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Sample records for endothelial madcam-1 expression

  1. An integrin alpha4beta7•IgG heterodimeric chimera binds to MAdCAM-1 on high endothelial venules in gut-associated lymphoid tissue.

    Science.gov (United States)

    Hoshino, Hitomi; Kobayashi, Motohiro; Mitoma, Junya; Sato, Yoshiko; Fukuda, Minoru; Nakayama, Jun

    2011-06-01

    Lymphocyte homing is regulated by a multistep process mediated by sequential adhesive interactions between circulating lymphocytes and high endothelial venules (HEVs). In gut-associated lymphoid tissue (GALT), the initial interactive step, "tethering and rolling," is partly mediated by integrin α4β7 expressed on GALT-homing lymphocytes and its ligand MAdCAM-1, which is exclusively expressed on HEVs in GALT. To probe functional MAdCAM-1 in tissue sections, we developed a soluble integrin α4β7 heterodimeric IgG chimera by joining the extracellular region of mouse integrin α4 and β7 subunits to a human IgG Fc domain. Western blot analysis revealed that co-transfection of HEK 293T cells with expression vectors encoding integrin α4•IgG and β7•IgG results in the formation of α4β7•IgG heterodimeric chimeras. This complex preferentially binds to CHO cells expressing MAdCAM-1 and, to a lesser extent, to cells expressing VCAM-1, but not to cells expressing ICAM-1. Moreover, α4β7•IgG specifically binds to HEVs in GALT in situ in a divalent cation-dependent fashion and inhibits lymphocyte binding to HEVs in GALT. These findings indicate that α4β7•IgG can be used as a probe for functional MAdCAM-1 expressed on HEVs in GALT and could potentially serve as an anti-inflammatory drug inhibiting GALT-specific lymphocyte migration.

  2. MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection.

    Science.gov (United States)

    Soderberg, Kelly A; Linehan, Melissa M; Ruddle, Nancy H; Iwasaki, Akiko

    2004-08-01

    The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.

  3. MAdCAM-1 is needed for diabetes development mediated by the T cell clone, BDC-2·5

    Science.gov (United States)

    Phillips, Jenny M; Haskins, Kathryn; Cooke, Anne

    2005-01-01

    The NOD-derived islet-reactive CD4+ T cell clone, BDC-2·5, is able to transfer diabetes to neonatal non-obese diabetic (NOD) mice but is unable to transfer disease to either adult NOD or NOD scid recipients. Transfer of diabetes to adult recipients by BDC-2·5 is only accomplished by cotransfer of CD8+ T cells from a diabetic donor. To understand why this CD4+ T cell clone is able to mediate diabetes in neonatal but not the adult recipients we examined the ability of the clone to traffic in the different recipients. Our studies showed that MAdCAM-1 has a very different expression pattern in the neonatal and adult pancreas. Blockade of this addressin prevents the clone from transferring diabetes to neonatal mice, suggesting that the differential pancreatic expression of MAdCAM-1 in neonatal and adult pancreas provides an explanation of the differences in diabetes development. PMID:16313366

  4. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  5. Reduced Ang2 expression in aging endothelial cells

    International Nuclear Information System (INIS)

    Hohensinner, P.J.; Ebenbauer, B.; Kaun, C.; Maurer, G.; Huber, K.; Wojta, J.

    2016-01-01

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  6. Reduced Ang2 expression in aging endothelial cells.

    Science.gov (United States)

    Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. A Small Molecule, Which Competes with MAdCAM-1, Activates Integrin α4β7 and Fails to Prevent Mucosal Transmission of SHIV-SF162P3.

    Directory of Open Access Journals (Sweden)

    Géraldine Arrode-Brusés

    2016-06-01

    Full Text Available Mucosal HIV-1 transmission is inefficient. However, certain viral and host characteristics may play a role in facilitating HIV acquisition and systemic expansion. Cells expressing high levels of integrin α4β7 have been implicated in favoring the transmission process and the infusion of an anti-α4β7 mAb (RM-Act-1 prior to, and during a repeated low-dose vaginal challenge (RLDC regimen with SIVmac251 reduced SIV acquisition and protected the gut-associated lymphoid tissues (GALT in the macaques that acquired SIV. α4β7 expression is required for lymphocyte trafficking to the gut lamina propria and gut inductive sites. Several therapeutic strategies that target α4β7 have been shown to be effective in treating inflammatory conditions of the intestine, such as inflammatory bowel disease (IBD. To determine if blocking α4β7 with ELN, an orally available anti-α4 small molecule, would inhibit SHIV-SF162P3 acquisition, we tested its ability to block MAdCAM-1 (α4β7 natural ligand and HIV-gp120 binding in vitro. We studied the pharmacokinetic profile of ELN after oral and vaginal delivery in macaques. Twenty-six macaques were divided into 3 groups: 9 animals were treated with ELN orally, 9 orally and vaginally and 8 were used as controls. All animals were challenged intra-vaginally with SHIV-SF162P3 using the RLDC regimen. We found that ELN did not protect macaques from SHIV acquisition although it reduced the SHIV-induced inflammatory status during the acute phase of infection. Notably, integrins can exist in different activation states and, comparing the effect of ELN and the anti-α4β7 mAb RM-Act-1 that reduced susceptibility to SIV infection, we determined that ELN induces the active conformation of α4β7, while RM-Act-1 inhibits its activation through an allosteric mechanism. These results suggest that inhibition of α4β7 activation may be necessary to reduce susceptibility to SIV/SHIV infection and highlight the complexity of anti

  8. Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

    Science.gov (United States)

    Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

    2008-08-01

    Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

  9. The effect of vascular endothelial growth factor-1 expression on ...

    African Journals Online (AJOL)

    Riyad Bendardaf

    2017-02-28

    Feb 28, 2017 ... The effect of vascular endothelial growth factor-1 expression on survival of ... Sharjah, Sharjah, United Arab Emirates; cFaculty of Medicine, Suez Canal University, ..... interleukin-6, and C-reactive protein level in colorectal.

  10. VE-Cadherin–Mediated Epigenetic Regulation of Endothelial Gene Expression

    Science.gov (United States)

    Morini, Marco F.; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I.; Conze, Lei L.; O’Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P.

    2018-01-01

    Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased

  11. VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

    Science.gov (United States)

    Morini, Marco F; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I; Conze, Lei L; O'Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P; Dejana, Elisabetta; Taddei, Andrea

    2018-01-19

    The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5 , vascular endothelial-protein tyrosine phosphatase ( VE-PTP ), and von Willebrand factor ( vWf ). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5 , VE-PTP , and vWf . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5 , VE-PTP , and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These

  12. Vascular endothelial growth factor ( VEGF ) receptor expression ...

    African Journals Online (AJOL)

    Avidin-biotin complex method was employed for immunohistochemical detection of VEGF. Results: VEGF immuno-expression was positive in 51.9% of CRC, while it was 18.2% in the normal colonic tissue (p<0.05). VEGF immunostaining was positively correlated with grade of colonic malignancy (p<0.05). Conclusion: ...

  13. Expression of vascular endothelial growth factor in Juvenile Angiofibroma.

    Science.gov (United States)

    Hota, Ashutosh; Sarkar, Chitra; Gupta, Siddhartha Datta; Kumar, Rakesh; Bhalla, Ashu Seith; Thakar, Alok

    2015-06-01

    To examine Juvenile Angiofibroma (JA) tissue for expression of vascular endothelial growth factor (VEGF), and to explore its relationship with puberty status, stage, recurrence and the intraoperative blood loss. Retrospective cohort study of 36 histologically proven cases of JA. Minimum follow up period was 3 years. VEGF expression on tumor cells assessed by immunohistochemistry and graded on two criteria--percentage of cells expressing positivity and the intensity of positivity. These two parameters assessed for impact on puberty status, stage, recurrence, and blood loss. VEGF expression noted on the tumor endothelial cells in 36/36, and on the tumor stromal cells in 34/36. The percentage of cells expressing VEGF and the intensity of expression were not significantly related to puberty status, tumor stage, recurrence, or intra-operative blood loss (p values 0.3-1.0). VEGF expression is near universal in JA. Such expression is independent of puberty status and stage, and does not impact on intra operative blood loss and recurrence. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

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    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  15. Decreased expression of serum and microvascular vascular endothelial growth factor receptor-2 in meningococcal sepsis*.

    NARCIS (Netherlands)

    Flier, M. van der; Baerveldt, E.M.; Miedema, A.; Hartwig, N.G.; Hazelzet, J.A.; Emonts, M.; Groot, R. de; Prens, E.P.; Vught, A.J. van; Jansen, N.J.

    2013-01-01

    OBJECTIVES: To determine the skin microvessel expression of vascular endothelial growth factor receptor 2 and serum-soluble vascular endothelial growth factor receptor 2 levels in children with meningococcal sepsis. DESIGN: Observational study. SETTING: Two tertiary academic children hospital PICUs.

  16. 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.

  17. Selective receptor expression restricts Nipah virus infection of endothelial cells

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    Diederich Sandra

    2008-11-01

    Full Text Available Abstract Nipah virus (NiV is a highly pathogenic paramyxovirus that causes severe diseases in animals and humans. Endothelial cell (EC infection is an established hallmark of NiV infection in vivo. Despite systemic virus spread via the vascular system, EC in brain and lung are preferentially infected whereas EC in other organs are less affected. As in vivo, we found differences in the infection of EC in cell culture. Only brain-derived primary or immortalized EC were found to be permissive to NiV infection. Using a replication-independent fusion assay, we could show that the lack of infection in non-brain EC was due to a lack of receptor expression. The NiV entry receptors ephrinB2 (EB2 or ephrinB3 were only expressed in brain endothelia. The finding that EB2 expression in previously non-permissive aortic EC rendered the cells permissive to infection then demonstrated that EB2 is not only necessary but also sufficient to allow the establishment of a productive NiV infection. This strongly suggests that limitations in receptor expression restrict virus entry in certain EC subsets in vivo, and are thus responsible for the differences in EC tropism observed in human and animal NiV infections.

  18. 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...

  19. The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

    Science.gov (United States)

    Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

    2008-11-01

    The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (PHRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer

  20. The Expression Profiles of Lysophospholipid Receptors (LPLRs in Different Endothelial Cells

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    Yu-Wei Lee

    2006-03-01

    Full Text Available Sphingosine-1-phosphate (S1P and lysophosphatidic acid (LPA are two bioactive lysophospholipids (LPLs, stored primarily in platelets and released during platelet activation. Both LPLs are capable of regulating endothelial cell functions. The physiological functions of S1P and LPA are mediated by interacting with eight different G-protein coupled receptors: S1P1 through 5 and LPA1 through 3, which activate three different heterotrimeric GTP proteins-including Gi、Gq and G(12/13. The expression of LPL receptors in endothelial cells would affect the responses of S1P and LPA to these cells. There is no previous report discussing the expression profiles of LPL receptors in different endothelial cells from various species. In this study, we aim to investigate the expression profiles of S1P and LPA receptors in different endothelial cells isolated from human, rat, mouse and bovine origin. We used RT-PCR to determine LPLs receptors expression profiles in different endothelial cells. Our results indicated that endothelial cells from various species express different LPL receptors. Endothelial cells isolated from the same source of different species also had different LPLs receptors expression profiles. Therefore, different endothelial cells should respond to LPLs in different manners.

  1. Cardiac microvascular endothelial cells express a functional Ca+ -sensing receptor.

    Science.gov (United States)

    Berra Romani, Roberto; Raqeeb, Abdul; Laforenza, Umberto; Scaffino, Manuela Federica; Moccia, Francesco; Avelino-Cruz, Josè Everardo; Oldani, Amanda; Coltrini, Daniela; Milesi, Veronica; Taglietti, Vanni; Tanzi, Franco

    2009-01-01

    The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores. Copyright 2008 S. Karger AG, Basel.

  2. 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

    in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis......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...

  3. Prognostic importance of vascular endothelial growth factor-A expression and vascular endothelial growth factor polymorphisms in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Smerdel, Maja; Waldstrøm, Marianne; Brandslund, Ivan

    2009-01-01

    INTRODUCTION: Vascular endothelial growth factors (VEGFs) play a central role in angiogenesis and consequently, in various steps of ovarian carcinogenesis. Gene polymorphisms within the VEGF system have revealed a correlation with prognosis in some malignancies. The aim of the present study...... was to examine the possible importance of 2 VEGF polymorphisms and VEGF-A expression in ovarian cancer. METHODS: We investigated 2 single nucleotide polymorphisms VEGF +405G/C and VEGF -460C/T by polymerase chain reaction and also analyzed VEGF-A expression by immunohistochemistry in 159 women with ovarian...... cancer. RESULTS: Vascular endothelial growth factor-A expression revealed a significant correlation with survival in a Cox proportional hazards regression model (P = 0.012). Germline polymorphisms were not correlated with clinicopathological parameters such as stage, type, and histology. Heterozygous...

  4. Increased vascular endothelial growth factor (VEGF) expression in ...

    African Journals Online (AJOL)

    User

    2011-05-16

    May 16, 2011 ... was quantified by means of western blot and immunohistochemistry technology. ... Key words: Vascular endothelial growth factor (VEGF), spinal cord injury, ... accordance with the National Institute of Health Guide for the Care.

  5. Increased vascular endothelial growth factor (VEGF) expression in ...

    African Journals Online (AJOL)

    User

    2011-05-16

    May 16, 2011 ... Vascular endothelial growth factor (VEGF), a well known angiogenic factor, has been shown to have direct and/or ... Endogenous repair efforts fail to repair ... Spinal cord injury model preparation and intramedullary spinal.

  6. Functional and gene expression analysis of hTERT overexpressed endothelial cells

    Directory of Open Access Journals (Sweden)

    Haruna Takano

    2008-09-01

    Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

  7. Endothelial Cell Migration and Vascular Endothelial Growth Factor Expression Are the Result of Loss of Breast Tissue Polarity

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Amy; Cuevas, Ileana; Kenny, Paraic A; Miyake, Hiroshi; Mace, Kimberley; Ghajar, Cyrus; Boudreau, Aaron; Bissell, Mina; Boudreau, Nancy

    2009-05-26

    Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1{alpha}-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

  8. The effect of vascular endothelial growth factor-1 expression on ...

    African Journals Online (AJOL)

    Colorectal cancer is third leading cause of cancer mortality. About 60% of patients had already developed metastasis at the time of diagnosis. Vascular endothelial growth factor (VEGF) is crucial for the development of neovascularization and hence metastasis. This study aimed at investigating the relation between the ...

  9. Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

    Science.gov (United States)

    Radwanska, Agata; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Ciais, Delphine; Rekima, Samah; Rupp, Tristan; Sudaka, Anne; Orend, Gertraud; Van Obberghen-Schilling, Ellen

    2017-10-06

    Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

  10. MicroRNA-147b regulates vascular endothelial barrier function by targeting ADAM15 expression.

    Directory of Open Access Journals (Sweden)

    Victor Chatterjee

    Full Text Available A disintegrin and metalloproteinase15 (ADAM15 has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS. An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3' UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.

  11. Alteration of human umbilical vein endothelial cell gene expression in different biomechanical environments.

    Science.gov (United States)

    Shoajei, Shahrokh; Tafazzoli-Shahdpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin

    2014-05-01

    Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm(2) , 1 Hz) and uniform (5 dyn/cm(2) ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm(2) ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes. © 2013 International Federation for Cell Biology.

  12. Expression and Function of the Homeostatic Molecule Del-1 in Endothelial Cells and the Periodontal Tissue

    Directory of Open Access Journals (Sweden)

    Jieun Shin

    2013-01-01

    Full Text Available Developmental endothelial locus-1 (Del-1 is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interfere with neutrophil recruitment and inflammation. Treatment of human endothelial cells with Del-1 did not affect the expression of endothelial molecules involved in the leukocyte adhesion cascade (ICAM-1, VCAM-1, and E-selectin. Moreover, genetic or age-associated Del-1 deficiency did not significantly alter the expression of these adhesion molecules in the murine periodontium, further ruling out altered adhesion molecule expression as a mechanism whereby Del-1 regulates leukocyte recruitment. Strikingly, Del-1 inhibited ICAM-1-dependent chemokine release (CXCL2, CCL3 by neutrophils. Therefore, Del-1 could potentially suppress the amplification of inflammatory cell recruitment mediated through chemokine release by infiltrating neutrophils. Interestingly, Del-1 was itself regulated by inflammatory stimuli, which generally exerted opposite effects on adhesion molecule expression. The reciprocal regulation between Del-1 and inflammation may contribute to optimally balance the protective and the potentially harmful effects of inflammatory cell recruitment.

  13. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  14. Mel-18, a mammalian Polycomb gene, regulates angiogenic gene expression of endothelial cells.

    Science.gov (United States)

    Jung, Ji-Hye; Choi, Hyun-Jung; Maeng, Yong-Sun; Choi, Jung-Yeon; Kim, Minhyung; Kwon, Ja-Young; Park, Yong-Won; Kim, Young-Myeong; Hwang, Daehee; Kwon, Young-Guen

    2010-10-01

    Mel-18 is a mammalian homolog of Polycomb group (PcG) genes. Microarray analysis revealed that Mel-18 expression was induced during endothelial progenitor cell (EPC) differentiation and correlates with the expression of EC-specific protein markers. Overexpression of Mel-18 promoted EPC differentiation and angiogenic activity of ECs. Accordingly, silencing Mel-18 inhibited EC migration and tube formation in vitro. Gene expression profiling showed that Mel-18 regulates angiogenic genes including kinase insert domain receptor (KDR), claudin 5, and angiopoietin-like 2. Our findings demonstrate, for the first time, that Mel-18 plays a significant role in the angiogenic function of ECs by regulating endothelial gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Expression of Vascular Endothelial Growth Factor in Odontogenic Cysts: Is There Any Impression on Clinical Outcome?

    Science.gov (United States)

    Sadri, Donia; Farhadi, Sareh; Shahabi, Zahra; Sarshar, Samaneh

    2016-01-01

    The recent scientific reports have shown that angiogenesis can affect biological behavior of pathologic lesions. Regarding unique clinical outcome of Odontogenic keratocyst (OKC), the present study was aimed to compare angiogenesis in Odontogenic keratocyst and Dentigerous cyst (DC). In this experimental study, tissue sections of 46 samples of OKC and DC were stained through immunohistochemical method using Vascular Endothelial Growth Factor (VEGF) antibody. VEGF expression was evaluated in epithelial cells, fibroblasts and endothelial cells. The average percentage of stained cells in any samples was categorized to 3 groups as follows: SCORE 0: 10% of cells or less are positive. SCORE 1: 10 to 50% of cells are positive. SCORE 2: more than 50% of cells are positive. Mann-U-Whitney, T-test and chi-square was used for statistical analysis. The average of VEGF expression in 24 samples of DC was 20.2% and in 22 samples of OKC was 52.6%, respectively. The average of VEGF expression in these two cysts had statistical significant differences. (PV= 0.045). There was significant statistical differences between two cysts in the terms of VEGF SCORE (PV= 0.000). OKC samples had significantly higher SCORE for the purpose of VEGF incidence than DC. Also, there were no differences between VEGF expression in epithelial cells of two cysts (PV= 0.268) there were significant statistical differences between two cysts in terms of endothelial cell staining. The endothelial cell staining was significantly higher in OKC than DC (PV= 0.037%). Regarding higher expression of Vascular Endothelial Growth factor in OKC than DC, it seems that angiogenesis may have great impression on clinical outcome of OKC.

  16. 2-Chlorohexadecanal and 2-chlorohexadecanoic acid induce COX-2 expression in human coronary artery endothelial cells

    OpenAIRE

    Messner, Maria C.; Albert, Carolyn J.; Ford, David A.

    2008-01-01

    2-Chlorohexadecanal (2-ClHDA), a 16-carbon chain chlorinated fatty aldehyde that is produced by reactive chlorinating species attack of plasmalogens, is elevated in atherosclerotic plaques, infarcted myocardium, and activated leukocytes. We tested the hypothesis that 2-ClHDA and its metabolites, 2-chlorohexadecanoic acid (2-ClHA) and 2-chlorohexadecanol (2-ClHOH), induce COX-2 expression in human coronary artery endothelial cells (HCAEC). COX-2 protein expression increased in response to 2-Cl...

  17. Expression of Toll-Like Receptor 4 in Glomerular Endothelial Cells under Diabetic Conditions

    International Nuclear Information System (INIS)

    Takata, Shunsuke; Sawa, Yoshihiko; Uchiyama, Takanobu; Ishikawa, Hiroyuki

    2013-01-01

    Diabetic conditions promote glomerulosclerosis by mesangial cells but the mechanisms are not fully elucidated. The present study evaluated the expression of toll-like receptor 4 in glomerular endothelial cells in the streptozotocin (STZ)-induced type 1 diabetic mouse (ICR-STZ) and the type 2 diabetic KK/TaJcl mouse which were fed a high fat diet feed (KK/Ta-HF). In the ICR-STZ and KK/Ta-HF almost glomeruli were immunostained with anti-TLR4 but there was no glomerulus immunostained by ani-TLR4 in the control ICR and KK/Ta. Laser-scanning confocal microscopy showed that the TLR4-positive region did not coincide with the podoplanin-positive region but coincide with the PECAM-1- and VE-cadherin-positive regions in the glomeruli of the ICR-STZ and KK/Ta-HF. The in situ hybridization showed that almost signals for TLR4 mRNA were present in the glomerulus of the ICR-STZ and KK/Ta-HF to a stronger extent than in the control ICR and KK/Ta. These suggest that glomerular endothelial cells usually express the TLR4 gene and hyperglycemia in the diabetic condition induces the TLR4 protein expression in the glomerular capillary endothelial cells. Cytokine productions through the TLR signaling pathway in glomerular endothelial cells may allow mesangial cells to produce extracellular matrix proteins in the diabetic milieu

  18. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...... germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth...

  19. Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

    Directory of Open Access Journals (Sweden)

    Konerding Moritz A

    2011-07-01

    Full Text Available Abstract Although blood vessel growth occurs readily in the systemic bronchial circulation, angiogenesis in the pulmonary circulation is rare. Compensatory lung growth after pneumonectomy is an experimental model with presumed alveolar capillary angiogenesis. To investigate the genes participating in murine neoalveolarization, we studied the expression of angiogenesis genes in lung endothelial cells. After left pneumonectomy, the remaining right lung was examined on days 3, 6, 14 and 21days after surgery and compared to both no surgery and sham thoracotomy controls. The lungs were enzymatically digested and CD31+ endothelial cells were isolated using flow cytometry cell sorting. The transcriptional profile of the CD31+ endothelial cells was assessed using quantitative real-time polymerase chain reaction (PCR arrays. Focusing on 84 angiogenesis-associated genes, we identified 22 genes with greater than 4-fold regulation and significantly enhanced transcription (p

  20. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Science.gov (United States)

    Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

  1. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Directory of Open Access Journals (Sweden)

    Delphine M Béziau

    Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

  2. Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis.

    Science.gov (United States)

    Gössl, Mario; Mödder, Ulrike I; Atkinson, Elizabeth J; Lerman, Amir; Khosla, Sundeep

    2008-10-14

    This study was designed to test whether patients with coronary atherosclerosis have increases in circulating endothelial progenitor cells (EPCs) expressing an osteogenic phenotype. Increasing evidence indicates a link between bone and the vasculature, and bone marrow and circulating osteogenic cells have been identified by staining for the osteoblastic marker, osteocalcin (OCN). Endothelial progenitor cells contribute to vascular repair, but repair of vascular injury may result in calcification. Using cell surface markers (CD34, CD133, kinase insert domain receptor [KDR]) to identify EPCs, we examined whether patients with coronary atherosclerosis had increases in the percentage of EPCs expressing OCN. We studied 72 patients undergoing invasive coronary assessment: control patients (normal coronary arteries and no endothelial dysfunction, n = 21) versus 2 groups with coronary atherosclerosis-early coronary atherosclerosis (normal coronary arteries but with endothelial dysfunction, n = 22) and late coronary atherosclerosis (severe, multivessel coronary artery disease, n = 29). Peripheral blood mononuclear cells were analyzed using flow cytometry. Compared with control patients, patients with early or late coronary atherosclerosis had significant increases (approximately 2-fold) in the percentage of CD34+/KDR+ and CD34+/CD133+/KDR+ cells costaining for OCN. Even larger increases were noted in the early and late coronary atherosclerosis patients in the percentage of CD34+/CD133-/KDR+ cells costaining for OCN (5- and 2-fold, p < 0.001 and 0.05, respectively). A higher percentage of EPCs express OCN in patients with coronary atherosclerosis compared with subjects with normal endothelial function and no structural coronary artery disease. These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary atherosclerosis.

  3. Endothelial marker-expressing stromal cells are critical for kidney formation.

    Science.gov (United States)

    Mukherjee, Elina; Maringer, Katherine; Papke, Emily; Bushnell, Daniel; Schaefer, Caitlin; Kramann, Rafael; Ho, Jacqueline; Humphreys, Benjamin D; Bates, Carlton; Sims-Lucas, Sunder

    2017-09-01

    Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice ( Flk1 fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1 fl/fl ( Flk1 ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1 ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1 ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1 ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1 ST-/- kidneys vs. Juvenile Flk1 ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1 ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages. Copyright © 2017 the American Physiological Society.

  4. 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

  5. Differential Gene Expression of Primary Cultured Lymphatic and Blood Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Gregory M. Nelson

    2007-12-01

    Full Text Available Blood vascular endothelial cells (BECs and the developmentally related lymphatic endothelial cells (LECs create complementary, yet distinct vascular networks. Each endothelial cell type interacts with flowing fluid and circulating cells, yet each vascular system has evolved specialized gene expression programs and thus both cell types display different phenotypes. BECs and LECs express distinct genes that are unique to their specific vascular microenvironment. Tumors also take advantage of the molecules that are expressed in these vascular systems to enhance their metastatic potential. We completed transcriptome analyses on primary cultured LECs and BECs, where each comparative set was isolated from the same individual. Differences were resolved in the expression of several major categories, such as cell adhesion molecules (CAMs, cytokines, cytokine receptors. We have identified new molecules that are associated with BECs (e.g., claudin-9, CXCL11, neurexin-1, neurexin-2, the neuronal growth factor regulator-1 and LECs (e.g., claudin-7, CD58, hyaluronan and proteoglycan link protein 1 (HAPLN1, the poliovirus receptor-related 3 molecule that may lead to novel therapeutic treatments for diseases of lymphatic or blood vessels, including metastasis of cancer to lymph nodes or distant organs.

  6. Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

  7. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

    Directory of Open Access Journals (Sweden)

    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  8. Regulation of S1P receptors and sphingosine kinases expression in acute pulmonary endothelial cell injury

    Directory of Open Access Journals (Sweden)

    Huiying Liu

    2016-12-01

    Full Text Available Background Acute lung injury and acute respiratory distress syndrome (ALI/ARDS is a severe clinical syndrome with mortality rate as high as 30–40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs exhibit make it possible to regulate the homeostatic control of S1P. Methods By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis. Results It was found that the down-regulation of TNF-α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes. Discussions The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1

  9. Regulation of S1P receptors and sphingosine kinases expression in acute pulmonary endothelial cell injury.

    Science.gov (United States)

    Liu, Huiying; Zhang, Zili; Li, Puyuan; Yuan, Xin; Zheng, Jing; Liu, Jinwen; Bai, Changqing; Niu, Wenkai

    2016-01-01

    Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is a severe clinical syndrome with mortality rate as high as 30-40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P) is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs) exhibit make it possible to regulate the homeostatic control of S1P. By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis. It was found that the down-regulation of TNF- α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes. The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1P and their possible synergistic mechanism.

  10. ALTERED EXPRESSION OF SURFACE RECEPTORS AT EA.HY926 ENDOTHELIAL CELL LINE INDUCED WITH PLACENTAL SECRETORY FACTORS

    Directory of Open Access Journals (Sweden)

    O. I. Stepanova

    2012-01-01

    Full Text Available Abstract. Placental cell populations produce a great variety of angiogenic factors and cytokines than control angiogenesis in placenta. Functional regulation of endothelial cells proceeds via modulation of endothelial cell receptors for endogenous angiogenic and apoptotic signals. Endothelial phenotype alteration during normal pregnancy and in cases of preclampsia is not well understood. The goal of this investigation was to evaluate altered expression of angiogenic and cytokine receptors at EA.hy926 endothelial cells under the influence of placental tissue supernatants. Normal placental tissue supernatants from 1st and 3rd trimesters, and pre-eclamptic placental tissue supernatants (3rd trimester stimulated angiogenic and cytokine receptors expression by the cultured endothelial cells, as compared with their background expression. Tissue supernatants from placental samples of 3rd trimester caused a decreased expression of angiogenic and cytokine receptors by endothelial cells, thus reflecting maturation of placental vascular system at these terms. Supernatants from preeclamptic placental tissue induced an increase of CD119 expression, in comparison with normal placental supernatants from the 3rd trimester. This finding suggests that IFNγ may be a factor of endothelial activation in pre-eclampsia. The study was supported by grants ГК №02.740.11.0711, НШ-3594.2010.7., and МД-150.2011.7.

  11. The anti-hypercholesterolemic effect of low p53 expression protects vascular endothelial function in mice.

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    Francois Leblond

    Full Text Available To demonstrate that p53 modulates endothelial function and the stress response to a high-fat western diet (WD.Three-month old p53+/+ wild type (WT and p53+/- male mice were fed a regular or WD for 3 months. Plasma levels of total cholesterol (TC and LDL-cholesterol were significantly elevated (p<0.05 in WD-fed WT (from 2.1±0.2 mmol/L to 3.1±0.2, and from 0.64±0.09 mmol/L to 1.25±0.11, respectively but not in p53+/- mice. The lack of cholesterol accumulation in WD-fed p53+/- mice was associated with high bile acid plasma concentrations (p53+/- =  4.7±0.9 vs. WT =  3.3±0.2 μmol/L, p<0.05 concomitant with an increased hepatic 7-alpha-hydroxylase mRNA expression. While the WD did not affect aortic endothelial relaxant function in p53+/- mice (WD =  83±5 and RD =  82±4% relaxation, it increased the maximal response to acetylcholine in WT mice (WD =  87±2 vs. RD =  62±5% relaxation, p<0.05 to levels of p53+/-. In WT mice, the rise in TC associated with higher (p<0.05 plasma levels of pro-inflammatory keratinocyte-derived chemokine, and an over-activation (p<0.05 of the relaxant non-nitric oxide/non-prostacyclin endothelial pathway. It is likely that in WT mice, activations of these pathways are adaptive and contributed to maintain endothelial function, while the WD neither promoted inflammation nor affected endothelial function in p53+/- mice.Our data demonstrate that low endogenous p53 expression prevents the rise in circulating levels of cholesterol when fed a WD. Consequently, the endothelial stress of hypercholesterolemia is absent in young p53+/- mice as evidenced by the absence of endothelial adaptive pathway over-activation to minimize stress-related damage.

  12. Quinapril treatment increases insulin-stimulated endothelial function and adiponectin gene expression in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Hermann, Thomas S; Li, Weijie; Dominguez, Helena

    2005-01-01

    OBJECTIVE: Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality and improve endothelial function in type 2 diabetic patients. We hypothesized that 2 months of quinapril treatment would improve insulin-stimulated endothelial function and glucose uptake in type 2 diabetic subjects...... and simultaneously increase the expression of genes that are pertinent for endothelial function and metabolism. METHODS: Twenty-four type 2 diabetic subjects were randomized to receive 2 months of quinapril 20 mg daily or no treatment in an open parallel study. Endothelium-dependent and -independent vasodilation...... occlusion plethysmography. Gene expression was measured by real-time PCR. RESULTS: Quinapril treatment increased insulin-stimulated endothelial function in the type 2 diabetic subjects (P = 0.005), whereas forearm glucose uptake was unchanged. Endothelial function was also increased by quinapril (P = 0...

  13. Deletion of Protein Tyrosine Phosphatase 1B (PTP1B Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction.

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    David J Herren

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM was induced in wild-type (WT and PTP1B-deficient mice (KO with streptozotocin (STZ injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384 ± 20 vs. Ko: 432 ± 29 mg/dL, cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

  14. Tumor necrosis factor-α regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells

    NARCIS (Netherlands)

    Giraudo, E.; Primo, L.; Audero, E.; Gerber, H.-P.; Koolwijk, P.; Soker, S.; Klagsbrun, M.; Ferrara, N.; Bussolino, F.

    1998-01-01

    Tumor necrosis factor-α (TNF-α) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-α does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show

  15. Orphan Nuclear Receptor Nur77 Is a Novel Negative Regulator of Endothelin-1 Expression In Vascular Endothelial Cells

    OpenAIRE

    Qin, Qing; Chen, Ming; Yi, Bing; You, Xiaohua; Yang, Ping; Sun, Jianxin

    2014-01-01

    Endothelin-1 (ET-1) produced by vascular endothelial cells plays essential roles in the regulation of vascular tone and development of cardiovascular diseases. The objective of this study is to identify novel regulators implicated in the regulation of ET-1 expression in vascular endothelial cells (ECs). By using quantitative real-time PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA), we show that either ectopic expression of orphan nuclear receptor Nur77 or pharmacological activati...

  16. Msx1 is expressed in retina endothelial cells at artery branching sites

    OpenAIRE

    Miguel Lopes; Olivier Goupille; Cécile Saint Cloment; Benoît Robert

    2012-01-01

    Summary Msx1 and Msx2 encode homeodomain transcription factors that play a role in several embryonic developmental processes. Previously, we have shown that in the adult mouse, Msx1lacZ is expressed in vascular smooth muscle cells (VSMCs) and pericytes, and that Msx2lacZ is also expressed in VSMCs as well as in a few endothelial cells (ECs). The mouse retina and choroid are two highly vascularized tissues. Vessel alterations in the retina are associated with several human diseases and the ret...

  17. The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

    Science.gov (United States)

    Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

    2002-09-15

    Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

  18. Date syrup-derived polyphenols attenuate angiogenic responses and exhibits anti-inflammatory activity mediated by vascular endothelial growth factor and cyclooxygenase-2 expression in endothelial cells.

    Science.gov (United States)

    Taleb, Hajer; Morris, R Keith; Withycombe, Cathryn E; Maddocks, Sarah E; Kanekanian, Ara D

    2016-07-01

    Bioactive components such as polyphenols, present in many plants, are purported to have anti-inflammatory and antiangiogenic properties. Date syrup, produced from date fruit of the date palm tree, has traditionally been used to treat a wide range of diseases with etiologies involving angiogenesis and inflammation. It was hypothesized that polyphenols in date syrup reduce angiogenic responses such as cell migration, tube formation, and matrix metalloproteinase activity in an inflammatory model by exhibiting anti-inflammatory activity mediated by vascular endothelial growth factor (VEGF) and the prostaglandin enzyme cyclooxygenase-2 (COX-2) in endothelial cells. Date syrup polyphenols at 60 and 600μg/mL reduced inflammation and suppressed several stages of angiogenesis, including endothelial cell migration, invasion, matrix metalloproteinase activity, and tube formation, without evidence of cytotoxicity. VEGF and COX-2 expression induced by tumor necrosis factor-alpha at both gene expression and protein level was significantly reduced by date syrup polyphenols in comparison to untreated cells. In conclusion, polyphenols in date syrup attenuated angiogenic responses and exhibited anti-inflammatory activity mediated by VEGF and COX-2 expression in endothelial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Tissue Factor-Expressing Tumor-Derived Extracellular Vesicles Activate Quiescent Endothelial Cells via Protease-Activated Receptor-1

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    Sara P. Y. Che

    2017-11-01

    Full Text Available Tissue factor (TF-expressing tumor-derived extracellular vesicles (EVs can promote metastasis and pre-metastatic niche formation, but the mechanisms by which this occurs remain largely unknown. We hypothesized that generation of activated factor X (FXa by TF expressed on tumor-derived EV could activate protease-activated receptors (PARs on non-activated endothelial cells to induce a pro-adhesive and pro-inflammatory phenotype. We obtained EV from TF-expressing breast (MDA-MB-231 and pancreatic (BxPC3 and Capan-1 tumor cell lines. We measured expression of E-selectin and secretion of interleukin-8 (IL-8 in human umbilical vein endothelial cells after exposure to EV and various immunologic and chemical inhibitors of TF, FXa, PAR-1, and PAR-2. After 6 h of exposure to tumor-derived EV (pretreated with factor VIIa and FX in vitro, endothelial cells upregulated E-selectin expression and secreted IL-8. These changes were decreased with an anti-TF antibody, FXa inhibitors (FPRCK and EGRCK, and PAR-1 antagonist (E5555, demonstrating that FXa generated by TF-expressing tumor-derived EV was signaling through endothelial PAR-1. Due to weak constitutive PAR-2 expression, these endothelial responses were not induced by a PAR-2 agonist peptide (SLIGKV and were not inhibited by a PAR-2 antagonist (FSLLRY after exposure to tumor-derived EV. In conclusion, we found that TF-expressing cancer-derived EVs activate quiescent endothelial cells, upregulating E-selectin and inducing IL-8 secretion through generation of FXa and cleavage of PAR-1. Conversion of resting endothelial cells to an activated phenotype by TF-expressing cancer-derived EV could promote cancer metastases.

  20. Salt-induced Na+/K+-ATPase-α/β expression involves soluble adenylyl cyclase in endothelial cells.

    Science.gov (United States)

    Mewes, Mirja; Nedele, Johanna; Schelleckes, Katrin; Bondareva, Olga; Lenders, Malte; Kusche-Vihrog, Kristina; Schnittler, Hans-Joachim; Brand, Stefan-Martin; Schmitz, Boris; Brand, Eva

    2017-10-01

    High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca 2+ - and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na + /K + -ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na + )-induced Na + /K + -ATPase-α and Na + /K + -ATPase-β protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na + /K + -ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na + /K + -ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na + /K + -ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na + /K + -ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na + /K + -ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

  1. Expression Profiling of Genes Related to Endothelial Cells Biology in Patients with Type 2 Diabetes and Patients with Prediabetes

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    Sara Moradipoor

    2016-01-01

    Full Text Available Endothelial dysfunction appears to be an early sign indicating vascular damage and predicts the progression of atherosclerosis and cardiovascular disorders. Extensive clinical and experimental evidence suggests that endothelial dysfunction occurs in Type 2 Diabetes Mellitus (T2DM and prediabetes patients. This study was carried out with an aim to appraise the expression levels in the peripheral blood of 84 genes related to endothelial cells biology in patients with diagnosed T2DM or prediabetes, trying to identify new genes whose expression might be changed under these pathological conditions. The study covered a total of 45 participants. The participants were divided into three groups: group 1, patients with T2DM; group 2, patients with prediabetes; group 3, control group. The gene expression analysis was performed using the Endothelial Cell Biology RT2 Profiler PCR Array. In the case of T2DM, 59 genes were found to be upregulated, and four genes were observed to be downregulated. In prediabetes patients, increased expression was observed for 49 genes, with two downregulated genes observed. Our results indicate that diabetic and prediabetic conditions change the expression levels of genes related to endothelial cells biology and, consequently, may increase the risk for occurrence of endothelial dysfunction.

  2. Arterial response to shear stress critically depends on endothelial TRPV4 expression.

    Directory of Open Access Journals (Sweden)

    Veronika Hartmannsgruber

    . CONCLUSIONS/SIGNIFICANCE: Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca(2+-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.

  3. Arterial Response to Shear Stress Critically Depends on Endothelial TRPV4 Expression

    Science.gov (United States)

    Kacik, Michael; Kaistha, Anuradha; Grgic, Ivica; Harteneck, Christian; Liedtke, Wolfgang; Hoyer, Joachim; Köhler, Ralf

    2007-01-01

    /Significance Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca2+-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction. PMID:17786199

  4. Suppression of endothelial t-PA expression by prolonged high laminar shear stress

    International Nuclear Information System (INIS)

    Ulfhammer, Erik; Carlstroem, Maria; Bergh, Niklas; Larsson, Pia; Karlsson, Lena; Jern, Sverker

    2009-01-01

    Primary hypertension is associated with an impaired capacity for acute release of endothelial tissue-type plasminogen activator (t-PA), which is an important local protective response to prevent thrombus extension. As hypertensive vascular remodeling potentially results in increased vascular wall shear stress, we investigated the impact of shear on regulation of t-PA. Cultured human endothelial cells were exposed to low (≤1.5 dyn/cm 2 ) or high (25 dyn/cm 2 ) laminar shear stress for up to 48 h in two different experimental models. Using real-time RT-PCR and ELISA, shear stress was observed to time and magnitude-dependently suppress t-PA transcript and protein secretion to approximately 30% of basal levels. Mechanistic experiments revealed reduced nuclear protein binding to the t-PA specific CRE element (EMSA) and an almost completely abrogated shear response with pharmacologic JNK inhibition. We conclude that prolonged high laminar shear stress suppresses endothelial t-PA expression and may therefore contribute to the enhanced risk of arterial thrombosis in hypertensive disease.

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

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    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.

  6. The coffee diterpene kahweol inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules in human endothelial cells

    International Nuclear Information System (INIS)

    Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang

    2006-01-01

    Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNFα-induced monocytes to endothelial cells and suppressed the TNFα-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNFα-induced JAK2-PI3K/Akt-NF-κB activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells

  7. Interleukin 6-Mediated Endothelial Barrier Disturbances Can Be Attenuated by Blockade of the IL6 Receptor Expressed in Brain Microvascular Endothelial Cells.

    Science.gov (United States)

    Blecharz-Lang, Kinga G; Wagner, Josephin; Fries, Alexa; Nieminen-Kelhä, Melina; Rösner, Jörg; Schneider, Ulf C; Vajkoczy, Peter

    2018-02-10

    Compromised blood-brain barrier (BBB) by dysregulation of cellular junctions is a hallmark of many cerebrovascular disorders due to the pro-inflammatory cytokines action. Interleukin 6 (IL6) is implicated in inflammatory processes and in secondary brain injury after subarachnoid hemorrhage (SAH) but its role in the maintenance of cerebral endothelium still requires a precise elucidation. Although IL6 has been shown to exert pro-inflammatory action on brain microvascular endothelial cells (ECs), the expression of one of the IL6 receptors, the IL6R is controversially discussed. In attempt to reach more clarity in this issue, we present here an evident baseline expression of the IL6R in BBB endothelium in vivo and in an in vitro model of the BBB, the cEND cell line. A significantly increased expression of IL6R and its ligand was observed in BBB capillaries 2 days after experimental SAH in mice. In vitro, we saw IL6 administration resulting in an intracellular and extracellular elevation of IL6 protein, which was accompanied by a reduced expression of tight and adherens junctions, claudin-5, occludin, and vascular-endothelial (VE-) cadherin. By functional assays, we could demonstrate IL6-incubated brain ECs to lose their endothelial integrity that can be attenuated by inhibiting the IL6R. Blockade of the IL6R by a neutralizing antibody has reconstituted the intercellular junction expression to the control level and caused a restoration of the transendothelial electrical resistance of the cEND cell monolayer. Our findings add depth to the current understanding of the involvement of the endothelial IL6R in the loss of EC integrity implicating potential therapy options.

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

    LENUS (Irish Health Repository)

    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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  11. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema

    DEFF Research Database (Denmark)

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten

    2011-01-01

    (VEGF) is an endothelial cell-specific mitogen and angiogen. VEGF-A protein, which is identical to vascular permeability factor, is a regulator of angiogenesis. In this study, 101 patients with meningiomas, and possible co-factors to PTBE, such as meningioma subtypes and tumor location, were examined....... Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression...... in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p

  12. Enhanced Vascular Endothelial Growth Factor Gene Expression in Ischaemic Skin of Critical Limb Ischaemia Patients

    Directory of Open Access Journals (Sweden)

    Silvia Bleda

    2012-01-01

    Full Text Available Objectives. To perform a quantitative analysis of the vascular endothelial growth factor (VEGF gene transcription in the skin of ischemic legs and provide information for VEGF in the pathogenesis in critical limb ischemia (CLI. Methods. Skin biopsies were obtained from 40 patients with CLI. Control samples came from 44 patients with chronic venous disease. VEGF gene expression was analysed using quantitative polymerase chain reaction. Results. Patients with CLI had higher skin VEGF expression than control group (RQ: 1.3 ± 0.1 versus 1, P=0.04. Conclusions. We found an association between ischemic skin and an elevated VEGF expression in legs from patients with CLI. These data support that the mechanism for VEGF upregulation in hypoxia conditions is intact and acts appropriately in the ischaemic limbs from patients with CLI.

  13. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema

    DEFF Research Database (Denmark)

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten

    2011-01-01

    Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor....... Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression...... in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p

  14. Anatomical specificity of vascular endothelial growth factor expression in glioblastomas: a voxel-based mapping analysis

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    Fan, Xing [Capital Medical University, Department of Neurosurgery, Beijing Tiantan Hospital, Beijing (China); Wang, Yinyan [Capital Medical University, Department of Neurosurgery, Beijing Tiantan Hospital, Beijing (China); Capital Medical University, Department of Neuropathology, Beijing Neurosurgical Institute, Beijing (China); Wang, Kai; Ma, Jun; Li, Shaowu [Capital Medical University, Department of Neuroradiology, Beijing Tiantan Hospital, Beijing (China); Liu, Shuai [Chinese Academy of Medical Sciences and Peking Union Medical College, Departments of Neurosurgery, Peking Union Medical College Hospital, Beijing (China); Liu, Yong [Chinese Academy of Sciences, Brainnetome Center, Institute of Automation, Beijing (China); Jiang, Tao [Capital Medical University, Department of Neurosurgery, Beijing Tiantan Hospital, Beijing (China); Beijing Academy of Critical Illness in Brain, Department of Clinical Oncology, Beijing (China)

    2016-01-15

    The expression of vascular endothelial growth factor (VEGF) is a common genetic alteration in malignant gliomas and contributes to the angiogenesis of tumors. This study aimed to investigate the anatomical specificity of VEGF expression levels in glioblastomas using voxel-based neuroimaging analysis. Clinical information, MR scans, and immunohistochemistry stains of 209 patients with glioblastomas were reviewed. All tumor lesions were segmented manually and subsequently registered to standard brain space. Voxel-based regression analysis was performed to correlate the brain regions of tumor involvement with the level of VEGF expression. Brain regions identified as significantly associated with high or low VEGF expression were preserved following permutation correction. High VEGF expression was detected in 123 (58.9 %) of the 209 patients. Voxel-based statistical analysis demonstrated that high VEGF expression was more likely in tumors located in the left frontal lobe and the right caudate and low VEGF expression was more likely in tumors that occurred in the posterior region of the right lateral ventricle. Voxel-based neuroimaging analysis revealed the anatomic specificity of VEGF expression in glioblastoma, which may further our understanding of genetic heterogeneity during tumor origination. This finding provides primary theoretical support for potential future application of customized antiangiogenic therapy. (orig.)

  15. Anatomical specificity of vascular endothelial growth factor expression in glioblastomas: a voxel-based mapping analysis

    International Nuclear Information System (INIS)

    Fan, Xing; Wang, Yinyan; Wang, Kai; Ma, Jun; Li, Shaowu; Liu, Shuai; Liu, Yong; Jiang, Tao

    2016-01-01

    The expression of vascular endothelial growth factor (VEGF) is a common genetic alteration in malignant gliomas and contributes to the angiogenesis of tumors. This study aimed to investigate the anatomical specificity of VEGF expression levels in glioblastomas using voxel-based neuroimaging analysis. Clinical information, MR scans, and immunohistochemistry stains of 209 patients with glioblastomas were reviewed. All tumor lesions were segmented manually and subsequently registered to standard brain space. Voxel-based regression analysis was performed to correlate the brain regions of tumor involvement with the level of VEGF expression. Brain regions identified as significantly associated with high or low VEGF expression were preserved following permutation correction. High VEGF expression was detected in 123 (58.9 %) of the 209 patients. Voxel-based statistical analysis demonstrated that high VEGF expression was more likely in tumors located in the left frontal lobe and the right caudate and low VEGF expression was more likely in tumors that occurred in the posterior region of the right lateral ventricle. Voxel-based neuroimaging analysis revealed the anatomic specificity of VEGF expression in glioblastoma, which may further our understanding of genetic heterogeneity during tumor origination. This finding provides primary theoretical support for potential future application of customized antiangiogenic therapy. (orig.)

