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Sample records for activate endothelial cells

  1. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    PubMed

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics. PMID:26068799

  2. Lymphatic endothelial cells actively regulate prostate cancer cell invasion.

    PubMed

    Shah, Tariq; Wildes, Flonne; Kakkad, Samata; Artemov, Dmitri; Bhujwalla, Zaver M

    2016-07-01

    Lymphatic vessels serve as the primary route for metastatic spread to lymph nodes. However, it is not clear how interactions between cancer cells and lymphatic endothelial cells (LECs), especially within hypoxic microenvironments, affect the invasion of cancer cells. Here, using an MR compatible cell perfusion assay, we investigated the role of LEC-prostate cancer (PCa) cell interaction in the invasion and degradation of the extracellular matrix (ECM) by two human PCa cell lines, PC-3 and DU-145, under normoxia and hypoxia, and determined the metabolic changes that occurred under these conditions. We observed a significant increase in the invasion of ECM by invasive PC-3 cells, but not poorly invasive DU-145 cells when human dermal lymphatic microvascular endothelial cells (HMVEC-dlys) were present. Enhanced degradation of ECM by PC-3 cells in the presence of HMVEC-dlys identified interactions between HMVEC-dlys and PCa cells influencing cancer cell invasion. The enhanced ECM degradation was partly attributed to increased MMP-9 enzymatic activity in PC-3 cells when HMVEC-dlys were in close proximity. Significantly higher uPAR and MMP-9 expression levels observed in PC-3 cells compared to DU-145 cells may be one mechanism for increased invasion and degradation of matrigel by these cells irrespective of the presence of HMVEC-dlys. Hypoxia significantly decreased invasion by PC-3 cells, but this decrease was significantly attenuated when HMVEC-dlys were present. Significantly higher phosphocholine was observed in invasive PC-3 cells, while higher glycerophosphocholine was observed in DU-145 cells. These metabolites were not altered in the presence of HMVEC-dlys. Significantly increased lipid levels and lipid droplets were observed in PC-3 and DU-145 cells under hypoxia reflecting an adaptive survival response to oxidative stress. These results suggest that in vivo, invasive cells in or near lymphatic endothelial cells are likely to be more invasive and degrade the ECM

  3. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    PubMed

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

    2016-07-01

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

  4. Effects of physical activity on endothelial progenitor cells (EPCs)

    PubMed Central

    De Biase, Chiara; De Rosa, Roberta; Luciano, Rossella; De Luca, Stefania; Capuano, Ernesto; Trimarco, Bruno; Galasso, Gennaro

    2014-01-01

    Physical activity has a therapeutic role in cardiovascular disease (CVD), through its beneficial effects on endothelial function and cardiovascular system. Circulating endothelial progenitor cells (EPCs) are bone marrow (BM) derived cells that represent a novel therapeutic target in CVD patients, because of their ability to home to sites of ischemic injury and repair the damaged vessels. Several studies show that physical activity results in a significant increase in circulating EPCs, and, in particular, there are some evidence of the beneficial exercise-induced effects on EPCs activity in CVD settings, including coronary artery disease (CAD), heart failure (HF), and peripheral artery disease (PAD). The aim of this paper is to review the current evidence about the beneficial effects of physical exercise on endothelial function and EPCs levels and activity in both healthy subjects and patients with CVD. PMID:24550833

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

    PubMed Central

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

    2016-01-01

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

  6. Virulent Treponema pallidum activates human vascular endothelial cells.

    PubMed

    Riley, B S; Oppenheimer-Marks, N; Hansen, E J; Radolf, J D; Norgard, M V

    1992-03-01

    Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium. PMID:1347056

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

    PubMed

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

    2015-12-01

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

  8. XIAP reverses various functional activities of FRNK in endothelial cells

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  9. Endothelial Activation by Platelets from Sickle Cell Anemia Patients

    PubMed Central

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T.; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  10. Endothelial activation by platelets from sickle cell anemia patients.

    PubMed

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  11. Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation

    PubMed Central

    Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

    2011-01-01

    Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

  12. Activated Brain Endothelial Cells Cross-Present Malaria Antigen

    PubMed Central

    Howland, Shanshan W.; Poh, Chek Meng; Rénia, Laurent

    2015-01-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. PMID:26046849

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

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated

  14. Matrix fibronectin disruption and altered endothelial cell adhesion induced by activated leukocytes

    SciTech Connect

    Vincent, P.; Richards, P.; Saba, T.; DelVecchio, P.

