3 endothelial-derived activated MPs (EDAc-MPs, and (4 endothelial-derived apoptotic MPs (EDAp-MPs, were measured just after the patient was enrolled into the study using flow cytometry. Patients who survived for more than one year were categorized into group 1 (n=56 (one-year survivors and patients who survived less than one year were categorized into group 2 (n=51 (one-year nonsurvivors. Male gender, incidence of liver metastasis, progression of disease after first-line treatment, poor performance status, and the Charlson comorbidity index were significantly higher in group 2 than in group 1 (all P<0.05. Additionally, as measured by flow cytometry, only the circulating level of EDAc-MPs was found to be significantly higher in group 2 than in group 1 (P=0.006. Multivariate analysis demonstrated that circulating level of EDAc-MPs along with brain metastasis and male gender significantly and independently predictive of one-year mortality (all P<0.035. Conclusion. Circulating EDAc-MPs may be a useful biomarker predictive of one-year morality in ES-NSCLC patients.

  17. Targeting Pulmonary Endothelial Hemoglobin α Improves Nitric Oxide Signaling and Reverses Pulmonary Artery Endothelial Dysfunction.

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    Alvarez, Roger A; Miller, Megan P; Hahn, Scott A; Galley, Joseph C; Bauer, Eileen; Bachman, Timothy; Hu, Jian; Sembrat, John; Goncharov, Dmitry; Mora, Ana L; Rojas, Mauricio; Goncharova, Elena; Straub, Adam C

    2017-12-01

    Pulmonary hypertension is characterized by pulmonary endothelial dysfunction. Previous work showed that systemic artery endothelial cells (ECs) express hemoglobin (Hb) α to control nitric oxide (NO) diffusion, but the role of this system in pulmonary circulation has not been evaluated. We hypothesized that up-regulation of Hb α in pulmonary ECs contributes to NO depletion and pulmonary vascular dysfunction in pulmonary hypertension. Primary distal pulmonary arterial vascular smooth muscle cells, lung tissue sections from unused donor (control) and idiopathic pulmonary artery (PA) hypertension lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Cocultures of human pulmonary microvascular ECs and distal pulmonary arterial vascular smooth muscle cells, lung tissue from control and pulmonary hypertensive lungs, and a mouse model of chronic hypoxia-induced PH were used. Immunohistochemical, immunoblot analyses, spectrophotometry, and blood vessel myography experiments were performed in this study. We find increased expression of Hb α in pulmonary endothelium from humans and mice with PH compared with controls. In addition, we show up-regulation of Hb α in human pulmonary ECs cocultured with PA smooth muscle cells in hypoxia. We treated pulmonary ECs with a Hb α mimetic peptide that disrupts the association of Hb α with endothelial NO synthase, and found that cells treated with the peptide exhibited increased NO signaling compared with a scrambled peptide. Myography experiments using pulmonary arteries from hypoxic mice show that the Hb α mimetic peptide enhanced vasodilation in response to acetylcholine. Our findings reveal that endothelial Hb α functions as an endogenous scavenger of NO in the pulmonary endothelium

  18. Characterizing human lung tissue microbiota and its relationship to epidemiological and clinical features.

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    Yu, Guoqin; Gail, Mitchell H; Consonni, Dario; Carugno, Michele; Humphrys, Michael; Pesatori, Angela C; Caporaso, Neil E; Goedert, James J; Ravel, Jacques; Landi, Maria Teresa

    2016-07-28

    The human lung tissue microbiota remains largely uncharacterized, although a number of studies based on airway samples suggest the existence of a viable human lung microbiota. Here we characterized the taxonomic and derived functional profiles of lung microbiota in 165 non-malignant lung tissue samples from cancer patients. We show that the lung microbiota is distinct from the microbial communities in oral, nasal, stool, skin, and vagina, with Proteobacteria as the dominant phylum (60 %). Microbiota taxonomic alpha diversity increases with environmental exposures, such as air particulates, residence in low to high population density areas, and pack-years of tobacco smoking and decreases in subjects with history of chronic bronchitis. Genus Thermus is more abundant in tissue from advanced stage (IIIB, IV) patients, while Legionella is higher in patients who develop metastases. Moreover, the non-malignant lung tissues have higher microbiota alpha diversity than the paired tumors. Our results provide insights into the human lung microbiota composition and function and their link to human lifestyle and clinical outcomes. Studies among subjects without lung cancer are needed to confirm our findings.

  19. Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

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    Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

    The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed

  20. High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells.

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    Iwata, Yasuhiro; Klaren, William D; Lebakken, Connie S; Grimm, Fabian A; Rusyn, Ivan

    Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.