  16. Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo

    DEFF Research Database (Denmark)

    Rømer, Johanne Lade

    2004-01-01

    Integrin alpha4beta 7 is the principal gut-homing receptor, and it is assumed that expression of this specific integrin directs lymphocytes to the gut in vivo. Adoptive cellular immunotherapy against inflammatory bowel disease (IBD) may depend on the expression of integrin alpha4beta 7...

  17. α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chung

    Full Text Available Endothelial cells (ECs express fibroblast growth factor (FGF receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs and human brain microvascular endothelial cells (HBMECs were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS expression, nitric oxide (NO production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

  18. Effect of irradiation on gene expression of rat liver adhesion molecules. In vivo and in vitro studies

    International Nuclear Information System (INIS)

    Moriconi, Federico; Malik, Ihtzaz; Ahmad, Ghayyor; Dudas, Joszef; Ramadori, Giuliano; Rave-Fraenk, Margret; Vorwerk, Hilke; Hille, Andrea; Hess, Clemens Friedrich; Christiansen, Hans

    2009-01-01

    Background and purpose: Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro. Material and methods: Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology. Results: Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β 1 -integrin, β 2 -integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β 1 -integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β 2 -integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1. Conclusion: The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver. (orig.)

  19. Correlation between matrix metalloproteinase-9 and vascular endothelial growth factor expression in lung adenocarcinoma.

    Science.gov (United States)

    Wen, Y L; Li, L

    2015-12-29

    The aim of this study was to investigate the correlation between the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) and clinicopathological features of lung adenocarcinoma. The expression of MMP-9 and VEGF was evaluated by immunohistochemistry of 30 samples from lung adenocarcinoma patients and 12 paratumoral (normal) tissue samples. In addition, the change in VEGF or MMP-9 expression after MMP-9 or VEGF blockade, respectively, was measured using western blot in lung adenocarcinoma A549 cells. High expression of MMP-9 was found in 63.3% of adenocarcinoma tissues versus 16.7% in normal tissues (P correlation was identified between MMP-9 and VEGF expression (correlation coefficient = 0.7094, P < 0.001), and their mutual overexpression was associated with clinical staging and lymph node status (P < 0.05). In addition, an decrease in VEGF protein expression was observed after MMP-9 blockade by an MMP-9-specific monoclonal antibody. Similarly, a decrease in MMP-9 protein expression was found after VEGF blockade by a VEGF-specific monoclonal antibody. In conclusion, VEGF and MMP-9 are overexpressed in lung adenocarcinoma tissues, and they have a synergistic effect on the invasion and metastasis of adenocarcinoma.

  20. HDAC Inhibition in Vascular Endothelial Cells Regulates the Expression of ncRNAs

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    Haloom Rafehi

    2016-05-01

    Full Text Available While clinical and pre-clinical trials indicate efficacy of histone deacetylase (HDAC inhibitors in disease mediated by dynamic lysine modification, the impact on the expression of non-coding RNAs (ncRNAs remains poorly understood. In this study, we investigate high throughput RNA sequencing data derived from primary human endothelial cells stimulated with HDAC inhibitors suberanilohydroxamic acid (SAHA and Trichostatin A (TSA. We observe robust regulation of ncRNA expression. Integration of gene expression data with histone 3 lysine 9 and 14 acetylation (H3K9/14ac and histone 3 lysine 4 trimethylation (H3K4me3 datasets identified complex and class-specific expression of ncRNAs. We show that EP300 target genes are subject to histone deacetylation at their promoter following HDAC inhibition. This deacetylation drives suppression of protein-coding genes. However, long intergenic non-coding RNAs (lincRNAs regulated by EP300 are activated following HDAC inhibition, despite histone deacetylation. This increased expression was driven by increased H3K4me3 at the gene promoter. For example, elevated promoter H3K4me3 increased lincRNA MALAT1 expression despite broad EP300-associated histone deacetylation. In conclusion, we show that HDAC inhibitors regulate the expression of ncRNA by complex and class-specific epigenetic mechanisms.

  1. Placental growth factor expression is reversed by antivascular endothelial growth factor therapy under hypoxic conditions.

    Science.gov (United States)

    Zhou, Ai-Yi; Bai, Yu-Jing; Zhao, Min; Yu, Wen-Zhen; Huang, Lv-Zhen; Li, Xiao-Xin

    2014-08-01

    Clinical trials have revealed that the antivascular endothelial growth factor (VEGF) therapies are effective in retinopathy of prematurity (ROP). But the low level of VEGF was necessary as a survival signal in healthy conditions, and endogenous placental growth factor (PIGF) is redundant for development. The purpose of this study was to elucidate the PIGF expression under hypoxia as well as the influence of anti-VEGF therapy on PIGF. CoCl2-induced hypoxic human umbilical vein endothelial cells (HUVECs) were used for an in vitro study, and oxygen-induced retinopathy (OIR) mice models were used for an in vivo study. The expression patterns of PIGF under hypoxic conditions and the influence of anti-VEGF therapy on PIGF were evaluated by quantitative reverse transcription-polymerase chain reaction (RTPCR). The retinal avascular areas and neovascularization (NV) areas of anti-VEGF, anti-PIGF and combination treatments were calculated. Retina PIGF concentration was evaluated by ELISA after treatment. The vasoactive effects of exogenous PIGF on HUVECs were investigated by proliferation and migration studies. PIGF mRNA expression was reduced by hypoxia in OIR mice, in HUVECs under hypoxia and anti-VEGF treatment. However, PIGF expression was reversed by anti-VEGF therapy in the OIR model and in HUVECs under hypoxia. Exogenous PIGF significantly inhibited HUVECs proliferation and migration under normal conditions, but it stimulated cell proliferation and migration under hypoxia. Anti-PIGF treatment was effective for neovascular tufts in OIR mice (P<0.05). The finding that PIGF expression is iatrogenically up-regulated by anti-VEGF therapy provides a consideration to combine it with anti-PIGF therapy.

  2. Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

    Science.gov (United States)

    Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

    2009-01-01

    Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

  3. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  4. Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

    DEFF Research Database (Denmark)

    Castillo, M B; Berchtold, M W; Rülicke, T

    1997-01-01

    To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

  5. Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

    Science.gov (United States)

    Cho, Young S; Kim, Chan H; Ha, Tae S; Ahn, Hee Y

    2015-11-18

    Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

  6. Elevated procoagulant endothelial and tissue factor expressing microparticles in women with recurrent pregnancy loss.

    Directory of Open Access Journals (Sweden)

    Rucha Patil

    Full Text Available BACKGROUND: 15% of reproducing couples suffer from pregnancy loss(PL and recurs in 2-3%. One of the most frequently hypothesized causes of unexplained PL refers to a defective maternal haemostatic response leading to uteroplacental thrombosis. Hereditary thrombophilia and antiphospholipid antibodies have been extensively described as risk factors for PL in women with unknown aetiology. Recently, a new marker has emerged: the cell-derived procoagulant circulating microparticles(MPs which have been reported to have a major role in many thrombosis complicated diseases. This study aims to analyze the significance of procoagulant MPs in women suffering from unexplained recurrent pregnancy loss(RPL, and characterize their cellular origin. METHOD AND FINDINGS: 115 women with RPL were analyzed for common thrombophilia markers and different cell derived MPs-total annexinV, platelet(CD41a, endothelial(CD146,CD62e, leukocyte(CD45, erythrocyte(CD235a and tissue factor(CD142(TF expressing MPs and were compared with 20 healthy non-pregnant women. Methodology for MP analysis was standardized by participating in the "Vascular Biology Scientific and Standardization Committee workshop". RESULTS: Total annexinV, TF and endothelial MPs were found significantly increased(p<0.05, 95% confidence interval in women with RPL. The procoagulant activity of MPs measured by STA-PPL clotting time assay was found in correspondence with annexinV MP levels, wherein the clot time was shortened in samples with increased MP levels. Differences in platelet, leukocyte and erythrocyte derived MPs were not significant. Thirty seven of 115 women were found to carry any of the acquired or hereditary thrombophilia markers. No significant differences were seen in the MP profile of women with and without thrombophilia marker. CONCLUSION: The presence of elevated endothelial, TF and phosphatidylserine expressing MPs at a distance (at least 3 months from the PL suggests a continued chronic

  7. Polychlorinated biphenyl-induced VCAM-1 expression is attenuated in aortic endothelial cells isolated from caveolin-1 deficient mice

    International Nuclear Information System (INIS)

    Han, Sung Gu; Eum, Sung Yong; Toborek, Michal; Smart, Eric; Hennig, Bernhard

    2010-01-01

    Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of cardiovascular diseases such as atherosclerosis. Vascular cell adhesion molecule-1 (VCAM-1) is a critical mediator for adhesion and uptake of monocytes across the endothelium in the early stages of atherosclerosis development. The upregulation of VCAM-1 by PCBs may be dependent on functional membrane domains called caveolae. Caveolae are particularly abundant in endothelial cell membranes and involved in trafficking and signal transduction. The objective of this study was to investigate the role of caveolae in PCB-induced endothelial cell dysfunction. Primary mouse aortic endothelial cells (MAECs) isolated from caveolin-1-deficient mice and background C57BL/6 mice were treated with coplanar PCBs, such as PCB77 and PCB126. In addition, siRNA gene silencing technique was used to knockdown caveolin-1 in porcine vascular endothelial cells. In MAECs with functional caveolae, VCAM-1 protein levels were increased after exposure to both coplanar PCBs, whereas expression levels of VCAM-1 were not significantly altered in cells deficient of caveolin-1. Furthermore, PCB-induced monocyte adhesion was attenuated in caveolin-1-deficient MAECs. Similarly, siRNA silencing of caveolin-1 in porcine endothelial cells confirmed the caveolin-1-dependent VCAM-1 expression. Treatment of cells with PCB77 and PCB126 resulted in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), and pharmacological inhibition of ERK1/2 diminished the observed PCB-induced increase in monocyte adhesion. These findings suggest that coplanar PCBs induce adhesion molecule expression, such as VCAM-1, in endothelial cells, and that this response is regulated by caveolin-1 and functional caveolae. Our data demonstrate a critical role of functional caveolae in the activation and dysfunction of endothelial cells by coplanar PCBs.

  8. VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

    Science.gov (United States)

    Fearnley, Gareth W; Odell, Adam F; Latham, Antony M; Mughal, Nadeem A; Bruns, Alexander F; Burgoyne, Nicholas J; Homer-Vanniasinkam, Shervanthi; Zachary, Ian C; Hollstein, Monica C; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2014-08-15

    Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways. © 2014 Fearnley et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

    Science.gov (United States)

    Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

    2005-06-01

    To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

  10. Time-course gene expression data on the transcriptional effects of Aminaphtone on ECV304 endothelial cells

    Directory of Open Access Journals (Sweden)

    Giulia Salazar

    2016-09-01

    Full Text Available We previously showed that Aminaphtone, a drug used in the treatment of chronic venous insufficiency, modulates several vasoactive factors, such as endothelin-1 and adhesion molecules. Here, we provide data of time-course experiments about the effects of Aminaphtone on gene expression at the genome-wide level in human endothelial cells undergoing cytokine stimulation in vitro. ECV-304 endothelial cells were incubated with interleukin-1β (IL-1β in the presence or absence of Aminaphtone for 1, 3, and 6 h. Gene expression profiles were analyzed by microarray. This article contains complete data on the genes significantly modulated by the drug over time. The data are supplemental to our original research article reporting detailed analysis of the actions of Aminaphtone on IL-1β stimulated endothelial cells at the molecular level, ''Gene expression profiling reveals novel protective effects of Aminaphtone on ECV304 endothelial cells'' (Salazar et al., 2016 [1]. Chemical compound studied in this article: Aminaphtone (PubChem CID: 84621, Keywords: Endothelial cells, Transcriptome, Inflammation, Vasoactive drug

  11. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  12. Hepatocellular hypoxia-induced vascular endothelial growth factor expression and angiogenesis in experimental biliary cirrhosis.

    Science.gov (United States)

    Rosmorduc, O; Wendum, D; Corpechot, C; Galy, B; Sebbagh, N; Raleigh, J; Housset, C; Poupon, R

    1999-10-01

    We tested the potential role of vascular endothelial growth factor (VEGF) and of fibroblast growth factor-2 (FGF-2) in the angiogenesis associated with experimental liver fibrogenesis induced by common bile duct ligation in Sprague-Dawley rats. In normal rats, VEGF and FGF-2 immunoreactivities were restricted to less than 3% of hepatocytes. One week after bile duct ligation, hypoxia was demonstrated by the immunodetection of pimonidazole adducts unevenly distributed throughout the lobule. After 2 weeks, hypoxia and VEGF expression were detected in >95% of hepatocytes and coexisted with an increase in periportal vascular endothelial cell proliferation, as ascertained by Ki67 immunolabeling. Subsequently, at 3 weeks the density of von Willebrand-labeled vascular section in fibrotic areas significantly increased. Semiquantitative reverse transcription polymerase chain reaction showed that VEGF(120) and VEGF(164) transcripts, that correspond to secreted isoforms, increased within 2 weeks, while VEGF(188) transcripts remained unchanged. FGF-2 mainly consisting of a 22-kd isoform, according to Western blot, was identified by immunohistochemistry in 49% and 100% of hepatocytes at 3 and 7 weeks, respectively. Our data provide evidence that in biliary-type liver fibrogenesis, angiogenesis is stimulated primarily by VEGF in response to hepatocellular hypoxia while FGF-2 likely contributes to the maintenance of angiogenesis at later stages.

  13. Expression of Metallothionein and Vascular Endothelial Growth Factor Isoforms in Breast Cancer Cells.

    Science.gov (United States)

    Wierzowiecka, Barbara; Gomulkiewicz, Agnieszka; Cwynar-Zajac, Lucja; Olbromski, Mateusz; Grzegrzolka, Jedrzej; Kobierzycki, Christopher; Podhorska-Okolow, Marzenna; Dziegiel, Piotr

    2016-01-01

    Metallothioneins (MTs) are low-molecular-weight and cysteine-rich proteins that bind heavy metal ions and oxygen-free radicals. MTs are commonly expressed in various tissues of mammals and are involved in regulation of cell proliferation and differentiation, and may be engaged in angiogenesis. Expression of MTs has been studied in many cancer types, especially breast cancer. The research results indicate that MTs may play important, although not yet fully known, roles in cancer angiogenesis. The aim of this study was to analyze the level of gene expression of selected MT isoforms induced with zinc ions in correlation with vascular endothelial growth factor (VEGF) isoforms in in vitro models of breast cancer. The studies were carried out in three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231). An epithelial cell line derived from normal breast tissue (Me16c) was used as a control. The levels of expression of selected MT isoforms and selected genes involved in angiogenesis were studied with real-time PCR. Expression of different MT isoforms was induced by zinc ions to differing degrees in individual breast cancer cell lines. An increase in the expression of some MT isoforms was associated with a slight increase in the level of expression of VEGFA. The research results may indicate certain correlation between an increased expression of selected MT isoforms and a pro-angiogenic factor VEGF in specific types of breast cancer cells. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  14. Vascular endothelial growth factor and basic fibroblast growth factor expression positively correlates with angiogenesis and peritumoural brain oedema in astrocytoma

    International Nuclear Information System (INIS)

    Jang, F.F.; Wei, W.

    2008-01-01

    Astrocytoma is the most malignant intracranial neoplasm and is characterized by high neovascularization and peritumoural brain oedema. Angiogenesis is a complicated process in oncogenesis regulated by the balance between angiogenic and antiangiogenic factors. The expression of two angiogenic growth factors, vascular endothelial growth factor and basic fibroblast growth factor were investigated using immunohistochemistry for astrocytoma from 82 patients and 11 normal human tissues. The expression of vascular endothelial growth factor and basic fibroblast growth factor positively correlate with the pathological grade of astrocytoma, microvessel density numbers and brain oedema, which may be responsible for the increased tumour neovascularization and peritumoural brain oedema. The results support the idea that inhibiting vascular endothelial growth factor and basic fibroblast growth factor are useful for the treatment of human astrocytoma and to improve patient's clinical outcomes and prognosis. (author)

  15. Msx1 is expressed in retina endothelial cells at artery branching sites

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    Miguel Lopes

    2012-02-01

    Msx1 and Msx2 encode homeodomain transcription factors that play a role in several embryonic developmental processes. Previously, we have shown that in the adult mouse, Msx1lacZ is expressed in vascular smooth muscle cells (VSMCs and pericytes, and that Msx2lacZ is also expressed in VSMCs as well as in a few endothelial cells (ECs. The mouse retina and choroid are two highly vascularized tissues. Vessel alterations in the retina are associated with several human diseases and the retina has been intensely used for angiogenesis studies, whereas the choroid has been much less investigated. Using the Msx1lacZ and Msx2lacZ reporter alleles, we observed that Msx2 is not expressed in the eye vascular tree in contrast to Msx1, for which we establish the spatial and temporal expression pattern in these tissues. In the retina, expression of Msx1 takes place from P3, and by P10, it becomes confined to a subpopulation of ECs at branching points of superficial arterioles. These branching sites are characterized by a subpopulation of mural cells that also show specific expression programs. Specific Msx gene inactivation in the endothelium, using Msx1 and Msx2 conditional mutant alleles together with a Tie2-Cre transgene, did not lead to conspicuous structural defects in the retinal vascular network. Expression of Msx1 at branching sites might therefore be linked to vessel physiology. The retinal blood flow is autonomously regulated and perfusion of capillaries has been proposed to depend on arteriolar precapillary structures that might be the sites for Msx1 expression. On the other hand, branching sites are subject to shear stress that might induce Msx1 expression. In the choroid vascular layer Msx1lacZ is expressed more broadly and dynamically. At birth Msx1lacZ expression takes place in the endothelium but at P21 its expression has shifted towards the mural layer. We discuss the possible functions of Msx1 in the eye vasculature.

  16. Msx1 is expressed in retina endothelial cells at artery branching sites.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Robert, Benoît

    2012-04-15

    Msx1 and Msx2 encode homeodomain transcription factors that play a role in several embryonic developmental processes. Previously, we have shown that in the adult mouse, Msx1(lacZ) is expressed in vascular smooth muscle cells (VSMCs) and pericytes, and that Msx2(lacZ) is also expressed in VSMCs as well as in a few endothelial cells (ECs). The mouse retina and choroid are two highly vascularized tissues. Vessel alterations in the retina are associated with several human diseases and the retina has been intensely used for angiogenesis studies, whereas the choroid has been much less investigated. Using the Msx1(lacZ) and Msx2(lacZ) reporter alleles, we observed that Msx2 is not expressed in the eye vascular tree in contrast to Msx1, for which we establish the spatial and temporal expression pattern in these tissues. In the retina, expression of Msx1 takes place from P3, and by P10, it becomes confined to a subpopulation of ECs at branching points of superficial arterioles. These branching sites are characterized by a subpopulation of mural cells that also show specific expression programs. Specific Msx gene inactivation in the endothelium, using Msx1 and Msx2 conditional mutant alleles together with a Tie2-Cre transgene, did not lead to conspicuous structural defects in the retinal vascular network. Expression of Msx1 at branching sites might therefore be linked to vessel physiology. The retinal blood flow is autonomously regulated and perfusion of capillaries has been proposed to depend on arteriolar precapillary structures that might be the sites for Msx1 expression. On the other hand, branching sites are subject to shear stress that might induce Msx1 expression. In the choroid vascular layer Msx1(lacZ) is expressed more broadly and dynamically. At birth Msx1(lacZ) expression takes place in the endothelium but at P21 its expression has shifted towards the mural layer. We discuss the possible functions of Msx1 in the eye vasculature.

  17. Air pollution upregulates endothelial cell procoagulant activity via ultrafine particle-induced oxidant signaling and tissue factor expression.

    Science.gov (United States)

    Snow, S J; Cheng, W; Wolberg, A S; Carraway, M S

    2014-07-01

    Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure. Published by Oxford University Press on behalf of Toxicological Sciences 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  18. Altered expressions of endothelial junction protein of placental capillaries in premature infants with intraventricular hemorrhage

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    Maria Ekawati

    2016-10-01

    Full Text Available Background: Placental hypoxia may lead to oxidative stress, which inflicts damage to capillary protein junction. The aim of this study was to evaluate altered expression of endothelial junction protein of capillaries in hypoxia condition and to observe its correlation with the incidence of  intraventricular hemorrhage in premature infants.Methods: A cross-sectional study was conducted by using placental tissues of premature infants as amodel of capillary integrity (29 hypoxic and 29 non-hypoxic. Hypoxia inducible factor (HIF-1α was measured to define placental tissue response to hypoxia; malondialdehyde (MDA and glutathione (GSH served as markers of oxidative stress. The expressions of junctional proteins, N-cadherin and occludin were analyzed by immunohistochemistry. Intraventricular hemorrhage (IVH was detected by cranial ultrasound at the third day. Unpaired t test, Mann-Whitney, and Chi-square tests were used to analyze the data.Results: The HIF-1α and MDA levels were slightly, but not significantly, higher in hypoxia group {13.64±8.70 pg/mg protein and 10.31 pmol/mg tissue (ranged 1.92–93.61, respectively}  compared to non- hypoxia group {10.65±5.35 pg/mg protein and 9.77 pmol/mg tissue (ranged 2.42–93.31}. GSH levels were not different in both groups (38.14 (ranged 9.44–118.91 and  38.47(ranged 16.49–126.76 ng/mg protein, respectively. mRNA expression of N-cadherin (0.13 and occludin (0.096 were significantly lower in hypoxia comparedto non-hypoxia group (p=0,001, while protein expression of  N-cadherin (3.4; 75.9; 6.9; 13.8% and occludin  (20.7; 3.4; 69.0; 3.4; 6.9%  in hypoxia group was not associated with IVH (p=0.783 and p=0.743.Conclusion: Hypoxia altered expression of endothelial junction protein in placental capillaries, but no association with intraventricular hemorrhage was observed.

  19. Ethanol stimulates tumor progression and expression of vascular endothelial growth factor in chick embryos.

    Science.gov (United States)

    Gu, Jian-Wei; Bailey, Amelia Purser; Sartin, Amanda; Makey, Ian; Brady, Ann L

    2005-01-15

    The mechanisms by which alcohol consumption causes cancer have not been established due to a lack of experimental studies. A chick embryo chorioallantoic membrane (CAM) model that bore human fibrosarcoma (HT1080) was used to determine whether the administration of physiologically relevant doses of ethanol could stimulate tumor growth, angiogenesis, metastasis, and vascular endothelial growth factor (VEGF) expression in tumors. HT1080 cells were inoculated onto the "upper CAM" on Day 8, saline or ethanol was administrated at a dose of 0.25 g/kg per day on the CAM, and the tumors were harvested on Day 17. VEGF mRNA and protein were determined by Northern blot analysis and enzyme-linked immunosorbent assay. Intratumoral vascular volume density (IVVD) was determined by point counting on periodic acid-Schiff-stained sections. Intravasation of HT1080 cells was determined using human-Alu polymerase chain reaction analysis. The effects of ethanol on VEGF expression and cell proliferation were examined in cultured HT1080 cells. Ethanol treatment for 9 days caused a 2.2-fold increase in tumor volume (867 +/- 138 mm(3) vs. 402 +/- 28 mm(3)), a 2.1-fold increase in IVVD (0.021 +/- 0.004 mm(3)/mm(3) vs. 0.010 mm(3)/mm(3) +/- 0.002 mm(3)/mm(3)), and a significant increase in VEGF mRNA or protein expression in tumors compared with a group of control embryos (n = 6 embryos; P 8-fold in the intravasated HT1080 cells in the CAM group compared with the control group (n = 6 embryos; P < 0.01). Physiologically relevant levels of ethanol (10 mM and 20 mM) caused a dose-related increase in VEGF mRNA and protein expression in cultured HT1080 cells. The ethanol-HT1080-conditioned media increased the proliferation of endothelial cells, but not of HT1080 cells. The findings suggest that the induction of angiogenesis and VEGF expression by ethanol represents an important mechanism of cancer progression associated with alcoholic beverage consumption. (c) 2004 American Cancer Society.

  20. Expression of Vascular Endothelial Growth Factor Receptors in Benign Vascular Lesions of the Orbit: A Case Series.

    Science.gov (United States)

    Atchison, Elizabeth A; Garrity, James A; Castillo, Francisco; Engman, Steven J; Couch, Steven M; Salomão, Diva R

    2016-01-01

    Vascular lesions of the orbit, although not malignant, can cause morbidity because of their location near critical structures in the orbit. For the same reason, they can be challenging to remove surgically. Anti-vascular endothelial growth factor (VEGF) drugs are increasingly being used to treat diseases with prominent angiogenesis. Our study aimed to determine to what extent VEGF receptors and their subtypes are expressed on selected vascular lesions of the orbit. Retrospective case series of all orbital vascular lesions removed by one of the authors (JAG) at the Mayo Clinic. A total of 52 patients who underwent removal of vascular orbital lesions. The pathology specimens from the patients were retrieved, their pathologic diagnosis was confirmed, demographic and clinical information were gathered, and sections from vascular tumors were stained with vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor type 1 (VEGFR1), vascular endothelial growth factor receptor type 2 (VEGFR2), and vascular endothelial growth factor receptor type 3 (VEGFR3). The existence and pattern of staining with VEGF and its subtypes on these lesions. There were 28 specimens of venous malformations, 4 capillary hemangiomas, 7 lymphatic malformations, and 6 lymphaticovenous malformations. All samples stained with VEGF, 55% stained with VEGFR1, 98% stained with VEGFR2, and 96% stained with VEGFR3. Most (94%) of the VEGFR2 staining was diffuse. Most orbital vascular lesions express VEGF receptors, which may suggest a future target for nonsurgical treatment. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  1. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

    NARCIS (Netherlands)

    Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

    1993-01-01

    An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

  2. Vascular expression of endothelial antigen PAL-E indicates absence of blood-ocular barriers in the normal eye

    NARCIS (Netherlands)

    Schlingemann, R. O.; Hofman, P.; Anderson, L.; Troost, D.; van der Gaag, R.

    1997-01-01

    The endothelium-specific antigen PAL-E is expressed in capillaries and veins throughout the body with the exception of the brain, where the antigen is absent from anatomical sites with a patent blood-brain barrier. In this study we determined vascular endothelial staining for PAL-E in the normal eye

  3. Suppression of vascular endothelial growth factor expression by cannabinoids in a canine osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Figueiredo AS

    2013-07-01

    Full Text Available Andreza S Figueiredo,1 Hiram J García-Crescioni,1 Sandra C Bulla,1 Matthew K Ross,2 Chelsea McIntosh,1 Kari Lunsford,3 Camilo Bulla11Department of Pathobiology and Population Medicine, 2Department of Basic Sciences, 3Department of Clinical Sciences and Animal Health Center, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USAAbstract: Vascular endothelial growth factor (VEGF is a key regulator in both physiologic and pathologic angiogenesis, and cannabinoids decrease VEGF release in human and murine cancer cells. The aim of this study was to assess the in vitro effects of a synthetic cannabinoid, WIN-55,212-2, on the expression of the proangiogenic factor VEGF-A in the canine osteosarcoma cell line 8. After analysis of gene expression by quantitative real-time polymerase chain reaction, the compound decreased VEGF-A expression by 35% ± 10% (P < 0.0001 as compared with the control. This synthetic cannabinoid shows promise as a potential inhibitor of angiogenesis, and further studies are warranted to investigate its in vivo effects and to explore the potential of this and related compounds as adjuvant cancer therapy in the dog.Keywords: dog, cancer, angiogenesis, cannabinoids

  4. In vitro and in situ intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells lining a polyester fabric.

    Science.gov (United States)

    Rémy, M; Valli, N; Brethes, D; Labrugère, C; Porté-Durrieu, M C; Dobrova, N B; Novikova, S P; Gorodkov, A J; Bordenave, L

    1999-02-01

    In order to improve long-term patency of vascular grafts, the promising concept of endothelial cell seeding is actually under investigation. Our laboratory tested a polyester coated with albumin and chitosan which permits a rapid colonization by human umbilical vein endothelial cells (HUVEC) and it seems relevant to test in vitro the expression of adhesive molecules expressed by cells with regard to the inflammatory process. We studied intercellular adhesion molecule-1 (ICAM-1) expression and focused our work on the determination of ICAM-1 sites expressed per adherent cell lining the biomaterial, thus in situ, in comparison to control HUVEC on plastic wells: the results obtained by binding experiments were correlated to flow cytometry analyses and showed that the polyester does not induce a proinflammatory state and that HUVEC covering the structure are able to respond to a stimulus.

  5. Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

    Science.gov (United States)

    Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

    2017-01-01

    Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

  6. Dynamic pattern of endothelial cell adhesion molecule expression in muscle and perineural vessels from patients with classic polyarteritis nodosa.

    Science.gov (United States)

    Coll-Vinent, B; Cebrián, M; Cid, M C; Font, C; Esparza, J; Juan, M; Yagüe, J; Urbano-Márquez, A; Grau, J M

    1998-03-01

    To investigate endothelial cell adhesion molecule expression in vessels from patients with classic polyarteritis nodosa (PAN). Frozen sections of 21 muscle and 16 nerve samples from 30 patients with biopsy-proven PAN and 12 histologically normal muscle and 2 histologically normal nerve samples from 12 controls were studied immunohistochemically, using specific monoclonal antibodies (MAb) that recognize adhesion molecules. Adhesion molecules identified were intercellular adhesion molecule 1 (ICAM-1), ICAM-2, ICAM-3, vascular cell adhesion molecule 1 (VCAM-1), platelet endothelial cell adhesion molecule 1 (PECAM-1), E-selectin, P-selectin, L-selectin, lymphocyte function-associated antigen 1 (LFA-1), and very late activation antigen 4 (VLA-4). Neutrophils were identified with a MAb recognizing neutrophil elastase. Endothelial cells were identified with the lectin ulex europaeus. In early lesions, expression of PECAM-1, ICAM-1, ICAM-2, and P-selectin was similar to that in control samples, and VCAM-1 and E-selectin were induced in vascular endothelium. In advanced lesions, immunostaining for adhesion molecules diminished or disappeared in luminal endothelium, whereas these molecules were clearly expressed in microvessels within and surrounding inflamed vessels. Staining in endothelia from vessels in a healing stage tended to be negative. A high proportion of infiltrating leukocytes expressed LFA-1 and VLA-4, and only a minority expressed L-selectin. No relationship between the expression pattern of adhesion molecules and clinical features, disease duration, or previous corticosteroid treatment was observed. Endothelial adhesion molecule expression in PAN is a dynamic process that varies according to the histopathologic stage of the vascular lesions. The preferential expression of constitutive and inducible adhesion molecules in microvessels suggests that angiogenesis contributes to the persistence of inflammatory infiltration in PAN.

  7. HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells.

    Science.gov (United States)

    Nauta, Tessa D; Duyndam, Monique C A; Weijers, Ester M; van Hinsbergh, Victor M W; Koolwijk, Pieter

    2016-01-01

    During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis. We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling. Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction. Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

  8. Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-ling; WEN Liang; CHEN Yan-jiong; ZHU Yi

    2009-01-01

    Background The vascular endothelial growth factor (VEGF) is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 (ICAM-1) is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells (ECs) is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs.Methods Bovine retinal ECs (BRECs) were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF (100 ng/ml) and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase (eNOS) were detected using Western blotting. Griess reaction was used to detect nitric oxide (NO).Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C (PKC) altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase (PI3K) or reactive oxygen species (ROS) significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased.Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

  9. [Expression profiles and bioinformatic analysis of miRNA in human dental pulp cells during endothelial differentiation].

    Science.gov (United States)

    Gong, Qimei; Jiang, Hongwei; Wang, Jinming; Ling, Junqi

    2014-05-01

    To investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation. DPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction. The relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway. The differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

  10. Regulation of endothelial cell adhesion molecule expression by mast cells, macrophages, and neutrophils.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    2011-01-01

    Full Text Available Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.Using bone marrow-derived mast cells from wild-type, Tnf(-/-, Ifng(-/-, Il6(-/- mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1, intercellular adhesion molecule-1 (ICAM-1, P-selectin, and E-selectin in murine heart endothelial cells (MHEC at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases.

  11. Induction stage-dependent expression of vascular endothelial growth factor and aquaporin-1 in diethylstilbestrol-treated rat pituitary

    Directory of Open Access Journals (Sweden)

    W Zhao

    2009-03-01

    Full Text Available The anterior pituitary gland undergoes tumourigenic changes in response to oestrogen treatment in several breeds of rats. We administered diethylstilbestrol (DES to female Wistar rats and assessed whether the expression of vascular endothelial growth factor (VEGF and aquaporin-1 (AQP-1 was altered at different time points following DES administration. In vivo magnetic resonance imaging (MRI scans showed that the mass index corresponding to the mid-sagittal area of DES-treated pituitary was significantly higher than the vehicle-controlled pituitary (p less than 0.01 at three specific time points, accompanied by a significant reduction in body weight. Haematoxylin and eosin (HE staining and immunohistochemical analysis demonstrated that during early stages of induction, DES increased cell proliferation and sprouting of endothelial cells, and VEGF expression transitioned from a vessel-surrounding pattern to a diffuse pattern. During later stages, angiogenesis was predominant, and VEGF expression decreased. In contrast to the early abundant expression of VEGF, endothelial expression of AQP- 1 increased during later stages. Our data indicated a dynamic scenario of biological alterations in DES-treated pituitary tissue, in which VEGF and AQP-1 exert their functions at different stages of induction, and we provide novel insights into understanding oestrogen-related tumourigenesis in the anterior pituitary gland.

  12. Induction stage-dependent expression of vascular endothelial growth factor and aquaporin-1 in diethylstilbestrol-treated rat pituitary

    Directory of Open Access Journals (Sweden)

    Z Wang

    2009-03-01

    Full Text Available The anterior pituitary gland undergoes tumourigenic changes in response to oestrogen treatment in several breeds of rats. We administered diethylstilbestrol (DES to female Wistar rats and assessed whether the expression of vascular endothelial growth factor (VEGF and aquaporin-1 (AQP-1 was altered at different time points following DES administration. In vivo magnetic resonance imaging (MRI scans showed that the mass index corresponding to the mid-sagittal area of DES-treated pituitary was significantly higher than the vehicle-controlled pituitary (p<0.01 at three specific time points, accompanied by a significant reduction in body weight. Haematoxylin and eosin (HE staining and immunohistochemical analysis demonstrated that during early stages of induction, DES increased cell proliferation and sprouting of endothelial cells, and VEGF expression transitioned from a vessel-surrounding pattern to a diffuse pattern. During later stages, angiogenesis was predominant, and VEGF expression decreased. In contrast to the early abundant expression of VEGF, endothelial expression of AQP- 1 increased during later stages. Our data indicated a dynamic scenario of biological alterations in DES-treated pituitary tissue, in which VEGF and AQP-1 exert their functions at different stages of induction, and we provide novel insights into understanding oestrogen-related tumourigenesis in the anterior pituitary gland.

  13. 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.

  14. Interleukin-6 and intercellular cell adhesion molecule-1 expression remains elevated in revived live endothelial cells following spaceflight.

    Science.gov (United States)

    Muid, S; Froemming, G R A; Ali, A M; Nawawi, H

    2013-12-01

    The effects of spaceflight on cardiovascular health are not necessarily seen immediately after astronauts have returned but can be delayed. It is important to investigate the long term effects of spaceflight on protein and gene expression of inflammation and endothelial activation as a predictor for the development of atherosclerosis and potential cardiovascular problems. The objectives of this study were to investigate the (a) protein and gene expression of inflammation and endothelial activation, (b) expression of nuclear factor kappa B (NFκB), signal transducer and activator of transcription-3 (STAT-3) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) 3 months post-space flight travel compared to ground controls. HUVEC cultured on microcarriers in fluid processing apparatus were flown to the International Space Station (ISS) by the Soyuz TMA-11 rocket. After landing, the cells were detached from microcarriers and recultured in T-25 cm(2) culture flasks (Revived HUVEC). Soluble protein expression of IL-6, TNF-α, ICAM-1, VCAM-1 and e-selectin were measured by ELISA. Gene expression of these markers and in addition NFκB, STAT-3 and eNOS were measured. Spaceflight induced IL-6 and ICAM-1 remain elevated even after 3 months post spaceflight travel and this is mediated via STAT-3 pathway. The downregulation of eNOS expression in revived HUVEC cells suggests a reduced protection of the cells and the surrounding vessels against future insults that may lead to atherosclerosis. It would be crucial to explore preventive measures, in relation to atherosclerosis and its related complications.

  15. Maternal obesity programs mitochondrial and lipid metabolism gene expression in infant umbilical vein endothelial cells.

    Science.gov (United States)

    Costa, S M R; Isganaitis, E; Matthews, T J; Hughes, K; Daher, G; Dreyfuss, J M; da Silva, G A P; Patti, M-E

    2016-11-01

    Maternal obesity increases risk for childhood obesity, but molecular mechanisms are not well understood. We hypothesized that primary umbilical vein endothelial cells (HUVEC) from infants of overweight and obese mothers would harbor transcriptional patterns reflecting offspring obesity risk. In this observational cohort study, we recruited 13 lean (pre-pregnancy body mass index (BMI) obese ('ov-ob', BMI⩾25.0 kg m -2 ) women. We isolated primary HUVEC, and analyzed both gene expression (Primeview, Affymetrix) and cord blood levels of hormones and adipokines. A total of 142 transcripts were differentially expressed in HUVEC from infants of overweight-obese mothers (false discovery rate, FDRmaternal BMI (FDRmaternal obesity, we analyzed the cord blood lipidome and noted significant increases in the levels of total free fatty acids (lean: 95.5±37.1 μg ml -1 , ov-ob: 124.1±46.0 μg ml -1 , P=0.049), palmitate (lean: 34.5±12.7 μg ml -1 , ov-ob: 46.3±18.4 μg ml -1 , P=0.03) and stearate (lean: 20.8±8.2 μg ml -1 , ov-ob: 29.7±17.2 μg ml -1 , P=0.04), in infants of overweight-obese mothers. Prenatal exposure to maternal obesity alters HUVEC expression of genes involved in mitochondrial and lipid metabolism, potentially reflecting developmentally programmed differences in oxidative and lipid metabolism.

  16. Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

    Science.gov (United States)

    Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

    2005-11-01

    Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in

  17. Orphan nuclear receptor Nur77 is a novel negative regulator of endothelin-1 expression in vascular endothelial cells.

    Science.gov (United States)

    Qin, Qing; Chen, Ming; Yi, Bing; You, Xiaohua; Yang, Ping; Sun, Jianxin

    2014-12-01

    Endothelin-1 (ET-1) produced by vascular endothelial cells plays essential roles in the regulation of vascular tone and development of cardiovascular diseases. The objective of this study is to identify novel regulators implicated in the regulation of ET-1 expression in vascular endothelial cells (ECs). By using quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), we show that either ectopic expression of orphan nuclear receptor Nur77 or pharmacological activation of Nur77 by 6-mercaptopurine (6-MP) substantially inhibits ET-1 expression in human umbilical vein endothelial cells (HUVECs), under both basal and thrombin-stimulated conditions. Furthermore, thrombin-stimulated ET expression is significantly augmented in both Nur77 knockdown ECs and aort from Nur77 knockout mice, suggesting that Nur77 is a negative regulator of ET-1 expression. Inhibition of ET-1 expression by Nur77 occurs at gene transcriptional levels, since Nur77 potently inhibits ET-1 promoter activity, without affecting ET-1 mRNA stability. As shown in electrophoretic mobility shift assay (EMSA), Nur77 overexpression markedly inhibits both basal and thrombin-stimulated transcriptional activity of AP-1. Mechanistically, we demonstrate that Nur77 specially interacts with c-Jun and inhibits AP-1 dependent c-Jun promoter activity, which leads to a decreased expression of c-Jun, a critical component involved in both AP-1 transcriptional activity and ET-1 expression in ECs. These findings demonstrate that Nur77 is a novel negative regulator of ET-1 expression in vascular ECs through an inhibitory interaction with the c-Jun/AP-1 pathway. Activation of Nur77 may represent a useful therapeutic strategy for preventing certain cardiovascular diseases, such as atherosclerosis and pulmonary artery hypertension. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Melatonin prevents human pancreatic carcinoma cell PANC-1-induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression.

    Science.gov (United States)

    Cui, Peilin; Yu, Minghua; Peng, Xingchun; Dong, Lv; Yang, Zhaoxu

    2012-03-01

    Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti-angiogenic capability is urgent in clinical practice. In this study, a co-culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC-1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co-cultured with PANC-1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC-1 cells. In addition, the VEGF mRNA expression was also down-regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co-culturing them with PANC-1 cells; this was associated with a suppression of VEGF expression in PANC-1 cells. © 2011 John Wiley & Sons A/S.

  19. Efficacy of Selenium Supplement on Gene Expression of Inflammatory Cytokines and Vascular Endothelial Growth Factor in Gestational Diabetes

    Directory of Open Access Journals (Sweden)

    Mehri Jamilian

    2018-01-01

    Full Text Available Abstract Background: Selenium supplement has multiple important effects, including anti-inflammatory effect. The aim of this study was to assess the effects of selenium supplement on gene expression of inflammatory cytokines and vascular endothelial growth factor in gestational diabetes. Materials and Methods: This randomized double blind placebo control trial was performed on 40 patients suffering from GDM aged 18–40 years old. Participants were randomly divided into interventional group receiving 200mg/day selenium supplements (n=20 and control group receiving placebo (n=20 for 6 weeks. Primary outcome was gene expression of inflammatory cytokines and VEGF which were assessed in lymphocyte of GDM patients by RT-PCR method. Results: After 6 weeks intervention, in comparison with the control group, interventional group showed down regulation of gene expression of tumor necrosis factor alpha (TNF–α (p=0.02 and transforming growth factor beta (TGF–β (p=0.01 and up-regulation of gene expression of vascular endothelial (VEGF (p = 0.03 in lymphocytes of GDM. There was not any significant change following intervention with selenium regarding gene expression of interleukin IL-1 β and IL-8 in lymphocytes of GDM patients. Conclusion: 6 weeks supplementation with selenium in patients with GDM can cause down regulated gene expression of TNF-α and TGF–β, and up regulated gene expression of VEGF. Selenium supplement had not any effect on gene expression of IL-1 β and IL-8.

  20. Influence of sex on the number of circulating endothelial microparticles and microRNA expression in middle-aged adults.

    Science.gov (United States)

    Bammert, Tyler D; Hijmans, Jamie G; Kavlich, Philip J; Lincenberg, Grace M; Reiakvam, Whitney R; Fay, Ryan T; Greiner, Jared J; Stauffer, Brian L; DeSouza, Christopher A

    2017-08-01

    What is the central question of this study? Are there sex-related differences in the number of circulating endothelial microparticles (EMPs) and microparticle microRNA expression in middle-aged adult humans? What is the main finding and its importance? Although the numbers of circulating endothelial microparticles do not differ between middle-aged men and women, there are sex-related differences in the expression of miR-125a in activation-derived EMPs and miR-34a in apoptosis-derived EMPs. Differences in circulating endothelial microparticle microRNA content may provide new insight into the sex-related disparity in the risk and prevalence of vascular disease in middle-aged adults. The aims of this study were to determine: (i) whether circulating concentrations of endothelial microparticles (EMPs) differ in middle-aged men compared with women; and (ii) whether there are sex-related differences in microRNA expression in EMPs. Peripheral blood was collected from 30 sedentary adults: 15 men (56 ± 6 years old) and 15 women (56 ± 5 years old). Endothelial microparticles were defined by markers of activation (CD62e + ) or apoptosis (CD31 + /CD42b - ) by flow cytometry. Expression of microRNA (miR-34a, 92a, 125a and 126) in activation- and apoptosis-derived EMPs was measured by RT-PCR. Circulating activation- (33 ± 31 versus 39 ± 35 microparticles μl -1 ) and apoptosis-derived EMPs (49 ± 54 versus 42 ± 43 microparticles μl -1 ) were not significantly different between men and women. Expression of miR-125a (2.23 ± 2.01 versus 6.95 ± 3.99 a.u.) was lower (∼215%; P < 0.05) in activation-derived EMPs, whereas expression of miR-34a (1.17 ± 1.43 versus 0.38 ± 0.35 a.u.) was higher (∼210%; P < 0.05) in apoptosis-derived EMPs from men compared with women. Expression of microRNA in circulating EMPs may provide new insight into sex-related differences in cardiovascular disease. © 2017 The Authors. Experimental Physiology © 2017 The

  1. Activated endothelial interleukin-1beta, -6, and -8 concentrations and intercellular adhesion molecule-1 expression are attenuated by lidocaine.

    LENUS (Irish Health Repository)

    Lan, Wei

    2012-02-03

    Endothelial cells play a key role in ischemia reperfusion injury. We investigated the effects of lidocaine on activated human umbilical vein endothelial cell (HUVEC) interleukin (IL)-1beta, IL-6, and IL-8 concentrations and intercellular adhesion molecule-1 (ICAM-1) expression. HUVECs were pretreated with different concentrations of lidocaine (0 to 0.5 mg\\/mL) for 60 min, thereafter tumor necrosis factor-alpha was added at a concentration of 2.5 ng\\/mL and the cells incubated for 4 h. Supernatants were harvested, and cytokine concentrations were analyzed by enzyme-linked immunosorbent assay. Endothelial ICAM-1 expression was analyzed by using flow cytometry. Differences were assessed using analysis of variance and post hoc unpaired Student\\'s t-test where appropriate. Lidocaine (0.5 mg\\/mL) decreased IL-1beta (1.89 +\\/- 0.11 versus 4.16 +\\/- 1.27 pg\\/mL; P = 0.009), IL-6 (65.5 +\\/- 5.14 versus 162 +\\/- 11.5 pg\\/mL; P < 0.001), and IL-8 (3869 +\\/- 785 versus 14,961 +\\/- 406 pg\\/mL; P < 0.001) concentrations compared with the control. IL-1beta, IL-6, and IL-8 concentrations in HUVECs treated with clinically relevant plasma concentrations of lidocaine (0.005 mg\\/mL) were similar to control. ICAM-1 expression on lidocaine-treated (0.05 mg\\/mL) HUVECs was less than on controls (198 +\\/- 52.7 versus 298 +\\/- 50.3; Mean Channel Fluorescence; P < 0.001). Activated endothelial IL-1beta, IL-6, and IL-8 concentrations and ICAM-1 expression are attenuated only by lidocaine at concentrations larger than clinically relevant concentrations.