    1986-03-01

    Sequestration of activated leukocytes (PMN) within the lung may contribute to pulmonary vascular injury following trauma, sepsis, or intravascular coagulation. Monolayers of cultured rat endothelial cells were utilized to evaluate the effect of activated PMNs on endothelial cell attachment and the extracellular fibronectin matrix over a 4 hr incubation interval. Rat endothelial cells were identified by immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with /sup 51/Cr in order to establish a cell injury assay in which the release of pelletable (cell associated) or non-pelletable activity was measured in the media. PMN activation was verified by chemiluminescence activity. Following phorbol myristate acetate (PMA) the leukocytes aggregated, chemiluminesced, and caused detachment of /sup 51/Cr endothelial cells. Endothelial detachment increased as a function of time with a plateau by 3 hrs. Immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin in association with endothelial cell disadhesion. Matrix fibronectin disruption was not seen with PMNs or PMA alone. Thus, disruption of the fibronectin matrix by released proteases may contribute to endothelial cell detachment.

  15. Tp17 membrane protein of Treponema pallidum activates endothelial cells in vitro.

    PubMed

    Zhang, Rui-Li; Wang, Qian-Qiu; Zhang, Jing-Ping; Yang, Li-Jia

    2015-04-01

    Tp17, a membrane immunogen of Treponema pallidum subsp. pallidum, was initially recognized as an inflammatory mediator of syphilis. Because the histopathology of syphilis is characterized by endothelial cell abnormalities, we investigated the effects of recombinant Tp17 (rTp17) on endothelial cell activation in vitro. Using real-time reverse transcription-PCR and whole-cell ELISA, we found that rTp17 activated endothelial cells, as demonstrated by the up-regulated expression and increased gene transcription of intercellular adhesion molecule 1 (ICAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1). rTp17 also enhanced the migration and subsequent adhesion of monocytes to endothelial cells as well as increased transendothelial migration of monocytes. These data suggest that the ability of Tp17 to activate endothelial cells may play an important role in the immunopathogenesis of syphilis. PMID:25744604

  16. Hypergravity and hypobaric hypoxic conditions promote endothelial cell and platelet activation.

    PubMed

    Rubenstein, David A; Yin, Wei

    2014-09-01

    Cardiovascular disease risk is heightened during exposure to altered gravity and/or altered barometric conditions. Previous work has suggested that this heightened cardiovascular risk is due to enhancements of endothelial cell inflammatory and/or thrombogenic responses. In recent work, the role of platelets on instigating or inhibiting endothelial cell responses associated with cardiovascular disease has been found to be dependent on both biochemical and biophysical factors. In this work, we aimed to determine how two biophysical forces, gravity and atmospheric pressure, alter endothelial cell and platelet functions and their interactions to instigate or inhibit cardiovascular disease responses. To address this aim, endothelial cells and platelets were subjected to a force 8 times greater than the normal gravitational force, for up to 30 minutes. In separate experiments, endothelial cells and platelets were subjected to 50% of normal atmospheric pressure. Endothelial cell and platelet responses, associated with cardiovascular diseases, were measured as a time course during exposure. In general, the exposure of endothelial cells to either hypergravity or hypobaric conditions enhanced cardiovascular disease responses. However, the presence of platelets generally inhibited endothelial cell responses. Platelet activation was, however, somewhat enhanced under both hypergravity and hypobaric conditions. Our data suggest that altered biophysical forces can modulate endothelial cell and platelet responses that are salient for cardiovascular disease progression. However, the interaction of these two cells tends to restrain the progression of the pro-cardiovascular disease responses. PMID:25211651

  17. Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes

    SciTech Connect

    Pakala, Rajbabu; Watanabe, Takuya; Benedict, Claude R

    2002-06-01

    Introduction: Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. Methods: Endothelial cell proliferation is determined by the amount of [{sup 3}H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels in the conditioned medium were determined by ELISA. Results: Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. Conclusion: Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors.