  2. INFLUENCE OF SOLUBLE PLACENTAL TISSUE-DERIVED MOLECULES UPON EXPRESSION OF ADHESION MOLECULES BY EA.HY926 ENDOTHELIAL CELLS

    Directory of Open Access Journals (Sweden)

    O. I. Stepanova

    2011-01-01

    Full Text Available Abstract.  Leukocyte  recruitment  to  placental  tissue  is  an  important  factor  of  its  development.  In  this respect, adhesion molecules at the endothelial cell surface represent a key determining factor of leukocyte adhesion and their trans-endothelial migration. The goal of investigation was to evaluate changed expression of adhesion molecules on the endothelial cells induced by supernates of placental tissue cultures. Placental tissue supernatants produced by the first- and third-trimester placental tissue from normal pregnancy, as well as from women with gestosis, induced higher expression of CD31, CD9, CD62E, CD62P, CD34, CD54, CD51/61, CD49d  and  integrin  β7  expression  by  endothelial  cells,  as  compared  with  their  baseline  levels.  However, the  supernates  from  pre-eclamptic  placental  tissue (3rd  trimester  caused  an  increased  CD9  expression by  endothelial  cells,  as  compared  with  effects  of placental  supernates  from  eclampsia-free  cases.  Our data  contribute  to  understanding  a  possible  role  of endothelial cell adhesion molecules in recruitment of leukocytes to placental tissue and possible participation of adhesion molecules in pathogenesis of pre-eclampsia. The work was supported by a grant from Russian Ministry of Education and Science ГК №02.740.11.0711 and Presidential grant № НШ-3594.2010.7 and МД-150.2011.7. (Med. Immunol., 2011, vol. 13, N 6, pp 589-596

  3. Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

    Science.gov (United States)

    Harris, Deshea L.

    2007-01-01

    Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that

  4. Three Cases of Organized Hematoma of the Maxillary Sinus: Clinical Features and Immunohistological Studies for Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor 2 Expressions

    Directory of Open Access Journals (Sweden)

    Shoichiro Imayoshi

    2015-01-01

    Full Text Available Objectives. Organized hematoma (OH is a rare, nonneoplastic, hemorrhagic lesion causing mucosal swelling and bone thinning, mainly in the maxillary sinus. We aimed to clarify the clinical presentation and treatment of OH. Methods. Three cases of maxillary sinus OH and a literature review are presented. Results. Three men aged 16–40 years complained of nasal obstruction, frequent epistaxis, and/or headache. Clinical and radiological examinations revealed a maxillary sinus OH. They were cured in a piecemeal fashion via endoscopic middle meatal antrostomy. Furthermore, vascular endothelial growth factor and its receptor were expressed in the lesion. Conclusions. The pathogenesis of OH is unclear and it presents various histological and imaging findings; however, it is not difficult to rule out malignant tumors. Minimally invasive surgery such as endoscopic sinus surgery can cure it completely. Thus, it is important to determine the diagnosis using CT and MRI and to quickly provide surgical treatment.

  5. Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

    Science.gov (United States)

    Basic-Jukic, Nikolina; Gulin, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Katalinic, Lea; Coric, Marijana; Veda, Marija Varnai; Ivkovic, Vanja; Kes, Petar; Jelakovic, Bojan

    2016-01-01

    Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF. © 2016 The Author(s) Published by S. Karger AG, Basel.

  6. Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules

    Directory of Open Access Journals (Sweden)

    Looareesuwan Sornchai

    2004-06-01

    Full Text Available Abstract Introduction Oxidative stress has been demonstrated in malaria. The potential oxidative modification of lipoproteins derived from malaria patients was studied. These oxidized lipids may have role in pathogenesis of malaria. Method The plasma lipid profile and existence of oxidized forms of very low density lipoprotein (VLDL, low density lipoprotein (LDL and high density lipoprotein (HDL were investigated in malaria (17 mild and 24 severe patients and 37 control subjects. Thiobarbituric acid reactive substances (TBARs, conjugated dienes, tryptophan fluorescence and fluidity of lipoproteins were determined as markers of oxidation. The biological effect of malarial lipoproteins was assessed by the expression of adhesion molecules on endothelial cells. Results Malarial lipoproteins had decreased cholesterol (except in VLDL and phospholipid. The triglyceride levels were unchanged. The cholesterol/phospholipid ratio of LDL was decreased in malaria, but increased in VLDL and HDL. TBARs and conjugate dienes were increased in malarial lipoproteins, while the tryptophan fluorescence was decreased. The fluidity of lipoproteins was increased in malaria. These indicated the presence of oxidized lipoproteins in malaria by which the degree of oxidation was correlated with severity. Of three lipoproteins from malarial patients, LDL displayed the most pronounced oxidative modification. In addition, oxidized LDL from malaria patients increased endothelial expression of adhesion molecules. Conclusion In malaria, the lipoproteins are oxidatively modified, and the degree of oxidation is related with severity. Oxidized LDL from malarial patients increases the endothelial expression of adhesion molecules. These suggest the role of oxidized lipoproteins, especially LDL, on the pathogenesis of disease.

  7. Tetrahydroxystilbene glucoside improves TNF-α-induced endothelial dysfunction: involvement of TGFβ/Smad pathway and inhibition of vimentin expression.

    Science.gov (United States)

    Yao, Wenjuan; Gu, Chengjing; Shao, Haoran; Meng, Guoliang; Wang, Huiming; Jing, Xiang; Zhang, Wei

    2015-01-01

    Endothelial dysfunction plays an important role in the pathogenesis of atherogenesis. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum (PM), exhibits significant anti-atherosclerotic activity. Here, we used human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-α (TNF-α) in vitro to investigate the cytoprotective effects of TSG on TNF-α-induced endothelial injury and the related mechanisms. Pretreatment with 50 and 100 μM TSG markedly attenuated TNF-α-induced loss of cell viability and release of lactate dehydrogenase (LDH) and inhibited TNF-α-induced cell apoptosis. The inhibition of vimentin expression was involved in the cytoprotection afforded by TSG. Using inhibitors for PI3K and TGFβ or siRNA for Akt and Smad2, we found that vimentin production in HUVECs is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by TNF-α. These results suggest that TSG protects HUVECs against TNF-α-induced cell damage by inhibiting vimentin expression via the interruption of the TGFβ/Smad signaling pathway.

  8. Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation

    Directory of Open Access Journals (Sweden)

    Nikolina Basic-Jukic

    2016-11-01

    Full Text Available Background/Aims: Delayed graft function (DGF is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2 is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF and DGF. Methods: 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients and DGF group (15 patients. BMP-2 expression in intima media (BMP2m and endothelium (BMP2e of epigastric artery was assessed by immunohistochemistry. Results: Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001 (Pst grade expression. Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477] and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]. Conclusions: Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF.

  9. TLQP-21 protects human umbilical vein endothelial cells against high-glucose-induced apoptosis by increasing G6PD expression.

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    Wei Zhang

    Full Text Available Hyperglycemia causes oxidative stress that could damage vascular endothelial cells, leading to cardiovascular complications. The Vgf gene was identified as a nerve growth factor-responsive gene, and its protein product, VGF, is characterized by the presence of partially cleaved products. One of the VGF-derived peptides is TLQP-21, which is composed of 21 amino acids (residues 556-576. Past studies have reported that TLQP-21 could stimulate insulin secretion in pancreatic cells and protect these cells from apoptosis, which suggests that TLQP-21 has a potential function in diabetes therapy. Here, we explore the protective role of TLQP-21 against the high glucose-mediated injury of vascular endothelial cells. Using human umbilical vascular endothelial cells (HUVECs, we demonstrated that TLQP-21 (10 or 50 nM dose-dependently prevented apoptosis under high-glucose (30 mmol/L conditions (the normal glucose concentration is 5.6 mmol/L. TLQP-21 enhanced the expression of NAPDH, resulting in upregulation of glutathione (GSH and a reduction in the levels of reactive oxygen species (ROS. TLQP-21 also upregulated the expression of glucose-6-phosphate dehydrogenase (G6PD, which is known as the main source of NADPH. Knockdown of G6PD almost completely blocked the increase of NADPH induced by TLQP-21, indicating that TLQP-21 functions mainly through G6PD to promote NADPH generation. In conclusion, TLQP-21 could increase G6PD expression, which in turn may increase the synthesis of NADPH and GSH, thereby partially restoring the redox status of vascular endothelial cells under high glucose injury. We propose that TLQP-21 is a promising drug for diabetes therapy.

  10. Over expression of vascular endothelial growth factor in correlation to Ki-67, grade and stage of breast cancer

    International Nuclear Information System (INIS)

    Al-Harris, Esraah S.; Al-Janabi, Asad A.; Al-Toriahi, Kaswer M.; Yasseen, Akeel A.

    2008-01-01

    Objective was to assess the significance of vascular endothelial growth factor (VEGF) protein over expression in human breast cancer, and its possible correlation with cell proliferation marker (Ki-67), grade and stage of breast cancer. We carried out this study at the Department of Pathology, Kufa University, between November 2006 and September 2007. A retrospective study was employed on paraffin-embedded blocks from 52 female patients with breast cancer. A group of 21 patients with benign breast lesions was included for comparison and 14 cases of normal breast tissue as control group. The investigation designed to employ immunohistochemistry using Avidin-Biotin Complex (ABC) method for detection of both VEGF and Ki-67. A total of 87 samples were included. Vascular endothelial growth factor immunoexpression was considered as positive in 61.5% of malignant and in 19% of benign breast lesions. No over expression sign has been noticed in normal breast tissue (p<0.005). No significant difference in VEGF over expression among different histological types of breast cancer (p<0.05). Vascular endothelial growth factor immunostaining was positively correlated with Ki-67, grade, stage, lymph node metastasis, and recurrence of breast cancer (p<0.05).No such correlation has been seen when the age of the patients has been considered. Vascular endothelial growth factor plays an important role in the pathogenesis of breast cancer evolution and supports the evidence of its role in angiogenesis and cell survival. This study recommended that the blocking of VEGF may be target for blocking angiogenesis and hence improving the efficacy of anti-cancer therapy. (author)

  11. Aging-induced dysregulation of dicer1-dependent microRNA expression impairs angiogenic capacity of rat cerebromicrovascular endothelial cells.

    Science.gov (United States)

    Ungvari, Zoltan; Tucsek, Zsuzsanna; Sosnowska, Danuta; Toth, Peter; Gautam, Tripti; Podlutsky, Andrej; Csiszar, Agnes; Losonczy, Gyorgy; Valcarcel-Ares, M Noa; Sonntag, William E; Csiszar, Anna

    2013-08-01

    Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol-catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.

  12. The influence of propofol on P-selectin expression and nitric oxide production in re-oxygenated human umbilical vein endothelial cells.

    LENUS (Irish Health Repository)

    Corcoran, T B

    2012-02-03

    BACKGROUND: Reperfusion injury is characterized by free radical production and endothelial inflammation. Neutrophils mediate much of the end-organ injury that occurs, requiring P-selectin-mediated neutrophil-endothelial adhesion, and this is associated with decreased endothelial nitric oxide production. Propofol has antioxidant properties in vitro which might abrogate this inflammation. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia and then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg\\/l or propofol 5 microg\\/l for 4 h after re-oxygenation and were then examined for P-selectin expression and supernatant nitric oxide concentrations for 24 h. P-selectin was determined by flow cytometry, and culture supernatant nitric oxide was measured as nitrite. RESULTS: In saline-treated cells, a biphasic increase in P-selectin expression was demonstrated at 30 min (P = 0.01) and 4 h (P = 0.023) after re-oxygenation. Propofol and Diprivan prevented these increases in P-selectin expression (P < 0.05). Four hours after re-oxygenation, propofol decreased endothelial nitric oxide production (P = 0.035). CONCLUSION: This is the first study to demonstrate an effect of propofol upon endothelial P-selectin expression. Such an effect may be important in situations of reperfusion injury such as cardiac transplantation and coronary artery bypass surgery. We conclude that propofol attenuates re-oxygenation-induced endothelial inflammation in vitro.

  13. Angiogenin and vascular endothelial growth factor expression in lungs of lung cancer patients.

    Science.gov (United States)

    Rozman, Ales; Silar, Mira; Kosnik, Mitja

    2012-12-01

    BACKGROUND.: Lung cancer is the leading cause of cancer deaths. Angiogenesis is crucial process in cancer growth and progression. This prospective study evaluated expression of two central regulatory molecules: angiogenin and vascular endothelial growth factor (VEGF) in patients with lung cancer. PATIENTS AND METHODS.: Clinical data, blood samples and broncho-alveolar lavage (BAL) from 23 patients with primary lung carcinoma were collected. BAL fluid was taken from part of the lung with malignancy, and from corresponding healthy side of the lung. VEGF and angiogenin concentrations were analysed by an enzyme-linked immunosorbent assay. Dilution of bronchial secretions in the BAL fluid was calculated from urea concentration ratio between serum and BAL fluid. RESULTS.: We found no statistical correlation between angiogenin concentrations in serum and in bronchial secretions from both parts of the lung. VEGF concentrations were greater in bronchial secretions in the affected side of the lung than on healthy side. Both concentrations were greater than serum VEGF concentration. VEGF concentration in serum was in positive correlation with tumour size (p = 0,003) and with metastatic stage of disease (p = 0,041). There was correlation between VEGF and angiogenin concentrations in bronchial secretions from healthy side of the lung and between VEGF and angiogenin concentrations in bronchial secretions from part of the lung with malignancy. CONCLUSION.: Angiogenin and VEGF concentrations in systemic, background and local samples of patients with lung cancer are affected by different mechanisms. Pro-angiogenic activity of lung cancer has an important influence on the levels of angiogenin and VEGF.

  14. Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

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    D'Orazi Gabriella

    2008-07-01

    Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

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

    Directory of Open Access Journals (Sweden)

    Hongxiu Wen

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

  16. VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells: a mechanism for feedback regulation of angiogenesis?

    International Nuclear Information System (INIS)

    Yao, Y.-G; Duh, Elia J.

    2004-01-01

    The Down syndrome critical region 1 (DSCR1) gene (also known as MCIP1, Adapt78) encodes a regulatory protein that binds to calcineurin catalytic A subunit and acts as a regulator of the calcineurin-mediated signaling pathway. We show in this study that DSCR1 is greatly induced in endothelial cells in response to VEGF, TNF-α, and A23187 treatment, and that this up-regulation is inhibited by inhibitors of the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway as well as by PKC inhibition and a Ca 2+ chelator. We hypothesized that the up-regulation of DSCR1 gene expression in endothelial cells could act as an endogenous feedback inhibitor for angiogenesis by regulating the calcineurin-NFAT signaling pathway. Our transient transfection analyses confirm that the overexpression of DSCR1 abrogates the up-regulation of reporter gene expression driven by both the cyclooxygenase 2 and DSCR1 promoters in response to stimulators. Our results indicate that DSCR1 up-regulation may represent a potential molecular mechanism underlying the regulation of angiogenic genes activated by the calcineurin-NFAT signaling pathway in endothelial cells

  17. (−-Epigallocatechin gallate inhibits endotoxin-induced expression of inflammatory cytokines in human cerebral microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Li Jieliang

    2012-07-01

    Full Text Available Abstract Background (−-Epigallocatechin gallate (EGCG is a major polyphenol component of green tea that has antioxidant activities. Lipopolysaccharide (LPS induces inflammatory cytokine production and impairs blood–brain barrier (BBB integrity. We examined the effect of EGCG on LPS-induced expression of the inflammatory cytokines in human cerebral microvascular endothelial cells (hCMECs and BBB permeability. Methods The expression of TNF-α, IL-1β and monocyte chemotactic protein-1 (MCP-1/CCL2 was determined by quantitative real time PCR (qRT-PCR and ELISA. Intercellular adhesion molecule 1 (ICAM-1 and vascular cell adhesion molecule (VCAM in hCMECs were examined by qRT-PCR and Western blotting. Monocytes that adhered to LPS-stimulated endothelial cells were measured by monocyte adhesion assay. Tight junctional factors were detected by qRT-PCR (Claudin 5 and Occludin and immunofluorescence staining (Claudin 5 and ZO-1. The permeability of the hCMEC monolayer was determined by fluorescence spectrophotometry of transmembrane fluorescin and transendothelial electrical resistance (TEER. NF-kB activation was measured by luciferase assay. Results EGCG significantly suppressed the LPS-induced expression of IL-1β and TNF-α in hCMECs. EGCG also inhibited the expression of MCP-1/CCL2, VCAM-1 and ICAM-1. Functional analysis showed that EGCG induced the expression of tight junction proteins (Occludin and Claudin-5 in hCMECs. Investigation of the mechanism showed that EGCG had the ability to inhibit LPS-mediated NF-κB activation. In addition, 67-kD laminin receptor was involved in the anti-inflammatory effect of EGCG. Conclusions Our results demonstrated that LPS induced inflammatory cytokine production in hCMECs, which could be attenuated by EGCG. These data indicate that EGCG has a therapeutic potential for endotoxin-mediated endothelial inflammation.

  18. Effects of anisodamine on the expressions of vascular endothelial growth factor and intercellular adhesion molecule 1 in experimental infusion phlebitis.

    Science.gov (United States)

    Zhang, Zhen-Xiang; Wang, Peng; Zhang, Qiu-Shi; Pan, Xue; Zhao, Qing-Xia; Wang, Xiao-Kai

    2012-01-01

    Infusion phlebitis is the most common side effect of clinical intravenous drug therapy and several clinical studies have demonstrated that anisodamine can effectively prevent the occurrence of infusion phlebitis. This study was designed to investigate effects of anisodamine on the expressions of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) in a rabbit model of infusion phlebitis and to analyze the mechanisms of anisodamine effect on the prevention and treatment of experimental infusion phlebitis. Twenty-four specific pathogen-free male Japanese white rabbits were randomly assigned to the control group, the model group, the magnesium sulfate group and the anisodamine group. The rabbit model of infusion phlebitis, induced by intravenous administration, was established and expressions of VEGF and ICAM-1 were determined and contrasted with the control group treated with normal saline. We evaluated expression by histopathology, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blotting assay. Pathohistological changes of the model group were observed, such as loss of venous endothelial cells, inflammatory cell infiltration, edema and thrombus. The magnesium sulfate group and the anisodamine group showed significant protective effects on vascular congestion, inflammatory cell infiltration, proliferation, swelling of endothelium and perivascular hemorrhage. The model group showed the highest expressions of VEGF and ICAM-1 of the four groups (P 0.05). Anisodamine alleviates inflammatory damage by significantly reducing the expressions of VEGF and ICAM-1, and shows significant protective effects in an animal model of infusion phlebitis.

  19. γ-Oryzanol reduces adhesion molecule expression in vascular endothelial cells via suppression of nuclear factor-κB activation.

    Science.gov (United States)

    Sakai, Satoshi; Murata, Takahisa; Tsubosaka, Yoshiki; Ushio, Hideki; Hori, Masatoshi; Ozaki, Hiroshi

    2012-04-04

    γ-Oryzanol (γ-ORZ) is a mixture of phytosteryl ferulates purified from rice bran oil. In this study, we examined whether γ-ORZ represents a suppressive effect on the lipopolysaccharide (LPS)-induced adhesion molecule expression on vascular endothelium. Treatment with LPS elevated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in bovine aortic endothelial cells (BAECs). Pretreatment with γ-ORZ dose-dependently decreased the LPS-mediated expression of these genes. Western blotting also revealed that pretreatment with γ-ORZ dose-dependently inhibited LPS-induced VCAM-1 expression in human umbilical vein endothelial cells. Consistently, pretreatment with γ-ORZ dose-dependently reduced LPS-induced U937 monocyte adhesion to BAECs. In immunofluorescence, LPS caused nuclear factor-κB (NF-κB) nuclear translocation in 40% of BAECs, which indicates NF-κB activation. Pretreatment with γ-ORZ, as well as its components (cycloartenyl ferulate, ferulic acid, or cycloartenol), dose-dependently inhibited LPS-mediated NF-κB activation. Collectively, our results suggested that γ-ORZ reduced LPS-mediated adhesion molecule expression through NF-κB inhibition in vascular endothelium.

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

    Directory of Open Access Journals (Sweden)

    Mathieu Barbier

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

  1. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

    Science.gov (United States)

    Lum, H; Jaffe, H A; Schulz, I T; Masood, A; RayChaudhury, A; Green, R D

    1999-09-01

    We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

  2. Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Christopher C.; Bloodworth, Jeffrey C. [Division of Pharmacology, Columbus, OH 43210 (United States); Mythreye, Karthikeyan [Duke University, Department of Medicine, Durham, NC 27708 (United States); Lee, Nam Y., E-mail: lee.5064@osu.edu [Division of Pharmacology, Columbus, OH 43210 (United States); Davis Heart and Lung Research Institute, Columbus, OH 43210 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.

  3. Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

    Science.gov (United States)

    Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

    2013-02-28

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Expression of vascular endothelial factor protein in the tumor tissues of patients with Stages I-II ovarian cancer

    Directory of Open Access Journals (Sweden)

    V. L. Karapetyan

    2010-01-01

    Full Text Available To define tumor markers is presently the most interesting and promising direction for the diagnosis of malignancies. The expression of the major angiogenesis factor vascular endothelial growth factor (VEGF in primary tumor tissue was studied in ovarian cancer (OC patients to define the prognostic value of the marker.The study enrolled 48 patients with OC. The immunohistochemical technique was used to examine VEGF expression in the primary tu- mor tissue. The frequency of VEGF expression, which was associated with lower relapse-free survival rates, was found to be high (85.4% in OC patients (p > 0.05.The tumor expression of the angiogenic factor VEGF was shown to provide prognostic information in early-stage ovarian epithelial cancer.

  5. Association of Vascular Endothelial Growth Factor Expression with Tumor Angiogenesis and with Early Relapse in Primary Breast Cancer

    Science.gov (United States)

    Hoshina, Seigo; Takayanagi, Toshiaki; Tominaga, Takeshi

    1994-01-01

    Angiogenesis is an independent prognostic indicator in breast cancer. In this report, the relationship between expression of vascular endothclial growth factor (VEGF; a selective mitogen for endothelial cells) and the microvessel density was examined in 103 primary breast cancers. The expression of VEGF was evaluated by immunocytochemical staining using anti‐VEGF antibody. The microvessel density, which was determined by immunostaining for factor VIII antigen, in VEGF‐rich tumors was clearly higher than that in VEGF‐poor tumors (P<0.01). There was a good correlation between VEGF expression and the increment of microvessel density. Furthermore, postoperative survey demonstrated that the relapse‐free survival rate of VEGF‐rich tumors was significantly worse than that of VEGF‐poor tumors. It was suggested that the expression of VEGF is closely associated with the promotion of angiogenesis and with early relapse in primary breast cancer. PMID:7525523

  6. Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

    LENUS (Irish Health Repository)

    Andrews, E J

    2012-02-03

    BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

  7. Antioxidant betalains from cactus pear (Opuntia ficus-indica) inhibit endothelial ICAM-1 expression.

    Science.gov (United States)

    Gentile, C; Tesoriere, L; Allegra, M; Livrea, M A; D'Alessio, P

    2004-12-01

    It has been suggested that some pigments would have antioxidant properties and that their presence in dietary constituents would contribute to reduce the risk of oxidative stress-correlated diseases. Among others, inflammatory response depends on redox status and may implicate oxidative stress. Vascular endothelial cells are a direct target of oxidative stress in inflammation. We have tested the impact of the free radical scavenger and antioxidant properties of betalains from the prickle pear in an in vitro model of endothelial cells. Here we show the capacity of betalains to protect endothelium from cytokine-induced redox state alteration, through ICAM-1 inhibition.

  8. Hypoxia upregulates Bcl-2 expression and suppresses interferon-gamma induced antiangiogenic activity in human tumor derived endothelial cells.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

    BACKGROUND: Hypoxia in solid tumors potentially stimulates angiogenesis by promoting vascular endothelial growth factor (VEGF) production and upregulating VEGF receptor expression. However, it is unknown whether hypoxia can modulate the effect of anti-angiogenic treatment on tumor-derived endothelium. METHODS: Human tumor-derived endothelial cells (HTDEC) were freshly isolated from surgically removed human colorectal tumors by collagenase\\/DNase digestion and Percol gradient sedimentation. Cell proliferation was assessed by measuring BrdU incorporation, and capillary tube formation was measured using Matrigel. Cell apoptosis was assessed by flow cytometry and ELISA, and Bcl-2 expression was detected by Western blot analysis. RESULTS: Under aerobic culture conditions (5% CO2 plus 21% O2) HTDEC expressed less Bcl-2 and were more susceptible to IFN-gamma-induced apoptosis with significant reductions in both cell proliferation and capillary tube formation, when compared with normal human macrovascular and microvascular EC. Following exposure of HTDEC to hypoxia (5% CO2 plus 2% O2), IFN-gamma-induced cell apoptosis, and antiangiogenic activity (i.e. an inhibition in cell proliferation and capillary tube formation) in HTDEC were markedly attenuated. This finding correlated with hypoxia-induced upregulation of Bcl-2 expression in HTDEC. CONCLUSIONS: These results indicate that hypoxia can protect HTDEC against IFN-gamma-mediated cell death and antiangiogenic activity, and suggest that improvement of tumor oxygenation may potentiate the efficacy of anti-cancer therapies specifically targeting the inhibition of tumor angiogenesis.

  9. Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

    Science.gov (United States)

    Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

    2010-09-01

    Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

  10. Tenascin in meningioma: expression is correlated with anaplasia, vascular endothelial growth factor expression, and peritumoral edema but not with tumor border shape.

    Science.gov (United States)

    Kiliç, Türker; Bayri, Yaşar; Ozduman, Koray; Acar, Melih; Diren, Semin; Kurtkaya, Ozlem; Ekinci, Gazanfer; Buğra, Kuyaş; Sav, Aydin; Ozek, M Memet; Pamir, M Necmettin

    2002-07-01

    Tenascin is an extracellular matrix glycoprotein that is expressed during embryogenesis, inflammation, angiogenesis, and carcinogenesis. The aim of this study was to investigate how tenascin expression relates to histological grade, angiogenesis, and radiological findings in meningiomas. Twenty typical, 20 atypical, and 5 malignant meningiomas were studied retrospectively. Tenascin expression and vascular endothelial growth factor (VEGF) expression in the tumor tissue were investigated by immunohistochemistry. Tenascin messenger ribonucleic acid expression was also studied by comparative reverse transcriptase-polymerase chain reaction. Magnetic resonance images from each case were assessed for peritumoral edema and tumor border shape. The atypical and malignant meningiomas showed higher levels of tenascin expression than the typical meningiomas. The more sensitive messenger ribonucleic acid-based methods confirmed this finding. Tenascin expression was correlated with peritumoral edema and VEGF expression but not with tumor border shape. In the 13 tumors with marked tenascin expression, peritumoral edema was Grade 0 in one, Grade 1 in three, and Grade 2 in nine specimens. In the same 13 tumors, VEGF expression was Grade 1 in five and Grade 2 in eight specimens, and the findings for tumor border shape were Grade 0 in seven, Grade 1 in four, and Grade 2 in two specimens. In meningiomas, tenascin expression is correlated with anaplasia, tumor-associated edema, and VEGF expression but not with tumor border shape. This protein may play a role in the neoplastic and/or angiogenic processes in atypical and malignant meningiomas and may thus be a potential target for meningioma therapy.

  11. Autoantibodies in dilated cardiomyopathy induce vascular endothelial growth factor expression in cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Saygili, Erol, E-mail: erol.saygili@med.uni-duesseldorf.de [Division of Cardiology, Pulmonology, and Vascular Medicine, University Hospital Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf (Germany); Noor-Ebad, Fawad; Schröder, Jörg W.; Mischke, Karl [Department of Cardiology, University RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen (Germany); Saygili, Esra [Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf (Germany); Rackauskas, Gediminas [Department of Cardiovascular Medicine, Vilnius University Hospital Santariskiu Klinikos, Vilnius University (Lithuania); Marx, Nikolaus [Department of Cardiology, University RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen (Germany); Kelm, Malte; Rana, Obaida R. [Division of Cardiology, Pulmonology, and Vascular Medicine, University Hospital Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf (Germany)

    2015-09-11

    Background: Autoantibodies have been identified as major predisposing factors for dilated cardiomyopathy (DCM). Patients with DCM show elevated serum levels of vascular endothelial growth factor (VEGF) whose source is unknown. Besides its well-investigated effects on angiogenesis, evidence is present that VEGF signaling is additionally involved in fibroblast proliferation and cardiomyocyte hypertrophy, hence in cardiac remodeling. Whether autoimmune effects in DCM impact cardiac VEGF signaling needs to be elucidated. Methods: Five DCM patients were treated by the immunoadsorption (IA) therapy on five consecutive days. The eluents from the IA columns were collected and prepared for cell culture. Cardiomyocytes from neonatal rats (NRCM) were incubated with increasing DCM-immunoglobulin-G (IgG) concentrations for 48 h. Polyclonal IgG (Venimmun N), which was used to restore IgG plasma levels in DCM patients after the IA therapy was additionally used for control cell culture purposes. Results: Elevated serum levels of VEGF decreased significantly after IA (Serum VEGF (ng/ml); DCM pre-IA: 45 ± 9.1 vs. DCM post–IA: 29 ± 6.7; P < 0.05). In cell culture, pretreatment of NRCM by DCM-IgG induced VEGF expression in a time and dose dependent manner. Biologically active VEGF that was secreted by NRCM significantly increased BNP mRNA levels in control cardiomyocytes and induced cell-proliferation of cultured cardiac fibroblast (Fibroblast proliferation; NRCM medium/HC-IgG: 1 ± 0.0 vs. NRCM medium/DCM-IgG 100 ng/ml: 5.6 ± 0.9; P < 0.05). Conclusion: The present study extends the knowledge about the possible link between autoimmune signaling in DCM and VEGF induction. Whether this observation plays a considerable role in cardiac remodeling during DCM development needs to be further elucidated. - Highlights: • Mechanisms of remodeling in dilated cardiomyopathy (DCM) are not fully understood. • Autoantibodies have been identified as major predisposing factors

  12. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    International Nuclear Information System (INIS)

    Ghislin, Stephanie; Obino, Dorian; Middendorp, Sandrine; Boggetto, Nicole; Alcaide-Loridan, Catherine; Deshayes, Frederique

    2012-01-01

    Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration

  13. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    Directory of Open Access Journals (Sweden)

    Ghislin Stephanie

    2012-10-01

    Full Text Available Abstract Background Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18 expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. Methods A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. Results We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Conclusion Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

  14. IL-27 inhibits lymphatic endothelial cell proliferation by STAT1-regulated gene expression

    DEFF Research Database (Denmark)

    Nielsen, Sebastian Rune; Hammer, Troels; Gibson, Josefine

    2013-01-01

    OBJECTIVE: IL-27 belongs to the IL-12 family of cytokines and is recognized for its role in Th cell differentiation and as an inhibitor of tumor-angiogenesis. The purpose of this study was to investigate the effect of IL-27 on proliferation of lymphatic endothelial cells to gain insight into the ...

  15. Increased expression of endothelial antigen PAL-E in human diabetic retinopathy correlates with microvascular leakage

    NARCIS (Netherlands)

    Schlingemann, R. O.; Hofman, P.; Vrensen, G. F.; Blaauwgeers, H. G.

    1999-01-01

    AIMS/HYPOTHESIS: The Pathologische Anatomie Leiden-Endothelium (PAL-E) antigen is a marker for loss of the blood-brain barrier function in brain tumours. It is endothelium specific and is associated with the endothelial plasmalemmal vesicles (caveolae) involved in transcellular transport. To test

  16. Endothelial-derived GM-CSF influences expression of oncostatin M

    Science.gov (United States)

    During and following transendothelial migration, neutrophils undergo a number of phenotypic changes resulting from encounters with endothelial-derived factors. This report uses an in vitro model with HUVEC and isolated human neutrophils to examine the effects of two locally-derived cytokines, granul...

  17. Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems.

    Science.gov (United States)

    Liu, Ju; Deutsch, Urban; Fung, Iris; Lobe, Corrinne G

    2014-11-01

    In the present study, the tetracycline-off and Cre/ loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP -flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

  18. Peroxisome Proliferator-Activated Receptor γ Regulates the Expression of Lipid Phosphate Phosphohydrolase 1 in Human Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Yazi Huang

    2014-01-01

    Full Text Available Lipid phosphate phosphohydrolase 1 (LPP1, a membrane ectophosphohydrolase regulating the availability of bioactive lipid phosphates, plays important roles in cellular signaling and physiological processes such as angiogenesis and endothelial migration. However, the regulated expression of LPP1 remains largely unknown. Here, we aimed to examine a role of peroxisome proliferator-activated receptor γ (PPARγ in the transcriptional control of LPP1 gene expression. In human umbilical vein endothelial cells (HUVECs, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR demonstrated that activation of PPARγ increased the mRNA level of LPP1. Chromatin immunoprecipitation assay showed that PPARγ binds to the putative PPAR-responsive elements (PPREs within the 5′-flanking region of the human LPP1 gene. Genomic fragment containing 1.7-kilobase of the promoter region was cloned by using PCR. The luciferase reporter assays demonstrated that overexpression of PPARγ and rosiglitazone, a specific ligand for PPARγ, could significantly upregulate the reporter activity. However, site-directed mutagenesis of the PPRE motif abolished the induction. In conclusion, our results demonstrated that PPARγ transcriptionally activated the expression of LPP1 gene in ECs, suggesting a potential role of PPARγ in the metabolism of phospholipids.

  19. Tanshinol suppresses endothelial cells apoptosis in mice with atherosclerosis via lncRNA TUG1 up-regulating the expression of miR-26a.

    Science.gov (United States)

    Chen, Chao; Cheng, Guangqing; Yang, Xiaoni; Li, Changsheng; Shi, Ran; Zhao, Ningning

    2016-01-01

    Endothelial cell (EC) apoptosis is a crucial process for the development of atherosclerosis. Tanshinol is reported to protect vascular endothelia and attenuate the formation of atherosclerosis. However, the potential molecule mechanism of the protective role of tanshinol in atherosclerosis need to be further investigated. ApoE(-/-)mice were fed with a high-fat diet and treated with tanshinol to detect the effect of tanshinol on endothelial cells apoptosis with TUNEL staining assay. qRT-PCR and Western blot were performed to examine the expression of TUG1 and miR-26a in endothelial cells. RNA-binding protein immunoprecipitation assay was performed to verify the relationship between TUG1 and miR-26a. It has been shown that tanshinol reduced the aortic atherosclerotic lesion area in the entire aorta and aortic sinus in a concentration dependent manner, and suppressed the endothelial cells apoptosis in ApoE(-/-) mice. We further found that the mRNA level of TUG1 was reduced and the expression of miR-26a was up-regulated by tanshinol in endothelial cells. In addition, TUG1 down-regulated the expression of miR-26a in ECV304 cells. Finally, it was shown that overexpression of TUG1 removed the reversed effect of tanshinol on oxidized low-density lipoprotein (ox-LDL)-induced endothelial cells apoptosis. Taken together, our study reveals that tanshinol could attenuate the endothelial cells apoptosis in atherosclerotic ApoE(-/-) mice. Moreover, low TUG1 expression and high level of miR-26a are associated with the endothelial protecting effect of tanshinol.

  20. Effect of penehyclidine hydrochloride on β-arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, J. [Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei (China); Xiao, F. [Department of Osteology, Pu Ai Hospital, Huazhong University of Science and Technology, Wuhan, Hubei (China); Zhang, Z.Z.; Wang, Y.P.; Chen, K.; Wang, Y.L. [Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei (China)

    2013-12-02

    β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M{sub 3} receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury.

  1. Effect of penehyclidine hydrochloride on β-arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

    International Nuclear Information System (INIS)

    Zhan, J.; Xiao, F.; Zhang, Z.Z.; Wang, Y.P.; Chen, K.; Wang, Y.L.

    2013-01-01

    β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M 3 receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury

  2. Infusion of hypertonic saline (7.5%) does not change neutrophil oxidative burst or expression of endothelial adhesion molecules after abdominal hysterectomy

    DEFF Research Database (Denmark)

    Kølsen-Petersen, Jens Aage; Rasmussen, Torsten Bøgh; Krog, Jan

    2006-01-01

    of leukocyte and differential count, neutrophil membrane expression of endothelial adhesion molecules by flow cytometry, and O2- -generation by superoxide dismutase-inhibitable reduction of cytochrome C. RESULTS: Surgery induced well-known changes in the number and distribution of white blood cells, reduced...... the expression of adhesion molecules, and halved the superoxide production unrelated to the tonicity or volume of the infused fluids. CONCLUSION: Infusion of a clinically relevant dose of hypertonic saline has no detectable effect on the membrane expression of endothelial adhesion molecules or O2- -generation...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-05

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

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

  6. Inhibition of endothelial cell expression of plasminogen activator inhibitor type-1 by gemfibrozil.

    Science.gov (United States)

    Fujii, S; Sawa, H; Sobel, B E

    1993-10-18

    Increased concentrations of plasminogen activator inhibitor type-1 (PAI-1) in plasma are associated with impaired fibrinolysis and venous and arterial thrombo-embolic disease. In pilot studies designed to identify pharmacologic approaches capable of diminishing such increases, we found that gemfibrozil attenuated the stimulation of synthesis of PAI-1 in a human, immortal, hepatoma cell line (Hep G2) induced by platelets. The present study was performed to determine whether it exerts analogous effects in non-immortal endothelial cells and whether it may therefore facilitate fibrinolysis locally in vivo. Human umbilical vein endothelial cells were incubated with pharmacologic concentrations of gemfibrozil. Gemfibrozil, 100 microM, suppressed basal PAI-1 production by 15% and attenuated the augmentation of synthesis of PAI-1 induced by lysates from platelets (4 x 10(7)/ml) by 36% over 24 h without inhibiting overall protein synthesis. In addition, the increases in PAI-1 mRNA otherwise induced by platelet lysates over 6 h were suppressed by 49% (Northern blots) without any demonstrable change in the intracellular half-life of PAI-1 mRNA. Pulse-chase experiments demonstrated diminution of PAI-1 protein synthesis in parallel with the changes observed in PAI-1 mRNA. To determine whether these effects of gemfibrozil on endothelial cells in vitro were paralleled by consistent changes in the concentrations of PAI-1 in plasma in vivo, we studied rabbits with induced carotid artery thrombosis and thrombolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Effect of prepro-calcitonin gene-related peptide-expressing endothelial progenitor cells on pulmonary hypertension.

    Science.gov (United States)

    Zhao, Qiang; Liu, Zixiong; Wang, Zhe; Yang, Cheng; Liu, Jun; Lu, Jun

    2007-08-01

    Calcitonin gene-related peptide (CGRP) is a potent smooth muscle cell proliferation inhibitor and vasodilator. It is now believed that CGRP plays an important role in maintaining a low pulmonary vascular resistance. We evaluated the therapeutic effect of intravenously administered CGRP-expressing endothelial progenitor cells (EPCs) on left-to-right shunt-induced pulmonary hypertension in rats. Endothelial progenitor cells were obtained from cultured human peripheral blood mononuclear cells. The genetic sequence for CGRP was subcloned into cultured EPCs by human expression plasmid. Pulmonary hypertension was established in immunodeficient rats with an abdominal aorta to inferior vena cava shunt operation. The transfected EPCs were injected through the left jugular vein at 10 weeks after the shunt operation. Mean pulmonary artery pressure and total pulmonary vascular resistance were detected with right cardiac catheterization at 4 weeks. The distribution of EPCs in the lung tissue was examined with immunofluorescence technique. Histopathologic changes in the structure of the pulmonary arteries was observed with electron microscopy and subjected to computerized image analysis. The lungs of rats transplanted with CGRP-expressing EPCs demonstrated a decrease in both mean pulmonary artery pressure (17.64 +/- 0.79 versus 22.08 +/- 0.95 mm Hg; p = 0.018) and total pulmonary vascular resistance (1.26 +/- 0.07 versus 2.45 +/- 0.18 mm Hg x min/mL; p = 0.037) at 4 weeks. Immunofluorescence revealed that intravenously administered cells were incorporated into the pulmonary vasculature. Pulmonary vascular remodeling was remarkably attenuated with the administration of CGRP-expressing EPCs. The transplantation of CGRP-expressing EPCs may effectively attenuate established pulmonary hypertension and exert reversal effects on pulmonary vascular remodeling. Our findings suggest that the therapy based on the combination of both CGRP transfection and EPCs may be a potentially useful

  8. Angiotensin II upregulates the expression of placental growth factor in human vascular endothelial cells and smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Guo Yingqiang

    2010-05-01

    Full Text Available Abstract Background Atherosclerosis is now recognized as a chronic inflammatory disease. Angiotensin II (Ang II is a critical factor in inflammatory responses, which promotes the pathogenesis of atherosclerosis. Placental growth factor (PlGF is a member of the vascular endothelial growth factor (VEGF family cytokines and is associated with inflammatory progress of atherosclerosis. However, the potential link between PlGF and Ang II has not been investigated. In the current study, whether Ang II could regulate PlGF expression, and the effect of PlGF on cell proliferation, was investigated in human vascular endothelial cells (VECs and smooth muscle cells (VSMCs. Results In growth-arrested human VECs and VSMCs, Ang II induced PlGF mRNA expression after 4 hour treatment, and peaked at 24 hours. 10-6 mol/L Ang II increased PlGF protein production after 8 hour treatment, and peaked at 24 hours. Stimulation with Ang II also induced mRNA expression of VEGF receptor-1 and -2(VEGFR-1 and -2 in these cells. The Ang II type I receptor (AT1R antagonist blocked Ang II-induced PlGF gene expression and protein production. Several intracellular signals elicited by Ang II were involved in PlGF synthesis, including activation of protein kinase C, extracellular signal-regulated kinase 1/2 (ERK1/2 and PI3-kinase. A neutralizing antibody against PlGF partially inhibited the Ang II-induced proliferation of VECs and VSMCs. However, this antibody showed little effect on the basal proliferation in these cells, whereas blocking antibody of VEGF could suppress both basal and Ang II-induced proliferation in VECs and VSMCs. Conclusion Our results showed for the first time that Ang II could induce the gene expression and protein production of PlGF in VECs and VSMCs, which might play an important role in the pathogenesis of vascular inflammation and atherosclerosis.

  9. Omentin inhibits TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via ERK/NF-{kappa}B pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Xia, E-mail: zhongxia1977@126.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Li, Xiaonan; Liu, Fuli; Tan, Hui [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Shang, Deya, E-mail: wenhuashenghuo1@163.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Black-Right-Pointing-Pointer Omentin reduces expression of ICAM-1 and VCAM-1 induced by TNF-{alpha} in HUVECs. Black-Right-Pointing-Pointer Omentin inhibits TNF-{alpha}-induced ERK and NF-{kappa}B activation in HUVECs. Black-Right-Pointing-Pointer Omentin supreeses TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 via ERK/NF-{kappa}B pathway. -- Abstract: In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha}) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-{alpha}-activated signal pathway of nuclear factor-{kappa}B (NF-{kappa}B) by preventing NF-{kappa}B inhibitory protein (I{kappa}B{alpha}) degradation and NF-{kappa}B/DNA binding activity. Omentin pretreatment significantly inhibited TNF-{alpha}-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-{alpha}-induced NF-{kappa}B activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-{alpha}. These results suggest that omentin may inhibit TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-{kappa}B pathway.

  10. Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression.

    Science.gov (United States)

    Calabriso, Nadia; Scoditti, Egeria; Massaro, Marika; Pellegrino, Mariangela; Storelli, Carlo; Ingrosso, Ilaria; Giovinazzo, Giovanna; Carluccio, Maria Annunziata

    2016-03-01

    The aim of the study was to evaluate the vascular anti-inflammatory effects of polyphenolic extracts from two typical South Italy red wines, the specific contribution of individual polyphenols and the underlying mechanisms of action. Human endothelial cells were incubated with increasing concentrations (1-50 μg/mL) of Primitivo and Negroamaro polyphenolic extracts (PWPE and NWPE, respectively) or pure polyphenols (1-25 μmol/L), including hydroxycinnamic acids (p-coumaric, caffeic and caftaric acids), flavonols (kaempferol, quercetin, myricetin) or stilbenes (trans-resveratrol, trans-piceid) before stimulation with lipopolysaccharide. Through multiple assays, we analyzed the endothelial-monocyte adhesion, the endothelial expression of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin), monocyte chemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), as well as ROS intracellular levels and the activation of NF-κB and AP-1. Both PWPE and NWPE, already at 1 μg/mL, inhibited monocyte adhesion to stimulated endothelial cells, a key event in triggering vascular inflammation. They down-regulated the expression of adhesion molecules, ICAM-1, VCAM-1, E-Selectin, as well as MCP-1 and M-CSF, at mRNA and protein levels. All polyphenols reduced intracellular ROS, and everything, except caftaric acid, inhibited the endothelial expression of adhesion molecules and MCP-1, although with different potency. Flavonols and resveratrol significantly reduced also the endothelial expression and release of M-CSF. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-κB and AP-1 activation but not to intracellular oxidative stress. This study showed multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts and indentified specific bioactive polyphenols which could counteract inflammatory diseases including atherosclerosis.

  11. Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

    Science.gov (United States)

    Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

    2005-01-01

    Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

  12. Increased expression of vascular endothelial growth factor attenuates contusion necrosis without influencing contusion edema after traumatic brain injury in rats.

    Science.gov (United States)

    Tado, Masahiro; Mori, Tatsuro; Fukushima, Masamichi; Oshima, Hideki; Maeda, Takeshi; Yoshino, Atsuo; Aizawa, Shin; Katayama, Yoichi

    2014-04-01

    To clarify the role of vascular endothelial growth factor (VEGF) in the formation of contusion edema and necrosis after traumatic brain injury, we examined the time course of changes in the VEGF expression (enzyme-linked immunosorbent assay), cerebrovascular permeability (extravasation of Evans blue), and water content (dry-wet weight method) of the contused brain tissue in a cortical impact injury model using rats. In addition, we tested the effects of administration of bevacizumab (VEGF monoclonal antibody) on changes in the cerebrovascular permeability and water content of the contused brain tissue, as well as the neurological deficits (rota rod test) and volume of contusion necrosis. Increased VEGF expression was maximal at 72 h after injury (pnecrosis at 21 days (pnecrosis. This is probably because of an increased angiogenesis and improved microcirculation in the areas surrounding the core of contusion.

  13. Expression of vascular endothelial growth factor in third-trimester placentas is not increased in growth-restricted fetuses.

    Science.gov (United States)

    Tse, J Y; Lao, T T; Chan, C C; Chiu, P M; Cheung, A N

    2001-01-01

    Vascular endothelial growth factor (VEGF) is considered the growth factor that stimulates vasculogenesis and angiogenesis. Recent studies have demonstrated its role in regulating placental growth and invasion. Its expression can be upregulated by hypoxia. Intrauterine growth restriction (IUGR) is thought to be associated with inadequate placental perfusion, which might result from a failure in the development of the villous vascular network. Our present study was undertaken to examine the relationship between VEGF expression and IUGR in pregnancies with preserved umbilical artery end-diastolic flow. VEGF Expression was determined by immunohistochemical analysis of placentas from 17 pregnancies with normal infant birth weight and 17 pregnancies complicated by IUGR. We found no significant differences in the expression of VEGF in villous syncytiotrophoblasts and intermediate trophoblasts in maternal decidua between IUGR and normal pregnancies. However, in both groups there was a strong correlation in the expression of VEGF with villous syncytiotrophoblasts and intermediate trophoblasts. In normal and IUGR pregnancies the infants' Apgar scores at birth were significantly correlated with VEGF staining in both syncytiotrophoblasts and intermediate trophoblasts (P < .05). A strong correlation also was found between cord hematocrit and VEGF staining in villous syncytiotrophoblasts (P < .05), but VEGF staining in intermediate trophoblasts was not correlated with cord hemoglobin or hematocrit. Our results suggest that VEGF acts in an autocrine and paracrine fashion in both normal and IUGR placentas, and its expression can have an effect on the well being of the infant at birth.

  14. Immunohistochemical Examination on the Distribution of Cells Expressed Lymphatic Endothelial Marker Podoplanin and LYVE-1 in the Mouse Tongue Tissue

    Science.gov (United States)

    Noda, Yuya; Amano, Ikuko; Hata, Minoru; Kojima, Hiroshi; Sawa, Yoshihiko

    2010-01-01

    The clinical study for lingual disease requires the detailed investigation of the lingual lymphatic network and lymphatic marker-positive cells. Recently, it has been reported that several tissue cells and leukocytes express lymphatic markers, LYVE-1 and podoplanin. This study was aimed to clarify the lingual distribution of cells expressing LYVE-1 and podoplanin. In the mouse tongue, podoplanin is expressed in nerve sheaths, lingual gland myoepithelial cells, and lymphatic vessels. LYVE-1 is expressed in the macrophage marker Mac-1-positive cells as well as lymphatic vessels, while factor-VIII was detected in only blood endothelial cells. α-SMA was detected in vascular smooth muscle and myoepithelial cells. Therefore, identification of lymphatic vessels in lingual glands, the combination of LYVE-1 and factor-VIII, or LYVE-1 and Mac-1 is useful because myoepithelial cells express podoplanin and α-SMA. The immunostaining of factor-VIII on lymphatic vessels was masked by the immunostaining to LYVE-1 or podoplanin because lymphatic vessels express factor-VIII to a far lesser extent than blood vessels. Therefore, except for the salivary glands, the combination of podoplanin and α-SMA, or factor-VIII is useful to identify lymphatic vessels and blood vessels with smooth muscle, or blood capillaries. PMID:20514293

  15. Expression of the growth factor progranulin in endothelial cells influences growth and development of blood vessels: a novel mouse model.

    Science.gov (United States)

    Toh, Huishi; Cao, Mingju; Daniels, Eugene; Bateman, Andrew

    2013-01-01

    Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5) and later (E15.5-17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in

  16. Expression of the growth factor progranulin in endothelial cells influences growth and development of blood vessels: a novel mouse model.

    Directory of Open Access Journals (Sweden)

    Huishi Toh

    Full Text Available Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5 and later (E15.5-17.5 developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with

  17. Inhibition of tumor necrosis factor-α-induced expression of adhesion molecules in human endothelial cells by the saponins derived from roots of Platycodon grandiflorum

    International Nuclear Information System (INIS)

    Kim, Ji Young; Kim, Dong Hee; Kim, Hyung Gyun; Song, Gyu-Yong; Chung, Young Chul; Roh, Seong Hwan; Jeong, Hye Gwang

    2006-01-01

    Adhesion molecules play an important role in the development of atherogenesis and are produced by endothelial cells after being stimulated with various inflammatory cytokines. This study examined the effect of saponins that were isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. CKS significantly inhibited the TNFα-induced increase in monocyte adhesion to endothelial cells as well as decreased the protein and mRNA expression levels of vascular adhesion molecule-1 and intercellular cell adhesion molecule-1 on endothelial cells. Furthermore, CKS significantly inhibited the TNFα-induced production of intracellular reactive oxygen species (ROS) and activation of NF-κB by preventing IκB degradation and inhibiting IκB kinase activity. Overall, CKS has anti-atherosclerotic and anti-inflammatory activity, which is least in part the result of it reducing the cytokine-induced endothelial adhesion to monocytes by inhibiting intracellular ROS production, NF-κB activation, and cell adhesion molecule expression in endothelial cells

  18. Alteration of protein expression pattern of vascular endothelial growth factor (VEGF) from soluble to cell-associated isoform during tumourigenesis

    International Nuclear Information System (INIS)

    Cressey, Ratchada; Wattananupong, Onusa; Lertprasertsuke, Nirush; Vinitketkumnuen, Usanee

    2005-01-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells, and its expression has been correlated with increased tumour angiogenesis. Although numerous publications dealing with the measurement of circulating VEGF for diagnostic and therapeutic monitoring have been published, the relationship between the production of tissue VEGF and its concentration in blood is still unclear. The aims of this study were to determine: 1) The expression pattern of VEGF isoforms at the protein level in colorectal and lung adenocarcinoma in comparison to the pattern in corresponding adjacent normal tissues 2) The relationship between the expression pattern of VEGF and total level of circulating VEGF in the blood to clarify whether the results of measuring circulating VEGF can be used to predict VEGF expression in tumour tissues. Ninety-four tissue samples were obtained from patients, 76 colorectal tumour tissues and 18 lung tumour tissues. VEGF protein expression pattern and total circulating VEGF were examined using western blot and capture ELISA, respectively. Three major protein bands were predominately detected in tumour samples with an apparent molecular mass under reducing conditions of 18, 23 and 26 kDa. The 18 kDa VEGF protein was expressed equally in both normal and colorectal tumour tissues and predominately expressed in normal tissues of lung, whereas the 23 and 26 kDa protein was only detected at higher levels in tumour tissues. The 18, 23 and 26 kDa proteins are believed to represent the VEGF 121 , the VEGF 165 and the VEGF 189 , respectively. There was a significant correlation of the expression of VEGF 165 with a smaller tumour size maximum diameter <5 cm (p < 0.05), and there was a significant correlation of VEGF 189 with advanced clinical stage of colorectal tumours. The measurement of total circulating VEGF in serum revealed that cancer patients significantly (p < 0.001) possessed a higher level of circulating VEGF (1081 ± 652 pg/ml in

  19. Interaction between the Stress Phase Angle (SPA and the Oscillatory Shear Index (OSI Affects Endothelial Cell Gene Expression.

    Directory of Open Access Journals (Sweden)

    Ronny Amaya

    Full Text Available Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS and solid circumferential stress (CS. Due to variations in impedance (global factors and geometric complexities (local factors in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle-SPA. Asynchronous flows (SPA close to -180° that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between non-reversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous

  20. Methylmercury Causes Blood-Brain Barrier Damage in Rats via Upregulation of Vascular Endothelial Growth Factor Expression.

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    Tetsuya Takahashi

    Full Text Available Clinical manifestations of methylmercury (MeHg intoxication include cerebellar ataxia, concentric constriction of visual fields, and sensory and auditory disturbances. The symptoms depend on the site of MeHg damage, such as the cerebellum and occipital lobes. However, the underlying mechanism of MeHg-induced tissue vulnerability remains to be elucidated. In the present study, we used a rat model of subacute MeHg intoxication to investigate possible MeHg-induced blood-brain barrier (BBB damage. The model was established by exposing the rats to 20-ppm MeHg for up to 4 weeks; the rats exhibited severe cerebellar pathological changes, although there were no significant differences in mercury content among the different brain regions. BBB damage in the cerebellum after MeHg exposure was confirmed based on extravasation of endogenous immunoglobulin G (IgG and decreased expression of rat endothelial cell antigen-1. Furthermore, expression of vascular endothelial growth factor (VEGF, a potent angiogenic growth factor, increased markedly in the cerebellum and mildly in the occipital lobe following MeHg exposure. VEGF expression was detected mainly in astrocytes of the BBB. Intravenous administration of anti-VEGF neutralizing antibody mildly reduced the rate of hind-limb crossing signs observed in MeHg-exposed rats. In conclusion, we demonstrated for the first time that MeHg induces BBB damage via upregulation of VEGF expression at the BBB in vivo. Further studies are required in order to determine whether treatment targeted at VEGF can ameliorate MeHg-induced toxicity.

  1. Increased Hepatic Expression of Endothelial Lipase Inhibits Cholesterol Diet-Induced Hypercholesterolemia and Atherosclerosis in Transgenic Rabbits.

    Science.gov (United States)

    Wang, Chuan; Nishijima, Kazutoshi; Kitajima, Shuji; Niimi, Manabu; Yan, Haizhao; Chen, Yajie; Ning, Bo; Matsuhisa, Fumikazu; Liu, Enqi; Zhang, Jifeng; Chen, Y Eugene; Fan, Jianglin

    2017-07-01

    Endothelial lipase (EL) is a key determinant in plasma high-density lipoprotein-cholesterol. However, functional roles of EL on the development of atherosclerosis have not been clarified. We investigated whether hepatic expression of EL affects plasma lipoprotein metabolism and cholesterol diet-induced atherosclerosis. We generated transgenic (Tg) rabbits expressing the human EL gene in the liver and then examined the effects of EL expression on plasma lipids and lipoproteins and compared the susceptibility of Tg rabbits with cholesterol diet-induced atherosclerosis with non-Tg littermates. On a chow diet, hepatic expression of human EL in Tg rabbits led to remarkable reductions in plasma levels of total cholesterol, phospholipids, and high-density lipoprotein-cholesterol compared with non-Tg controls. On a cholesterol-rich diet for 16 weeks, Tg rabbits exhibited significantly lower hypercholesterolemia and less atherosclerosis than non-Tg littermates. In Tg rabbits, gross lesion area of aortic atherosclerosis was reduced by 52%, and the lesions were characterized by fewer macrophages and smooth muscle cells compared with non-Tg littermates. Increased hepatic expression of EL attenuates cholesterol diet-induced hypercholesterolemia and protects against atherosclerosis. © 2017 American Heart Association, Inc.

  2. Improvement of insulin sensitivity in response to exercise training in type 2 diabetes mellitus is associated with vascular endothelial growth factor A expression.

    Science.gov (United States)

    Wagner, Henrik; Fischer, Helene; Degerblad, Marie; Alvarsson, Michael; Gustafsson, Thomas

    2016-09-01

    Insulin sensitivity changes in response to exercise training demonstrate a large variation. Vascular endothelial growth factor A could promote increased insulin sensitivity through angiogenesis. We investigated associations between changes in expression of key genes and insulin sensitivity, aerobic capacity and glycaemic control following exercise training in diabetes mellitus type 2. Subjects with diabetes mellitus type 2 underwent 12 weeks of structured exercise. Euglycaemic clamp, exercise test and HbA1c were performed. Muscle biopsies were obtained for mRNA expression. A total of 16 subjects completed the study. Change in vascular endothelial growth factor A expression was positively associated with an increase in insulin sensitivity (p = 0.004) and with a decrease in HbA1c (p = 0.034). Vascular endothelial growth factor A receptor-1 expression showed similar associations. The variation in physical adaptation to exercise training in diabetes mellitus type 2 was associated with changes in expression of vascular endothelial growth factor A in muscle. This difference in induced gene expression could contribute to the variation in exercise training effects on insulin sensitivity. Measures of capillary blood flow need to be assessed in future studies. © The Author(s) 2016.

  3. Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering.

    Science.gov (United States)

    Jabbarzadeh, Ehsan; Jiang, Tao; Deng, Meng; Nair, Lakshmi S; Khan, Yusuf M; Laurencin, Cato T

    2007-12-01

    Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.

  4. Establishment of canine hemangiosarcoma xenograft models expressing endothelial growth factors, their receptors, and angiogenesis-associated homeobox genes

    International Nuclear Information System (INIS)

    Kodama, Atsushi; Yanai, Tokuma; Sakai, Hiroki; Matsuura, Satoko; Murakami, Mami; Murai, Atsuko; Mori, Takashi; Maruo, Kouji; Kimura, Tohru; Masegi, Toshiaki

    2009-01-01

    Human hemangiosarcoma (HSA) tends to have a poor prognosis; its tumorigenesis has not been elucidated, as there is a dearth of HSA clinical specimens and no experimental model for HSA. However, the incidence of spontaneous HSA is relatively high in canines; therefore, canine HSA has been useful in the study of human HSA. Recently, the production of angiogenic growth factors and their receptors in human and canine HSA has been reported. Moreover, the growth-factor environment of HSA is very similar to that of pathophysiological angiogenesis, which some homeobox genes regulate in the transcription of angiogenic molecules. In the present study, we established 6 xenograft canine HSA tumors and detected the expression of growth factors, their receptors, and angiogenic homeobox genes. Six primary canine HSAs were xenografted to nude mice subcutaneously and serially transplanted. Subsequently, the expressions of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factors (bFGF), flt-1 and flk-1 (receptors of VEGF-A), FGFR-1, and angiogenic homeobox genes HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 were investigated in original and xenograft tumors by histopathology, immunostaining, and reverse transcription polymerase chain reaction (RT-PCR), using canine-specific primer sets. Histopathologically, xenograft tumors comprised a proliferation of neoplastic cells that were varied in shape, from spindle-shaped and polygonal to ovoid; some vascular-like structures and vascular clefts of channels were observed, similar to those in the original tumors. The expression of endothelial markers (CD31 and vWF) was detected in xenograft tumors by immunohistochemistry and RT-PCR. Moreover, the expression of VEGF-A, bFGF, flt-1, flk-1, FGFR-1, HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 was detected in xenograft tumors. Interestingly, expressions of bFGF tended to be higher in 3 of the xenograft HSA tumors than in the other tumors. We established 6 xenograft canine HSA

  5. Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.

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    Silvia Dragoni

    Full Text Available BACKGROUND: An increase in the frequency of circulating endothelial colony forming cells (ECFCs, the only subset of endothelial progenitor cells (EPCs truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF. Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs and subjects suffering from renal cellular carcinoma (RCC-ECFCs. SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1. METHODOLOGY/PRINCIPAL FINDINGS: We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs. SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA or physiological (i.e. ATP stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective. CONCLUSIONS: Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3

  6. Plasma factors in severe early-onset preeclampsia do not substantially alter endothelial gene expression in vitro

    NARCIS (Netherlands)

    Donker, RB; Asgeirsdottir, SA; Gerbens, F; van Pamus, MG; Kallenberg, CGM; Meerman, GJT; Aarnoudse, JG; Molema, G

    OBJECTIVE: Systemic endothelial dysfunction is a central feature in the pathophysiology of preeclampsia. Its cell biologic and molecular basis is poorly understood. One leading hypothesis argues that endothelial dysfunction is caused by (at present largely unknown) circulating factors released from

  7. Gene expression patterns of vascular endothelial growth factor (VEGF-A) in human placenta from pregnancies with intrauterine growth restriction.

    Science.gov (United States)

    Szentpéteri, Imre; Rab, Attila; Kornya, László; Kovács, Péter; Joó, József Gábor

    2013-07-01

    In this study, we describe changes in gene expression pattern of vascular endothelial growth factor (VEGF)-A in human placenta obtained from pregnancies with intrauterine growth restriction using placenta from normal pregnancies as control. We compared gene expression of VEGF-A in placental samples from Intrauterine growth restriction (IUGR) pregnancies versus placenta obtained from normal pregnancies. Among potential confounders, important clinical informations were also analyzed. In the IUGR group, the VEGF-A gene was overexpressed compared to the normal pregnancy group (Ln 2(α)β-actin: 1.32; Ln 2(α)GADPH: 1.56). There was no correlation between the degree of growth restriction and VEGF-A gene expression (Ln 2(α)(0-5)percentile: 0.58; Ln 2(α)(5-10)percentile: 0.64). Within the IUGR group, there was a trend toward a positive correlation between placental VEGF-A gene activity and gestational age at delivery (Ln 2(α) 37 weeks: 1.35). Our findings suggest that the increase in placental expression of the VEGF-A gene and the resultant stimulation of angiogenesis are a response to hypoxic environment developing in the placental tissue in IUGR. Thus, it appears to be a secondary event rather than a primary factor in the development of IUGR There is a trend toward a positive correlation between gestational age and placental VEGF-A gene activity.

  8. Pulmonary microvascular hyperpermeability and expression of vascular endothelial growth factor in smoke inhalation- and pneumonia-induced acute lung injury.

    Science.gov (United States)

    Lange, Matthias; Hamahata, Atsumori; Traber, Daniel L; Connelly, Rhykka; Nakano, Yoshimitsu; Traber, Lillian D; Schmalstieg, Frank C; Herndon, David N; Enkhbaatar, Perenlei

    2012-11-01

    Acute lung injury (ALI) and sepsis are major contributors to the morbidity and mortality of critically ill patients. The current study was designed further evaluate the mechanism of pulmonary vascular hyperpermeability in sheep with these injuries. Sheep were randomized to a sham-injured control group (n=6) or ALI/sepsis group (n=7). The sheep in the ALI/sepsis group received inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. These groups were monitored for 24 h. Additional sheep (n=16) received the injury and lung tissue was harvested at different time points to measure lung wet/dry weight ratio, vascular endothelial growth factor (VEGF) mRNA and protein expression as well as 3-nitrotyrosine protein expression in lung homogenates. The injury induced severe deterioration in pulmonary gas exchange, increases in lung lymph flow and protein content, and lung water content (P<0.01 each). These alterations were associated with elevated lung and plasma nitrite/nitrate concentrations, increased tracheal blood flow, and enhanced VEGF mRNA and protein expression in lung tissue as well as enhanced 3-nitrotyrosine protein expression (P<0.05 each). This study describes the time course of pulmonary microvascular hyperpermeability in a clinical relevant large animal model and may improve the experimental design of future studies. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

  9. ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells.

    Science.gov (United States)

    Zhang, Hu; Zhang, Shuhong; Zhang, Jilin; Liu, Dongxin; Wei, Jiayi; Fang, Wengang; Zhao, Weidong; Chen, Yuhua; Shang, Deshu

    2018-05-01

    The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer's disease, and multiple sclerosis. We previously reported that in Alzheimer's disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood-brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood-brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

  10. Endoglin (CD105) expression on microvessel endothelial cells in juvenile nasopharyngeal angiofibroma: tissue microarray analysis and association with prognostic significance.

    Science.gov (United States)

    Wang, Jing-Jing; Sun, Xi-Cai; Hu, Li; Liu, Zhuo-Fu; Yu, Hua-Peng; Li, Han; Wang, Shu-Yi; Wang, De-Hui

    2013-12-01

    The purpose of this study was to examine endoglin (CD105) expression on microvessel endothelial cells (ECs) in juvenile nasopharyngeal angiofibroma (JNA) and its relationship with recurrence. Immunohistochemistry was performed to detect CD105 expression in a tissue microarray from 70 patients with JNA. Correlation between CD105 expression on microvessel ECs and clinicopathological features, as well as tumor recurrence, were analyzed. Immunohistochemistry revealed CD105 expression on ECs but not in stroma of patients with JNA. Chi-square analysis indicated CD105-based microvessel density (MVD) was correlated with JNA recurrence (p = .013). Univariate and multivariate analyses determined that MVD was a significant predictor of time to recurrence (p = .009). The CD105-based MVD was better for predicting disease recurrence (AUROC: 0.673; p = .036) than other clinicopathological features. MVD is a useful predictor for poor prognosis of patients with JNA after curative resection. Angiogenesis, which may play an important role in the occurrence and development of JNA, is therefore a potential therapeutic target for JNA. Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.

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

    Science.gov (United States)

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

    2006-11-01

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    The blood-brain barrier (BBB) represents the interface between the blood and the brain parenchyma and consists of endothelial cells which are tightly sealed together by tight junction proteins. The endothelial cells are in addition supported by pericytes, which are embedded in the vascular basement...... of the present study was to create four different in vitro constructs of the murine BBB to characterise if the expression and secretion of basement membrane proteins by the murine brain capillary endothelial cells (mBCECs) was affected by co-culturing with pericytes, mixed glial cells, or both. Primary m......BCECs and pericytes were isolated from brains of adult mice. Mixed glial cells were prepared from cerebral cortices of newborn mice. The mBCECs were grown as mono-culture, or co-cultured with pericytes, mixed glial cells, or both. To study the expression of basement membrane proteins RT-qPCR, mass spectrometry...

  13. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    -alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  14. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

    Science.gov (United States)

    Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

    2015-01-01

    The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

  15. Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

    Science.gov (United States)

    Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

    2016-10-01

    Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

  16. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    International Nuclear Information System (INIS)

    Zuo, Luning; Li, Qiang; Sun, Bei; Xu, Zhiying; Ge, Zhiming

    2013-01-01

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  17. The effect of lidocaine on in vitro neutrophil and endothelial adhesion molecule expression induced by plasma obtained during tourniquet-induced ischaemia and reperfusion.

    LENUS (Irish Health Repository)

    Lan, W

    2012-02-03

    BACKGROUND: Changes in neutrophil and endothelial adhesion molecule expression occur during perioperative ischaemia and reperfusion (I\\/R) injury. We investigated the effects of lidocaine on neutrophil-independent changes in neutrophil and endothelial adhesion molecule expression associated with tourniquet-induced I\\/R. METHODS: Plasma was obtained from venous blood samples (tourniquet arm) taken before (baseline), during, 15 min, 2 and 24 h following tourniquet release in seven patients undergoing elective upper limb surgery with tourniquet application. Isolated neutrophils from healthy volunteers (n = 7) were pretreated in the presence or absence of lidocaine (0.005, 0.05 and 0.5 mg mL(-1) for 1 h, and then incubated with I\\/R plasma for 2 h. Human umbilical vein endothelial cells (HUVECs) were pretreated in the presence or absence of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)) for 1 h, and then incubated with the plasma for 4 h. Adhesion molecule expression was estimated using flow cytometry. Data were analysed using ANOVA and post hoc Student-Newman-Keuls tests. RESULTS: I\\/R plasma (withdrawn 15 min following tourniquet release) increased isolated neutrophil CD11b (P = 0.03), CD18 (P = 0.01) and endothelial intercellular adhesion molecule-1 (ICAM-1) (P = 0.008) expression compared to baseline. CD11b, CD18 and ICAM-1 expression on lidocaine (0.005 mg mL(-1)) treated neutrophils was similar to control. CD11b (P < 0.001), CD18 (P = 0.03) and ICAM-1 (P = 0.002) expression on lidocaine (0.05 mg mL(-1)) treated neutrophils and HUVECs was less than that on controls. CONCLUSION: Increased in vitro neutrophil and endothelial cell adhesion molecule expression on exposure to plasma obtained during the early reperfusion phase is diminished by lidocaine at greater than clinically relevant plasma concentrations.

  18. Myocardial regeneration by transplantation of modified endothelial progenitor cells expressing SDF-1 in a rat model

    DEFF Research Database (Denmark)

    Schuh, A.; Kroh, A.; Konschalla, S.

    2012-01-01

    Cell based therapy has been shown to attenuate myocardial dysfunction after myocardial infarction (MI) in different acute and chronic animal models. It has been further shown that stromal-cell derived factor-1a (SDF-1a) facilitates proliferation and migration of endogenous progenitor cells...... into injured tissue. The aim of the present study was to investigate the role of exogenously applied and endogenously mobilized cells in a regenerative strategy for MI therapy. Lentivirally SDF-1a-infected endothelial progenitor cells (EPCs) were injected after 90 min. of ligation and reperfusion of the left...... compared to medium control. Intracoronary application of cells did not lead to significant differences compared to medium injected control hearts. Histology showed a significantly elevated rate of apoptotic cells and augmented proliferation after transplantation of EPCs and EPCs + SDF-1 alpha in infarcted...

  19. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  20. Triglyceride-rich lipoprotein modulates endothelial vascular cell adhesion molecule (VCAM-1 expression via differential regulation of endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Ying I Wang

    Full Text Available Circulating triglyceride-rich lipoproteins (TGRL from hypertriglyceridemic subjects exacerbate endothelial inflammation and promote monocyte infiltration into the arterial wall. We have recently reported that TGRL isolated from human blood after a high-fat meal can elicit a pro- or anti-atherogenic state in human aortic endothelial cells (HAEC, defined as up- or down-regulation of VCAM-1 expression in response to tumor necrosis factor alpha (TNFα stimulation, respectively. A direct correlation was found between subjects categorized at higher risk for cardiovascular disease based upon serum triglycerides and postprandial production of TGRL particles that increased VCAM-1-dependent monocyte adhesion to inflamed endothelium. To establish how TGRL metabolism is linked to VCAM-1 regulation, we examined endoplasmic reticulum (ER stress and the unfolded protein response (UPR pathways. Regardless of its atherogenicity, the rate and extent of TGRL internalization and lipid droplet formation by HAEC were uniform. However, pro-atherogenic TGRL exacerbated ER membrane expansion and stress following TNFα stimulation, whereas anti-atherogenic TGRL ameliorated such effects. Inhibition of ER stress with a chemical chaperone 4-phenylbutyric acid decreased TNFα-induced VCAM-1 expression and abrogated TGRL's atherogenic effect. Activation of ER stress sensors PKR-like ER-regulated kinase (PERK and inositol requiring protein 1α (IRE1α, and downstream effectors including eukaryotic initiation factor-2α (eIF2α, spliced X-box-binding protein 1 (sXBP1 and C/EBP homologous protein (CHOP, directly correlated with the atherogenic activity of an individual's TGRL. Modulation of ER stress sensors also correlated with changes in expression of interferon regulatory factor 1 (IRF-1, a transcription factor of Vcam-1 responsible for regulation of its expression. Moreover, knockdown studies using siRNA defined a causal relationship between the PERK/eIF2α/CHOP pathway and

  1. Effects of Chinese yellow wine on nitric oxide synthase and intercellular adhesion molecule-1 expressions in rat vascular endothelial cells.

    Science.gov (United States)

    Zhao, Fei; Ji, Zheng; Chi, Jufang; Tang, Weiliang; Zhai, Xiaoya; Meng, Liping; Guo, Hangyuan

    2016-02-01

    The objective of this study was to determine similarities in the effect of yellow wine as compared to statin and the possibility that yellow wine inhibits tumour necrosis factor-α (TNF-α)-induced nitric oxide (NO) synthesis, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) in cultured rat vascular endothelial cells (VECs). We isolated VECs, and cultivated and purified Sprague Dawley (SD) rat thoracic aortas in vitro. We selected the optimal wine concentration using clonogenic and MTT assays to measure cell survival. Next, we divided the cells into 9 groups: (1) control, (2) TNF-α, (3) TNF-α + rosuvastatin (10 μmol/L), (4) TNF-α + ethanol 0.5%, (5) TNF-α + yellow wine 0.5%, (6) TNF-α + ethanol 1.0%, (7) TNF-α + yellow wine 1.0%, (8) TNF-α + ethanol 1.5%, and (9) TNF-α + yellow wine 1.5% and they were given the corresponding treatment for 24 h. We determined NO production with nitrate reductase. We then measured eNOS activity, and detected eNOS, iNOS, and ICAM-1 protein levels by Western blotting. Compared with the TNF-α group, NO production, eNOS activity, and eNOS protein expression in the rosuvastatin, and yellow wine 1.0%, and 1.5% groups were significantly increased. Protein expression of iNOS and ICAM-1 in the rosuvastatin, yellow wine 1.0%, and 1.5% groups were significantly decreased. Compared with the rosuvastatin group, eNOS, iNOS, and ICAM-1 protein expression in the yellow wine (0.5% -1.5%) groups were significantly different. Treatment with yellow wine increased NO production, eNOS activity, and eNOS protein expression, which decreases iNOS and ICAM-1 protein expression. We conclude that yellow wine may have similar beneficial effects as rosuvastatin on the cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions.

  2. Expression of the vascular endothelial growth factor receptor neuropilin-1 at the human embryo-maternal interface.

    Science.gov (United States)

    Baston-Buest, Dunja M; Porn, Anne C; Schanz, Andrea; Kruessel, Jan-S; Janni, Wolfgang; Hess, Alexandra P

    2011-02-01

    Angiogenesis is required for successful implantation of the invading blastocyst. Vascular endothelial growth factor (VEGF) is an important key player in angiogenesis and vascular remodeling during the implantation process. Besides its well-characterized receptors VEGFR1 and VEGFR2, neuropilin-1 (NRP-1) has been shown to play an additional role in the signaling process of angiogenesis in human endometrium during the menstrual cycle, as a co-receptor of VEGF. These findings led to the hypothesis that NRP-1 might play a role in the vascular remodeling process during embryo implantation and the establishment of a pregnancy. NRP-1 mRNA transcript and protein expression were investigated in human choriocarcinoma cell lines (JEG-3, Jar and BeWo) aiming to evaluate the expression of NRP-1 in vitro, as well as in human decidua of all three trimesters of pregnancy, by western blot analysis (three samples of each trimester of pregnancy). The localization of NRP-1 in human decidua of all three trimesters of pregnancy was analyzed by immunohistochemistry (five samples of each trimester of pregnancy). NRP-1 transcript and protein were expressed in all cell lines examined. Corresponding to the analysis of human tissue by western blot and the localization by immunohistochemistry, NRP-1 protein higher expressed in samples of early pregnancy in comparison to the end of pregnancy. NRP-1 was expressed in the decidua, villi and invading cytotrophoblast of all samples investigated. This is the first study clearly showing the expression of NRP-1 in human decidua and trophoblast, suggesting an important role for the VEGF co-receptor NRP-1 besides the established receptor VEGFR2 at the embryo-maternal interface during embryonic implantation and placentation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  3. Vascular endothelial growth factor C promotes cervical cancer cell invasiveness via regulation of microRNA-326/cortactin expression.

    Science.gov (United States)

    Cheng, Yang; Jiang, Shuyi; Yuan, Jin; Liu, Junxiu; Simoncini, Tommaso

    2018-04-16

    Vascular endothelial growth factor C (VEGF-C) accelerates cervical cancer metastasis, while the detailed mechanism remains largely unknown. Recent evidence indicates that microRNA play a crucial role in controlling cancer cell invasiveness. In the present study, we investigated the role of miR-326 in VEGF-C-induced cervical cancer cell invasion. VEGF-C expression was higher and miR-326 was much lower in primary cervical cancer specimens than that in non-cancerous specimens, and a negative correlation between VEGF-C and miR-326 was found. On cervical carcinoma cell line SiHa cells, treatment with VEGF-C downregulated miR-326 level and increased cortactin protein expression. Transfection with miR-326 mimic reversed cortactin expression induced by VEGF-C, suggesting that VEGF-C increased cortactin via downregulation of miR-326. VEGF-C activated c-Src and c-Src inhibitor PP2 abolished VEGF-C effect on miR-326 and cortactin expression, implying that VEGF-C regulated miR-326/cortactin via c-Src signaling. VEGF-C promoted SiHa cell invasion index, which was largely inhibited by transfection with miR-326 antagonist or by siRNA against cortactin. In conclusion, our findings implied that VEGF-C reduced miR-326 expression and increased cortactin expression through c-Src signaling, leading to enhanced cervical cancer invasiveness. This may shed light on potential therapeutic strategies for cervical cancer therapy.

  4. Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    ML Rossi

    2009-06-01

    Full Text Available Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs and impaired bioavailabilty of nitric oxide (NO are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5’ nuclease assays (TaqMan™ PCRs to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA and 15 with acute coronary syndromes (ACS without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001 in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype.

  5. Effect of systemic piracetam treatment on flap survival and vascular endothelial growth factor expression after ischemia-reperfusion injury.

    Science.gov (United States)

    Tuncer, Serhan; Ayhan, Suhan; Findikcioglu, Kemal; Ergun, Hakan; Tuncer, Ilhan

    2011-09-01

    The effects of piracetam on flap survival, ischemia-reperfusion (I/R) injury, and vascular endothelial growth factor (VEGF) expression were evaluated in this study. Unipedicled epigastric flap model was used in 36 rats and was evaluated within 4 groups. The flap was elevated and untreated in Group 1. Postoperative piracetam treatment was given for 7 days in Group 2. In Group 3, 4 hours of ischemia and 2 hours of reperfusion were applied. I/R was applied to Group 4 and piracetam was given 30 minutes before reperfusion and postoperatively for 7 days. Laser Doppler flowmetry was used to measure blood flow changes. VEGF expression was determined using immunohistochemical methods on tissue samples taken after the completion of 2 hours reperfusion in groups 3 and 4. Flap necrosis was measured on the day 7 in all groups. Blood flow rates did not show significant difference between piracetam treated and untreated I/R groups. Piracetam significantly reduced necrosis area both in ischemic and nonischemic flaps ( P piracetam-treated Group 4 compared with Group 3 ( P = 0.005). This experimental study demonstrates that systemic piracetam treatment improves survival of pedicled flaps, reduces necrosis amounts, and increases VEGF expression in I/R induced flaps. © Thieme Medical Publishers.

  6. Effects of hyperthyroidism on expression of vascular endothelial growth factor (VEGF) and apoptosis in fetal adrenal glands.

    Science.gov (United States)

    Karaca, T; Hulya Uz, Y; Karabacak, R; Karaboga, I; Demirtas, S; Cagatay Cicek, A

    2015-11-26

    This study investigated the expression of vascular endothelial growth factor (VEGF), vascular density, and apoptosis in fetal rat adrenal glands with hyperthyroidism in late gestation. Twelve mature female Wistar albino rats with the same biological and physiological features were used for this study. Rats were divided into two groups: control and hyperthyroidism. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (250 μg/kg) before pregnancy for 21 days and during pregnancy. Rats in the control and hyperthyroidism groups were caged according to the number of male rats. Zero day of pregnancy (Day 0) was indicated when the animals were observed to have microscopic sperm in vaginal smears. Pregnant rats were sacrificed on the 20th day of pregnancy; blood from each animal was collected to determine the concentrations of maternal adrenocorticotropic hormone and thyroxine. Rat fetuses were then quickly removed from the uterus, and the adrenal glands of the fetuses were dissected. VEGF expression, vascular density, and apoptosis were analyzed in fetal rat adrenal glands. Maternal serum levels of the adrenocorticotropic hormone and free thyroxine were significantly higher in the hyperthyroidism group than in the control group. Immunohistochemistry revealed that the number of VEGF positive cells and vessel density significantly increased in the hyperthyroidism rat fetal adrenal group compared with the control group. Hyperthyroidism did not change the fetal and placental weights and the number of fetuses. This study demonstrates that hyperthyroidism may have an effect on the development of rat adrenal glands mediated by VEGF expression, angiogenesis, and apoptosis.

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

    Science.gov (United States)

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

    2017-03-08

    Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

  8. Gene expression profiles of glucose toxicity-exposed islet microvascular endothelial cells.

    Science.gov (United States)

    Liu, Mingming; Lu, Wenbao; Hou, Qunxing; Wang, Bing; Sheng, Youming; Wu, Qingbin; Li, Bingwei; Liu, Xueting; Zhang, Xiaoyan; Li, Ailing; Zhang, Honggang; Xiu, Ruijuan

    2018-03-25

    Islet microcirculation is mainly composed by IMECs. The aim of the study was to investigate the differences in gene expression profiles of IMECs upon glucose toxicity exposure and insulin treatment. IMECs were treated with 5.6 mmol L -1 glucose, 35 mmol L -1 glucose, and 35 mmol L -1 glucose plus 10 -8  mol L -1 insulin, respectively. Gene expression profiles were determined by microarray and verified by qPCR. GO terms and KEGG analysis were performed to assess the potential roles of differentially expressed genes. The interaction and expression tendency of differentially expressed genes were analyzed by Path-Net algorithm. Compared with glucose toxicity-exposed IMECs, 1574 mRNAs in control group and 2870 mRNAs in insulin-treated IMECs were identified with differential expression, respectively. GO and KEGG pathway analysis revealed that these genes conferred roles in regulation of apoptosis, proliferation, migration, adhesion, and metabolic process etc. Additionally, MAPK signaling pathway and apoptosis were the dominant nodes in Path-Net. IMECs survival and function pathways were significantly changed, and the expression tendency of genes from euglycemia and glucose toxicity exposure to insulin treatment was revealed and enriched in 7 patterns. Our study provides a microcirculatory framework for gene expression profiles of glucose toxicity-exposed IMECs. © 2018 John Wiley & Sons Ltd.

  9. Immunohistochemical properties of the expression of endothelial nitric oxide synthase in placenta in its dysfunction in women with iron deficiency anemia

    Directory of Open Access Journals (Sweden)

    I. A. Ancheva

    2014-08-01

    Full Text Available Aim. The pertinence of the study is related to a large prevalence of dysfunction of the placenta in pregnant women suffering from anemia. To evaluate the immunohistochemical expression characteristics of endothelial NO-synthase (eNOS in the placenta during its dysfunction in women against the background of anemia 30 samples of placental tissue were studied with the use of immunohistochemical and micrometer methods. Materials and results. It was found that the expression of eNOS in patients with iron defi ciency anemia in the cytoplasm of syncytium of villi and fetal capillary endothelium as well as decidual vessels decreases, with the most pronounced changes in the expression of eNOS observed in the presence of the combination of placental dysfunction and iron defi ciency anemia in the form of paradoxical increase in expression. Conclusion. This indicates the necessity for correction of endothelial function in women with anemia during pregnancy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Kui; Fan, Wendong; Wang, Xing; Ke, Xiao [Division of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080 (China); Wu, Guifu, E-mail: eecpchina@yahoo.com.cn [Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou 510080 (China); Hu, Chengheng, E-mail: huchenghengpci@yahoo.com.cn [Division of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080 (China)

    2012-10-12

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

  11. The effect of lidocaine on neutrophil CD11b/CD18 and endothelial ICAM-1 expression and IL-1beta concentrations induced by hypoxia-reoxygenation.

    LENUS (Irish Health Repository)

    Lan, W

    2012-02-03

    BACKGROUND: Lidocaine has actions potentially of benefit during ischaemia-reperfusion. Neutrophils and endothelial cells have an important role in ischaemia-reperfusion injury. METHODS: Isolated human neutrophil CD11b and CD18, and human umbilical vein endothelial cell (HUVEC) ICAM-1 expression and supernatant IL-1beta concentrations in response to hypoxia-reoxygenation were studied in the presence or absence of different concentrations of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)). Adhesion molecule expression was quantified by flow cytometry and IL- 1beta concentrations by ELISA. Differences were assessed with analysis of variance and Student-Newman-Keuls as appropriate. Data are presented as mean+\\/-SD. RESULTS: Exposure to hypoxia-reoxygenation increased neutrophil CD11b (94.33+\\/-40.65 vs. 34.32+\\/-6.83 mean channel fluorescence (MCF), P = 0.02), CD18 (109.84+\\/-35.44 vs. 59.05+\\/-6.71 MCF, P = 0.03) and endothelial ICAM-1 (146.62+\\/-16.78 vs. 47.29+\\/-9.85 MCF, P < 0.001) expression compared to normoxia. Neutrophil CD18 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (71.07+\\/-10.14 vs. 109.84+\\/-35.44 MCF, P = 0.03). Endothelial ICAM-1 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (133.25+\\/-16.05 vs. 146.62+\\/-16.78 MCF, P = 0.03). Hypoxia-reoxygenation increased HUVEC supernatant IL-1beta concentrations compared to normoxia (3.41+\\/-0.36 vs. 2.65+\\/-0.21 pg mL(-1), P = 0.02). Endothelial supernatant IL-1beta concentrations in lidocaine-treated HUVECs were similar to controls. CONCLUSIONS: Lidocaine at clinically relevant concentrations decreased neutrophil CD18 and endothelial ICAM-1 expression but not endothelial IL-1beta concentrations.

  12. Chronic aerobic exercise associated to dietary modification improve endothelial function and eNOS expression in high fat fed hamsters.

    Directory of Open Access Journals (Sweden)

    Beatriz C S Boa

    Full Text Available Obesity is epidemic in the western world and central adipose tissue deposition points to increased cardiovascular morbidity and mortality, independently of any association between obesity and other cardiovascular risk factors. Physical exercise has been used as non-pharmacological treatment to significantly reverse/attenuate obesity comorbidities. In this study we have investigated effects of exercise and/or dietary modification on microcirculatory function, body composition, serum glucose, iNOS and eNOS expression on 120 male hamsters treated for 12 weeks with high fat chow (HF, n = 30 starting on the 21st day of birth. From week 12 to 20, animals were randomly separated in HF (no treatment change, return to standard chow (HFSC, n = 30, high fat chow associated to an aerobic exercise training program (AET (HFEX, n = 30 and return to standard chow+AET (HFSCEX, n = 30. Microvascular reactivity in response to acetylcholine and sodium nitroprusside and macromolecular permeability increase induced by 30 minutes ischemia followed by reperfusion were assessed on the cheek pouch preparation. Total body fat and aorta eNOS and iNOS expression by immunoblotting assay were evaluated on the experimental day. Compared to HFSC and HFSCEX groups, HF and HFEX ones presented increased visceral fat [(mean±SEM (HF4.9±1.5 g and (HFEX4.7±0.9 g vs. (HFSC*3.0±0.7 g and (HFSCEX*1.9±0.4 g/100 g BW]; impaired endothelial-dependent vasodilatation [Ach 10(-8 M (HF87.9±2.7%; (HFSC*116.7±5.9%; (HFEX*109.1±4.6%; (HFSCEX*105±2.8%; Ach10(-6 M (HF95.3±3.1%; (HFSC*126±6.2%; (HFEX*122.5±2.8%; (HFSCEX*118.1±4.3% and Ach10(-4 M (HF109.5±4.8%; (HFSC*149.6±6.6%; (HFEX*143.5±5.4% and (HFSCEX*139.4±5.2%], macromolecular permeability increase after ischemia/reperfusion [(HF40.5±4.2; (HFSC*19.0±1.6; (HFEX*18.6±2.1 and (HFSCEX* 21.5±3.7 leaks/cm2, decreased eNOS expression, increased leptin and glycaemic levels. Endothelial-independent microvascular

  13. Expression, purification, and characterization of a diabody against the most important angiogenesis cell receptor: Vascular endothelial growth factor receptor 2

    Directory of Open Access Journals (Sweden)

    Mahdi Behdani

    2012-01-01

    Full Text Available Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2 is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.

  14. PDE7B is a novel, prognostically significant mediator of glioblastoma growth whose expression is regulated by endothelial cells.

    Directory of Open Access Journals (Sweden)

    Michael D Brooks

    Full Text Available Cell-cell interactions between tumor cells and constituents of their microenvironment are critical determinants of tumor tissue biology and therapeutic responses. Interactions between glioblastoma (GBM cells and endothelial cells (ECs establish a purported cancer stem cell niche. We hypothesized that genes regulated by these interactions would be important, particularly as therapeutic targets. Using a computational approach, we deconvoluted expression data from a mixed physical co-culture of GBM cells and ECs and identified a previously undescribed upregulation of the cAMP specific phosphodiesterase PDE7B in GBM cells in response to direct contact with ECs. We further found that elevated PDE7B expression occurs in most GBM cases and has a negative effect on survival. PDE7B overexpression resulted in the expansion of a stem-like cell subpopulation in vitro and increased tumor growth and aggressiveness in an in vivo intracranial GBM model. Collectively these studies illustrate a novel approach for studying cell-cell interactions and identifying new therapeutic targets like PDE7B in GBM.

  15. Coffee induces vascular endothelial growth factor (VEGF) expression in human neuroblastama SH-SY5Y cells.

    Science.gov (United States)

    Kakio, Shota; Funakoshi-Tago, Megumi; Kobata, Kenji; Tamura, Hiroomi

    2017-07-01

    Recent evidence indicates that hypoxia-inducible vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective effects on neuronal and glial cells. On the other hand, recent epidemiological studies showed that daily coffee consumption has been associated with a lower risk of several neuronal disorders. Therefore, we investigated the effect of coffee on VEGF expression in human neuroblastoma SH-SY5Y cells. We found that even low concentration of coffee (coffee was attributed to the coffee-dependent inhibition of prolyl hydroxylation of HIF1α, which is essential for proteolytic degradation of HIF-1α. However, no inhibition was observed at the catalytic activity in vitro. Coffee component(s) responsible for the activation of HIF-1α was not major constituents such as caffeine, caffeic acid, chlorogenic acid, and trigonelline, but was found to emerge during roasting process. The active component(s) was extractable with ethyl acetate. Our results suggest that daily consumption of coffee may induce VEGF expression in neuronal cells. This might be related to protective effect of coffee on neural disorders such as Alzheimer's disease and Parkinson's disease.

  16. Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

    Science.gov (United States)

    De Meyer, Simon F.; Vanhoorelbeke, Karen; Chuah, Marinee K.; Pareyn, Inge; Gillijns, Veerle; Hebbel, Robert P.; Collen, Désiré; Deckmyn, Hans; VandenDriessche, Thierry

    2006-01-01

    Von Willebrand disease (VWD) is an inherited bleeding disorder, caused by quantitative (type 1 and 3) or qualitative (type 2) defects in von Willebrand factor (VWF). Gene therapy is an appealing strategy for treatment of VWD because it is caused by a single gene defect and because VWF is secreted into the circulation, obviating the need for targeting specific organs or tissues. However, development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA (8.4 kb [kilobase]) and the inherent complexity of the VWF protein that requires extensive posttranslational processing. In this study, a gene-based approach for VWD was developed using lentiviral transduction of blood-outgrowth endothelial cells (BOECs) to express functional VWF. A lentiviral vector encoding complete human VWF was used to transduce BOECs isolated from type 3 VWD dogs resulting in high-transduction efficiencies (95.6% ± 2.2%). Transduced VWD BOECs efficiently expressed functional vector-encoded VWF (4.6 ± 0.4 U/24 hour per 106 cells), with normal binding to GPIbα and collagen and synthesis of a broad range of multimers resulting in phenotypic correction of these cells. These results indicate for the first time that gene therapy of type 3 VWD is feasible and that BOECs are attractive target cells for this purpose. PMID:16478886

  17. NFAT regulation of cystathionine γ-lyase expression in endothelial cells is impaired in rats exposed to intermittent hypoxia.

    Science.gov (United States)

    Gonzalez Bosc, Laura V; Osmond, Jessica M; Giermakowska, Wieslawa K; Pace, Carolyn E; Riggs, Jennifer L; Jackson-Weaver, Olan; Kanagy, Nancy L

    2017-04-01

    Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O 2 -5% CO 2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H 2 S)-induced vasodilation in rats. The enzyme that produces H 2 S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca 2+ concentration ([Ca 2+ ]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca 2+ ] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca 2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation. Copyright © 2017 the American Physiological Society.

  18. Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel.

    Science.gov (United States)

    Sandovici, Maria; Henning, Robert H; Hut, Roelof A; Strijkstra, Arjen M; Epema, Anne H; van Goor, Harry; Deelman, Leo E

    2004-09-01

    Hibernating animals transiently reduce renal function during their hypothermic periods (torpor), while completely restoring it during their periodical rewarming to euthermia (arousal). Moreover, structural integrity of the kidney is preserved throughout the hibernation. Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) is a crucial vasodilatory mediator and a protective factor in the kidney. We investigated renal NOS expression in hibernating European ground squirrels after 1 day and 7 days of torpor (torpor short, TS, and torpor long, TL, respectively), at 1.5 and at 10 h of rewarming (arousal short, AS, and arousal long, AL, respectively), and in continuously euthermic animals after hibernation (EU). For that purpose, we performed NOS activity assay, immunohistochemistry and real-time PCR analysis. Immunohistochemistry revealed a decreased glomerular eNOS expression in hibernating animals (TS, TL, AS, and AL) compared to non-hibernating animals (EU, p EU. In all methods used, torpid and aroused squirrels did not differ. These results demonstrate differential regulation of eNOS in glomeruli and interstitium of hibernating animals, which is unaffected during arousal. The differential regulation of eNOS may serve to reduce ultrafiltration without jeopardizing tubular structures during hibernation.

  19. 7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

    Science.gov (United States)

    Duran, M J; Pierre, S V; Lesnik, P; Pieroni, G; Bourdeaux, M; Dignat-Georges, F; Sampol, J; Maixent, J M

    2010-11-09

    As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

  20. The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

    Science.gov (United States)

    Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

    2018-01-01

    The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

  1. Cytotoxicity, oxidative stress and expression of adhesion molecules in human umbilical vein endothelial cells exposed to dust from paints with or without nanoparticles

    DEFF Research Database (Denmark)

    Mikkelsen, Lone; Jensen, Keld A; Koponen, Ismo K

    2013-01-01

    Abstract Nanoparticles in primary form and nanoproducts might elicit different toxicological responses. We compared paint-related nanoparticles with respect to effects on endothelial oxidative stress, cytotoxicity and cell adhesion molecule expression. Primary human umbilical vein endothelial cells...... were exposed to primary nanoparticles (fine, photocatalytic or nanosized TiO(2), aluminium silicate, carbon black, nano-silicasol or axilate) and dust from sanding reference- or nanoparticle-containing paints. Most of the samples increased cell surface expressions of vascular cell adhesion molecule-1...... (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), but paint sanding dust samples generally generated less response than primary particles of TiO(2) and carbon black. We found no relationship between the expression of adhesion molecules, cytotoxicity and production of reactive oxygen species...

  2. Vascular endothelial dysfunction in β-thalassemia occurs despite increased eNOS expression and preserved vascular smooth muscle cell reactivity to NO.

    Directory of Open Access Journals (Sweden)

    Ekatherina Stoyanova

    Full Text Available The hereditary β-thalassemia major condition requires regular lifelong blood transfusions. Transfusion-related iron overloading has been associated with the onset of cardiovascular complications, including cardiac dysfunction and vascular anomalies. By using an untransfused murine model of β-thalassemia major, we tested the hypothesis that vascular endothelial dysfunction, alterations of arterial structure and of its mechanical properties would occur despite the absence of treatments.Vascular function and structure were evaluated ex vivo. Compared to the controls, endothelium-dependent vasodilation with acetylcholine was blunted in mesenteric resistance arteries of β-thalassemic mice while the endothelium-independent vasodilator (sodium nitroprusside produced comparable vessel dilation, indicating endothelial cell impairment with preserved smooth muscle cell reactivity to nitric oxide (NO. While these findings suggest a decrease in NO bioavailability, Western blotting showed heightened expression of aortic endothelial NO synthase (eNOS in β-thalassemia. Vascular remodeling of the common carotid arteries revealed increased medial elastin content. Under isobaric conditions, the carotid arteries of β-thalassemic mice exhibited decreased wall stress and softening due to structural changes of the vessel wall.A complex vasculopathy was identified in untransfused β-thalassemic mice characterized by altered carotid artery structure and endothelial dysfunction of resistance arterioles, likely attributable to reduced NO bioavailability despite enhanced vascular eNOS expression.

  3. Application of chitosan scaffolds on vascular endothelial growth factor and fibroblast growth factor 2 expressions in tissue engineering principles

    Directory of Open Access Journals (Sweden)

    Ariyati Retno Pratiwi

    2015-12-01

    Full Text Available Background: Tissue engineering has given satisfactory results as biological tissue substitutes to restore, replace, or regenerate tissues that have a defect. Chitosan is an organic biomaterial often used in the biomedical field. Chitosan has biocompatible, antifungal, and antibacterial properties. Chitosan is osteoconductive, suitable for bone regeneration applications. Bone defect healing begins with inflammatory phase as a response to the presence of vascular injury, so new vascularization is required. Vascular endothelial growth factor (VEGF and basic fibroblast growth factor-2 (FGF2 are indicators of the beginning of bone regeneration process, playing an important role in angiogenesis. Purpose: This research was aimed to determine the effects of chitosan scaffold application on the expressions of VEGF and FGF2 in tissue engineering principles. Method: Chitosan was dissolved in CH3COOH and NaOH to form a gel. Chitosan gel was then printed in mould to freeze dry for 24 hours. Those rats with defected bones were divided into two groups. Group 1 was the control group which defected bones were not administrated with chitosan scaffolds. Group 2 was the treatment group which defected bones were administrated with chitosan scaffolds. Those rats were sacrificed on day 14. Tissue preparations were made, and then immunohistochemical staining was conducted. Finally, a statistical analysis was conducted using Kruskal Wallis test. Result: There was no significant difference in the expressions of VEGF and FGF2 between the control group and the treatment group (p>0.05. Conclusion: Chitosan scaffolds do not affect the expressions of VEGF and FGF2 during bone regeneration process on day 14 in tissue engineering principles

  4. Effects of hyperthyroidism on expression of vascular endothelial growth factor (VEGF and apoptosis in fetal adrenal glands

    Directory of Open Access Journals (Sweden)

    T. Karaca

    2015-11-01

    Full Text Available This study investigated the expression of vascular endothelial growth factor (VEGF, vascular density, and apoptosis in fetal rat adrenal glands with hyperthyroidism in late gestation. Twelve mature female Wistar albino rats with the same biological and physiological features were used for this study. Rats were divided into two groups: control and hyperthyroidism. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (250 μg/kg before pregnancy for 21 days and during pregnancy. Rats in the control and hyperthyroidism groups were caged according to the number of male rats. Zero day of pregnancy (Day 0 was indicated when the animals were observed to have microscopic sperm in vaginal smears. Pregnant rats were sacrificed on the 20th day of pregnancy; blood from each animal was collected to determine the concentrations of maternal adrenocorticotropic hormone and thyroxine. Rat fetuses were then quickly removed from the uterus, and the adrenal glands of the fetuses were dissected. VEGF expression, vascular density, and apoptosis were analyzed in fetal rat adrenal glands. Maternal serum levels of the adrenocorticotropic hormone and free thyroxine were significantly higher in the hyperthyroidism group than in the control group. Immunohistochemistry revealed that the number of VEGF positive cells and vessel density significantly increased in the hyperthyroidism rat fetal adrenal group compared with the control group. Hyperthyroidism did not change the fetal and placental weights and the number of fetuses. This study demonstrates that hyperthyroidism may have an effect on the development of rat adrenal glands mediated by VEGF expression, angiogenesis, and apoptosis. 

  5. Immunohistochemical expression of vascular endothelial growth factor in keratocystic odontogenic tumor, dentigerous cyst, and radicular cyst: A comparative study.

    Science.gov (United States)

    Khajuria, Nidhi; Metgud, Rashmi; Naik, Smitha; Lerra, Sahul; Tiwari, Priya; Mamta; Katakwar, Payal; Tak, Anirudh

    2016-01-01

    Cyst and tumors arise from tissue remains of odontogenesis, these interactions have been considered to play an important role in the tumorigenesis of odontogenic lesions. The connective tissue stroma has an essential role in the preservation of epithelial tissues and minor alterations in the epithelium are followed by corresponding changes in the stroma, such as angiogenesis. Vascular endothelial growth factor (VEGF) is considered the first factor which maintains its position as the most critical driver of vascular formation and is required to initiate the formation of immature vessels, with this aim, present study was executed to evaluate VEGF expression in kertocystic odontogenic tumor, dentigerous cyst and radicular cyst (RC). A retrospective study was carried out comprising a total of 31 cases; 13 cases of keratocystic odontogenic tumor (KCOT), nine cases of dentigerous cyst (DC) and nine cases of RC. The sections were stained immunohistochemically with VEGF antibody and were evaluated for the presence and intensity of the immuno reactive cells. Statistical analysis was carried out using Chi-square test to inter-compare the VEGF expression between KCOT, DC, and RC. VEGF expression in the epithelium and connective tissue was significantly higher in KCOT compared to dentigerous and RC. One case of KCOT with carcinomatous change also revealed positive results for the VEGF expression in the dysplastic epithelium, tumor islands, and connective tissue. The significant difference was observed on inter-comparison of the VEGF expression in the connective tissue of KCOT and DC, whereas no significant difference was observed in the VEGF expression in the connective tissue of KCOT and DC. The present study data supports the literature finding that angiogenesis can be important in the progression and enlargement of odontogenic cysts similarly to what occurs in neoplastic conditions and further it can be concluded that the higher positivity for VEGF of KCOT could help to

  6. N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

    Science.gov (United States)

    Okada, Takashi; Morino, Katsutaro; Nakagawa, Fumiyuki; Tawa, Masashi; Kondo, Keiko; Sekine, Osamu; Imamura, Takeshi; Okamura, Tomio; Ugi, Satoshi; Maegawa, Hiroshi

    2017-06-24

    N -3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n -3 PUFAs, increased in n -3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n -3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n -3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n -3 PUFAs may contribute to their cardio-protective effect.

  7. Influence of the structure of poly (L-lactic acid) electrospun fibers on the bioactivity of endothelial cells: proliferation and inflammatory cytokines expression.

    Science.gov (United States)

    Liu, Xiaoyan; Zhang, Xiazhi; Wu, Keke; Yang, Wufeng; Jiao, Yanpeng; Zhou, Changren

    2017-02-01

    Electrospinning has been used to fabricate random and aligned poly (L-lactic acid) (PLLA) fibers with three kinds of diameter under optimal conditions. The main purpose of this paper was to investigate the influence of the diameter and orientation of fibers on the bioactivity of endothelial cells, especially on the inflammatory cytokines expression. The morphology of electrospun fibers and the cells on the fibers after 3 and 6 days culture were observed by scanning electron microscopy. Also the cell proliferation activity and cell cycle were tested and the results showed that the random fibers were more favorable for endothelial cells growth. The effect of PLLA film (served as a control) and six kinds of PLLA fibers mats on the inflammatory cytokines expression after cells incubated for 2 and 4 days were investigated. It was concluded that there was more intense inflammatory cytokines expression by cells on flat PLLA film than that on electrospun fiber mats. Also the fiber diameter has greater effect on the activity and inflammatory cytokines expression of endothelial cells than the fiber orientation, in which fibers with smaller size has weaker inflammatory reaction.

  8. Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

    Science.gov (United States)

    Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2008-01-01

    Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

  9. Substance P ameliorates tumor necrosis factor-alpha-induced endothelial cell dysfunction by regulating eNOS expression in vitro.

    Science.gov (United States)

    Piao, Jiyuan; Hong, Hyun Sook; Son, Youngsook

    2018-04-01

    The aim of this study was to explore the beneficial effects of SP on NO production and inflammation-induced vascular endothelium cell death. To mimic the inflammatory environment, TNF-α was treated with HUVECs, and SP was added prior to TNF-α to determine its protective effect. WST-1 assay was performed to detect cell viability. NO level in conditioned medium was measured by Griess Reagent System. The protein level of cleaved caspase-3, eNOS, and phosphorylated Akt was detected by Western blot analysis. TNF-α declined endothelial cell viability by downregulating Akt and NO production. TNF-α-induced cell death was reliably restored by NO, confirming the requirement of NO for cell survival. By contrast, pretreatment of SP attenuated TNF-α-induced cellular apoptosis, accompanied by an increase in the phosphorylation of Akt, eNOS expression, and NO production. Blockage of NK-1R, phosphorylated Akt or eNOS by CP-96345, A6730, or L-NAME entirely eliminated the effect of SP. SP can protect the vascular endothelium against inflammation-induced damage through modulation of the Akt/eNOS/NO signaling pathway. © 2018 John Wiley & Sons Ltd.

  10. [Expression and clinical significance of kisspeptin-1, matrix metalloproteinase-2 and vascular endothelial growth factor in tissue of colon cancer].

    Science.gov (United States)

    Wang, Wenhui; Qi, Yuanling; Xu, Qian; Ren, Haipeng

    2016-03-01

    To detect the expression of kisspeptin-1 (KISS-1), matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in the tissue of colon cancer, and analyze the relativity between KISS-1, MMP-2, VEGF and pathological characteristics of colon cancer. A total of 60 colon cancer patients and 60 patients with benign colorectal disease who received surgical treatment in our hospital from January 2009 to June 2010 were selected as observation group and control group respectively. The cancer tissue samples and excision samples collected from them were used to detect KISS-1, MMP-2 and VEGF with immunohistochemistry. The positive rates of KISS-1, MMP-2 and VEGF were 31.7%, 58.3% and 78.3% in observation group, and 73.3%, 16.7% and 33.3% in control group. The positive rate of KISS-1 in observation group was lower than that in control group (χ(2)=23.489, Pcolon cancer (χ(2)=8.997, P=0.011; χ(2)=6.163, P=0.013; χ(2)=8.519, P=0.014; χ(2)=9.160, P=0.002; χ(2)=16.577, Pclinical stage of colon cancer and provide evidence for clinical diagnosis and prognosis prediction by detecting KISS-1, MMP-2 and VEGF.

  11. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  12. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    International Nuclear Information System (INIS)

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-01-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

  13. Expression pattern of vascular endothelial growth factor 2 during sea urchin development.

    Science.gov (United States)

    Kipryushina, Yulia O; Yakovlev, Konstantin V; Kulakova, Milana A; Odintsova, Nelly A

    2013-12-01

    The VEGF family in the sea urchin is comprised of three members designated Vegf1 through Vegf3. In this study, we found a high level of similarity between the PDGF/VEGF domain of the predicted gene Sp-Vegf2 in the sea urchin Strongylocentrotus purpuratus and the same domain of a gene that we found in a closely related sea urchin, Strongylocentrotus intermedius. The sequence of the Si-Vegf2 cDNA was determined, and the expression of the Si-Vegf2 mRNA throughout early sea urchin development was studied by RT-PCR and in situ hybridization. Also we analyzed phylogenetic relationships of Si-Vegf2 and other members of the PDGF and VEGF families. We have found that the Si-Vegf2 present during the time span from the egg to the 4-arm pluteus stage. This mRNA is uniformly distributed in eggs, cleaving embryos and early blastulae. At the gastrula stage, the Si-Vegf2 transcripts are localized in the ventrolateral clusters of primary mesenchyme cells, and later, at the prism stage, they are detected in the forming apex. At the early pluteus stage, Si-Vegf2 mRNAs are found in two groups of mesenchyme cells in the scheitel region on the apical pole. We have determined that Si-Vegf2 is a mesenchyme-expressed factor but its developmental function is unknown. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: hara-tk@igakuken.or.jp [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)

    2016-06-03

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  15. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    International Nuclear Information System (INIS)

    Kawaguchi, Manami; Kitajima, Kenji; Kanokoda, Mai; Suzuki, Hidenori; Miyashita, Kazuya; Nakajima, Marino; Nuriya, Hideko; Kasahara, Kohji; Hara, Takahiko

    2016-01-01

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit − Tie2 − CD41 + Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41 + CD42b + CD61 + platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit − Tie2 − CD41 + . •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  16. Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes

    International Nuclear Information System (INIS)

    Shibata, Toru; Akiyama, Nobutake; Noda, Makoto; Sasai, Keisuke; Hiraoka, Masahiro

    1998-01-01

    Purpose: Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy. Materials and Methods: Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed. Results: The construct pGL3/VEGF containing the 385 bp fragment of the 5' flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3-4, and 8-12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3' flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness. Conclusions: The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness

  17. Cadmium induces matrix metalloproteinase-9 expression via ROS-dependent EGFR, NF-kB, and AP-1 pathways in human endothelial cells

    International Nuclear Information System (INIS)

    Lian, Sen; Xia, Yong; Khoi, Pham Ngoc; Ung, Trong Thuan; Yoon, Hyun Joong; Kim, Nam Ho; Kim, Kyung Keun; Jung, Young Do

    2015-01-01

    Highlights: • Cadmium induces MMP-9 expression through NADPH oxidase-derived ROS. • Cadmium induces MMP-9 through EGFR-mediated Akt, Erk1/2 and JNK1/2 signaling pathways. • Akt, MAPKs (Erk1/2 and JNK1/2) functioned as upstream signals of NF-kB and AP-1 respectively, in cadmium-induced MMP-9 in endothelial cells. • ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression in ECV304 cells. - Abstract: Cadmium (Cd), a widespread cumulative pollutant, is a known human carcinogen, associated with inflammation and tumors. Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in tumor metastasis; however, the mechanisms underlying the MMP-9 expression induced by Cd remain obscure in human endothelial cells. Here, Cd elevated MMP-9 expression in dose- and time-dependent manners in human endothelial cells. Cd increased ROS production and the ROS-producing NADPH oxidase. Cd translocates p47 phox , a key subunit of NADPH oxidase, to the cell membrane. Cd also activated the phosphorylation of EGFR, Akt, Erk1/2, and JNK1/2 in addition to promoting NF-kB and AP-1 binding activities. Specific inhibitor and mutagenesis studies showed that EGFR, Akt, Erk1/2, JNK1/2 and transcription factors NF-κB and AP-1 were related to Cd-induced MMP-9 expression in endothelial cells. Akt, Erk1/2, and JNK1/2 functioned as upstream signals in the activation of NF-κB and AP-1, respectively. In addition, N-acetyl-L-cystein (NAC), diphenyleneiodonium chloride (DPI) and apocynin (APO) inhibited the Cd-induced activation of EGFR, Akt, Erk1/2, JNK1/2, and p38 MAPK, indicating that ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression. At present, it states that Cd displayed marked invasiveness in ECV304 cells, which was partially abrogated by MMP-9 neutralizing antibodies. These results demonstrated that Cd induces MMP-9 expression via ROS-dependent EGFR- > Erk1/2, JNK1/2- > AP-1 and EGFR- > Akt- > NF-κB signaling pathways and, in turn

  18. Favorable prognosis of operable non-small cell lung cancer (NSCLC) patients harboring an increased expression of tumor endothelial markers (TEMs).

    Science.gov (United States)

    Pircher, Andreas; Fiegl, Michael; Untergasser, Gerold; Heidegger, Isabel; Medinger, Michael; Kern, Johann; Hilbe, Wolfgang

    2013-08-01

    Genome analyses of endothelial cells identified genes specifically expressed by tumor endothelial cells, called tumor endothelial markers (TEMs). Currently there are no data available concerning the role of TEMs in non-small cell lung cancer (NSCLC). Therefore, the aim of this study was to investigate the role of TEMs in NSCLC in vitro and in vivo. First we evaluated the expression of various TEMs (Robo4, Clec14 and ECSCR) by qRT-PCR and Western blot analyses in three NSCLC cell lines (A549, Calu1, Colo699) and compared them to human umbilical vein endothelial cells (HUVECs), endothelial colony forming cells (ECFCs) and human bronchial epithelial cells (HBEpCs). Next the expression of TEMs was measured in resected tumor tissue of NSCLC patients (n = 63) by qRT-PCR and compared to adjacent non-cancerous lung tissue (n = 52). Further, immunohistochemical analysis of Robo4 expression in tumor tissue (n = 33) and adjacent non-cancerous tissue (n = 27) was performed. We found that NSCLC cell lines and HBEpC did not express TEMs on the mRNA level compared to HUVECs (p = 0.001). In the contrary, a significant up-regulation of Robo4 and Clec14 was found in tumor samples (Robo4 p = 0.03, Clec14 p = 0.002). Both facts clearly indicate that these proteins are allocated to the tumor stromal department. Correlation with clinical data showed that increased TEM expression correlated with prolonged overall survival of operated NSCLC patients (Robo4 high 120.5 vs. Robo4 low 47.6 months, Clec14 high 108.1 vs. Clec14 low 54.5 months and ECSCR high 120.5 vs. ECSCR low 42.2 months). In summary, we found that TEMs are overexpressed in NSCLC stromal tissue and that an increased TEM expression correlated with an increased overall survival in early stage NSCLC. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Expression of inducible nitric oxide synthase, caspase-3 and production of reactive oxygen intermediate on endothelial cells culture (HUVECs treated with P. falciparum infected erythrocytes and tumour necrosis factor-α

    Directory of Open Access Journals (Sweden)

    Loeki E. Fitri

    2006-09-01

    Full Text Available Cytoadherence of P. falciparum infected erythrocytes on endothelial cells is a key factor in development of severe malaria. This process may associated with the activation of local immune that was enhanced by tumour necrosis factor-α (TNF-α. This study was conducted to see the influence of P.falciparum infected erythrocytes cytoadherence and TNF-α treatment in inducing endothelial cells activation in vitro. inducible nitric oxide synthase (iNOS and caspase-3 expression, also reactive oxygen intermediate (ROI production were used as parameters. An Experimental laboratory study had been done to observe endothelial cells activation (HUVECs after treatment with TNF-α for 20 hours or P. falciparum infected erythrocytes for 1 hour or both of them. Normal endothelial cells culture had been used as a control. Using immunocytochemistry local immune activation of endothelial cells was determined by iNOS and caspase-3 expression. Nitro Blue Tetrazolium reduction-assay was conducted to see the ROI production semi quantitatively. inducible nitric oxide synthase expression only found on endothelial cells culture treated with P. falciparum infected erythrocytes or both P. falciparum infected erythrocytes and TNF-α. Caspase-3 expression found slightly on normal endothelial cells culture. This expression increased significantly on endothelial cells culture treated with both P.falciparum infected erythrocytes and TNF-α (p=0.000. The normal endothelial cells release low level of ROI in the presence of non-specific trigger, PMA. In the presence of P. falciparum infected erythrocytes or TNF-α or both of them, some cells showed medium to high levels of ROI. Cytoadherence of P. falciparum infected erythrocytes and TNF α treatment on endothelial cells can induce activation of local immune marked by increase inducible nitric oxide synthase and release of free radicals that cause cell damage. (Med J Indones 2006; 15:151-6 Keywords: P.falciparum ,HUVECs, TNF-α, i

  20. Differential intracellular calcium influx, nitric oxide production, ICAM-1 and IL8 expression in primary bovine endothelial cells exposed to nonesterified fatty acids.

    Science.gov (United States)

    Loaiza, Anitsi; Carretta, María D; Taubert, Anja; Hermosilla, Carlos; Hidalgo, María A; Burgos, Rafael A

    2016-02-25

    Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. However, the mechanisms underlying these effects are still unknown. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca(2+) i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs). Myristic (MA), palmitic (PA), stearic (SA), oleic (OA) and linoleic acid (LA) rapidly increased Ca(2+) i. The calcium response to all tested NEFAs showed an extracellular calcium dependence and only the LA response was significantly inhibited until the intracellular calcium was chelated. The EC50 values for MA and LA were 125 μM and 37 μM, respectively, and the MA and LA effects were dependent on calcium release from the endoplasmic reticulum stores and on the L-type calcium channels. Only the calcium response to MA was significantly reduced by GW1100, a selective G-protein-coupled free fatty acid receptor (GPR40) antagonist. We also detected a functional FFAR1/GPR40 protein in BUVECs by using western blotting and the FFAR1/GPR40 agonist TAK-875. Only LA increased the cellular nitric oxide levels in a calcium-dependent manner. LA stimulation but not MA stimulation increased ICAM-1 and IL-8-expression in BUVECs. This effect was inhibited by GW1100, an antagonist of FFAR1/GPR40, but not by U-73122, a phospholipase C inhibitor. These findings strongly suggest that each individual NEFA stimulates endothelial cells in a different way, with clearly different effects on intracellular calcium mobilization, NO production, and IL-8 and ICAM-1 expression in primary BUVECs. These findings not only extend our understanding of NEFA-mediated diseases in ruminants, but also provide new insight into the different molecular mechanisms involved during endothelial cell activation by NEFAs.

  1. Eriodictyol Protects Endothelial Cells against Oxidative Stress-Induced Cell Death through Modulating ERK/Nrf2/ARE-Dependent Heme Oxygenase-1 Expression.

    Science.gov (United States)

    Lee, Seung Eun; Yang, Hana; Son, Gun Woo; Park, Hye Rim; Park, Cheung-Seog; Jin, Young-Ho; Park, Yong Seek

    2015-06-26

    The pathophysiology of cardiovascular diseases is complex and may involve oxidative stress-related pathways. Eriodictyol is a flavonoid present in citrus fruits that demonstrates anti-inflammatory, anti-cancer, neurotrophic, and antioxidant effects in a range of pathophysiological conditions including vascular diseases. Because oxidative stress plays a key role in the pathogenesis of cardiovascular disease, the present study was designed to verify whether eriodictyol has therapeutic potential. Upregulation of heme oxygenase-1 (HO-1), a phase II detoxifying enzyme, in endothelial cells is considered to be helpful in cardiovascular disease. In this study, human umbilical vein endothelial cells (HUVECs) treated with eriodictyol showed the upregulation of HO-1 through extracellular-regulated kinase (ERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathways. Further, eriodictyol treatment provided protection against hydrogen peroxide-provoked cell death. This protective effect was eliminated by treatment with a specific inhibitor of HO-1 and RNA interference-mediated knockdown of HO-1 expression. These data demonstrate that eriodictyol induces ERK/Nrf2/ARE-mediated HO-1 upregulation in human endothelial cells, which is directly associated with its vascular protection against oxidative stress-related endothelial injury, and propose that targeting the upregulation of HO-1 is a promising approach for therapeutic intervention in cardiovascular disease.

  2. Increased expression of microRNA-221 inhibits PAK1 in endothelial progenitor cells and impairs its function via c-Raf/MEK/ERK pathway

    International Nuclear Information System (INIS)

    Zhang, Xiaoping; Mao, Haian; Chen, Jin-yuan; Wen, Shengjun; Li, Dan; Ye, Meng; Lv, Zhongwei

    2013-01-01

    Highlights: ► MicroRNA-221 is upregulated in the endothelial progenitor cells of atherosclerosis patients. ► PAK1 is a direct target of microRNA-221. ► MicroRNA-221 inhibits EPCs proliferation through c-Raf/MEK/ERK pathway. -- Abstract: Coronary artery disease (CAD) is associated with high mortality and occurs via endothelial injury. Endothelial progenitor cells (EPCs) restore the integrity of the endothelium and protect it from atherosclerosis. In this study, we compared the expression of microRNAs (miRNAs) in EPCs in atherosclerosis patients and normal controls. We found that miR-221 expression was significantly up-regulated in patients compared with controls. We predicted and identified p21/Cdc42/Rac1-activated kinase 1 (PAK1) as a novel target of miR-221 in EPCs. We also demonstrated that miR-221 targeted a putative binding site in the 3′UTR of PAK1, and absence of this site was inversely associated with miR-221 expression in EPCs. We confirmed this relationship using a luciferase reporter assay. Furthermore, overexpression of miR-221 in EPCs significantly decreased EPC proliferation, in accordance with the inhibitory effects induced by decreased PAK1. Overall, these findings demonstrate that miR-221 affects the MEK/ERK pathway by targeting PAK1 to inhibit the proliferation of EPCs

  3. Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of β1, β2 and β3-adrenergic receptors in retinal endothelial cells

    Science.gov (United States)

    2014-01-01

    Background Aberrant epigenetic profiles are concomitant with a spectrum of developmental defects and diseases. Role of methylation is an increasingly accepted factor in the pathophysiology of diabetes and its associated complications. This study aims to examine the correlation between oxidative stress and methylation of β1, β2 and β3-adrenergic receptors and to analyze the differential variability in the expression of these genes under hyperglycemic conditions. Methods Human retinal endothelial cells were cultured in CSC complete medium in normal (5 mM) or high (25 mM) glucose to mimic a diabetic condition. Reverse transcription PCR and Western Blotting were performed to examine the expression of β1, β2 and β3-adrenergic receptors. For detections, immunocytochemistry was used. Bisulfite sequencing method was used for promoter methylation analysis. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure reactive oxygen species (ROS) production in the cells. Results β1 and β3-adrenergic receptors were expressed in retinal endothelial cells while β2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on β1 and β2-adrenergic receptors methylation and expression however β3-adrenergic receptors showed a significantly higher expression (p adrenergic receptors methylation with no significant effect on β1 and β2-adrenergic receptors. β2-adrenergic receptor was hypermethylated with halted expression. Conclusion Our study demonstrates that β1 and β3-adrenergic receptors expressed in human retinal endothelial cells. Oxidative stress and apoptosis are inversely proportional to the extent of promoter methylation, suggesting that methylation loss might be due to oxidative stress-induced DNA damage. PMID:24885710

  4. Lycopene inhibits NF-κB activation and adhesion molecule expression through Nrf2-mediated heme oxygenase-1 in endothelial cells.

    Science.gov (United States)

    Yang, Po-Min; Chen, Huang-Zhi; Huang, Yu-Ting; Hsieh, Chia-Wen; Wung, Being-Sun

    2017-06-01

    The endothelial expression of cell adhesion molecules plays a leading role in atherosclerosis. Lycopene, a carotenoid with 11 conjugated double bonds, has been shown to have anti-inflammatory properties. In the present study, we demonstrate a putative mechanism for the anti-inflammatory effects of lycopene. We demonstrate that lycopene inhibits the adhesion of tumor necrosis factor α (TNFα)-stimulated monocytes to endothelial cells and suppresses the expression of intercellular cell adhesion molecule-1 (ICAM-1) at the transcriptional level. Moreover, lycopene was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein, IκBα, following 6 h of pre-treatment. In TNFα-stimulated endothelial cells, nuclear factor-κB (NF-κB) nuclear translocation and transcriptional activity were abolished by up to 12 h of lycopene pre-treatment. We also found that lycopene increased the intracellular glutathione (GSH) level and glutamate-cysteine ligase expression. Subsequently, lycopene induced nuclear factor-erythroid 2 related factor 2 (Nrf2) activation, leading to the increased expression of downstream of heme oxygenase-1 (HO-1). The use of siRNA targeting HO-1 blocked the inhibitory effects of lycopene on IκB degradation and ICAM-1 expression. The inhibitory effects of lycopene thus appear to be mediated through its induction of Nrf2-mediated HO-1 expression. Therefore, the findings of the present study indicate that lycopene suppresses the activation of TNFα-induced signaling pathways through the upregulation of Nrf2-mediated HO-1 expression.

  5. Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport

    Directory of Open Access Journals (Sweden)

    Szilvia Veszelka

    2018-05-01

    Full Text Available Cell culture-based blood-brain barrier (BBB models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC, ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA. As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L, and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1 and influx transporters (GLUT-1, LAT-1 were present in all models at mRNA levels. The transcript of BCRP (ABCG2 was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which

  6. Upregulation of endothelial heme oxygenase-1 expression through the activation of the JNK pathway by sublethal concentrations of acrolein.

    Science.gov (United States)

    Wu, C C; Hsieh, C W; Lai, P H; Lin, J B; Liu, Y C; Wung, B S

    2006-08-01

    Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde that is present in cigarette smoke. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various such electrophilic compounds. In this study, the regulatory effects of acrolein upon the expression of HO-1 were investigated in endothelial cells (ECs). We demonstrate that acrolein induces the elevation of HO-1 protein levels, and subsequent enzyme activity, at non-cytotoxic concentrations. An additional alpha,beta-unsaturated aldehyde, cinnamaldehyde, was also found to increase HO-1 expression and have less cytotoxicity than acrolein. Moreover, acrolein-mediated HO-1 induction is abrogated in the presence of actinomycin D and cycloheximide. Nrf2 is a transcription factor involved in the induction of HO-1 through an antioxidant response element (ARE) in the promoter region of the HO-1 gene. We show that acrolein induces Nrf2 translocation and ARE-luciferase reporter activity. Acrolein was also found to induce the production of both superoxide and H2O2 at levels greater than 100 microM. However, with the exception of NAC, no antioxidant generated any effect upon acrolein-dependent HO-1 expression in ECs. Our present findings suggest that reactive oxygen species (ROS) may not be a major modulator for HO-1 induction. Using buthionine sulfoximine to deplete the intracellular GSH levels further enhanced the effects of acrolein. We also found that cellular GSH level was rapidly reduced after both 10 and 100 microM acrolein treatment. However, after 6 h of exposure to ECs, only 10 microM acrolein treatment increases GSH level. In addition, only the JNK inhibitor SP600125 and tyrosine kinase inhibitor genistein had any significant inhibitory impact upon the upregulation of HO-1 by acrolein. Pretreatment with a range of other PI3 kinase inhibitors, including wortmannin and LY294002, showed no effects. Hence, we show in our current experiments that a sublethal concentration of acrolein is in fact a

  7. Upregulation of endothelial heme oxygenase-1 expression through the activation of the JNK pathway by sublethal concentrations of acrolein

    International Nuclear Information System (INIS)

    Wu, C.C.; Hsieh, C.W.; Lai, P.H.; Lin, J.B.; Liu, Y.C.; Wung, B.S.

    2006-01-01

    Acrolein is a highly electrophilic α,β-unsaturated aldehyde that is present in cigarette smoke. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various such electrophilic compounds. In this study, the regulatory effects of acrolein upon the expression of HO-1 were investigated in endothelial cells (ECs). We demonstrate that acrolein induces the elevation of HO-1 protein levels, and subsequent enzyme activity, at non-cytotoxic concentrations. An additional α,β-unsaturated aldehyde, cinnamaldehyde, was also found to increase HO-1 expression and have less cytotoxicity than acrolein. Moreover, acrolein-mediated HO-1 induction is abrogated in the presence of actinomycin D and cycloheximide. Nrf2 is a transcription factor involved in the induction of HO-1 through an antioxidant response element (ARE) in the promoter region of the HO-1 gene. We show that acrolein induces Nrf2 translocation and ARE-luciferase reporter activity. Acrolein was also found to induce the production of both superoxide and H 2 O 2 at levels greater than 100 μM. However, with the exception of NAC, no antioxidant generated any effect upon acrolein-dependent HO-1 expression in ECs. Our present findings suggest that reactive oxygen species (ROS) may not be a major modulator for HO-1 induction. Using buthionine sulfoximine to deplete the intracellular GSH levels further enhanced the effects of acrolein. We also found that cellular GSH level was rapidly reduced after both 10 and 100 μM acrolein treatment. However, after 6 h of exposure to ECs, only 10 μM acrolein treatment increases GSH level. In addition, only the JNK inhibitor SP600125 and tyrosine kinase inhibitor genistein had any significant inhibitory impact upon the upregulation of HO-1 by acrolein. Pretreatment with a range of other PI3 kinase inhibitors, including wortmannin and LY294002, showed no effects. Hence, we show in our current experiments that a sublethal concentration of acrolein is in fact a novel HO-1 inducer

  8. Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1.

    Science.gov (United States)

    Yang, Xinyu; Li, Lin; Liu, Jin; Lv, Ben; Chen, Fangping

    2016-01-01

    Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection – a Technical Report

    Science.gov (United States)

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  10. Effect of Puumala hantavirus infection on Human Umbilical Vein Endothelial Cell hemostatic function: platelet interactions, increased tissue factor expression and fibrinolysis regulator release

    Directory of Open Access Journals (Sweden)

    Marco Goeijenbier

    2015-03-01

    Full Text Available Puumala virus (PUUV infection causes over 5000 cases of hemorrhagic fever in Europe annually and can influence the hemostatic balance extensively. Infection might lead to hemorrhage, while a recent study showed an increased risk of myocardial infarction during or shortly after PUUV infection. The mechanism by which this hantavirus influences the coagulation system remains unknown. Therefore we aimed to elucidate mechanisms explaining alterations seen in primary and secondary hemostasis during PUUV infection. By using low passage PUUV isolates to infect primary human umbilical vein endothelial cells (HUVECs we were able to show alterations in the regulation of primary- and secondary hemostasis and in the release of fibrinolysis regulators. Our main finding was an activation of secondary hemostasis due to increased tissue factor expression leading to increased thrombin generation in a functional assay. Furthermore, we showed that during infection platelets adhered to HUVECs and subsequently specifically to PUUV virus particles. Infection of HUVECs with PUUV did not result in increased von Willebrand factor while they produced more plasminogen activator inhibitor type-1 (PAI-1 compared to controls. The PAI-1 produced in this model formed complexes with vitronectin. This is the first report that reveals a potential mechanism behind the pro-coagulant changes in PUUV patients, which could be the result of increased thrombin generation due to an increased tissue factor expression on endothelial cells during infection. Furthermore, we provide insight into the contribution of endothelial cell responses regarding hemostasis in PUUV pathogenesis.

  11. Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

    Science.gov (United States)

    Fazal, Fabeha; Bijli, Kaiser M; Minhajuddin, Mohd; Rein, Theo; Finkelstein, Jacob N; Rahman, Arshad

    2009-07-31

    Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.

  12. Essential Role of Cofilin-1 in Regulating Thrombin-induced RelA/p65 Nuclear Translocation and Intercellular Adhesion Molecule 1 (ICAM-1) Expression in Endothelial Cells*

    Science.gov (United States)

    Fazal, Fabeha; Bijli, Kaiser M.; Minhajuddin, Mohd; Rein, Theo; Finkelstein, Jacob N.; Rahman, Arshad

    2009-01-01

    Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-κB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-κB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser3 phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-κB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-κB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-κB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-κB activity and ICAM-1 expression occurred downstream of IκBα degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells. PMID:19483084

  13. Let-7i attenuates human brain microvascular endothelial cell damage in oxygen glucose deprivation model by decreasing toll-like receptor 4 expression.

    Science.gov (United States)

    Xiang, Wei; Tian, Canhui; Peng, Shunli; Zhou, Liang; Pan, Suyue; Deng, Zhen

    2017-11-04

    The let-7 family of microRNAs (miRNAs) plays an important role on endothelial cell function. However, there have been few studies on their role under ischemic conditions. In this study, we demonstrate that let-7i, belonging to the let-7 family, rescues human brain microvascular endothelial cells (HBMECs) in an oxygen-glucose deprivation (OGD) model. Our data show that the expression of let-7 family miRNAs was downregulated after OGD. Overexpression of let-7i significantly alleviated cell death and improved survival of OGD-treated HBMECs. Let-7i also protected permeability in an in vitro blood brain barrier (BBB) model. Further, let-7i downregulated the expression of toll-like receptor 4 (TLR4), an inflammation trigger. Moreover, overexpression of let-7i decreased matrix metallopeptidase 9 (MMP9) and inducible nitric oxide synthase (iNOS) expression under OGD. Upon silencing TLR4 expression in HBMECs, the anti-inflammatory effect of let-7i was abolished. Our research suggests that let-7i promotes OGD-induced inflammation via downregulating TLR4 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Sodium-dependent vitamin C transporter 2 (SVCT2 expression and activity in brain capillary endothelial cells after transient ischemia in mice.

    Directory of Open Access Journals (Sweden)

    Burkhard Gess

    Full Text Available Expression and transport activity of Sodium-dependent Vitamin C Transporter 2 (SVCT2 was shown in various tissues and organs. Vitamin C was shown to be cerebroprotective in several animal models of stroke. Data on expression, localization and transport activity of SVCT2 after cerebral ischemia, however, has been scarce so far. Thus, we studied the expression of SVCT2 after middle cerebral artery occlusion (MCAO in mice by immunohistochemistry. We found an upregulation of SVCT2 after stroke. Co-stainings with Occludin, Von-Willebrand Factor and CD34 demonstrated localization of SVCT2 in brain capillary endothelial cells in the ischemic area after stroke. Time-course analyses of SVCT2 expression by immunohistochemistry and western blots showed upregulation in the subacute phase of 2-5 days. Radioactive uptake assays using (14C-labelled ascorbic acid showed a significant increase of ascorbic acid uptake into the brain after stroke. Taken together, these results provide evidence for the expression and transport activity of SVCT2 in brain capillary endothelial cells after transient ischemia in mice. These results may lead to the development of novel neuroprotective strategies in stroke therapy.

  15. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

    International Nuclear Information System (INIS)

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E.; Detmar, Michael; Halin, Cornelia

    2009-01-01

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity

  16. Protein kinase A-alpha directly phosphorylates FoxO1 in vascular endothelial cells to regulate expression of vascular cellular adhesion molecule-1 mRNA.

    Science.gov (United States)

    Lee, Ji-Won; Chen, Hui; Pullikotil, Philomena; Quon, Michael J

    2011-02-25

    FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase α (PKA-α). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-α. Using an immune complex kinase assay with [γ-(32)P]ATP, purified PKA-α directly phosphorylated wild-type FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-α in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-α and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-α decreased forskolin- or prostaglandin E(2)-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-α, catalytically inactive mutant PKA-α, or siRNA against PKA-α caused corresponding increases or decreases in transactivation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-α or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-α treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-α in vascular endothelial cells.

  17. Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma.

    Science.gov (United States)

    Yhee, J Y; Yu, C H; Kim, J H; Im, K S; Kim, N H; Brodersen, B W; Doster, A R; Sur, J-H

    2012-01-01

    The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Lysophosphatidic Acid Up-regulates MT1-MMP Expression through a Gi –dependent Pathway in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Po-Wei Lin

    2009-11-01

    Full Text Available Lysophosphatidic acid (LPA is a low molecular weight lysophospholipid (LPL. Through binding to its specific G protein-coupled receptor family, LPA regulates various cellular functions, including proliferation, migration, invasion, and differentiation. Matrix-metalloproteinases (MMPs are zinc-dependent protease and play important roles in regulating the interaction between cells and extracellular matrix (ECM. Among these MMPs, membrane type 1-metalloproteinase (MT1-MMP not only degrades ECM protein but also activates metalloproteinase-2 (MMP-2, Gelatinase A, which are important to endothelial cell migration. Our previous study showed that LPA enhances MMP-2 expression and activity in human umbilical vein endothelial cells (HUVECs. In this study, we further revealed that LPA also induce MT1-MMP mRNA and protein expressions in HUVECs through real-time PCR and Western blotting, respectively. Furthermore, by applying chemical inhibitors, we found that LPA-induced MT1-MMP expression is mainly through a Gi- and partially through a Gq-dependent pathway. Our results provide new evidence that LPA might modulate ECM through regulating the expression of MT1-MMP.

  19. Hypoxia-Inducible Factor-1α (HIF-1α) Expression on Endothelial Cells in Juvenile Nasopharyngeal Angiofibroma: A Review of 70 cases and Tissue Microarray Analysis.

    Science.gov (United States)

    Song, Xiaole; Yang, Chenhe; Zhang, Huankang; Wang, Jingjing; Sun, Xicai; Hu, Li; Liu, Zhuofu; Wang, Dehui

    2018-06-01

    To examine the expression of hypoxia-inducible factor-1α (HIF-1α) and its related molecules (cellular repressor of E1A-stimulated genes [CREG], osteopontin [OPN], proto-oncogene tyrosine-protein kinase Src [c-Src], and vascular endothelial growth factor [VEGF]) in juvenile nasopharyngeal angiofibroma (JNA) and explore the correlation between clinical prognosis and HIF-1α expression. The study performed a retrospective review of the clinical records of patients with JNA treated between 2003 and 2007. Specimens were analyzed by immunohistochemistry for HIF-1α, CREG, OPN, c-Src, and VEGF expression, and microvessel density (MVD) was assessed by tissue microarray. The correlation between expression levels and clinicopathological features including age, tumor stage, intraoperative blood loss, and recurrence was analyzed. HIF-1α, CREG, OPN, c-Src, and VEGF were upregulated in endothelial cells (ECs) of patients with JNA, and strong correlations in the expression of these molecules were observed. HIF-1α expression was higher in young patients ( P = .032) and in recurrent cases ( P = .01). Survival analysis showed that low HIF-1α levels in ECs predicted longer time to recurrence (log rank test P = .006). Receiver operating characteristic curve analysis showed that HIF-1α was a prognostic factor for recurrence (area under the curve = 0.690, P = .019). No correlation was found between the expression of molecules and Radkowski stage or intraoperative blood loss. In cases of JNA treated surgically, HIF-1α expression in ECs is a useful prognostic factor for tumor recurrence.

  20. Macrophages in lung tissue from patients with pulmonary emphysema express both inducible and endothelial nitric oxide synthase

    NARCIS (Netherlands)

    van Straaten, JFM; Postma, DS; Coers, W; Noordhoek, JA; Kauffman, HF; Timens, W

    To provide information concerning a possible biologic role of nitric oxide (NO) in smoking-related emphysema, we performed immunohistochemical studies in lung tissue from control subjects and patients with mild and severe emphysema We studied the presence of inducible and endothelial NO synthases

  1. Capillarization and vascular endothelial growth factor expression in hypertrophying anterior latissimus dorsi muscle of the Japanese quail.

    NARCIS (Netherlands)

    Degens, H.; Anderson, R.K.; Alway, S.E.

    2003-01-01

    Hypertrophy may increase the diffusion distances from capillaries to the interior of the muscle fibers. We hypothesized that capillary proliferation occurs during hypertrophy, which is accompanied by an up-regulation of vascular endothelial growth factor (VEGF). Hypertrophy of the left anterior

  2. ICAM-1 and VCAM-1 expression by endothelial cells grown on fibronectin-coated TCPS and PS

    NARCIS (Netherlands)

    van der Zijpp, Y.J.T.; Poot, Andreas A.; Feijen, Jan

    2003-01-01

    Small-diameter vascular grafts rapidly fail as a result of blood coagulation and platelet deposition. Endothelial cells lining the inner side of blood vessels can provide the graft lumen with an antithrombogenic surface. One of the remaining problems is cell detachment after restoration of blood

  3. Parasites causing cerebral falciparum malaria bind multiple endothelial receptors and express EPCR and ICAM-1-binding PfEMP1

    DEFF Research Database (Denmark)

    Tuikue Ndam, Nicaise; Moussiliou, Azizath; Lavstsen, Thomas

    2017-01-01

    Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1...

  4. Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

    Directory of Open Access Journals (Sweden)

    Ingo Ahrens

    Full Text Available Endothelial progenitor cells (EPCs can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP, PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15 or pro-angiogenic (galectin-3 properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP was the most up-regulated gene.

  5. Partial contribution of the Keap1–Nrf2 system to cadmium-mediated metallothionein expression in vascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Shinkai, Yasuhiro [Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575 (Japan); Kimura, Tomoki [Faculty of Science and Engineering, Setsunan University, 17-8 Ikedanaka-machi, Neyagawa, Osaka 572-8508 (Japan); Itagaki, Ayaka [Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa, Kanazawa, 920-1181, Ishikawa (Japan); Yamamoto, Chika [Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa, Kanazawa, 920-1181, Ishikawa (Japan); Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510 (Japan); Taguchi, Keiko; Yamamoto, Masayuki [Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Kumagai, Yoshito, E-mail: yk-em-tu@md.tsukuba.ac.jp [Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575 (Japan); Kaji, Toshiyuki, E-mail: t-kaji@rs.tus.ac.jp [Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa, Kanazawa, 920-1181, Ishikawa (Japan); Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan)

    2016-03-15

    Cadmium is an environmental electrophile that modifies protein reactive thiols such as Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear factor-erythroid 2-related factor 2 (Nrf2). In the present study, we investigated a role of the Keap1–Nrf2 system in cellular response to cadmium in vascular endothelial cells. Exposure of bovine aortic endothelial cells to cadmium resulted in modification of Keap1 and Nrf2 activation, thereby up-regulating not only its typical downstream proteins but also metallothionein-1/2. Experiments with siRNA-mediated knockdown of Nrf2 or Keap1 supported participation of the Keap1–Nrf2 system in the modulation of metallothionein-1/2 expression. Furthermore, chromatin immunoprecipitation assay showed that Nrf2 was recruited to the antioxidant response element of the promoter region of the bovine metallothionein-2 gene in the presence of cadmium. These results suggest that the transcription factor Nrf2 plays, at least in part, a role in the changes in metallothionein expression mediated by exposure to cadmium. - Highlights: • Role of the Keap1–Nrf2 system in cellular response to cadmium was examined. • We used bovine aortic endothelial cells as a model of the vascular endothelium. • Exposure of cells to cadmium resulted in modification of Keap1 and Nrf2 activation. • Keap1–Nrf2 system participated in the modulation of metallothionein-1/2 expression. • Nrf2 was recruited to the antioxidant response element of MT2 promoter region.

  6. Quantitative gene-expression of the tumor angiogenesis markers vascular endothelial growth factor, integrin alphaV and integrin beta3 in human neuroendocrine tumors

    DEFF Research Database (Denmark)

    Oxboel, Jytte; Binderup, Tina; Knigge, Ulrich

    2009-01-01

    , in neuroendocrine tumors. We used quantitative real-time PCR for measuring mRNA gene-expression of vascular endothelial growth factor (VEGF), integrin alphaV, and integrin beta3, and CD34 for a group of patients with neuroendocrine tumors (n=13). Tissue from patients with colorectal cancer liver metastases (n=14...... compared to both colorectal liver metastases (p=0.10) and normal liver tissue (p=0.06). In neuroendocrine tumors, gene-expression was highly variable of VEGF (530-fold), integrin alphaV (23-fold) and integrin beta3 (106-fold). Quantitative gene-expression levels of the key angiogenesis molecules VEGF......Anti-angiogenesis treatment is a promising new therapy for cancer that recently has also been suggested for patients with neuroendocrine tumors. The aim of the present study was therefore to investigate the level of tumor angiogenesis, and thereby the molecular basis for anti-angiogenesis treatment...

  7. 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.

  8. Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Ekhtear [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Karnan, Sivasundaram; Damdindorj, Lkhagvasuren [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Takahashi, Miyuki [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan)

    2013-12-15

    Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.

  9. Intermittent Hypoxia Is Associated With High Hypoxia Inducible Factor-1α but Not High Vascular Endothelial Growth Factor Cell Expression in Tumors of Cutaneous Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Isaac Almendros

    2018-04-01

    Full Text Available Epidemiological associations linking between obstructive sleep apnea and poorer solid malignant tumor outcomes have recently emerged. Putative pathways proposed to explain that these associations have included enhanced hypoxia inducible factor (HIF-1α and vascular endothelial growth factor (VEGF cell expression in the tumor and altered immune functions via intermittent hypoxia (IH. Here, we examined relationships between HIF-1α and VEGF expression and nocturnal IH in cutaneous melanoma (CM tumor samples. Prospectively recruited patients with CM tumor samples were included and underwent overnight polygraphy. General clinical features, apnea–hypopnea index (AHI, desaturation index (DI4%, and CM characteristics were recorded. Histochemical assessments of VEGF and HIF-1α were performed, and the percentage of positive cells (0, <25, 25–50, 51–75, >75% was blindly tabulated for VEGF expression, and as 0, 0–5.9, 6.0–10.0, >10.0% for HIF-1α expression, respectively. Cases with HIF-1α expression >6% (high expression were compared with those <6%, and VEGF expression >75% of cells was compared with those with <75%. 376 patients were included. High expression of VEGF and HIF-1α were seen in 88.8 and 4.2% of samples, respectively. High expression of VEGF was only associated with increasing age. However, high expression of HIF-1α was significantly associated with age, Breslow index, AHI, and DI4%. Logistic regression showed that DI4% [OR 1.03 (95% CI: 1.01–1.06] and Breslow index [OR 1.28 (95% CI: 1.18–1.46], but not AHI, remained independently associated with the presence of high HIF-1α expression. Thus, IH emerges as an independent risk factor for higher HIF-1α expression in CM tumors and is inferentially linked to worse clinical CM prognostic indicators.

  10. Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Keegan Achsah D

    2011-05-01

    Full Text Available Abstract Background Semaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined. Results Allergen treatment and lung-specific vascular endothelial growth factor (VEGF expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly

  11. Allergen-stimulated T lymphocytes from allergic patients induce vascular cell adhesion molecule-1 (VCAM-1) expression and IL-6 production by endothelial cells.

    Science.gov (United States)

    Delneste, Y; Jeannin, P; Gosset, P; Lassalle, P; Cardot, E; Tillie-Leblond, I; Joseph, M; Pestel, J; Tonnel, A B

    1995-01-01

    Adhesion of inflammatory cells to endothelium is a critical step for their transvascular migration to inflammatory sites. To evaluate the relationship between T lymphocytes (TL) and vascular endothelium, supernatants from allergen-stimulated TL obtained from patients sensitive to Dermatophagoides pteronyssinus (Dpt) versus healthy subjects were added to endothelial cell (EC) cultures. TL were stimulated by autologous-activated antigen-presenting cells (APC) previously fixed in paraformaldehyde to prevent monokine secretion. Two parameters were measured: the expression of adhesion molecule and the production of IL-6. Related allergen-stimulated TL supernatants from allergic patients induced an increase of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) expression when supernatants of the control groups (TL exposed to an unrelated allergen or not stimulated or TL obtained from healthy subjects) did not. E-selectin expression was not modulated whatever the supernatant added to EC culture. IL-6 production by EC was significantly enhanced after activation with related allergen-stimulated TL supernatants from allergics compared with control supernatants. Induction of VCAM-1 expression was inhibited by adding neutralizing antibodies against IL-4, whereas IL-6 production and ICAM-1 expression were inhibited by anti-interferon-gamma (IFN-gamma) antibodies. Enhanced production of IL-4 and IFN-gamma was detected in related allergen-stimulated TL supernatants from allergic subjects compared with the different supernatants. These data suggest that allergen-specific TL present in the peripheral blood of allergic patients are of Th1 and Th2 subtypes. Their stimulation in allergic patients may lead to the activation of endothelial cells and thereby participate in leucocyte recruitment towards the inflammatory site. PMID:7542574

  12. WISP-3 inhibition of miR-452 promotes VEGF-A expression in chondrosarcoma cells and induces endothelial progenitor cells angiogenesis.

    Science.gov (United States)

    Lin, Chih-Yang; Tzeng, Huey-En; Li, Te-Mao; Chen, Hsien-Te; Lee, Yi; Yang, Yi-Chen; Wang, Shih-Wei; Yang, Wei-Hung; Tang, Chih-Hsin

    2017-06-13

    Chondrosarcoma is the second most prevalent general primary tumor of bone following osteosarcoma. Chondrosarcoma development may be linked to angiogenesis, which is principally elicited by vascular endothelial growth factor-A (VEGF-A). VEGF-A level has been recognized as a prognostic marker in angiogenesis. WNT1-inducible signaling pathway protein-3 (WISP)-3/CCN6 belongs to the CCN family and is involved in regulating several cellular functions, including cell proliferation, differentiation, and migration. Nevertheless, the effect of WISP-3 on VEGF-A production and angiogenesis in human chondrosarcoma remains largely unknown. This current study shows that WISP-3 promoted VEGF-A production and induced angiogenesis of human endothelial progenitor cells. Moreover, WISP-3-enhanced VEGF-A expression and angiogenesis involved the c-Src and p38 signaling pathways, while miR-452 expression was negatively affected by WISP-3 via the c-Src and p38 pathways. Our results illustrate the clinical significance of WISP-3, VEGF-A and miR-452 in human chondrosarcoma patients. WISP-3 may illustrate a novel therapeutic target in the metastasis and angiogenesis of chondrosarcoma.

  13. Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer

    Directory of Open Access Journals (Sweden)

    Stacker Steven A

    2010-07-01

    Full Text Available Abstract Background It has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression. Methods We initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3 and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells. Results Lymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium, although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating

  14. Expression of human olfactory receptor 10J5 in heart aorta, coronary artery, and endothelial cells and its functional role in angiogenesis.

    Science.gov (United States)

    Kim, Sung-Hee; Yoon, Yeo Cho; Lee, Ae Sin; Kang, NaNa; Koo, JaeHyung; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-05-01

    ORs are ectopically expressed in non-chemosensory tissues including muscle, kidney, and keratinocytes; however, their physiological roles are largely unknown. We found that human olfactory receptor 10J5 (OR10J5) is expressed in the human aorta, coronary artery, and umbilical vein endothelial cells (HUVEC). Lyral induces Ca(2+) and phosphorylation of AKT in HUVEC. A knockdown study showed the inhibition of the lyral-induced Ca(2+) and the phosphorylation AKT and implied that these processes are mediated by OR10J5. In addition, lyral enhanced migration of HUVEC, which were also inhibited by RNAi in a migration assay. In addition, matrigel plug assay showed that lyral enhanced angiogenesis in vivo. Together these data demonstrate the physiological role of OR10J5 in angiogenesis and represent roles of ORs in HUVEC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

    OpenAIRE

    Chu, Chih-Sheng; Wang, Yu-Chen; Lu, Long-Sheng; Walton, Brian; Yilmaz, H. Ramazan; Huang, Roger Y.; Sawamura, Tatsuya; Dixon, Richard A. F.; Lai, Wen-Ter; Chen, Chu-Huang; Lu, Jonathan

    2013-01-01

    Objectives Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. Methods and Results Plasma LDL sa...

  16. [Characteristics of sublingual vein and expressions of vascular endothelial growth factor and hypoxia-inducible factor 1alpha proteins in sublingual tissues of Beagle dogs with portal hypertension].

    Science.gov (United States)

    Li, Bai-yu; Wang, Li-na; Yue, Xiao-qiang; Li, Bai

    2009-05-01

    To observe sublingual vein characteristics and the expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) proteins in sublingual tissues of Beagle dogs with cirrhotic portal hypertension. Twelve Beagle dogs were randomly divided into normal control group and cirrhotic portal hypertension group. There were 6 dogs in each group. A canine model of cirrhosis portal hypertension was established by injecting dimethylnitrosamine (DMN) into portal vein once a week for 7 weeks. The characteristics of sublingual vein were observed. Portal venous pressure was measured by using bioelectric recording techniques. The expressions of VEGF and HIF-1alpha proteins in sublingual vein were detected by immunohistochemical method. The shape and color of sublingual vein in beagle dogs in the cirrhotic portal hypertension group changed obviously as compared with the normal control group. Immunohistochemical results showed that there were almost no expressions of VEGF and HIF-1alpha proteins in sublingual tissues in the normal control group; however, the expressions of VEGF and HIF-1alpha proteins in sublingual tissues in the cirrhotic portal hypertension group significantly increased. Changes of portal pressure may lead to the formation of the abnormal sublingual vein by increasing the expressions of VEGF and HIF-1alpha proteins in sublingual tissues in Beagle dogs with portal hypertension.

  17. Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

    DEFF Research Database (Denmark)

    Hansen, Torben Frøstrup; Andersen, Claus Lindbjerg; Nielsen, Boye Schnack

    2011-01-01

    was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were...... the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24‑0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR...... analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA‑126-expressing using...

  18. Silencing of osteopontin promotes the radiosensitivity of breast cancer cells by reducing the expression of hypoxia inducible factor 1 and vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    YANG Li; ZHAO Wei; ZUO Wen-shu; WEI Ling; SONG Xian-rang; WANG Xing-wu; ZHENG Gang; ZHENG Mei-zhu

    2012-01-01

    Background Osteopontin (OPN) is a secreted phosphoglycoprotein (SSP) that is overexpressed in a variety of tumors and was regarded as a molecular marker of tumors.In this study,we intended to demonstrate the role of OPN in human breast cancer cell line MDA-MB-231.Methods Recombinant plasmid expressing small interfering RNA (siRNA) specific to OPN mRNA was transfected into MDA-MB-231 cells to generate the stable transfected cell line MDA-MB-343,and the empty plasmid tansfected cells (MDA-MB-neg) or wildtype MDA-MB-231 cells were used as control cells respectively.Expression of OPN,hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) proteins was analyzed by Western blotting analysis.The radiosensitivity of cells was determined by detecting cell apoptosis,cell proliferation and cell senescence.Results HIF-1 and VEGF proteins in MDA-MB-343 cells were significantly downregulated upon the efficient knockdown of OPN expression under either hypoxia or normoxia environment.Moreover,expression of OPN protein was upregualted upon hypoxic culture.Stable OPN-silencing also decreased cell invasion,increased cell apoptosis and cell senescence,as well as reduced clonogenic survival,resulting in increase radiation tolerance.Conclusions Suppression of OPN gene expression can enhance radiosensitivity and affect cell apoptosis in breast cancer cells.OPN seems to be an attractive target for the improvement of radiotherapy.

  19. Identification and expression of troponin T, a new marker on the surface of cultured tumor endothelial cells by aptamer ligand

    International Nuclear Information System (INIS)

    Ara, Mst Naznin; Hyodo, Mamoru; Ohga, Noritaka; Akiyama, Kosuke; Hida, Kyoko; Hida, Yasuhiro; Shinohara, Nobuo; Harashima, Hideyoshi

    2014-01-01

    The identification of a specific biomarker involves the development of new clinical diagnostic tools, and an in-depth understanding of the disease at the molecular level. When new blood vessels form in tumor cells, endothelial cell production is induced, a process that plays a key role in disease progression and metastasis to distinct organs for solid tumor types. The present study reports on the identification of a new biomarker on primary cultured mouse tumor endothelial cells (mTECs) using our recently developed high-affinity DNA aptamer AraHH001 (K d = 43 nmol/L) assisted proteomics approach. We applied a strategy involving aptamer-facilitated biomarker discovery. Biotin-tagged AraHH001 was incubated with lysates of mTECs and the aptamer-proteins were then conjugated with streptavidin magnetic beads. Finally, the bound proteins were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. We identified troponin T via matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, the molecular target of aptamer AraHH001, and its presence was confirmed by measuring mRNA, protein levels, western blot, immunostaining, a gel shift assay of AraHH001 with troponin T. We first report here on the discovery of troponin T on mTECs, a promising and interesting diagnostic tool in the development of antiangiogenic therapy techniques the involves the targeting of the tumor vasculature

  20. TNAP and EHD1 are over-expressed in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties.

    Directory of Open Access Journals (Sweden)

    Barbara Deracinois

    Full Text Available Although the physiological properties of the blood-brain barrier (BBB are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established in vitro BBB model developed in our laboratory, we performed a differential nano-LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial cells. Due to the heterogeneity of the crude extract, we increased identification yields by applying a repeatable, reproducible fractionation process based on the proteins' relative hydrophobicity. We present proteomic and biochemical evidence to show that tissue non-specific alkaline phosphatase (TNAP and Eps15 homology domain-containing protein 1(EDH1 are over-expressed by bovine BCECs after the re-induction of BBB properties. We discuss the impact of these findings on current knowledge of endothelial and BBB permeability.

  1. Selenium modulates MMP2 expression through the TGFβ1/Smad signalling pathway in human umbilical vein endothelial cells and rabbits following lipid disturbance.

    Science.gov (United States)

    Xu, Chenggui; Lu, Guihua; Li, Qinglang; Zhang, Juhong; Huang, Zhibin; Gao, Xiuren

    2017-07-01

    A high-fat diet is a major risk factor for coronary heart diseases. Matrix metalloprotease (MMP) expression is changed in many cardiovascular diseases. Selenium, which is an important trace element in animals, has a close relationship with cardiovascular diseases. The TGFβ1/Smad signalling pathway is ubiquitous in diverse tissues and cells, and it is also associated with the occurrence and development of cardiovascular diseases. Therefore, in this study, we aimed to determine selenium's effect on lipid metabolism, atherosclerotic plaque formation, and MMP2 expression, as well as the underlying functional mechanism. In vivo tests: 24 male New Zealand white rabbits were randomly divided into 4 groups: regular diet, high-fat diet, high-fat diet+selenium and regular diet+selenium groups. The high-fat diet induced the lipid disturbances of rabbits at week 12. Selenium supplementation lowered total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels (pSelenium supplementation also suppressed MMP2 over-expression in thoracic aortas. In vitro tests: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of selenium or ox-LDL. Ox-LDL promoted MMP2 expression by increasing TGFβ1, pSmad2, pSmad3 and Smad3 expression (pSelenium attenuated MMP2 over-expression by regulating the TGFβ1/Smad signalling pathway. Selenium suppressed high-fat diet-induced MMP2 over-expression in vivo by improving lipid metabolism. In vitro, selenium attenuated MMP2 over-expression through the TGFβ1/Smad signalling pathway. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

    International Nuclear Information System (INIS)

    Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

    2013-01-01

    Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis

  3. Expression of vascular endothelial growth factor and matrix metalloproteinase-9 in Apis mellifera Lawang propolis extract gel-treated traumatic ulcers in diabetic rats

    Directory of Open Access Journals (Sweden)

    Diah Savitri Ernawati

    2018-03-01

    Full Text Available Aim: The aim of this study was to determine the effect of Apis mellifera propolis extract gel on vascular endothelial growth factor (VEGF and matrix metalloproteinase-9 (MMP-9 expression in the traumatic ulcers of rats afflicted with diabetes mellitus (DM. Materials and Methods: The study was conducted on 24 male Wistar rats (Rattus norvegicus induced with DM by injecting 50 mg/kg of Streptozotocin, intraperitoneally, and a traumatic ulcer on their lower lip mucosa. These were divided into eight groups: Four each for control and treatment groups. Each control and treatment group consisted of three rats. The control groups treated with hydroxypropyl methylcellulose 5% gel and treatment groups were administered with propolis extract gel. The expression of VEGF and MMP-9 was observed on days 3, 5, 7, and 9. Furthermore, mice sacrificed and the lower lip labial mucosa tissue of mice has been taken to make the histopathology anatomy preparation by means of immunohistochemical examination with monoclonal antibodies anti-VEGF and anti-MMP-9. Results: This experiment revealed higher VEGF expression and lower MMP-9 expression in the treatment group as compared to that of the control group. Analysis of Variance showed significant differences (p<0.01 of both VEGF expression and MMP-9 expression between the two groups. A Tukey's analysis did not find strong contrasts in VEGF and MMP-9 expressions between various treatment groups. However, those between treatment and control groups were found to be considerable. Conclusion: Propolis extract gel increased the expression of VEGF and decreased that of MMP-9 during the healing process of traumatic ulcers on the oral mucosa of diabetes afflicted Wistar rats (R. norvegicus.

  4. Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

    Science.gov (United States)

    Pantazatos, S P; Huang, Y-Y; Rosoklija, G B; Dwork, A J; Arango, V; Mann, J J

    2017-05-01

    Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted Pdepression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted Psuicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted Pdepression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

  5. Perfusion parameters of dynamic contrast-enhanced magnetic resonance imaging in patients with rectal cancer: Correlation with microvascular density and vascular endothelial growth factor expression

    International Nuclear Information System (INIS)

    Kim, Yeo Eun; Lim, Joon Seok; Kim, Myeong Jin; Kim, Ki Whang; Choi, Jun Jeong; Kim, Dae Hong; Myoung, Sung Min

    2013-01-01

    To determine whether quantitative perfusion parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) correlate with immunohistochemical markers of angiogenesis in rectal cancer. Preoperative DCE-MRI was performed in 63 patients with rectal adenocarcinoma. Transendothelial volume transfer (K trans ) and fractional volume of the extravascular-extracellular space (Ve) were measured by Interactive Data Language software in rectal cancer. After surgery, microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression scores were determined using immunohistochemical staining of rectal cancer specimens. Perfusion parameters (K trans , Ve) of DCE-MRI in rectal cancer were found to be correlated with MVD and VEGF expression scores by Spearman's rank coefficient analysis. T stage and N stage (negative or positive) were correlated with perfusion parameters and MVD. Significant correlation was not found between any DCE-MRI perfusion parameters and MVD (rs = -0.056 and p 0.662 for K trans ; rs = -0.103 and p = 0.416 for Ve), or between any DCE-MRI perfusion parameters and the VEGF expression score (rs = -0.042, p 0.741 for K trans ; r = 0.086, p = 0.497 for Ve) in rectal cancer. TN stage showed no significant correlation with perfusion parameters or MVD (p > 0.05 for all). DCE-MRI perfusion parameters, K trans and Ve, correlated poorly with MVD and VEGF expression scores in rectal cancer, suggesting that these parameters do not simply denote static histological vascular properties.

  6. Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration

    Directory of Open Access Journals (Sweden)

    Sung Hwan Kim

    2016-12-01

    Full Text Available Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF, secreted by the retinal pigment epithelium (RPE, in pathological angiogenesis and the development of choroidal neovascularization (CNV in age-related macular degeneration (AMD. RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH, which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS. Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

  7. Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation.

    Science.gov (United States)

    Park, Yoon Shin; Kim, Gyungah; Jin, Yoon Mi; Lee, Jee Young; Shin, Jong Wook; Jo, Inho

    2016-01-08

    We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5'-deletions from -3040 to -1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Both high expression of pyruvate kinase M2 and vascular endothelial growth factor-C predicts poorer prognosis in human breast cancer.

    Science.gov (United States)

    Lin, Yang; Liu, Fangfang; Fan, Yu; Qian, Xiaolong; Lang, Ronggang; Gu, Feng; Gu, Jun; Fu, Li

    2015-01-01

    Pyruvate kinase M2 (PKM2) and vascular endothelial growth factor-C (VEGF-C) have been known to play an important role in tumorigenesis and tumor progression in breast cancer. However, the association between PKM2 and VEGF-C in breast cancer remains unclear. In the present study, a total of 218 specimens from breast cancer patients and 26 paired breast tumors with adjacent normal tissues as well as two breast cancer cell lines were enrolled to investigate the correlation between PKM2 and VEGF-C. We found that PKM2 and VEGF-C mRNA levels were both significantly increasing in breast tumors compared with adjacent normal tissues. Knockdown of PKM2 mRNA expression resulted in VEGF-C mRNA and protein down-regulated as well as cell proliferation inhibited. A positive correlation between PKM2 and VEGF-C expression was identified by immunohistochemical analyses of 218 specimens of patients with breast cancer (P=0.023). PKM2 high expression was significantly correlated with histological grade (P=0.030), lymph node stage (P=0.001), besides VEGF-C high expression was significantly associated with lymphovascular invasion (P=0.012). While combined high expression of PKM2 and VEGF-C was found to be associated with worse histological grade, more lymph node metastasis, more lymphovascular invasion, shorter progression free survival (PFS), and poorer overall survival (OS) in human breast cancer. The results of the present study suggested that PKM2 expression was correlated with VEGF-C expression, and combination of PKM2 and VEGF-C levels had the better prognostic significance in predicting the poor outcome of patients with breast cancer.

  9. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

    Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  10. Endothelial RIG-I activation impairs endothelial function

    International Nuclear Information System (INIS)

    Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2012-01-01

    Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  11. Tumor microenvironment conditions alter Akt and Na+/H+ exchanger NHE1 expression in endothelial cells more than hypoxia alone

    DEFF Research Database (Denmark)

    Pedersen, Anna-Kathrine; Mendes Lopes de Melo, Joana; Mørup, Nina

    2017-01-01

    Background Chronic angiogenesis is a hallmark of most tumors and takes place in a hostile tumor microenvironment (TME) characterized by hypoxia, low nutrient and glucose levels, elevated lactate and low pH. Despite this, most studies addressing angiogenic signaling use hypoxia as a proxy for tumor...... cells, Akt1 most abundantly. Akt1 protein expression was reduced by TME yet unaffected by hypoxia, while Akt phosphorylation was increased by TME. The Akt loss was partly reversed by MCF-7 human breast cancer cell conditioned medium, suggesting that in vivo, the cancer cell secretome may compensate....../inhibition. Conclusions NHE1 and Akt are downregulated by TME conditions, more potently than by hypoxia alone. This inhibits endothelial cell migration and growth in a manner likely modulated by the cancer cell secretome....

  12. Endothelial glutathione-S-transferase A4-4 protects against oxidative stress and modulates iNOS expression through NF-κB translocation

    International Nuclear Information System (INIS)

    Yang Yongzhen; Yang Yusong; Xu Ya; Lick, Scott D.; Awasthi, Yogesh C.; Boor, Paul J.

    2008-01-01

    Our recent work in endothelial cells and human atherosclerotic plaque showed that overexpression of glutathione-S-tranferases (GSTs) in endothelium protects against oxidative damage from aldehydes such as 4-HNE. Nuclear factor (NF)-κB plays a crucial role during inflammation and immune responses by regulating the expression of inducible genes such as inducible nitric oxide synthase (iNOS). 4-HNE induces apoptosis and affects NF-κB mediated gene expression, but conflicting results on 4-HNE's effect on NF-κB have been reported. We compared the effect of 4-HNE on iNOS and the NF-κB pathway in control mouse pancreatic islet endothelial (MS1) cells and those transfected with mGSTA4, a α-class GST with highest activity toward 4-HNE. When treated with 4-HNE, mGSTA4-transfected cells showed significant upregulation of iNOS and nitric oxide (NO) through (NF)-κB (p65) translocation in comparison with wild-type or vector-transfected cells. Immunohistochemical studies of early human plaques showed lower 4-HNE content and upregulation of iNOS, which we take to indicate that GSTA4-4 induction acts as an enzymatic defense against high levels of 4-HNE, since 4-HNE accumulated in more advanced plaques, when detoxification and exocytotic mechanisms are likely to be overwhelmed. These studies suggest that GSTA4-4 may play an important defensive role against atherogenesis through detoxification of 4-HNE and upregulation of iNOS

  13. d(− Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

    Directory of Open Access Journals (Sweden)

    Pablo Alarcón

    2017-08-01

    Full Text Available Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(− lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(− lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(− lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET production (NETosis in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(− lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(− lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1. d(− lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(− lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(− lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

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

    Directory of Open Access Journals (Sweden)

    Tahanian E

    2011-05-01

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

  15. Resveratrol: A Multifunctional Compound Improving Endothelial Function

    OpenAIRE

    Li, Huige; F?rstermann, Ulrich

    2009-01-01

    The red wine polyphenol resveratrol boosts endothelium-dependent and -independent vasorelaxations. The improvement of endothelial function by resveratrol is largely attributable to nitric oxide (NO) derived from endothelial NO synthase (eNOS). By stimulating eNOS expression, eNOS phosphorylation and eNOS deacetylation, resveratrol enhances endothelial NO production. By upregulating antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and suppressing the expression a...

  16. Low-energy extracorporeal shock wave therapy for promotion of vascular endothelial growth factor expression and angiogenesis and improvement of locomotor and sensory functions after spinal cord injury.

    Science.gov (United States)

    Yahata, Kenichiro; Kanno, Haruo; Ozawa, Hiroshi; Yamaya, Seiji; Tateda, Satoshi; Ito, Kenta; Shimokawa, Hiroaki; Itoi, Eiji

    2016-12-01

    OBJECTIVE Extracorporeal shock wave therapy (ESWT) is widely used to treat various human diseases. Low-energy ESWT increases expression of vascular endothelial growth factor (VEGF) in cultured endothelial cells. The VEGF stimulates not only endothelial cells to promote angiogenesis but also neural cells to induce neuroprotective effects. A previous study by these authors demonstrated that low-energy ESWT promoted expression of VEGF in damaged neural tissue and improved locomotor function after spinal cord injury (SCI). However, the neuroprotective mechanisms in the injured spinal cord produced by low-energy ESWT are still unknown. In the present study, the authors investigated the cell specificity of VEGF expression in injured spinal cords and angiogenesis induced by low-energy ESWT. They also examined the neuroprotective effects of low-energy ESWT on cell death, axonal damage, and white matter sparing as well as the therapeutic effect for improvement of sensory function following SCI. METHODS Adult female Sprague-Dawley rats were divided into the SCI group (SCI only) and SCI-SW group (low-energy ESWT applied after SCI). Thoracic SCI was produced using a New York University Impactor. Low-energy ESWT was applied to the injured spinal cord 3 times a week for 3 weeks after SCI. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan open-field locomotor score for 42 days after SCI. Mechanical and thermal allodynia in the hindpaw were evaluated for 42 days. Double staining for VEGF and various cell-type markers (NeuN, GFAP, and Olig2) was performed at Day 7; TUNEL staining was also performed at Day 7. Immunohistochemical staining for CD31, α-SMA, and 5-HT was performed on spinal cord sections taken 42 days after SCI. Luxol fast blue staining was performed at Day 42. RESULTS Low-energy ESWT significantly improved not only locomotion but also mechanical and thermal allodynia following SCI. In the double staining, expression of VEGF was observed in Neu

  17. CCL5 promotes vascular endothelial growth factor expression and induces angiogenesis by down-regulating miR-199a in human chondrosarcoma cells.

    Science.gov (United States)

    Liu, Guan-Ting; Huang, Yuan-Li; Tzeng, Huey-En; Tsai, Chun-Hao; Wang, Shih-Wei; Tang, Chih-Hsin

    2015-02-28

    Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis. Angiogenesis is a critical step in tumor growth and metastasis. Chemokine CCL5 (previously called RANTES) has been shown to facilitate tumor progression and metastasis. However, the relationship of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study, CCL5 increased VEGF expression and also promoted chondrosarcoma medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. MicroRNA analysis was performed in CCL5-treated chondrosarcoma cells versus control cells to investigate the mechanism of CCL5-mediated promotion of chondrosarcoma angiogenesis. Among the miRNAs regulated by CCL5, miR-199a was the most downregulated miRNA after CCL5 treatment. In addition, co-transfection with miR-199a mimic reversed the CCL5-mediated VEGF expression and angiogenesis in vitro and in vivo. Moreover, overexpression of CCL5 increased tumor-associated angiogenesis and tumor growth by downregulating miR-199a in the xenograft tumor angiogenesis model. Taken together, these results demonstrated that CCL5 promotes VEGF-dependent angiogenesis in human chondrosarcoma cells by downregulating miR-199a. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.

    Science.gov (United States)

    Al-Ahmad, Abraham J

    2017-10-01

    Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells. Copyright © 2017 the American Physiological Society.

  19. Autoantibodies Targeting AT1 Receptor from Patients with Acute Coronary Syndrome Upregulate Proinflammatory Cytokines Expression in Endothelial Cells Involving NF-κB Pathway

    Directory of Open Access Journals (Sweden)

    Weijuan Li

    2014-01-01

    Full Text Available Our study intended to prove whether agonistic autoantibodies to angiotensin II type 1 receptor (AT1-AAs exist in patients with coronary heart disease (CHD and affect the human endothelial cell (HEC by upregulating proinflammatory cytokines expression involved in NF-κB pathway. Antibodies were determined by chronotropic responses of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists (valsartan and AT1-EC2 as described previously. Interleukin-6 (IL-6, vascular cell adhesion molecule-1 (VCAM-1, and monocyte chemotactic protein-1 (MCP-1 expression were improved at both mRNA and protein levels in HEC, while NF-κB in the DNA level was improved detected by electrophoretic mobility shift assays (EMSA. These improvements could be inhibited by specific AT1 receptor blocker valsartan, NF-κB blocker pyrrolidine dithiocarbamate (PDTC, and specific short peptides from the second extracellular loop of AT1 receptor. These results suggested that AT1-AAs, via the AT1 receptor, induce expression of proinflammatory cytokines involved in the activation of NF-κB. AT1-AAs may play a great role in the pathogenesis of the acute coronary syndrome by mediating vascular inflammatory effects involved in the NF-κB pathway.

  20. The expression of vascular endothelial growth factor is affected by hypoxia inducible factor-1α in peritoneum of endometriosis mice treated with genistein

    Directory of Open Access Journals (Sweden)

    Sylvi Wafda Nur Amellia

    2016-09-01

    Full Text Available This study aimed to investigate whether the genistein is able to decrease the expressions of vascular endothelial growth factor-A (VEGF-A and hypoxia inducible factor-1α (HIF-1α in mouse model of endometriosis. Forty female mice (Mus musculus were divided into eight groups (n = 5 each, including the control (untreated group, endometriosis group, and endometriosis groups treated with various doses of genistein (50; 100; 200; 300; 400; and 500 mg/day. VEGF-A and HIF-1α analyses were performed by immunohistochemistry. We found significant increases in the VEGF-A and HIF-α expressions in endometriosis group compared to the control group. The increased expressions of VEGF-A and HIF-1α were significantly (p < 0.05 attenuated by the administration of all doses of genistein. In conclusion, in mouse model of endometriosis, genistein potentially inhibits the increase in angiogenesis in peritoneal tissue. Therefore, this result may provide a novel anti-angiogenic treatment strategy for the therapy of endometriosis.

  1. Notch-RBP-J signaling regulates the mobilization and function of endothelial progenitor cells by dynamic modulation of CXCR4 expression in mice.

    Directory of Open Access Journals (Sweden)

    Lin Wang

    Full Text Available Bone marrow (BM-derived endothelial progenitor cells (EPC have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.

  2. 1α,25-Dihydroxyvitamin D(3) inhibits vascular cellular adhesion molecule-1 expression and interleukin-8 production in human coronary arterial endothelial cells.

    Science.gov (United States)

    Kudo, Keiko; Hasegawa, Shunji; Suzuki, Yasuo; Hirano, Reiji; Wakiguchi, Hiroyuki; Kittaka, Setsuaki; Ichiyama, Takashi

    2012-11-01

    Kawasaki disease is an acute febrile vasculitis of childhood that is associated with elevated production of inflammatory cytokines, causing damage to the coronary arteries. The production of proinflammatory cytokines and expression of adhesion molecules in human coronary arterial endothelial cells (HCAECs) is regulated by nuclear transcription factor-κB (NF-κB) activation. We have previously reported that the active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)), inhibits tumor necrosis factor-α (TNF-α)-induced NF-κB activation. In this study, we examined the anti-inflammatory effects of 1α,25-(OH)(2)D(3) on TNF-α-induced adhesion molecule expression (vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1)) and cytokine production (interleukin-6 (IL-6) and IL-8) in HCAECs. Pretreatment with 1α,25-(OH)(2)D(3) significantly inhibited TNF-α-induced VCAM-1 expression and IL-8 production in HCAECs. Our results suggest that adjunctive 1α,25-(OH)(2)D(3) therapy may modulate the inflammatory response during Kawasaki disease vasculitis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Elevated AKR1C3 expression promotes prostate cancer cell survival and prostate cell-mediated endothelial cell tube formation: implications for prostate cancer progressioan

    International Nuclear Information System (INIS)

    Dozmorov, Mikhail G; Lin, Hsueh-Kung; Azzarello, Joseph T; Wren, Jonathan D; Fung, Kar-Ming; Yang, Qing; Davis, Jeffrey S; Hurst, Robert E; Culkin, Daniel J; Penning, Trevor M

    2010-01-01

    Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR1C enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we propose a novel pathological function of AKR1C3 in tumor angiogenesis and its potential role in promoting PCa progression. To recapitulate elevated AKR1C3 expression in cancerous prostate, the human PCa PC-3 cell line was stably transfected with an AKR1C3 expression construct to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analysis were performed to identify AKR1C3-mediated pathways of activation and their potential biological consequences in PC-3 cells. Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and an in vitro Matrigel angiogenesis assays were applied to validate the pro-angiogenic activity of PC3-AKR1C3 transfectants identified by bioinformatics analysis. Microarray and bioinformatics analysis suggested that overexpression of AKR1C3 in PC-3 cells modulates estrogen and androgen metabolism, activates insulin-like growth factor (IGF)-1 and Akt signaling pathways, as well as promotes tumor angiogenesis and aggressiveness. Levels of IGF-1 receptor (IGF-1R) and Akt activation as well as vascular endothelial growth factor (VEGF) expression and secretion were significantly elevated in PC3-AKR1C3 transfectants in comparison to PC3-mock transfectants. PC3-AKR1C3 transfectants also promoted endothelial cell (EC) tube formation on Matrigel as compared to the AKR1C3-negative parental PC-3 cells and PC3-mock transfectants. Pre-treatment of PC3-AKR1C3 transfectants with a selective IGF-1R kinase inhibitor (AG1024) or a non-selective phosphoinositide 3-kinases (PI3K) inhibitor (LY294002) abolished ability of the cells to promote EC tube formation. Bioinformatics

  4. Activation of endothelial nitric oxide synthase by dietary isoflavones: role of NO in Nrf2-mediated antioxidant gene expression.

    Science.gov (United States)

    Mann, Giovanni E; Rowlands, David J; Li, Francois Y L; de Winter, Patricia; Siow, Richard C M

    2007-07-15

    The endothelium plays a key role in the maintenance of vascular homeostasis, and increased oxidative stress in vascular disease leads to reduced nitric oxide bioavailability and impaired endothelium-dependent relaxation of resistance vessels. Although epidemiological evidence suggests that diets containing high amounts of natural antioxidants afford protection against coronary heart disease (CHD), antioxidant supplementation trials have largely reported only marginal health benefits. There is controversy concerning the cardiovascular benefits of prolonged estrogen/progestin or soy isoflavone therapy for postmenopausal women and patients with an increased risk of CHD. Research on the potential health benefits of soy isoflavones and other polyphenols contained in red wine, green and black tea and dark chocolate developed rapidly during the 1990's, and recent clinical trials and studies in animal models and cultured endothelial cells provide important and novel insights into the mechanisms by which dietary polyphenols afford protection against oxidative stress. In this review, we highlight that NO and reactive oxygen radicals may mediate dietary polyphenol induced activation of Nrf2, which in turn triggers antioxidant response element (ARE) driven transcription of phase II detoxifying and antioxidant defense enzymes in vascular cells.

  5. The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

    Science.gov (United States)

    Appukuttan, Binoy; McFarland, Trevor J.; Stempel, Andrew; Kassem, Jean B.; Hartzell, Matthew; Zhang, Yi; Bond, Derek; West, Kelsey; Wilson, Reid; Stout, Andrew; Pan, Yuzhen; Ilias, Hoda; Robertson, Kathryn; Klein, Michael L.; Wilson, David; Smith, Justine R.; Stout, J. Timothy

    2012-01-01

    Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases. PMID:22761647

  6. Effect of high ovarian response on the expression of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in peri-implantation endometrium in IVF women.

    Science.gov (United States)

    Xu, Li-Zhen; Gao, Min-Zhi; Yao, Li-Hua; Liang, A-Juan; Zhao, Xiao-Ming; Sun, Zhao-Gui

    2015-01-01

    To investigate the effect of ovarian stimulation on the expression of EG-VEGF mRNA and protein in peri-implantation endometrium in women undergoing IVF and its relation with endometrial receptivity (ER). Prospective laboratory study. University hospital. Eighteen women in stimulated cycles (SC) as study subjects and 18 women in natural cycles (NC) as controls. Women in SC group were classified with two subgroups, high ovarian response (SC1, n=9) with peak serum E2>5,000 pg/mL and moderate ovarian response (SC2, n=9) with peak serum E2 1,000-5,000 pg/mL. Endometrial biopsies were collected 6 days after ovulation in NC or after oocyte retrieval in SC. Endometrium histological dating was observed with HE staining. EG-VEGF mRNA expression levels determined by real-time polymerase chain reaction analysis, and protein levels by immunohistochemistry. All endometrial samples were in the secretory phase. The endometrial development in SC1 was 1 to 2 days advanced to NC, and with dyssynchrony between glandular and stromal tissue. Immunohistochemistry analysis showed that EG-VEGF protein was predominantly expressed in the glandular epithelial cells and endothelial cells of vessels, and also presented in the stroma. The image analysis confirmed that both the gland and stroma of endometrium in SC1 had a significantly lower EG-VEGF protein expression than that in SC2 and NC endometrium. Moreover, EG-VEGF mRNA levels were significantly lower in SC1 than in NC. Both EG-VEGF protein and mRNA levels had no significant difference between SC2 and NC. Decreased expression of EG-VEGF in the peri-implantation is associated with high ovarian response, which may account for the impaired ER and lower implantation rate in IVF cycles.

  7. Molecular cloning, expression analysis and miRNA prediction of vascular endothelial growth factor A (VEGFAa and VEGFAb) in pond loach Misgurnus anguillicaudatus, an air-breathing fish.

    Science.gov (United States)

    Luo, Weiwei; Liang, Xiao; Huang, Songqian; Cao, Xiaojuan

    2016-12-01

    Vascular endothelial growth factor A (VEGFA) is the most studied and the best characterized member of the VEGF family and is a key regulator of angiogenesis via its ability to affect the proliferation, migration, and differentiation of endothelial cells. In this study, the full-length cDNAs encoding VEGFAa and VEGFAb from pond loach, Misgurnus anguillicaudatus, were isolated. The VEGFAa is constituted by an open reading frame (ORF) of 570bp encoding for a peptide of 189 amino acid residues, a 639bp 5'-untranslated region (UTR) and a 2383bp 3' UTR. The VEGFAb is constituted by an ORF of 687bp encoding for a peptide of 228 amino acid residues, a 560bp 5' UTR and a 1268bp 3' UTR. Phylogenetic analysis indicated that the VEGFAa and VEGFAb of pond loach were conserved in vertebrates. Expression levels of VEGFAa and VEGFAb were detected by RT-qPCR at different development stages of pond loach and in different tissues of 6-month-old, 12-month-old and 24-month-old pond loach. Moreover, eight predicted miRNAs (miR-200, miR-29, miR-218, miR-338, miR-103, miR-15, miR-17 and miR-223) targeting VEGFAa and VEGFAb were validated by an intestinal air-breathing inhibition experiment. This study will be of value for further studies into the function of VEGFA and its corresponding miRNAs, which will shed a light on the vascularization and accessory air-breathing process in pond loach. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Reported Prestroke Physical Activity Is Associated with Vascular Endothelial Growth Factor Expression and Good Outcomes after Stroke.

    Science.gov (United States)

    López-Cancio, Elena; Ricciardi, Ana Clara; Sobrino, Tomás; Cortés, Jordi; de la Ossa, Natalia Pérez; Millán, Mónica; Hernández-Pérez, María; Gomis, Meritxell; Dorado, Laura; Muñoz-Narbona, Lucía; Campos, Francisco; Arenillas, Juan F; Dávalos, Antoni

    2017-02-01

    Physical activity (PhA) prior to stroke has been associated with good outcomes after the ischemic insult, but there is scarce data on the involved molecular mechanisms. We studied consecutive acute ischemic stroke patients admitted to a single tertiary stroke center. Prestroke PhA was evaluated with the International Physical Activity Questionnaire (metabolic equivalent of minutes/week). We studied several circulating angiogenic and neurogenic factors at different time points: vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), and brain-derived neurotrophic factor (BDNF) at admission, day 7, and at 3 months. We considered good functional outcome at 3 months (modified Rankin scale  ≤ 2) as primary end point, and final infarct volume as secondary outcome. We studied 83 patients with at least 2 time point serum determinations (mean age 69.6 years, median National Institutes of Health Stroke Scale 17 at admission). Patients more physically active before stroke had a significantly higher increment of serum VEGF on the seventh day when compared to less active patients. This increment was an independent predictor of good functional outcome at 3 months and was associated with smaller infarct volume in multivariate analyses adjusted for relevant covariates. We did not find independent associations of G-CSF or BDNF levels neither with level of prestroke PhA nor with stroke outcomes. Although there are probably more molecular mechanisms by which PhA exerts its beneficial effects in stroke outcomes, our observation regarding the potential role of VEGF is plausible and in line with previous experimental studies. Further research in this field is needed. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  9. Aqueous extract of Allium sativum L bulbs offer nephroprotection by attenuating vascular endothelial growth factor and extracellular signal-regulated kinase-1 expression in diabetic rats.

    Science.gov (United States)

    Shiju, T M; Rajkumar, R; Rajesh, N G; Viswanathan, Pragasam

    2013-02-01

    To investigate the nephroprotective effect of garlic and elucidate the mechanism by which it prevents the progression of diabetic nephropathy in diabetic rats, diabetes was induced by a single ip injection of streptozotocin (45 mg/kg body weight). Garlic extract (500 mg/kg body weight) and aminoguanidine (1 g/L) were supplemented in the treatment groups. Histopathological examination using H&E, PAS staining and the immunohistochemical analysis of vascular endothelial growth factor (VEGF) and extracellular signal-regulated kinase-1 (ERK-1) expression were performed on kidney sections at the end of 12 weeks. Significant change in both, the urine and serum biochemistry confirmed kidney damage in diabetic animals which was further confirmed by the histological changes such as mesangial expansion, glomerular basement membrane thickening, glycosuria and proteinuria. However, the diabetic animals treated with garlic extract showed a significant change in urine and serum biochemical parameters such as albumin, urea nitrogen and creatinine compared to that of diabetic rats. Further, the garlic supplemented diabetic rats showed a significant decrease in the expression of VEGF and ERK-1 compared to diabetic rats, attenuating mesangial expansion and glomerulosclerosis. Thus, garlic extract rendered nephroprotection in diabetic rats.

  10. Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

    Science.gov (United States)

    Niimi, Kenta; Ueda, Mizuha; Fukumoto, Moe; Kohara, Misaki; Sawano, Toshinori; Tsuchihashi, Ryo; Shibata, Satoshi; Inagaki, Shinobu; Furuyama, Tatsuo

    2017-08-05

    Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. YKL-40-Induced Inhibition of miR-590-3p Promotes Interleukin-18 Expression and Angiogenesis of Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Te-Mao Li

    2017-04-01

    Full Text Available YKL-40, also known as human cartilage glycoprotein-39 or chitinase-3-like-1, is a pro-inflammatory protein that is highly expressed in rheumatoid arthritis (RA patients. Angiogenesis is a critical step in the pathogenesis of RA, promoting the infiltration of inflammatory cells into joints and providing oxygen and nutrients to RA pannus. In this study, we examined the effects of YKL-40 in the production of the pro-inflammatory cytokine interleukin-18 (IL-18, and the stimulation of angiogenesis and accumulation of osteoblasts. We observed that YKL-40 induces IL-18 production in osteoblasts and thereby stimulates angiogenesis of endothelial progenitor cells (EPCs. We found that this process occurs through the suppression of miR-590-3p via the focal adhesion kinase (FAK/PI3K/Akt signaling pathway. YKL-40 inhibition reduced angiogenesis in in vivo models of angiogenesis: the chick embryo chorioallantoic membrane (CAM and Matrigel plug models. We report that YKL-40 stimulates IL-18 expression in osteoblasts and facilitates EPC angiogenesis.

  12. Adenovirus Vector E4 Gene Regulates Connexin 40 and 43 Expression in Endothelial Cells via PKA and PI3K Signal Pathways

    Science.gov (United States)

    Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K.; Vincent, Loïc; Hackett, Neil R.; Wang, Shiyang; Young, Lauren M.; Hempstead, Barbara; Crystal, Ronald G.; Rafii, Shahin

    2010-01-01

    Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4−, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intratracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways. PMID:15831817

  13. Wine and endothelial function.

    Science.gov (United States)

    Caimi, G; Carollo, C; Lo Presti, R

    2003-01-01

    In recent years many studies have focused on the well-known relationship between wine consumption and cardiovascular risk. Wine exerts its protective effects through various changes in lipoprotein profile, coagulation and fibrinolytic cascades, platelet aggregation, oxidative mechanisms and endothelial function. The last has earned more attention for its implications in atherogenesis. Endothelium regulates vascular tone by a delicate balancing among vasorelaxing (nitric oxide [NO]) and vasoconstrincting (endothelins) factors produced by endothelium in response to various stimuli. In rat models, wine and other grape derivatives exerted an endothelium-dependent vasorelaxing capacity especially associated with the NO-stimulating activity of their polyphenol components. In experimental conditions, reservatrol (a stilbene polyphenol) protected hearts and kidneys from ischemia-reperfusion injury through antioxidant activity and upregulation of NO production. Wine polyphenols are also able to induce the expression of genes involved in the NO pathway within the arterial wall. The effects of wine on endothelial function in humans are not yet clearly understood. A favorable action of red wine or dealcoholized wine extract or purple grape juice on endothelial function has been observed by several authors, but discrimination between ethanol and polyphenol effects is controversial. It is, however likely that regular and prolonged moderate wine drinking positively affects endothelial function. The beneficial effects of wine on cardiovascular health are greater if wine is associated with a healthy diet. The most recent nutritional and epidemiologic studies show that the ideal diet closely resembles the Mediterranean diet.

  14. Vascular endothelial growth factor polymorphisms and a synchronized examination of plasma and tissue expression in epithelial ovarian cancers.

    Science.gov (United States)

    Bhaskari, J; Premalata, C S; Shilpa, V; Rahul, B; Pallavi, V R; Ramesh, G; Krishnamoorthy, Lakshmi

    2016-01-01

    In this study, we have analyzed six genetic polymorphisms of the VEGF-A gene and correlated the genetic data with plasma and tissue expression of VEGF-A in epithelial ovarian carcinomas. A total of 130 cases including 95 malignant carcinomas, 17 low malignant potential and 18 benign tumours were studied. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were studied by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma levels of VEGF-A were estimated by enzyme-linked immunosorbent assay (ELISA) and tissue expression of VEGF-A by immunohistochemistry (IHC). Four polymorphisms of the above excluding rs699947 and rs3025039 showed significant association with malignancy, and we observed the presence of positive correlation between haplotype CCGGCC and increased expression of VEGF-A in both plasma and tissues which also correlated with poor prognosis and recurrence suggesting a probable increase in resistance to treatment in such carriers. Highly upregulated tissue expression of VEGF-A was seen in all epithelial ovarian carcinomas with intensity of expression increasing from benign to malignant cases. ELISA data from our study showed an increase in circulating levels of VEGF-A in malignancies. VEGF-A plasma levels can be employed as a biomarker for high-grade malignancy in epithelial ovarian cancers alongside tissue expression and CA-125 levels. This study is unique due to the fact that a simultaneous analysis of plasma and tissue expression has been demonstrated and is a first such study in epithelial ovarian cancers and representing the Indian population (South-east Asian) synchronized with genetic polymorphism data as well.

  15. Nucleotide-binding oligomerization domain 1 regulates Porphyromonas gingivalis-induced vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression in endothelial cells through NF-κB pathway.

    Science.gov (United States)

    Wan, M; Liu, J; Ouyang, X

    2015-04-01

    Porphyromonas gingivalis has been shown to actively invade endothelial cells and induce vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) overexpression. Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition reporter, and its involvement in this process was unknown. This study focused on endothelial cells infected with P. gingivalis, the detection of NOD1 expression and the role that NOD1 plays in the upregulation of VCAM-1 and ICAM-1. The human umbilical vein endothelial cell line (ECV-304) was intruded by P. gingivalis W83, and cells without any treatment were the control group. Expression levels of NOD1, VCAM-1, ICAM-1, phosphorylated P65 between cells with and without treatment on both mRNA and protein levels were compared. Then we examined whether mesodiaminopimelic acid (NOD1 agonist) could increase VCAM-1 and ICAM-1 expression, meanwhile, NOD1 gene silence by RNA interference could reduce VCAM-1, ICAM-1 and phosphorylated P65 release. At last, we examined whether inhibition of NF-κB by Bay117082 could reduce VCAM-1 and ICAM- 1 expression. The mRNA levels were measured by real-time polymerase chain reaction, and protein levels by western blot or electrophoretic mobility shift assays (for phosphorylated P65). P. gingivalis invasion showed significant upregulation of NOD1, VCAM-1 and ICAM-1. NOD1 activation by meso-diaminopimelic acid increased VCAM-1 and ICAM-1 expression, and NOD1 gene silence reduced VCAM-1 and ICAM-1 release markedly. The NF-κB signaling pathway was activated by P. gingivalis, while NOD1 gene silence decreased the activation of NF-κB. Moreover, inhibition of NF-κB reduced VCAM-1 and ICAM-1 expression induced by P. gingivalis in endothelial cells. The results revealed that P. gingivalis induced NOD1 overexpression in endothelial cells and that NOD1 played an important role in the process of VCAM-1 and ICAM-1 expression in endothelial cells infected with P

  16. The transcription factor SOX18 regulates the expression of matrix metalloproteinase 7 and guidance molecules in human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Martina Hoeth

    Full Text Available Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail.SOX18 was expressed in HUVEC using a recombinant adenoviral vector and the altered gene expression profile was analyzed using microarrays. Expression of several regulated candidate SOX18 target genes was verified by real-time PCR. Knock-down of SOX18 using RNA interference was then used to confirm the effect of the transcription factor on selected genes that included the guidance molecules ephrin B2 and semaphorin 3G. One gene, MMP7, was chosen for further analysis, including detailed promoter studies using reporter gene assays, electrophoretic mobility shift analysis and chromatin-immunoprecipitation, revealing that it responds directly to SOX18. Immunohistochemical analysis demonstrated the co-expression of SOX18 and MMP7 in blood vessels of human skin.The identification of MMP7 as a direct SOX18 target gene as well as other potential candidates including guidance molecules provides a molecular basis for the proposed function of this transcription factor in the regulation of vessel formation.

  17. Tanshinone II A Attenuates TNF-α-Induced Expression of VCAM-1 and ICAM-1 in Endothelial Progenitor Cells by Blocking Activation of NF-κB

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    Jin-Xiu Yang

    2016-11-01

    Full Text Available Background/Aims: Tanshinone IIA (Tan IIA is effective in the treatment of inflammation and atherosclerosis. The adhesion of inflammatory cells to vascular endothelium plays important role in atherogenic processes. This study examined the effects of Tan IIA on expression of adhesion molecules in tumor necrosis factor-α (TNF-α-induced endothelial progenitor cells (EPCs. Methods: EPCs were pretreated with Tan IIA and stimulated with TNF-α. Mononuclear cell (MNC adhesion assay was performed to assess the effects of Tan IIA on TNF-α-induced MNC adhesion. Expression of vascular cell adhesion molecule-1 (VCAM-1/intracellular adhesion molecule-1 (ICAM-1 and activation of Nuclear factor κB (NF-κB signaling pathway were measured. Results: The results showed that the adhesion of MNCs to TNF-α-induced EPCs and expression of VCAM-1/ICAM-1 in EPCs were promoted by TNF-α, which were reduced by Tan IIA. TNF-α increased the amount of phosphorylation of NF-κB, IκB-α and IKKα/β in cytosolic fractions and NF-κB p65 in nucleus, while Tan IIA reduced its amount. Conclusion: This study demonstrated a novel mechanism for the anti-inflammatory/anti-atherosclerotic activity of Tan IIA, which may involve down-regulation of VCAM-1 and ICAM-1 through partial blockage of TNF-α-induced NF-κB activation and IκB-α phosphorylation by the inhibition of IKKα/β pathway in EPCs.

  18. Expression and clinical significance of extracellular matrix protein 1 and vascular endothelial growth factor-C in lymphatic metastasis of human breast cancer

    International Nuclear Information System (INIS)

    Wu, Qiu-Wan; She, Hong-Qiang; Liang, Jing; Huang, Yu-Fan; Yang, Qing-Mo; Yang, Qiao-Lu; Zhang, Zhi-Ming

    2012-01-01

    Extracellular matrix protein 1 (ECM1) and vascular endothelial growth factor-C (VEGF-C) are secretory glycoproteins that are associated with lymphangiogenesis; these proteins could, therefore, play important roles in the lymphatic dissemination of tumors. However, very little is known about their potential roles in lymphangiogenesis. The aim of this study was to investigate whether correlations exist between ECM1 and VEGF-C in human breast cancer, lymphangiogenesis, and the clinicopathological characteristics of the disease. ECM1 and VEGF-C mRNA and protein expression levels in 41 patients were investigated using real-time reverse transcriptase polymerase chain reaction (RT-PCR), or immunohistochemical (IHC) staining of breast cancer tissue, matched noncancerous breast epithelial tissues, and suspicious metastatic axillary lymph nodes. D2-40 labelled lymph vessels and lymphatic microvessel density (LMVD) were counted. Correlations between ECM1 or VEGF-C protein expression levels, LMVD, and clinicopathological parameters were statistically tested. The rate of ECM1 positive staining in breast cancer tissues was higher (31/41, 75.6%) than that in the corresponding epithelial tissues (4/41, 9.8%, P < 0.001) and lymph nodes (13/41, 31.7%, P < 0.001). Similarly, the VEGF-C expression rate in cancer specimens was higher (33/41, 80.5%) than in epithelial tissues (19/41, 46.3%, P < 0.01) or lymph nodes (15/41, 36.6%, P < 0.01). Higher ECM1 and VEGF-C mRNA expression levels were also detected in the tumor tissues, compared to the non-cancerous tissue types or lymph nodes (P < 0.05). ECM1 protein expression was positively correlated with the estrogen receptor status (P < 0.05) and LMVD (P < 0.05). LMVD in the ECM1- and VEGF-C-positive tumor specimens was higher than that in the tissue types with negative staining (P < 0.05). Both ECM1 and VEGF-C were overexpressed in breast cancer tissue samples. ECM1 expression was positively correlated with estrogen responsiveness and the

  19. Receptor for advanced glycation end products - membrane type1 matrix metalloproteinase axis regulates tissue factor expression via RhoA and Rac1 activation in high-mobility group box-1 stimulated endothelial cells.

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    Koichi Sugimoto

    Full Text Available BACKGROUND: Atherosclerosis is understood to be a blood vessel inflammation. High-mobility group box-1 (HMGB-1 plays a key role in the systemic inflammation. Tissue factor (TF is known to lead to inflammation which promotes thrombus formation. Membrane type1 matrix metalloprotease (MT1-MMP associates with advanced glycation endproducts (AGE triggered-TF protein expression and phosphorylation of NF-κB. However, it is still unclear about the correlation of MT1-MMP and HMBG-1-mediated TF expression. In this study, we investigated the molecular mechanisms of TF expression in response to HMGB-1 stimulation and the involvement of MT1-MMP in endothelial cells. METHODS AND RESULTS: Pull-down assays and Western blotting revealed that HMGB-1 induced RhoA/Rac1 activation and NF-kB phosphorylation in cultured human aortic endothelial cells. HMGB-1 increased the activity of MT1-MMP, and inhibition of RAGE or MT1-MMP by siRNA suppressed HMGB-1-induced TF upregulation as well as HMGB-1-triggered RhoA/Rac1 activation and NF-kB phosphorylation. CONCLUSIONS: The present study showed that RAGE/MT1-MMP axis modified HMBG-1-mediated TF expression through RhoA and Rac1 activation and NF-κB phosphorylation in endothelial cells. These results suggested that MT1-MMP was involved in vascular inflammation and might be a good target for treating atherosclerosis.

  20. Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing.

    Science.gov (United States)

    Khaki, Mohsen; Salmanian, Ali Hatef; Mosayebi, Ghasem; Baazm, Maryam; Babaei, Saeed; Molaee, Neda; Abtahi, Hamid

    2017-07-01

    Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli ( E. coli ) system and then biological activity of this protein was evaluated in animal wound healing. E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced. Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Recombinant VEGF-A produced by pET32a in E. coli , possesses acceptable structure and has wound healing capability.

  1. Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

    Directory of Open Access Journals (Sweden)

    Mohsen Khaki

    2017-07-01

    Full Text Available Objective(s: Vascular endothelial growth factor (VEGF is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli system and then biological activity of this protein was evaluated in animal wound healing. Materials and Methods: E. coli BL21 (DE3 competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG. The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w was used for external wound (25×15mm thickness healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: The recombinant protein with molecular weight of 45 kilodaltons (kDa and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Conclusion: Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.

  2. Antibacterial activity of defensin PaDef from avocado fruit (Persea americana var. drymifolia) expressed in endothelial cells against Escherichia coli and Staphylococcus aureus.

    Science.gov (United States)

    Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Suárez-Rodríguez, Luis M; Salgado-Garciglia, Rafael; Rodríguez-Zapata, Luis C; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2013-01-01

    Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 μg/mL total protein (27-38%) but was more evident at 100 μg/mL (52-65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

  3. Antibacterial Activity of Defensin PaDef from Avocado Fruit (Persea americana var. drymifolia Expressed in Endothelial Cells against Escherichia coli and Staphylococcus aureus

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    Jaquelina Julia Guzmán-Rodríguez

    2013-01-01

    Full Text Available Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP represent attractive control agents. Mexican avocado (Persea americana var. drymifolia is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp encoding a protein (78 aa homologous with plant defensins (>80%. We expressed the defensin PaDef cDNA (pBME3 in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%. Also, S. aureus viability was inhibited from 50 μg/mL total protein (27–38% but was more evident at 100 μg/mL (52–65%. This inhibition was higher than the effect showed by polyclonal population (~23%. Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

  4. Low-energy extracorporeal shock wave therapy promotes vascular endothelial growth factor expression and improves locomotor recovery after spinal cord injury.

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    Yamaya, Seiji; Ozawa, Hiroshi; Kanno, Haruo; Kishimoto, Koshi N; Sekiguchi, Akira; Tateda, Satoshi; Yahata, Kenichiro; Ito, Kenta; Shimokawa, Hiroaki; Itoi, Eiji

    2014-12-01

    Extracorporeal shock wave therapy (ESWT) is widely used for the clinical treatment of various human diseases. Recent studies have demonstrated that low-energy ESWT upregulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis and functional recovery in myocardial infarction and peripheral artery disease. Many previous reports suggested that VEGF produces a neuroprotective effect to reduce secondary neural tissue damage after spinal cord injury (SCI). The purpose of the present study was to investigate whether low-energy ESWT promotes VEGF expression and neuroprotection and improves locomotor recovery after SCI. Sixty adult female Sprague-Dawley rats were randomly divided into 4 groups: sham group (laminectomy only), sham-SW group (low-energy ESWT applied after laminectomy), SCI group (SCI only), and SCI-SW group (low-energy ESWT applied after SCI). Thoracic spinal cord contusion injury was inflicted using an impactor. Low-energy ESWT was applied to the injured spinal cord 3 times a week for 3 weeks. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) Scale (open field locomotor score) at different time points over 42 days after SCI. Hematoxylin and eosin staining was performed to assess neural tissue damage in the spinal cord. Neuronal loss was investigated by immunostaining for NeuN. The mRNA expressions of VEGF and its receptor, Flt-1, in the spinal cord were assessed using real-time polymerase chain reaction. Immunostaining for VEGF was performed to evaluate VEGF protein expression in the spinal cord. In both the sham and sham-SW groups, no animals showed locomotor impairment on BBB scoring. Histological analysis of H & E and NeuN stainings in the sham-SW group confirmed that no neural tissue damage was induced by the low-energy ESWT. Importantly, animals in the SCI-SW group demonstrated significantly better locomotor improvement than those in the SCI group at 7, 35, and 42 days after injury (p

  5. The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

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    Ricarda Cortés-Vieyra

    Full Text Available Glycogen synthase kinase 3 (GSK3 is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN, one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65 at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor. Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

  6. Hyperglycemia Induces Toll-Like Receptor-2 and -4 Expression and Activity in Human Microvascular Retinal Endothelial Cells: Implications for Diabetic Retinopathy

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    Uthra Rajamani

    2014-01-01

    Full Text Available Diabetic retinopathy (DR causes visual impairment in working age adults and hyperglycemia-mediated inflammation is central in DR. Toll-like receptors (TLRs play a key role in innate immune responses and inflammation. However, scanty data is available on their role in DR. Hence, in this study, we examined TLR2 and TLR4 mRNA and protein expression and activity in hyperglycemic human retinal endothelial cells (HMVRECs. HMVRECs were treated with hyperglycemia (HG or euglycemia and mRNA and protein levels of TLR-2, TLR-4, MyD88, IRF3, and TRIF as well as NF-κB p65 activation were measured. IL-8, IL-1β, TNF-α and MCP-1, ICAM-1, and VCAM-1 as well as monocyte adhesion to HMVRECs were also assayed. HG (25 mM significantly induced TLR2 and TLR4 mRNA and protein in HMVRECs. It also increased both MyD88 and non-MyD88 pathways, nuclear factor-κB (NF-κB, biomediators, and monocyte adhesion. This inflammation was attenuated by TLR-4 or TLR-2 inhibition, and dual inhibition by a TLR inhibitory peptide as well as TLR2 and 4 siRNA. Additionally, antioxidant treatment reduced TLR-2 and TLR4 expression and downstream inflammatory markers. Collectively, our novel data suggest that hyperglycemia induces TLR-2 and TLR-4 activation and downstream signaling mediating increased inflammation possibly via reactive oxygen species (ROS and could contribute to DR.

  7. Hypoxia-inducible factor-1α, vascular endothelial growth factor, inducible nitric oxide synthase, and endothelin-1 expression correlates with angiogenesis in congenital heart disease

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    Hsin-Ling Yin

    2016-07-01

    Full Text Available In Taiwan, the average prevalence of congenital heart disease (CHD is 13.08/1000 live births. Most children with CHD die before the age of 5 years; therefore, identifying treatment methods to extend the life of CHD patients is an important issue in clinical practice. The objective of this study is to evaluate the roles of hypoxia-inducible factor-1α (HIF-1α, vascular endothelial growth factor (VEGF, inducible nitric oxide synthase (iNOS, endothelin-1 (ET-1, and CD34 in CHD autopsy cases in comparison with autopsy cases without CHD. The study included 19 autopsy cases, which were divided into the following four groups: acyanotic CHD (n = 11, cyanotic CHD (n = 3, CHD associated with chromosomal abnormalities (n = 3, and complex CHD (n = 2. Heart specimens obtained from 10 autopsy cases without CHD were included as controls. Our results indicated that high percentages of HIF-1α (100%, VEGF (89.5%, iNOS (78.9%, and ET-1 (84.2% expressions were observed in CHD autopsy cases and this was found to be significant. HIF-1α induced by hypoxia could play a potential role in relating downstream gene expressions in CHD patients. Upregulation of VEGF by HIF-1α could play an important role in triggering angiogenesis to protect myocardial cell survival in a hypoxic microenvironment. Therefore, HIF-1α could be a significant prognosis marker in CHD and be a prospective candidate in the development of target therapy in cardiovascular diseases.

  8. Increased expression of C-reactive protein gene in inflamed gingival tissues could be derived from endothelial cells stimulated with interleukin-6.

    Science.gov (United States)

    Maekawa, Tomoki; Tabeta, Koichi; Kajita-Okui, Keiko; Nakajima, Takako; Yamazaki, Kazuhisa

    2011-11-01

    Epidemiological studies have suggested periodontitis as a risk factor for ischemic heart disease. High sensitive C-reactive protein (hs-CRP), a predictor of cardiovascular risk, is elevated in periodontitis patients. Therefore, local infection-induced elevation of systemic CRP could account for the relationship between the 2 diseases. However, the underlying mechanism of CRP production in the periodontal tissues has not been fully elucidated. Therefore, the aim of the present study was to clarify the mechanism of CRP production in periodontal tissues. Gene expression of CRP in gingival biopsies was analysed by quantitative PCR. Human gingival epithelial cells (HGECs), human gingival fibroblasts (HGFBs), and human coronary artery endothelial cells (HCAECs) were characterized for CRP-producing ability by incubating with interleukin (IL)-1β, IL-6, soluble IL-6 receptor (sIL-6R), and Porphyromonas gingivalis strain W83. Gene expression of CRP is significantly elevated in periodontitis lesions compared with gingivitis lesions. HCAECs, but not HGECs and HGFBs, produced CRP in response to IL-6 and IL-1β in the presence of sIL-6R. In contrast to IL-6, the effect of IL-1β on CRP production was indirect via induction of IL-6. IL-1β was produced by HGECs and HGFBs with stimulation of P. gingivalis antigens. These results suggest that CRP induced locally by periodontal infection may play another role in the pathogenesis of periodontal disease, and to a much lesser extent, has the potential to modulate systemic CRP level by extra-hepatic CRP production. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Immunohistochemical expression of Insulin-like growth factor-1, Transforming growth factor-beta1, and Vascular endothelial growth factor in parathyroid adenoma and hyperplasia

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    Hamide Sayar

    2014-01-01

    Full Text Available Background: Insulin-like growth factor (IGF, transforming growth factor-beta1 (TGF-β1, and vascular endothelial growth factor (VEGF are commonly studied growth factors, but little data are available on the immunohistochemical expression of these factors in parathyroid lesions. Materials and Methods: Tissue specimens from 36 patients with primary hyperparathyroidism (P-HPT (26 adenomas and 10 primary hyperplasias were examined. Normal parathyroid tissue adjacent to the adenoma or area of hyperplasia was used as control tissue. Preoperative laboratory testing [serum Ca and P, creatinine and parathormone levels (PTH] which led to the diagnosis of P-HPT had been performed, the size and weight of the parathyroid glands measured, and postoperative serum PTH levels determined. Paraffin-embedded parathyroid tissue specimens were stained with antibodies to IGF-1, VEGF, and TGF-β1 using standard immunohistochemical procedures. Results: IGF-1 immunoreactivity was seen in 50% of hyperplasia and in 46% of adenoma samples, but in 87% of normal parathyroid tissue in the vicinity of the adenomas (P = 0.005. TGF-β1 immunoreactivity was observed in 90% of hyperplasia, in 92% of adenoma samples, and in 95% of normal tissues around adenomas. VEGF immunoreactivity was observed in 70% of hyperplastic and 65% of adenomatous tissues, as well as in 54% of normal tissues in the vicinity of the adenoma. No significant differences in the expression of IGF-1, TGF-β1, and VEGF were observed between primary adenomas compared to hyperplasia samples (P > 0.05. Conclusions: Parathyroid tissue is clearly a site for production of IGF-1, TGF-β1, and VEGF. IGF-1 receptor activity was higher in normal parathyroid tissue compared to hyperplastic and adenomatous tissue.

  10. Surface chemistry of gold nanoparticles determines the biocorona composition impacting cellular uptake, toxicity and gene expression profiles in human endothelial cells.

    Science.gov (United States)

    Chandran, Parwathy; Riviere, Jim E; Monteiro-Riviere, Nancy A

    2017-05-01

    This study investigated the role of nanoparticle size and surface chemistry on biocorona composition and its effect on uptake, toxicity and cellular responses in human umbilical vein endothelial cells (HUVEC), employing 40 and 80 nm gold nanoparticles (AuNP) with branched polyethyleneimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coatings. Proteomic analysis identified 59 hard corona proteins among the various AuNP, revealing largely surface chemistry-dependent signature adsorbomes exhibiting human serum albumin (HSA) abundance. Size distribution analysis revealed the relative instability and aggregation inducing potential of bare and corona-bound BPEI-AuNP, over LA- and PEG-AuNP. Circular dichroism analysis showed surface chemistry-dependent conformational changes of proteins binding to AuNP. Time-dependent uptake of bare, plasma corona (PC) and HSA corona-bound AuNP (HSA-AuNP) showed significant reduction in uptake with PC formation. Cell viability studies demonstrated dose-dependent toxicity of BPEI-AuNP. Transcriptional profiling studies revealed 126 genes, from 13 biological pathways, to be differentially regulated by 40 nm bare and PC-bound BPEI-AuNP (PC-BPEI-AuNP). Furthermore, PC formation relieved the toxicity of cationic BPEI-AuNP by modulating expression of genes involved in DNA damage and repair, heat shock response, mitochondrial energy metabolism, oxidative stress and antioxidant response, and ER stress and unfolded protein response cascades, which were aberrantly expressed in bare BPEI-AuNP-treated cells. NP surface chemistry is shown to play the dominant role over size in determining the biocorona composition, which in turn modulates cell uptake, and biological responses, consequently defining the potential safety and efficacy of nanoformulations.

  11. The anti-inflammatory effect of low-dose radiation therapy involves a diminished CCL20 chemokine expression and granulocyte/endothelial cell adhesion

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    Roedel, F. [Dept. of Radiotherapy and Oncology, Univ. of Frankfurt/Main (Germany); Hofmann, D.; Auer, J.; Roellinghoff, M.; Beuscher, H.U. [Inst. of Microbiology and Immunology, Univ. of Erlangen-Nuremberg, Erlangen (Germany); Keilholz, L. [Dept. of Radiotherapy, Clinical Center Bayreuth (Germany); Sauer, R. [Dept. of Radiooncology, Univ. of Erlangen-Nuremberg, Erlangen (Germany)

    2008-01-15

    Background and purpose: low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, however, the underlying molecular mechanisms are not fully understood. The manipulation of polymorphonuclear neutrophil (PMN) function and/or recruitment may be one mechanism. Chemokines contribute to this process by creating a chemotactic gradient and by activating integrins. This study aimed to characterize the effect of LD-RT on CCL20 chemokine production and PMN/endothelial cell (EC) adhesion. Material and methods: the EC line EA.hy.926 was irradiated with doses ranging from 0 to 3 Gy and was co-cultured with PMNs from healthy donors either by direct cell contact or separated by transwell membrane chambers. CXCL8, CCL18, CCL20 chemokine and tumor necrosis factor-(TNF-){alpha} cytokine levels in supernatants were determined by ELISA and adhesion assays were performed. The functional impact of the cytokines transforming growth factor-(TGF-){beta}{sub 1} and TNF-{alpha} and of the intercellular adhesion molecule-(ICAM-)1 on CCL20 expression was analyzed by using neutralizing antibodies. Results: as compared to CXCL8 and CCL18, CCL20 chemokine secretion was found to be exclusively induced by a direct cell-cell contact between PMNs and EA.hy.926 ECs in a TNF-{alpha}-dependent, but ICAM-1-independent manner. Furthermore, irradiation with doses between 0.5 and 1 Gy resulted in a significant reduction of CCL20 release which was dependent on TGF-{beta}{sub 1} (p < 0.01). The decrease of CCL20 paralleled with a significant reduction in PMN/EA.hy.926 EC adhesion (p < 0.001). Conclusion: the modulation of CCL20 chemokine expression and PMN/EC adhesion adds a further facet to the plethora of mechanisms contributing to the anti-inflammatory efficacy of LD-RT. (orig.)

  12. Effects of Chinese medicinal herbs on expression of brain-derived Neurotrophic factor (BDNF) and its interaction with human breast cancer MDA-MB-231 cells and endothelial HUVECs.

    Science.gov (United States)

    Chiu, Jen-Hwey; Chen, Fang-Pey; Tsai, Yi-Fang; Lin, Man-Ting; Tseng, Ling-Ming; Shyr, Yi-Ming

    2017-08-12

    Our previous study demonstrated that an up-regulation of the Brain-Derived Neurotrophic Factor (BDNF) signaling pathway is involved the mechanism causing the recurrence of triple negative breast cancer. The aim of this study is to investigate the effects of commonly used Chinese medicinal herbs on MDA-MB-231 and HUVEC cells and how they interact with BDNF. Human TNBC MDA-MB-231 cells and human endothelial HUVEC cells were used to explore the effect of commonly used Chinese herbal medicines on cancer cells alone, on endothelial cells alone and on cancer cell/endothelial cell interactions; this was done via functional studies, including migration and invasion assays. Furthermore, Western blot analysis and real-time PCR investigations were also used to investigate migration signal transduction, invasion signal transduction, and angiogenic signal transduction in these systems. Finally, the effect of the Chinese medicinal herbs on cancer cell/endothelial cell interactions was assessed using co-culture and ELISA. In terms of autoregulation, BDNF up-regulated TrkB gene expression in both MDA-MB-231 and HUVEC cells. Furthermore, BDNF enhanced migration by MDA-MB-231 cells via Rac, Cdc42 and MMP, while also increasing the migration of HUVEC cells via MMP and COX-2 expression. As measured by ELISA, the Chinese herbal medicinal herbs A. membranaceus, P. lactiflora, L. chuanxiong, P. suffruticosa and L. lucidum increased BDNF secretion by MDA-MB-231 cells. Similarly, using a co-culture system with MDA-MB-231 cells, A. membranaceus and L. lucidum modulated BDNF-TrkB signaling by HUVEC cells. We conclude that BDNF plays an important role in the metastatic interaction between MDA-MB-231 and HUVEC cells. Some Chinese medicinal herbs are able to enhance the BDNF-related metastatic potential of the interaction between cancer cells and endothelial cells. These findings provide important information that should help with the development of integrated medical therapies for breast

  13. Expression of Toll-Like Receptor 2 in Glomerular Endothelial Cells and Promotion of Diabetic Nephropathy by Porphyromonas gingivalis Lipopolysaccharide

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    Hatakeyama, Yuji; Ishikawa, Hiroyuki; Tsuruga, Eichi

    2014-01-01

    The toll-like receptor (TLR) has been suggested as a candidate cause for diabetic nephropathy. Recently, we have reported the TLR4 expression in diabetic mouse glomerular endothelium. The study here investigates the effects of the periodontal pathogen Porphyromonas gingivalis lipopolysaccharide (LPS) which is a ligand for TLR2 and TLR4 in diabetic nephropathy. In laser-scanning microscopy of glomeruli of streptozotocin- and a high fat diet feed-induced type I and type II diabetic mice, TLR2 localized on the glomerular endothelium and proximal tubule epithelium. The TLR2 mRNA was detected in diabetic mouse glomeruli by in situ hybridization and in real-time PCR of the renal cortex, the TLR2 mRNA amounts were larger in diabetic mice than in non-diabetic mice. All diabetic mice subjected to repeated LPS administrations died within the survival period of all of the diabetic mice not administered LPS and of all of the non-diabetic LPS-administered mice. The LPS administration promoted the production of urinary protein, the accumulation of type I collagen in the glomeruli, and the increases in IL-6, TNF-α, and TGF-β in the renal cortex of the glomeruli of the diabetic mice. It is thought that blood TLR ligands like Porphyromonas gingivalis LPS induce the glomerular endothelium to produce cytokines which aid glomerulosclerosis. Periodontitis may promote diabetic nephropathy. PMID:24835775

  14. Anti-vascular endothelial growth factor therapy-induced glioma invasion is associated with accumulation of Tie2-expressing monocytes

    Science.gov (United States)

    Hossain, Mohammad B.; Conrad, Charles A.; Aldape, Kenneth D.; Fuller, Gregory N.; Marini, Frank C.; Alonso, Marta M.; Idoate, Miguel Angel; Gilbert, Mark R.; Fueyo, Juan; Gomez-Manzano, Candelaria

    2014-01-01

    The addition of anti-angiogenic therapy to the few treatments available to patients with malignant gliomas was based on the fact that these tumors are highly vascularized and on encouraging results from preclinical and clinical studies. However, tumors that initially respond to this therapy invariably recur with the acquisition of a highly aggressive and invasive phenotype. Although several myeloid populations have been associated to this pattern of recurrence, a specific targetable population has not been yet identified. Here, we present evidence for the accumulation of Tie2-expressing monocytes/macrophages (TEMs) at the tumor/normal brain interface of mice treated with anti-VEGF therapies in regions with heightened tumoral invasion. Furthermore, we describe the presence of TEMs in malignant glioma surgical specimens that recurred after bevacizumab treatment. Our studies showed that TEMs enhanced the invasive properties of glioma cells and secreted high levels of gelatinase enzymatic proteins. Accordingly, Tie2+MMP9+ monocytic cells were consistently detected in the invasive tumor edge upon anti-VEGF therapies. Our results suggest the presence of a specific myeloid/monocytic subpopulation that plays a pivotal role in the mechanism of escape of malignant gliomas from anti-VEGF therapies and therefore constitutes a new cellular target for combination therapies in patients selected for anti-angiogenesis treatment. PMID:24809734

  15. Expression of p53, inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions: correlation with clinical features

    International Nuclear Information System (INIS)

    Feng, Chang Wei; Wang, Li Dong; Jiao, Lian Hua; Liu, Bin; Zheng, Shu; Xie, Xin Ji

    2002-01-01

    The growth and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis. Recent studies indicate that the inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF) and the tumor suppressor p53 are fundamental play-markers of the angiogenic process. Overexpression of iNOS and VEGF has been shown to induce angiogenesis in tumors. P53 suppresses angiogenesis by down-regulating VEGF and iNOS. The correlation of expression of p53, VEGF and iNOS and clinical features in gastric carcinogenesis, however, has not been well characterized. The expression of p53, iNOS and VEGF in gastric precancerous and cancerous lesions and its relation with the clinical features was determined with immunohistochemistry (avidin-biotin-peroxidase complex method) on 55 randomly selected GC patients and 60 symptom-free subjects from the mass survey in the high-incidence area for GC in Henan, northern China. The positive immunostainig rates for p53, iNOS and VEGF in gastric carcinomas were 51%, 44% and 51%, respectively, and correlated well with TNM stages, but did not show significant difference among the groups with different degrees of gastric wall invasion depth by GC. A positive immunostaining reaction for the iNOS protein was significantly correlated with lymph node metastasis (p = 0.019; Spearman correlation coefficient). P53 protein accumulation was higher in the poorly-differentiated gastric carcinoma than in well-differentiated one. In gastric biopsies, no positive immunosatining was observed for p53, iNOS and VEGF in the histologically normal tissue and chronic superficial gastritis (CSG). However, p53, iNOS and VEGF positive immunostaining was observed in the tissues with different severities of lesions of chronic atrophic gastritis (CAG), intestinal metaplasia (IM) and dysplasia (DYS), and the positive rates increased with the lesion progression from CAG to IM to DYS. A high coincidental positive and negative immunostaining

  16. The influence of propofol on the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in reoxygenated human umbilical vein endothelial cells.

    LENUS (Irish Health Repository)

    Corcoran, T B

    2012-02-03

    BACKGROUND: Leucocytes are a pivotal component of the inflammatory cascade that results in tissue injury in a large group of disorders. Free radical production and endothelial activation promote leucocyte-endothelium interactions via endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) which augment these processes, particularly in the setting of reperfusion injury. Propofol has antioxidant properties which may attenuate the increased expression of these molecules that is observed. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia, then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg mL(-1) or propofol 5 microg mL(-1), for 4 h after reoxygenation and were examined for ICAM-1 and VCAM-1 expression. RESULTS: Hypoxia did not increase the expression of ICAM-1\\/VCAM-1. ICAM-1 expression peaked 12 h after reoxygenation (21.75(0.6) vs. 9.6(1.3), P = 0.02). Propofol, but not Diprivan, prevented this increase (8.2(2.9) vs. 21.75(0.6), P = 0.009). VCAM-1 expression peaked 24 h after reoxygenation (9.8(0.9) vs. 6.6(0.6), P = 0.03). Propofol and Diprivan prevented this increase, with no difference between the two treatments observed (4.3(0.3) and 6.4(0.5) vs. 9.8(0.9), P = 0.001, 0.02, respectively). CONCLUSION: These effects are likely to be attributable to the antioxidant properties of propofol, and suggest that propofol may have a protective role in disorders where free radical mediated injury promotes leucocyte-endothelium adhesive interactions.

  17. Correlation of emmprin expression in vascular endothelial cells with blood-brain-barrier function: a study using magnetic resonance imaging enhanced by Gd-DTPA and immunohistochemistry in brain tumors.

    Science.gov (United States)

    Sameshima, Tetsuro; Nabeshima, Kazuki; Toole, Bryan P; Inoue, Teruhiko; Yokogami, Kiyotaka; Nakano, Shinichi; Ohi, Takekazu; Wakisaka, Shinichiro

    2003-06-01

    In a previous study, we demonstrated that the expression levels in tumor cells of emmprin (CD147) correlated with the grade of astrocytic tumors. Also, we found that emmprin was expressed in vascular endothelial cells of the non-neoplastic brain and hypothesized that emmprin expression could be associated with normal blood-brain-barrier (BBB) function of vascular endothelial cells. In this study, this possibility was examined in non-neoplastic brain, glioma and metastatic carcinoma tissues by comparing emmprin immunohistochemistry with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) enhancement of magnetic resonance imaging (MRI), which is a clinical indicator of the BBB function. This study included 10 cases of non-neoplastic brain tissues, 7 of metastatic carcinoma, 7 of diffuse astrocytoma, 4 of anaplastic astrocytoma and 13 of glioblastoma multiforme. In all the cases, MRI with administration of Gd-DTPA was performed. The lesions were resected using the microdissection method with the help of ultrasonography and a neuronavigator. The tissues from Gd-DTPA-enhanced or non-enhanced areas were processed into frozen sections and subjected to immunohistochemistry with anti-emmprin antibody. The expression of emmprin in brain vascular endothelial cells inversely correlated with Gd-DTPA-enhancement of MRI: emmprin was positive in tissues not enhanced by Gd-DTPA and was negative in DTPA-enhanced tissues. Since BBB function presumably remains unimpaired in regions in which MR images are not Gd-DTPA-enhanced, emmprin expression appears to be associated with unimpaired BBB function. This is the first report to demonstrate a possible correlation between emmprin expression and BBB function in humans.

  18. p38 MAPK and JNK antagonistically control senescence and cytoplasmic p16INK4A expression in doxorubicin-treated endothelial progenitor cells.

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    Paolo Spallarossa

    Full Text Available Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16( INK4A localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16(INK4A, suggests that doxorubicin-induced p16( INK4A does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16( INK4A expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

  19. Decrease of PECAM-1-gene-expression induced by proinflammatory cytokines IFN-γ and IFN-α is reversed by TGF-β in sinusoidal endothelial cells and hepatic mononuclear phagocytes

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    Ramadori Giuliano

    2008-05-01

    Full Text Available Abstract Background and aim The mechanisms of transmigration of inflammatory cells through the sinusoids are still poorly understood. This study aims to identify in vitro conditions (cytokine treatment which may allow a better understanding of the changes in PECAM (platelet endothelial cell adhesion molecule-1-gene-expression observed in vivo. Methods and results In this study we show by immunohistochemistry, that there is an accumulation of ICAM-1 (intercellular cell adhesion molecule-1 and ED1 positive cells in necrotic areas of livers of CCl4-treated rats, whereas there are few PECAM-1 positive cells observable. After the administration of CCl4, we could detect an early rise of levels of IFN-γ followed by an enhanced TGF-β protein level. As shown by Northern blot analysis and surface protein expression analysed by flow cytometry, IFN-γ-treatment decreased PECAM-1-gene-expression in isolated SECs (sinusoidal endothelial cells and mononuclear phagocytes (MNPs in parallel with an increase in ICAM-1-gene-expression in a dose and time dependent manner. In contrast, TGF-β-treatment increased PECAM-1-expression. Additional administration of IFN-γ to CCl4-treated rats and observations in IFN-γ-/- mice confirmed the effect of IFN-γ on PECAM-1 and ICAM-1-expression observed in vitro and increased the number of ED1-expressing cells 12 h after administration of the toxin. Conclusion The early decrease of PECAM-1-expression and the parallel increase of ICAM-1-expression following CCl4-treatment is induced by elevated levels of IFN-γ in livers and may facilitate adhesion and transmigration of inflammatory cells. The up-regulation of PECAM-1-expression in SECs and MNPs after TGF-β-treatment suggests the involvement of PECAM-1 during the recovery after liver damage.

  20. The endothelial αENaC contributes to vascular endothelial function in vivo

    DEFF Research Database (Denmark)

    Tarjus, Antoine; Maase, Martina; Jeggle, Pia

    2017-01-01

    The Epithelial Sodium Channel (ENaC) is a key player in renal sodium homeostasis. The expression of α β γ ENaC subunits has also been described in the endothelium and vascular smooth muscle, suggesting a role in vascular function. We recently demonstrated that endothelial ENaC is involved in aldo......-mediated dilation. Our data suggest that endothelial αENaC contributes to vascular endothelial function in vivo....

  1. Hepatopulmonary syndrome induced by common bile duct ligation in a rabbit model: correlation between pulmonary vascular dilatation on thin-section CT and angiography and serum nitrite concentration or endothelial nitric oxide synthase (eNOS)1 expression

    International Nuclear Information System (INIS)

    Lee, Ki Nam; Yoon, Seong Kuk; Lee, Jin Wha; Kim, Ki Nam; Park, Byung Ho; Kwak, Jong Young; Jeong, Jin Sook; Kim, Young Hoon

    2004-01-01

    To investigate the correlation between radiologic vascular dilatation and serum nitrite concentration and eNOS expression in the endothelial cell and pneumocyte in a rabbit model of hepatopulmonary syndrome induced by common bile duct ligation (CBDL). Thin-section CT scans of the lung and pulmonary angiography were obtained 3 weeks after CBDL (n=6), or a sham operation (n=4), and intrapulmonary vasodilatation was assessed. The diameter and tortuosity of peripheral vessels in the right lower lobe by thin-section CT and angiography at the same level of the right lower lobe in all subjects were correlated to serum nitrite concentration and eNOS (endothelial nitric oxide synthase) expression as determined by immunostaining. The diameters of pulmonary vessels on thin-section CT were well correlated with nitrite concentrations in serum (r=0.92, ρ < 0.001). Dilated pulmonary vessels were significantly correlated with an increased eNOS expression (r=0.94, ρ < 0.0001), and the severity of pulmonary vessel tortuosity was found to be well correlated with serum nitrite concentration (r=0.90, ρ < 0.001). The peripheral pulmonary vasculature in hepatopulmonary syndrome induced by CBLD was dilated on thin-section CT and on angiographs. Our findings suggest that peripheral pulmonary vascular dilatations are correlated with serum nitrite concentrations and pulmonary eNOS expression

  2. Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

    Science.gov (United States)

    Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

    2015-01-01

    ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

  3. Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease

    NARCIS (Netherlands)

    Kranenburg, A. R.; de Boer, W. I.; Alagappan, V. K. T.; Sterk, P. J.; Sharma, H. S.

    2005-01-01

    BACKGROUND: Ongoing inflammatory processes resulting in airway and vascular remodelling characterise chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) could play a role in tissue remodelling and

  4. Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease

    NARCIS (Netherlands)

    A.R. Kranenburg (Andor); W.I. de Boer (Pim); V.K.T. Alagappan (Vijay Kumar Thyagarajan); P.J. Sterk (Peter); H.S. Sharma (Hari)

    2005-01-01

    textabstractBACKGROUND: Ongoing inflammatory processes resulting in airway and vascular remodelling characterise chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) could play a role in

  5. Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

    NARCIS (Netherlands)

    Engelse, M.A.; Laurens, N.; Verloop, R.E.; Koolwijk, P.; Hinsbergh, V.W.M. van

    2008-01-01

    The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA

  6. Expression of adhesion molecules, monocyte interactions and oxidative stress in human endothelial cells exposed to wood smoke and diesel exhaust particulate matter

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Loft, Steffen; Roursgaard, Martin

    2012-01-01

    Toxicological effects of wood smoke particles are less investigated than traffic-related combustion particles. We investigated the effect of wood smoke particles, generated by smouldering combustion conditions, on human umbilical endothelial cells (HUVECs) co-cultured with or without monocytic TH...

  7. Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: Implications for capillary-like tube formation in a fibrin matrix

    NARCIS (Netherlands)

    Kroon, M.E.; Koolwijk, P.; Vecht, B. van der; Hinsbergh, V.W.M. van

    2000-01-01

    Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth

  8. Abscisic acid ameliorates experimental IBD by downregulating cellular adhesion molecule expression and suppressing immune cell infiltration.

    Science.gov (United States)

    Guri, Amir J; Hontecillas, Raquel; Bassaganya-Riera, Josep

    2010-12-01

    Abscisic acid (ABA) has shown effectiveness in ameliorating inflammation in obesity, diabetes and cardiovascular disease models. The objective of this study was to determine whether ABA prevents or ameliorates experimental inflammatory bowel disease (IBD). C57BL/6J mice were fed diets with or without ABA (100mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily. Colonic mucosal lesions were evaluated by histopathology, and cellular adhesion molecular and inflammatory markers were assayed by real-time quantitative PCR. Flow cytometry was used to quantify leukocyte populations in the blood, spleen, and mesenteric lymph nodes (MLN). The effect of ABA on cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression in splenocytes was also investigated. ABA significantly ameliorated disease activity, colitis and reduced colonic leukocyte infiltration and inflammation. These improvements were associated with downregulation in vascular cell adhesion marker-1 (VCAM-1), E-selectin, and mucosal addressin adhesion marker-1 (MAdCAM-1) expression. ABA also increased CD4(+) and CD8(+) T-lymphocytes in blood and MLN and regulatory T cells in blood. In vitro, ABA increased CTLA-4 expression through a PPAR γ-dependent mechanism. We conclude that ABA ameliorates gut inflammation by modulating T cell distribution and adhesion molecule expression. Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Duban-Deweer Sophie

    2010-11-01

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

  10. Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

    DEFF Research Database (Denmark)

    Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

    2018-01-01

    PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

  11. Polyphenols in preventing endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Sylwia Biegańska-Hensoldt

    2017-03-01

    Full Text Available One of the main causes of mortality in developed countries is atherosclerosis. The pathogenesis of atherosclerosis is associated with endothelial dysfunction. Consumption of food rich in natural antioxidants including polyphenols significantly improves endothelial cells functions.Polyphenols have a beneficial effect on the human body and play an important part in protecting the cardiovascular system. Polyphenols present in food have antioxidant, anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. Catechins cause an increase in the activity of endothelial nitric oxide synthase (eNOS and increased production of nitric oxide (NO and decrease in blood pressure. Catechins also reduce platelet adhesion, lower the concentration of C-reactive protein and tumor necrosis factor alpha and interleukin-6. Resveratrol inhibits NADPH oxidase expression, increases the expression of eNOS and NO production as well as decreases the expression of proinflammatory cytokines, and also lowers the concentration of the soluble forms of adhesion molecules – sICAM-1 and sVCAM-1 in blood. Quercetin reduces the blood level of low density lipoprotein cholesterol, lowers blood pressure, reduces the concentration of C-reactive protein and F2-isoprostane level. Curcumin has antagonistic activity to homocysteine. Curcumin increases the expression of eNOS and reduces oxidative DNA damage in rat cardiomyocytes. Numerous attempts are taken for improving the bioavailability of polyphenols in order to increase their use in the body.

  12. Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

    Science.gov (United States)

    Schäfer, Nicola; Lohmann, Christine; Winnik, Stephan; van Tits, Lambertus J; Miranda, Melroy X; Vergopoulos, Athanasios; Ruschitzka, Frank; Nussberger, Jürg; Berger, Stefan; Lüscher, Thomas F; Verrey, François; Matter, Christian M

    2013-12-01

    Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

  13. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pourgholami, Mohammad H., E-mail: mh.pourgholami@unsw.edu.au [University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney (Australia); Khachigian, Levon M.; Fahmy, Roger G. [Centre for Vascular Research, The University of New South Wales, Department of Haematology, The Prince of Wales Hospital, Sydney (Australia); Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson [University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney (Australia)

    2010-07-09

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  14. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    International Nuclear Information System (INIS)

    Pourgholami, Mohammad H.; Khachigian, Levon M.; Fahmy, Roger G.; Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson

    2010-01-01

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  15. Cinnamaldehyde inhibits the tumor necrosis factor-α-induced expression of cell adhesion molecules in endothelial cells by suppressing NF-κB activation: Effects upon IκB and Nrf2

    International Nuclear Information System (INIS)

    Liao, B.-C.; Hsieh, C.-W.; Liu, Y.-C.; Tzeng, T.-T.; Sun, Y.-W.; Wung, B.-S.

    2008-01-01

    The production of adhesion molecules and subsequent attachment of leukocytes to endothelial cells (ECs) are critical early events in atherogenesis. These adhesion molecules thus play an important role in the development of this disease. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of cinnamaldehyde, a Cinnamomum cassia Presl-specific diterpene. In our current study, we have examined the effects of both cinnamaldehyde and extracts of C. cassia on cytokine-induced monocyte/human endothelial cell interactions. We find that these compounds inhibit the adhesion of TNFα-induced monocytes to endothelial cells and suppress the expression of the cell adhesion molecules, VCAM-1 and ICAM-1, at the transcriptional level. Moreover, in TNFα-treated ECs, the principal downstream signal of VCAM-1 and ICAM-1, NF-κB, was also found to be abolished in a time-dependent manner. Interestingly, cinnamaldehyde exerts its anti-inflammatory effects by blocking the degradation of the inhibitory protein IκB-α, but only in short term pretreatments, whereas it does so via the induction of Nrf2-related genes, including heme-oxygenase-1 (HO-1), over long term pretreatments. Treating ECs with zinc protoporphyrin, a HO-1 inhibitor, partially blocks the anti-inflammatory effects of cinnamaldehyde. Elevated HO-1 protein levels were associated with the inhibition of TNFα-induced ICAM-1 expression. In addition to HO-1, we also found that cinnamaldehyde can upregulate Nrf2 in nuclear extracts, and can increase ARE-luciferase activity and upregulate thioredoxin reductase-1, another Nrf2-related gene. Moreover, cinnamaldehyde exposure rapidly reduces the cellular GSH levels in ECs over short term treatments but increases these levels after 9 h exposure. Hence, our present findings indicate that cinnamaldehyde suppresses TNF-induced singling pathways via two distinct mechanisms that are activated by different pretreatment periods

  16. Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.

    Science.gov (United States)

    Meisen, Iris; Rosenbrück, Regina; Galla, Hans-Joachim; Hüwel, Sabine; Kouzel, Ivan U; Mormann, Michael; Karch, Helge; Müthing, Johannes

    2013-06-01

    Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

  17. Inhibition of hypoxia inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis.

    Science.gov (United States)

    Terzuoli, Erika; Donnini, Sandra; Giachetti, Antonio; Iñiguez, Miguel A; Fresno, Manuel; Melillo, Giovanni; Ziche, Marina

    2010-08-15

    2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE-2). DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 mumol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1beta-mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1alpha. Moreover, DPE blocked mPGEs-1-dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1alpha, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal-related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1alpha expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1alpha translation. We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1alpha/mPGEs-1/VEGF axis.

  18. Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

    DEFF Research Database (Denmark)

    Wang, X; Häring, M-F; Rathjen, Thomas

    2017-01-01

    in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...... adhesion in quiescent tumour endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumour necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules...

  19. Andrographolide inhibits hypoxia-induced HIF-1α-driven endothelin 1 secretion by activating Nrf2/HO-1 and promoting the expression of prolyl hydroxylases 2/3 in human endothelial cells.

    Science.gov (United States)

    Lin, Hung-Chih; Su, Shih-Li; Lu, Chia-Yang; Lin, Ai-Hsuan; Lin, Wan-Chun; Liu, Chin-San; Yang, Ya-Chen; Wang, Hsiu-Miao; Lii, Chong-Kuei; Chen, Haw-Wen

    2017-03-01

    Andrographolide, the main bioactive component of the medicinal plant Andrographis paniculata, has been shown to possess potent anti-inflammatory activity. Endothelin 1 (ET-1), a potent vasoconstrictor peptide produced by vascular endothelial cells, displays proinflammatory property. Hypoxia-inducible factor 1α (HIF-1α), the regulatory member of the transcription factor heterodimer HIF-1α/β, is one of the most important molecules that responds to hypoxia. Changes in cellular HIF-1α protein level are the result of altered gene transcription and protein stability, with the latter being dependent on prolyl hydroxylases (PHDs). In this study, inhibition of pro-inflammatory ET-1 expression and changes of HIF-1α gene transcription and protein stability under hypoxia by andrographolide in EA.hy926 endothelial-like cells were investigated. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2. We found that hypoxia stimulated the production of reactive oxygen species (ROS), the expression of HIF-1α mRNA and protein, and the expression and secretion of ET-1. These effects, however, were attenuated by co-exposure to andrographolide, bilirubin, and RuCO. Silencing Nrf2 and heme oxygenase 1 (HO-1) reversed the inhibitory effects of andrographolide on hypxoia-induced HIF-1α mRNA and protein expression. Moreover, andrographolide increased the expression of prolyl hydroxylases (PHD) 2/3, which hydroxylate HIF-1α and promotes HIF-1α proteasome degradation, with an increase in HIF-1α hydroxylation was noted under hypoxia. Inhibition of p38 MAPK abrogated the hypoxia-induced increases in HIF-1α mRNA and protein expression as well as ET-1 mRNA expression and secretion. Taken together, these results suggest that andrographolide suppresses hypoxia-induced pro-inflammatory ET-1 expression by activating Nrf2/HO-1, inhibiting p38 MAPK signaling, and promoting PHD2/3 expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 918-930, 2017. © 2016 Wiley

  20. Prognostic values of tumor endothelial markers in patients with colorectal cancer

    OpenAIRE

    Rmali, KA; Puntis, MCA; Jiang, WG

    2005-01-01

    AIM: Tumor endothelial markers (TEMs) are a newly discovered family of endothelial markers associated with tumor specific angiogenesis. This study sought to examine the levels of expression (qualitatively and quantitatively) for TEMs in human colon cancer.

  1. Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.

    Science.gov (United States)

    Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C

    1997-01-01

    Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  3. The bio-complex "reaction pattern in vertebrate cells" reduces cytokine-induced cellular adhesion molecule mRNA expression in human endothelial cells by attenuation of NF-kappaB translocation.

    Science.gov (United States)

    Rönnau, Cindy; Liebermann, Herbert E H; Helbig, Franz; Staudt, Alexander; Felix, Stephan B; Ewert, Ralf; Landsberger, Martin

    2009-02-28

    The bio-complex "reaction pattern in vertebrate cells" (RiV) is mainly represented by characteristic exosome-like particles--probably as reaction products of cells to specific stress. The transcription factor NF-kappaB plays a central role in inflammation. We tested the hypothesis that RiV particle preparations (RiV-PP) reduce cellular adhesion molecule (CAM) expression (ICAM-1, VCAM-1, E-selectin) by the attenuation of NF-kappaB translocation in human umbilical vein endothelial cells (HUVEC). After 4 hours, pre-incubation of HUVEC with RiV-PP before stimulation with TNF-alpha significantly reduced ICAM-1 (65.5+/-10.3%) and VCAM-1 (71.1+/-12.3%) mRNA expression compared to TNF-alpha-treated cells (100%, n=7). ICAM-1 surface expression was significantly albeit marginally reduced in RiV/TNF-alpha- treated cells (92.0+/-5.6%, n=4). No significant effect was observed on VCAM-1 surface expression. In RiV/TNF-alpha-treated cells (n=4), NF-kappaB subunits p50 (85.7+/-4.1%) and p65 (85.0+/-1.8%) nuclear translocation was significantly reduced. RiV-PP may exert an anti-inflammatory effect in HUVEC by reducing CAM mRNA expression via attenuation of p50 and p65 translocation.

  4. Potential of pomegranate fruit extract (Punica granatum Linn.) to increase vascular endothelial growth factor and platelet-derived growth factor expressions on the post-tooth extraction wound of Cavia cobaya.

    Science.gov (United States)

    Nirwana, Intan; Rachmadi, Priyawan; Rianti, Devi

    2017-08-01

    Pomegranates fruit extracts have several activities, among others, anti-inflammatory, antibacterial, and antioxidants that have the main content punicalagin and ellagic acid. Pomegranate has the ability of various therapies through different mechanisms. Vascular endothelial growth factor (VEGF) function was to form new blood vessels produced by various cells one of them was macrophages. Platelet-derived growth factor (PDGF) was a growth factor proven chemotactic, increased fibroblast proliferation and collagen matrix production. In addition, VEGF and PDGF synergize in their ability to vascularize tissues. The PDGF function was to stabilize and regulate maturation of new blood vessels. Activities of pomegranate fruit extract were observed by measuring the increased of VEGF and PDGF expression as a marker of wound healing process. To investigate the potential of pomegranate extracts on the tooth extraction wound to increase the expression of VEGF and PDGF on the 4 th day of wound healing process. This study used 12 Cavia cobaya , which were divided into two groups, namely, the provision of 3% sodium carboxymethyl cellulose and pomegranate extract. The 12 C. cobaya would be executed on the 4 th day, the lower jaw of experimental animals was taken, decalcified about 30 days. The expression of VEGF and PDGF was examined using immunohistochemical techniques. The differences of VEGF and PDGF expression were evaluated statistically using t-test. Statistically analysis showed that there were significant differences between control and treatment groups (p<0.05). Pomegranate fruit extract administration increased VEGF and PDGF expression on post-tooth extraction wound.

  5. Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

    Science.gov (United States)

    Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

    2017-11-01

    Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The Gene Expression of Human Endothelial Cells Is Modulated by Subendothelial Extracellular Matrix Proteins: Short-Term Response to Laminar Shear Stress

    Czech Academy of Sciences Publication Activity Database

    Chlupáč, Jaroslav; Filová, Elena; Havlíková, Jana; Matějka, R.; Riedel, Tomáš; Houska, Milan; Brynda, Eduard; Pamula, E.; Rémy, M.; Bareille, R.; Fernandez, P.; Daculsi, R.; Bourget, Ch.; Bačáková, Lucie; Bordenave, L.

    2014-01-01

    Roč. 20, 15-16 (2014), s. 2253-2264 ISSN 2152-4947 R&D Projects: GA ČR(CZ) GAP108/10/1106; GA ČR(CZ) GAP108/11/1857; GA ČR(CZ) GA305/08/0108; GA ČR GAP205/12/1702; GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109; GA MZd(CZ) NT11270 Grant - others:GA MŠk(CZ) Barrande 2005-06-036-1 Institutional support: RVO:67985823 ; RVO:61389013 Keywords : atherosclerosis * endothelial cells * extracellular matrix proteins * tissue engineering Subject RIV: EI - Biotechnology ; Bionics; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 4.448, year: 2014

  7. Analysis of expression of von Willebrand factor, endothelin-1, vascular endothelial growth factor in Budd-Chiari syndrome with membranous obstruction

    International Nuclear Information System (INIS)

    Han Xinqiang; Zu Maoheng

    2011-01-01

    Objective: To investigate whether the injured vascular endothelial plays an important role in the membranous formation of the inferior vena cava (IVC). Methods: There were 40 cases of membranous obstruction of inferior vena cava (MOVC) in the experimental group and 40 arrhythmic inpatients in the control group from affiliated hospital. There were 23 males and 17 females in experimental group while 21 males and 19 females in control group, and the age were (41.8±8.1) yrs and (43.2± 7.6) yrs respectively. All of them had no anti-coagulation (clotting) drug history and smoking history, no hypertension, no pulmonary artery hypertension, no coronary heart disease, no valvular disease, no myocardial disease, no blood disease, no diabetes, no connective tissue disease and malignancy, and liver and renal function must be normal. And then the serum concentrations of von Willebrand factor (vWF), endothelin-l (ET-1), vascular endothelial growth factor (VEGF) were defined by enzyme-linked immunosorbent assay (ELISA). The results were analyzed with independent sample t-test. Results: In MOVC patients, the serum concentrations of vWF, ET-1 and VEGF [(37.8±6.6) μg/L, (31.9± 6.0) ng/L, (20.84±5.78) μg/L] were higher than those in the control group [(3.3±1.3) μg/L, (5.3±1.8) ng/L, (4.2±1.2) μg/L. t=32.65, 26.70, 17.85, P<0.01, respectively]. Conclusions: The injury of vascular endothelium is related to the formation of membrane in the IVC. (authors)

  8. Functional expression of a proton-coupled organic cation (H+/OC antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model

    Directory of Open Access Journals (Sweden)

    Shimomura Keita

    2013-01-01

    Full Text Available Abstract Background Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. Methods Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions. Results Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters. The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. Conclusions The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.

  9. Expression of angiopoietin-2 and vascular endothelial growth factor receptor-3 correlates with lymphangiogenesis and angiogenesis and affects survival of oral squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Chao Li

    Full Text Available BACKGROUND: Both Ang-2 and VEGFR-3 are major regulators of angiogenesis and lymphangiogenesis, respectively, and thus may affect prognosis of OSCC. We sought to determine the associations between Ang-2 and VEGFR-3 expression and survival of OSCC. METHODS: Ang-2 and VEGFR-3 expression was determined immunohistochemically in tumor tissues from 112 patients with OSCC; OSCC-adjacent noncancerous oral tissue from 85 OSCC patients; and normal oral mucosa from 37 cancer-free individuals. A log-rank test and Cox proportional hazard models were used to compare survival among different groups with expression of Ang-2 and VEGFR-3. RESULTS: Ang-2 and VEGFR-3 expression was upregulated in OSCC compared to nontumor tissue (all P<0.05. High Ang-2 expression positively correlated with microvessel density (MVD (P<0.01, and high VEGFR-3 expression positively correlated with lymph node metastasis (P<0.01 and lymphatic vessel density (LVD (P<0.01. The patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly worse survival than in patients with low expression of Ang-2 or any other co-expression status (all P<0.05, respectively. Furthermore, multivariable analysis showed that patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly increased risk of death compared with those with low expression of Ang-2 or any other co-expression status (HR, 2.7, 95% CI, 1.1-6.2 and 5.0, 1.3-15.4, respectively. CONCLUSIONS: These results suggest that increased expression in tumors of Ang-2 may individually, or in combination with VEGFR-3, predict poor prognosis of OSCC.

  10. Endothelial remodelling and intracellular calcium machinery.

    Science.gov (United States)

    Moccia, F; Tanzi, F; Munaron, L

    2014-05-01

    Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

  11. Resveratrol induces mitochondrial biogenesis in endothelial cells.

    Science.gov (United States)

    Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

    2009-07-01

    Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

  12. Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

    Science.gov (United States)

    Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

    2017-09-15

    Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

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

    Science.gov (United States)

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

    2013-10-29

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

  14. PTEN differentially regulates expressions of ICAM-1 and VCAM-1 through PI3K/Akt/GSK-3β/GATA-6 signaling pathways in TNF-α-activated human endothelial cells.

    Science.gov (United States)

    Tsoyi, Konstantin; Jang, Hwa Jin; Nizamutdinova, Irina Tsoy; Park, Kyungok; Kim, Young Min; Kim, Hye Jung; Seo, Han Geuk; Lee, Jae Heun; Chang, Ki Churl

    2010-11-01

    Phosphotase and tensin homolog deleted on chromosome 10 (PTEN) is a potent negative regulator of PI3K/Akt pathway. Here, we tried to elucidate the role of PTEN in the regulation of endothelial adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1, induced by TNF-α in human endothelial cells (ECs). Transfection with PTEN overexpressing vector resulted in the significant decrease in phosphorylation of Akt in TNF-α-treated ECs. PTEN strongly inhibited VCAM-1 but not ICAM-1, however this inhibitory effect was reversed by co-transfection with constitutively active-Akt (CA-Akt-HA) in TNF-α-stimulated ECs. Additionally, silencing of PTEN with specific siRNA showed significant increase of phosphor-Akt compared with TNF-α alone treated ECs. siPTEN significantly upregulated VCAM-1 but was indifferent to ICAM-1 in TNF-α-treated cells. Further, chromatin immunoprecipitation (ChIP) assay showed that PTEN targets GATA-6 but not IRF-1 binding to VCAM-1 promoter. In addition, GATA-6 is associated with glycogen synthesis kinase-3beta (GSK-3β) which is in turn regulated by PTEN-dependent Akt activity. Finally, PTEN significantly prevented monocyte adhesion to TNF-α-induced ECs probably through VCAM-1 regulation. It is concluded that PTEN selectively inhibits expression of VCAM-1 but not ICAM-1 through modulation of PI3K/Akt/GSK-3β/GATA-6 signaling cascade in TNF-α-treated ECs. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  15. Targeted adenovirus mediated inhibition of NF-kappa B-dependent inflammatory gene expression in endothelial cells in vitro and in vivo

    NARCIS (Netherlands)

    Kuldo, J. M.; Asgeirsdottir, S. A.; Zwiers, P. J.; Bellu, A. R.; Rots, M. G.; Schalk, J. A. C.; Ogawara, K. I.; Trautwein, C.; Banas, B.; Haisma, H. J.; Molema, G.; Kamps, J. A. A. M.

    2013-01-01

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor kappa B signal transduction could silence the proinflammatory activation status of

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

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

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

  17. The related transcriptional enhancer factor-1 isoform, TEAD4(216, can repress vascular endothelial growth factor expression in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Binoy Appukuttan

    Full Text Available Increased cellular production of vascular endothelial growth factor (VEGF is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4 protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4(216, which represses VEGF promoter activity. The TEAD4(216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE, which is the sequence critical to hypoxia inducible factor (HIF-mediated effects. The TEAD4(216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4(216 isoform can competitively repress the stimulatory activity of the TEAD4(434 and TEAD4(148 enhancers. Synthesis of the native VEGF(165 protein and cellular proliferation is suppressed by the TEAD4(216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4(216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.

  18. Electroacupuncture modulates stromal cell-derived factor-1α expression and mobilization of bone marrow endothelial progenitor cells in focal cerebral ischemia/reperfusion model rats.

    Science.gov (United States)

    Xie, Chenchen; Gao, Xiang; Luo, Yong; Pang, Yueshan; Li, Man

    2016-10-01

    Stromal cell-derived factor-1α(SDF-1α) plays a crucial role in regulating the mobilization, migration and homing of endothelial progenitor cells(EPCs). Electroacupuncture(EA), a modern version of Traditional Chinese Medicine, can improve neurological recovery and angiogenesis in cerebral ischemic area. This study aimed to investigate the effects of electroacupuncture(EA) on the mobilization and migration of bone marrow EPCs and neurological functional recovery in rats model after focal cerebral ischemia/reperfusion and the potentially involved mechanisms. Sprague-Dawley rats received filament occlusion of the right middle cerebral artery for 2h followed by reperfusion for 12h, 1d, 2d, 3d, 7d respectively. Rats were randomly divided into sham group, model group and EA group. After 2h of the reperfusion, EA was given at the "Baihui" (GV 20)/Siguan ("Hegu" (LI 4)/"Taichong" (LR 3)) acupoints in the EA group. Modified neurological severity score (mNSS) was used to assess the neurological functional recovery. EPCs number and SDF-1α level in bone marrow(BM) and peripheral blood(PB) were detected by using fluorescence-activated cell sorting (FACS) analysis and quantitative real time polymerase chain reaction (qRT-PCR) respectively. An mNSS test showed that EA treatment significantly improved the neurological functional outcome. EPCs number in PB and BM were obviously increased in the EA group. After cerebral ischemia, the SDF-1α level was decreased in BM while it was increased in PB, which implied a gradient of SDF-1α among BM and PB after ischemia. It suggested that the forming of SDF-1α concentration gradient can induce the mobilization and homing of EPCs. Eletroacupuncture as a treatment can accelerate and increase the forming of SDF-1α concentration gradient to further induce the mobilization of EPCs and angiogenesis in ischemic brain and improve the neurological function recovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

    National Research Council Canada - National Science Library

    Huang, Shuang

    2004-01-01

    .... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

  20. The role of neoangiogenesis and vascular endothelial growth factor ...

    African Journals Online (AJOL)

    2015-10-28

    Oct 28, 2015 ... Significant neovascularization was seen in diabetic group for VEGF, CD31, ... VEGF expression similar to changes in diabetic nephropathy and retinopathy in the .... Immunohistochemical staining was detected in endothelial.

  1. Expression and Function of Hypoxia Inducible Factor-1α and Vascular Endothelial Growth Factor in Pulp Tissue of Teeth under Orthodontic Movement

    Directory of Open Access Journals (Sweden)

    Fulan Wei

    2015-01-01

    Full Text Available Orthodontic force may lead to cell damage, circulatory disturbances, and vascular changes of the dental pulp, which make a hypoxic environment in pulp. In order to maintain the homeostasis of dental pulp, hypoxia will inevitably induce the defensive reaction. However, this is a complex process and is regulated by numerous factors. In this study, we established an experimental animal model of orthodontic tooth movement to investigate the effects of mechanical force on the expression of VEGF and HIF-1α in dental pulp. Histological analysis of dental pulp and expressions of HIF-1α and VEGF proteins in dental pulp were examined. The results showed that inflammation and vascular changes happened in dental pulp tissue in different periods. Additionally, there were significant changes in the expression of HIF-1α and VEGF proteins under orthodontic force. After application of mechanical load, expression of HIF-1α and VEGF was markedly positive in 1, 3, 7 d, and 2 w groups, and then it weakened in 4 w group. These findings suggested that the expression of HIF-1α and VEGF was enhanced by mechanical force. HIF-1α and VEGF may play an important role in retaining the homeostasis of dental pulp during orthodontic tooth movement.

  2. Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells

    NARCIS (Netherlands)

    Chui, Amy; Gunatillake, Tilini; Brennecke, Shaun P.; Ignjatovic, Vera; Monagle, Paul T.; Whitelock, John M.; van Zanten, Dagmar E.; Eijsink, Jasper; Wang, Yao; Deane, James; Borg, Anthony J.; Stevenson, Janet; Erwich, Jan Jaap; Said, Joanne M.; Murthi, Padma

    Objective-Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced

  3. The expression of endothelial and inducible nitric oxide synthase and apoptosis in intestinal ischemia and reperfusion injury under the action of ischemic preconditioning and pentoxifylline.

    Science.gov (United States)

    Oliveira, Teresinha Regina Ribeiro de; Oliveira, Geraldo Ferreira de; Simões, Ricardo Santos; Feitosa, Suellen Maurim; Tikazawa, Eduardo Hiroshi; Monteiro, Hugo Pequeno; Fagundes, Djalma José; Taha, Murched Omar

    2017-11-01

    To investigate the expression of nitric oxide synthase (NOS) and apoptosis associated with ischemic preconditioning (IPC) and pentoxifylline (PTX) in intestinal ischemia (I) and reperfusion (R) injury. Thirty male rats were assigned to 5 groups: (CG), no clamping of the superior mesenteric artery (90 minutes); (IR-SS) saline + ischemia (30 minutes) + reperfusion (60 minutes); (IR-PTX) PTX + ischemia (30 minutes) + reperfusion (60 minutes); (IPC-IR-SS) 5 minutes of ischemia + 5 minutes of reperfusion (IPC) + saline + I(30 minutes)+R(60 minutes); and (IPC-IR-PTX) IPC + PTX + I(30 minutes)+ R(60 minutes). The application of IPC and PTX showed a significantly lower immunohistochemistry reaction for active caspase-3 (P0.05). The NOS-2 expression (qRTPCR) in the IR-PTX group (P<0.05) was higher than the values for the IPC+IR-SS and IPC-IR-PTX groups. The NOS-3 expression was significantly upper in the IPC-IR-PTX group than in the CG (P<0.05), the IR-SS (P<0.05) and the IR-PTX (P<0.05) groups. The BCL-2 and active caspase-3 showed beneficial effects on PTX and IPC. The expression of NOS-2 and NOS-3 in the IPC and IPC-PTX groups showed no synergistic effect.

  4. Vascular endothelial genes that are responsive to tumor necrosis factor-alpha in vitro are expressed in atherosclerotic lesions, including inhibitor of apoptosis protein-1, stannin, and two novel genes

    NARCIS (Netherlands)

    Horrevoets, A. J.; Fontijn, R. D.; van Zonneveld, A. J.; de Vries, C. J.; ten Cate, J. W.; Pannekoek, H.

    1999-01-01

    Activation and dysfunction of endothelial cells play a prominent role in patho-physiological processes such as atherosclerosis. We describe the identification by differential display of 106 cytokine-responsive gene fragments from endothelial cells, activated by monocyte conditioned medium or tumor

  5. Hypoxia-induced factor-1 alpha, vascular endothelial growth factor expression in BRCA1-related breast cancer: A prospective study in tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Manisha Sharma

    2017-01-01

    Full Text Available Introduction: Breast cancer is the commonest cause of death among middle aged women. BRCA1 associated tumors carry a poor prognosis. Angiogenesis is considered necessary for tumor growth and for its metastasis. Hypoxia stimulates HIF-1α which then activates transcription of various proangiogenic cytokines like VEGF. In the present study we examined HIF-1α expression, sVEGF levels and BRCA1 mutations and their relation with clinicopathological parameters. We also determined whether the angiogenic markers have different role in angiogenesis in BRCA1 related cancers as compared to sporadic breast cancers. Materials and Methods: The study was conducted on 50 cases of breast cancer specimens. Histopathological typing and grading was done followed by immunohistochemistry for BRCA1 and HIF-1α. VEGF was done in the serum by ELISA. Results: All the tumors were infiltrating ductal carcinoma NOS. 16 cases were reported grade II and 34 cases as grade III. On immunohistochemistry, 27 cases showed BRCA1 positivity and HIF-1α was positive in 39 cases. sVEGF levels were increased in 21 cases (42%. BRCA1 positivity, HIF-1α expression and increased VEGF levels were significantly associated with higher grade and lymph node metastasis. There was significant correlation of BRCA1 positivity with increased HIF-1α expression (P = 0.009 and increased sVEGF levels (P = 0.005. Conclusion: Our findings suggest that BRCA 1 positive tumors have unique molecular profile and different mechanism of tumorigenesis. Such tumors are associated with increased HIF-1α expression and VEGF levels.

  6. Inhibition of Endothelial p53 Improves Metabolic Abnormalities Related to Dietary Obesity

    Directory of Open Access Journals (Sweden)

    Masataka Yokoyama

    2014-06-01

    Full Text Available Accumulating evidence has suggested a role for p53 activation in various age-associated conditions. Here, we identified a crucial role of endothelial p53 activation in the regulation of glucose homeostasis. Endothelial expression of p53 was markedly upregulated when mice were fed a high-calorie diet. Disruption of endothelial p53 activation improved dietary inactivation of endothelial nitric oxide synthase that upregulated the expression of peroxisome proliferator-activated receptor-γ coactivator-1α in skeletal muscle, thereby increasing mitochondrial biogenesis and oxygen consumption. Mice with endothelial cell-specific p53 deficiency fed a high-calorie diet showed improvement of insulin sensitivity and less fat accumulation, compared with control littermates. Conversely, upregulation of endothelial p53 caused metabolic abnormalities. These results indicate that inhibition of endothelial p53 could be a novel therapeutic target to block the vicious cycle of cardiovascular and metabolic abnormalities associated with obesity.

  7. Evolution of endothelial keratoplasty.

    Science.gov (United States)

    Price, Francis W; Price, Marianne O

    2013-11-01

    Endothelial keratoplasty has evolved into a popular alternative to penetrating keratoplasty (PK) for the treatment of endothelial dysfunction. Although the earliest iterations were challenging and were not widely adopted, the iteration known as Descemet stripping endothelial keratoplasty (DSEK) has gained widespread acceptance. DSEK combines a simplified technique for stripping dysfunctional endothelium from the host cornea and microkeratome dissection of the donor tissue, a step now commonly completed in advance by eye bank technicians. Studies show that a newer endothelial keratoplasty iteration, known as Descemet membrane endothelial keratoplasty (DMEK), provides an even faster and better visual recovery than DSEK does. In addition, DMEK significantly reduces the risk of immunologic graft rejection episodes compared with that in DSEK or in PK. Although the DMEK donor tissue, consisting of the bare endothelium and Descemet membrane without any stroma, is more challenging to prepare and position in the recipient eye, recent improvements in instrumentation and surgical techniques are increasing the ease and the reliability of the procedure. DSEK successfully mitigates 2 of the main liabilities of PK: ocular surface complications and structural problems (including induced astigmatism and perpetually weak wounds), whereas DMEK further mitigates the 2 principal remaining liabilities of PK: immunologic graft reactions and secondary glaucoma from prolonged topical corticosteroid use.

  8. Trifluoperazine: corneal endothelial phototoxicity

    International Nuclear Information System (INIS)

    Hull, D.S.; Csukas, S.; Green, K.

    1983-01-01

    Trifluoperazine is used for the treatment of psychiatric disorders. Perfusion of corneal endothelial cells with trifluoperazine-HC1 concurrent with exposure to long wavelength ultraviolet light resulted in a corneal swelling rate greater than that found in perfused corneas not exposed to ultraviolet light. Exposure of endothelial cells to 25 W incandescent light during perfusion with trifluoperazine-HC1 did not result in a higher corneal swelling rate compared to those perfused in the dark. The increased corneal swelling rate could be produced by pre-exposure of the trifluoperazine-HC1 perfusing solution to ultraviolet light suggesting the production of toxic photoproducts during exposure of trifluoperazine-HC1 to ultraviolet light. Perfusion of corneal endothelial cells with non-ultraviolet illuminated trifluoperazine-HC1 had no effect on endothelial cell membranes or ultrastructure. This is in contrast to cells perfused with trifluoperazine-HC1 that had been exposed to ultraviolet light in which there was an alteration of mitochondria and a loss of cytoplasmic homogeneity. The data imply that the trifluoperazine-HC1 photoproduct had an adverse effect on cellular transport mechanisms. The study also further demonstrates the value of the corneal endothelial cell model for identifying the physiological and anatomical changes occuring in photo-induced toxic reactions. (author)

  9. Mitochondria and Endothelial Function

    Science.gov (United States)

    Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

    2013-01-01

    In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease. PMID:23580773

  10. Endothelial cell oxidative stress and signal transduction

    Directory of Open Access Journals (Sweden)

    ROCIO FONCEA

    2000-01-01

    Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

  11. 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.

  12. Injuries to the vascular endothelium: vascular wall and endothelial dysfunction.

    Science.gov (United States)

    Fisher, Mark

    2008-01-01

    Vascular endothelial injury has multiple elements, and this article focuses on ischemia-related processes that have particular relevance to ischemic stroke. Distinctions between necrotic and apoptotic cell death provide a basic science context in which to better understand the significance of classical core and penumbra concepts of acute stroke, with apoptotic processes particularly prominent in the penumbra. The mitochondria are understood to serve as a reservoir of proteins that mediate apoptosis. Oxidative stress pathways generating reactive oxygen species (ROS) are prominent in endothelial injury, both ischemic and nonischemic, with prominent roles of enzyme- and nonenzymemediated pathways; mitochondria once again have a critical role, particularly in the nonenzymatic pathways generating ROS. Inflammation also contributes to vascular endothelial injury, and endothelial cells have the capacity to rapidly increase expression of inflammatory mediators following ischemic challenge; this leads to enhanced leukocyte-endothelial interactions mediated by selectins and adhesion molecules. Preconditioning consists of a minor version of an injurious event, which in turn may protect vascular endothelium from injury following a more substantial event. Presence of the blood-brain barrier creates unique responses to endothelial injury, with permeability changes due to impairment of endothelial-matrix interactions compounding altered vasomotor tone and tissue perfusion mediated by nitric oxide. Pharmacological protection against vascular endothelial injury can be provided by several of the phosphodiesterases (cilostazol and dipyridamole), along with statins. Optimal clinical responses for protection of brain vascular endothelium may use preconditioning as a model, and will likely require combined protection against apoptosis, ROS, and inflammation.

  13. Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature

    Directory of Open Access Journals (Sweden)

    Soufiane El Hallani

    2014-01-01

    Full Text Available Background. Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM. Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM. Materials and Methods. We used EGFR FISH Probe to detect EGFR amplification and anti-CD31, CD105, VE-cadherin, and vWF to identify endothelial cells. Endothelial and GBM cells were grown separately, labeled with GFP and DsRed lentiviruses, and then cocultured with or without contact. Results. In a subset of GBM tissues, we found that several tumor endothelial cells carry EGFR amplification, characteristic of GBM tumor cells. This observation was reproduced in vitro: when tumor stem cells derived from GBM were grown in the presence of human endothelial cells, a fraction of them acquired endothelial markers (CD31, CD105, VE-cadherin, and vWF. By transduction with GFP and DsRed expressing lentiviral vectors, we demonstrate that this phenomenon is due to cell fusion and not transdifferentiation. Conclusion. A fraction of GBM stem cells thus has the capacity to fuse with endothelial cells and the resulting hybrids may participate in tumor microvascular proliferation and in treatment resistance.

  14. Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Both Anton

    2001-08-01

    Full Text Available Abstract Background Activation of nuclear factor-κB (NF-κB is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1 can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50. Results Smooth muscle cells (SMC from human coronary plaque material (HCPSMC, plaque material of 52 patients, SMC from the human coronary media (HCMSMC, human endothelial cells (EC from umbilical veins (HUVEC, and human coronary EC (HCAEC were successfully isolated (HCPSMC, HUVEC, identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC. 12 hrs prior to TNF-α stimulus (20 ng/mL, 6 hrs RelA p65 and NF-κB1 p50 (1, 2, 4, 10, 20, and 30 μM and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-κB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-κB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-κB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-κB1 p50. Conclusions The data point out that differences exist in the NF-κB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-κB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.

  15. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

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    Maskomani Silambarasan

    2016-04-01

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

  16. PECAM-1 polymorphism affects monocyte adhesion to endothelial cells.

    Science.gov (United States)

    Goodman, Reyna S; Kirton, Christopher M; Oostingh, Gertie J; Schön, Michael P; Clark, Michael R; Bradley, J Andrew; Taylor, Craig J

    2008-02-15

    Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

  17. Human umbilical vein endothelium-derived exosomes play a role in foetoplacental endothelial dysfunction in gestational diabetes mellitus

    NARCIS (Netherlands)

    Sáez, Tamara; Salsoso, Rocío; Leiva, Andrea; Toledo, Fernando; de Vos, Paul; Faas, Marijke; Sobrevia, Luis

    2017-01-01

    Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric

  18. Human umbilical vein endothelium-derived exosomes play a role in foetoplacental endothelial dysfunction in gestational diabetes mellitus

    NARCIS (Netherlands)

    Sáez, Tamara; Salsoso, Rocío; Leiva, Andrea; Toledo, Fernando; de Vos, Paul; Faas, Marijke; Sobrevia, Luis

    Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric

  19. Identification of derlin-1 as a novel growth factor-responsive endothelial antigen by suppression subtractive hybridization

    International Nuclear Information System (INIS)

    Ran Yuliang; Jiang Yangfu; Zhong Xing; Zhou Zhuan; Liu Haiyan; Hu Hai; Lou Jinning; Yang Zhihua

    2006-01-01

    Endothelial cells play an important regulatory role in embryonic development, reproductive functions, tumor growth and progression. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify differentially expressed genes between non-stimulated endothelial cells and activated endothelial cells. Following mRNA isolation of non-stimulated and hepatocellular carcinoma homogenate-stimulated cells, cDNAs of both populations were prepared and subtracted by suppressive PCR. Sequencing of the enriched cDNAs identified a couple of genes differentially expressed, including derlin-1. Derlin-1 was significantly up-regulated by tumor homogenates, VEGF, and endothelial growth supplements in a dose-dependent manner. Knock-down of derlin-1 triggered endothelial cell apoptosis, inhibited endothelial cell proliferation, and blocked the formation of a network of tubular-like structures. Our data reveal that derlin-1 is a novel growth factor-responsive endothelial antigen that promotes endothelial cell survival and growth

  20. Infections and endothelial cells

    NARCIS (Netherlands)

    Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

    2003-01-01

    Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

  1. Resveratrol and Endothelial Nitric Oxide

    Directory of Open Access Journals (Sweden)

    Ning Xia

    2014-10-01

    Full Text Available Nitric oxide (NO derived from the endothelial NO synthase (eNOS has antihypertensive, antithrombotic, anti-atherosclerotic and antiobesogenic properties. Resveratrol is a polyphenol phytoalexin with multiple cardiovascular and metabolic effects. Part of the beneficial effects of resveratrol are mediated by eNOS. Resveratrol stimulates NO production from eNOS by a number of mechanisms, including upregulation of eNOS expression, stimulation of eNOS enzymatic activity and reversal of eNOS uncoupling. In addition, by reducing oxidative stress, resveratrol prevents oxidative NO inactivation by superoxide thereby enhancing NO bioavailability. Molecular pathways underlying these effects of resveratrol involve SIRT1, AMPK, Nrf2 and estrogen receptors.

  2. Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

    Science.gov (United States)

    Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

    2014-04-01

    In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

  3. Endothelial microparticles (EMP in physiology and pathology

    Directory of Open Access Journals (Sweden)

    Ewa Sierko

    2015-08-01

    Full Text Available Endothelial microparticles (EMP are released from endothelial cells (ECs in the process of activation and/or apoptosis. They harbor adhesive molecules, enzymes, receptors and cytoplasmic structures and express a wide range of various constitutive antigens, typical for ECs, at their surface. Under physiological conditions the concentration of EMP in the blood is clinically insignificant. However, it was reported that under pathological conditions EMP concentration in the blood might slightly increase and contribute to blood coagulation, angiogenesis and inflammation. It has been shown that EMP directly and indirectly contribute to the activation of blood coagulation. Endothelial microparticles directly participate in blood coagulation through their surface tissue factor (TF – a major initiator of blood coagulation. Furthermore, EMP exhibit procoagulant potential via expression of negatively charged phospholipids at their surface, which may promote assembly of coagulation enzymes (TF/VII, tenases and prothrombinase complexes, leading to thrombus formation. In addition, they provide a binding surface for coagulation factors: IXa, VIII, Va and IIa. Moreover, it is possible that EMP transfer TF from TF-bearing EMP to activated platelets and monocytes by binding them through adhesion molecules. Also, EMP express von Willebrand factor, which may facilitate platelet aggregation. Apart from their procoagulant properties, it was demonstrated that EMP may express adhesive molecules and metalloproteinases (MMP-2, MMP-9 at their surface and release growth factors, which may contribute to angiogenesis. Additionally, surface presence of C3 and C4 – components of the classical pathway – suggests pro-inflammatory properties of these structures. This article contains a summary of available data on the biology and pathophysiology of endothelial microparticles and their potential role in blood coagulation, angiogenesis and inflammation.

  4. Endothelial actions of atrial and B-type natriuretic peptides.

    Science.gov (United States)

    Kuhn, Michaela

    2012-05-01

    The cardiac hormone atrial natriuretic peptide (ANP) is critically involved in the maintenance of arterial blood pressure and intravascular volume homeostasis. Its cGMP-producing GC-A receptor is densely expressed in the microvascular endothelium of the lung and systemic circulation, but the functional relevance is controversial. Some studies reported that ANP stimulates endothelial cell permeability, whereas others described that the peptide attenuates endothelial barrier dysfunction provoked by inflammatory agents such as thrombin or histamine. Many studies in vitro addressed the effects of ANP on endothelial proliferation and migration. Again, both pro- and anti-angiogenic properties were described. To unravel the role of the endothelial actions of ANP in vivo, we inactivated the murine GC-A gene selectively in endothelial cells by homologous loxP/Cre-mediated recombination. Our studies in these mice indicate that ANP, via endothelial GC-A, increases endothelial albumin permeability in the microcirculation of the skin and skeletal muscle. This effect is critically involved in the endocrine hypovolaemic, hypotensive actions of the cardiac hormone. On the other hand the homologous GC-A-activating B-type NP (BNP), which is produced by cardiac myocytes and many other cell types in response to stressors such as hypoxia, possibly exerts more paracrine than endocrine actions. For instance, within the ischaemic skeletal muscle BNP released from activated satellite cells can improve the regeneration of neighbouring endothelia. This review will focus on recent advancements in our understanding of endothelial NP/GC-A signalling in the pulmonary versus systemic circulation. It will discuss possible mechanisms accounting for the discrepant observations made for the endothelial actions of this hormone-receptor system and distinguish between (patho)physiological and pharmacological actions. Lastly it will emphasize the potential therapeutical implications derived from the

  5. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

    Science.gov (United States)

    Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

    2002-09-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

  6. COPD as an endothelial disorder: endothelial injury linking lesions in the lungs and other organs? (2017 Grover Conference Series)

    Science.gov (United States)

    Polverino, Francesca; Celli, Bartolome R.

    2018-01-01

    Chronic obstructive pulmonary disease (COPD) is characterized by chronic expiratory airflow obstruction that is not fully reversible. COPD patients develop varying degrees of emphysema, small and large airway disease, and various co-morbidities. It has not been clear whether these co-morbidities share common underlying pathogenic processes with the pulmonary lesions. Early research into the pathogenesis of COPD focused on the contributions of injury to the extracellular matrix and pulmonary epithelial cells. More recently, cigarette smoke-induced endothelial dysfunction/injury have been linked to the pulmonary lesions in COPD (especially emphysema) and systemic co-morbidities including atherosclerosis, pulmonary hypertension, and chronic renal injury. Herein, we review the evidence linking endothelial injury to COPD, and the pathways underlying endothelial injury and the “vascular COPD phenotype” including: (1) direct toxic effects of cigarette smoke on endothelial cells; (2) generation of auto-antibodies directed against endothelial cells; (3) vascular inflammation; (4) increased oxidative stress levels in vessels inducing increases in lipid peroxidation and increased activation of the receptor for advanced glycation end-products (RAGE); (5) reduced activation of the anti-oxidant pathways in endothelial cells; (6) increased endothelial cell release of mediators with vasoconstrictor, pro-inflammatory, and remodeling activities (endothelin-1) and reduced endothelial cell expression of mediators that promote vasodilation and homeostasis of endothelial cells (nitric oxide synthase and prostacyclin); and (7) increased endoplasmic reticular stress and the unfolded protein response in endothelial cells. We also review the literature on studies of drugs that inhibit RAGE signaling in other diseases (angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), or vasodilators developed for idiopathic pulmonary arterial hypertension that have been tested

  7. Melatonin inhibits endothelin-1 and induces endothelial nitric oxide ...

    African Journals Online (AJOL)

    Although, I/R augmented the endothelin-1 (ET-1) gene expression and the level of big endothelin-1 (big ET-1) in liver tissue, melatonin attenuated these increases. Conversely, non-significant decrease in endothelial nitric oxide synthase (eNOS) mRNA expression in I/R group was significantly elevated by melatonin in ...

  8. 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...

  9. Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

    Directory of Open Access Journals (Sweden)

    Mareike Florek

    Full Text Available The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs in murine models of allogeneic hematopoietic cell transplantation (HCT has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L, which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

  10. Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells.

    Science.gov (United States)

    Lu, Chia-Yang; Yang, Ya-Chen; Li, Chien-Chun; Liu, Kai-Li; Lii, Chong-Kuei; Chen, Haw-Wen

    2014-09-01

    Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox), and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47(phox) abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent. Copyright © 2014. Published by Elsevier Inc.

  11. Endothelial cell energy metabolism, proliferation, and apoptosis in pulmonary hypertension.

    Science.gov (United States)

    Xu, Weiling; Erzurum, Serpil C

    2011-01-01

    Pulmonary arterial hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary hemodynamics and excessive growth and dysfunction of the endothelial cells that line the arteries in PAH lungs. Establishment of methods for culture of pulmonary artery endothelial cells from PAH lungs has provided the groundwork for mechanistic translational studies that confirm and extend findings from model systems and spontaneous pulmonary hypertension in animals. Endothelial cell hyperproliferation, survival, and alterations of biochemical-metabolic pathways are the unifying endothelial pathobiology of the disease. The hyperproliferative and apoptosis-resistant phenotype of PAH endothelial cells is dependent upon the activation of signal transducer and activator of transcription (STAT) 3, a fundamental regulator of cell survival and angiogenesis. Animal models of PAH, patients with PAH, and human PAH endothelial cells produce low nitric oxide (NO). In association with the low level of NO, endothelial cells have reduced mitochondrial numbers and cellular respiration, which is associated with more than a threefold increase in glycolysis for energy production. The shift to glycolysis is related to low levels of NO and likely to the pathologic expression of the prosurvival and proangiogenic signal transducer, hypoxia-inducible factor (HIF)-1, and the reduced mitochondrial antioxidant manganese superoxide dismutase (MnSOD). In this article, we review the phenotypic changes of the endothelium in PAH and the biochemical mechanisms accounting for the proliferative, glycolytic, and strongly proangiogenic phenotype of these dysfunctional cells, which consequently foster the panvascular progressive pulmonary remodeling in PAH. © 2011 American Physiological Society.

  12. Endothelial ERK signaling controls lymphatic fate specification

    Science.gov (United States)

    Deng, Yong; Atri, Deepak; Eichmann, Anne; Simons, Michael

    2013-01-01

    Lymphatic vessels are thought to arise from PROX1-positive endothelial cells (ECs) in the cardinal vein in response to induction of SOX18 expression; however, the molecular event responsible for increased SOX18 expression has not been established. We generated mice with endothelial-specific, inducible expression of an RAF1 gene with a gain-of-function mutation (RAF1S259A) that is associated with Noonan syndrome. Expression of mutant RAF1S259A in ECs activated ERK and induced SOX18 and PROX1 expression, leading to increased commitment of venous ECs to the lymphatic fate. Excessive production of lymphatic ECs resulted in lymphangiectasia that was highly reminiscent of abnormal lymphatics seen in Noonan syndrome and similar “RASopathies.” Inhibition of ERK signaling during development abrogated the lymphatic differentiation program and rescued the lymphatic phenotypes induced by expression of RAF1S259A. These data suggest that ERK activation plays a key role in lymphatic EC fate specification and that excessive ERK activation is the basis of lymphatic abnormalities seen in Noonan syndrome and related diseases. PMID:23391722

  13. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    Science.gov (United States)

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

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. In smokers, Sonic hedgehog modulates pulmonary endothelial function through vascular endothelial growth factor.

    Science.gov (United States)

    Henno, Priscilla; Grassin-Delyle, Stanislas; Belle, Emeline; Brollo, Marion; Naline, Emmanuel; Sage, Edouard; Devillier, Philippe; Israël-Biet, Dominique

    2017-05-23

    Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The Sonic hedgehog (SHH) pathway is involved in vascular physiology. We sought to establish whether the SHH pathway has a role in pulmonary endothelial dysfunction in smokers. The ex vivo endothelium-dependent relaxation of pulmonary artery rings in response to acetylcholine (Ach) was compared in 34 current or ex-smokers and 8 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of SHH inhibitors (GANT61 and cyclopamine), an SHH activator (SAG) and recombinant VEGF on the Ach-induced relaxation. The level of VEGF protein in the pulmonary artery ring was measured in an ELISA. SHH pathway gene expression was quantified in reverse transcriptase-quantitative polymerase chain reactions. Ach-induced relaxation was much less intense in smokers than in never-smokers (respectively 24 ± 6% and 50 ± 7% with 10 -4 M Ach; p = 0.028). All SHH pathway genes were expressed in pulmonary artery rings from smokers. SHH inhibition by GANT61 reduced Ach-induced relaxation and VEGF gene expression in the pulmonary artery ring. Recombinant VEGF restored the ring's endothelial function. VEGF gene and protein expression levels in the pulmonary artery rings were positively correlated with the degree of Ach-induced relaxation and negatively correlated with the number of pack-years. SHH pathway genes and proteins are expressed in pulmonary artery rings from smokers, where they modulate endothelial function through VEGF.

  15. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    Science.gov (United States)

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  16. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  17. Nipah virus infection and glycoprotein targeting in endothelial cells

    Directory of Open Access Journals (Sweden)

    Maisner Andrea

    2010-11-01

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

  18. Endogenous Vascular Endothelial Growth Factor-A (VEGF-A) Maintains Endothelial Cell Homeostasis by Regulating VEGF Receptor-2 Transcription*

    Science.gov (United States)

    E, Guangqi; Cao, Ying; Bhattacharya, Santanu; Dutta, Shamit; Wang, Enfeng; Mukhopadhyay, Debabrata

    2012-01-01

    Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis. PMID:22167188

  19. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    International Nuclear Information System (INIS)

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-01-01

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

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

    Science.gov (United States)

    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

  1. Functional inhibition of NF-kappa B signal transduction in alpha v alpha beta 3 integrin expressing endothelial cells by using RGD-PEG-modified adenovirus with a mutant I kappa B gene

    NARCIS (Netherlands)

    Ogawara, K; Kuldo, JM; Oosterhuis, K; Kroesen, BJ; Rots, MG; Trautwein, C; Kimura, T; Haisma, HJ; Molema, G

    2006-01-01

    In order to selectively block nuclear factor kappa B (NF-kappa B)-dependent signal transduction in angiogenic endothelial cells, we constructed an alpha v beta 3 integrin specific adenovirus encoding dominant negative I kappa B (dnI kappa B) as a therapeutic gene. By virtue of RGD modification of

  2. Expression and localization of insulin-like growth factor system in corpus luteum during different stages of estrous cycle in water buffaloes (Bubalus bubalis) and the effect of insulin-like growth factor I on production of vascular endothelial growth factor and progesterone in luteal cells cultured in vitro.

    Science.gov (United States)

    Uniyal, S; Panda, R P; Chouhan, V S; Yadav, V P; Hyder, I; Dangi, S S; Gupta, M; Khan, F A; Sharma, G T; Bag, S; Sarkar, M

    2015-01-01

    This study investigated the expression and localization of insulin-like growth factor (IGF) system at different stages of buffalo CL and the role of IGF-I in stimulating vascular endothelial growth factor (VEGF) and progesterone (P4) production in cultured luteal cells. The mRNA expression of IGF system, VEGF, steroidogenic acute regulatory protein, P450scc, and hydroxysteroid dehydrogenase (HSD) was investigated by quantitative real-time polymerase chain reaction (PCR). Protein expression of IGF was demonstrated by Western blot and localization by immunohistochemistry. Progesterone and VEGF production was assayed using RIA and ELISA. A relatively high mRNA expression of IGF-I and IGF-II in early, mid- and late luteal phases with immunoreactivity mostly restricted to cytoplasm of large luteal cells indicates their autocrine role, whereas very weak immunoreactivity in endothelial cells during the mid-luteal phase indicates their paracrine role. Insulin-like growth factor receptors, IGF-IR and IGF-IIR, were restricted to large luteal cells with high mRNA and protein expressions in the mid-luteal phase. The significantly higher expression of insulin-like growth factor binding protein (IGFBP)-1, -3, -5, and -6 in the early or mid-luteal phase suggested their stimulatory role, whereas that of IGFBP-2 and -4 in mid-, late, and regressive luteal stages implied their inhibitory role. The mRNA expressions of key steroidogenic factors and VEGF were signif