  18. Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway

    PubMed Central

    Uemura, Mami; Nagasawa, Ayumi; Terai, Kenta

    2016-01-01

    Osteogenesis is categorized into two groups based on developmental histology, intramembranous and endochondral ossification. The role of blood vessels during endochondral ossification is well known, while their role in intramembranous ossification, especially the intertissue pathway, is poorly understood. Here, we demonstrate endothelial Yap/Taz is a novel regulator of intramembranous ossification in zebrafish. Appropriate blood flow is required for Yap/Taz transcriptional activation in endothelial cells and intramembranous ossification. Additionally, Yap/Taz transcriptional activity in endothelial cells specifically promotes intramembranous ossification. BMP expression by Yap/Taz transactivation in endothelial cells is also identified as a bridging factor between blood vessels and intramembranous ossification. Furthermore, the expression of Runx2 in pre-osteoblast cells is a downstream target of Yap/Taz transcriptional activity in endothelial cells. Our results provide novel insight into the relationship between blood flow and ossification by demonstrating intertissue regulation. PMID:27273480

  19. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.

    PubMed

    Jin, Benjamin Y; Sartoretto, Juliano L; Gladyshev, Vadim N; Michel, Thomas

    2009-10-13

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells. PMID:19805165

  20. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

    PubMed Central

    Jin, Benjamin Y.; Sartoretto, Juliano L.; Gladyshev, Vadim N.; Michel, Thomas

    2009-01-01

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H2O2 treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC50 for H2O2-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H2O2 concentrations. The Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H2O2-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H2O2 biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H2O2 generation, which is blocked by PEG-catalase. eNOS−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H2O2 generation in endothelial cells. PMID:19805165

  1. Markers of endothelial cell activation and injury in childhood haemolytic uraemic syndrome.

    PubMed

    Nevard, C H; Blann, A D; Jurd, K M; Haycock, G B; Hunt, B J

    1999-08-01

    Diarrhoea-associated haemolytic uraemic syndrome (D+ HUS) is usually caused by verotoxin-producing Escherichia coli. Histology shows endothelial swelling with localised thrombus. Activation of coagulation and fibrinolysis also occurs. These facts, combined with the knowledge that recovery usually follows within weeks, led us to hypothesise that verotoxin causes localised endothelial cell activation but not injury. Markers of endothelial cell activation and injury were measured serially in 30 children with acute D+ HUS, healthy children, and children receiving chronic dialysis. Interpretation of markers was complicated by the renal dysfunction characteristic of D+ HUS. Nevertheless there was no evidence for endothelial cell injury, as soluble tissue factor levels were not increased and soluble thrombomodulin levels were lower than dialysed controls (P<0.001). In the acute phase, soluble vascular cell adhesion molecule levels were raised above normal (P<0.001), but were lower than dialysed controls (P<0.001), and soluble E-selectin levels were not significantly increased compared with normal controls (P=0.2). Hence, there was no evidence for endothelial cell damage or endothelial cell activation by the time children reached hospital; but this study did not exclude the possibility that endothelial cell activation occurred prior to hospital admission. PMID:10452276

  2. Activation of the canonical Wnt/{beta}-catenin pathway enhances monocyte adhesion to endothelial cells

    SciTech Connect

    Lee, Dong Kun . E-mail: leedk@memorialhealthsource.com; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-08-18

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/{beta}-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3{beta} or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/{beta}-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/{beta}-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules.

  3. LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-β phosphorylation

    PubMed Central

    Menden, Heather; Tate, Everett; Hogg, Neil

    2013-01-01

    Lipopolysaccharide (LPS)-mediated endothelial activation contributes to lung inflammation and alveolar remodeling seen in premature infants with bronchopulmonary dysplasia (BPD). The mechanisms underlying LPS-mediated oxidative stress and proinflammatory signaling in human pulmonary microvascular endothelial cells (HPMEC) remain unclear. We hypothesized that NADPH oxidase (Nox) mediates LPS-induced endothelial activation in HPMEC by regulating phosphorylation of Toll-like receptor (TLR) pathway proteins. LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) was associated with increased 2-OH-E+ (marker for superoxide formation) levels and was attenuated by apocynin and the Nox inhibitor, VAS2870. LPS triggered membrane translocation of p67phox, suggesting activation of Nox2. Silencing Nox2, but not Nox4, suppressed LPS-induced ICAM-1 expression in HPMEC. Immunoprecipitation studies showed that inhibitor of κ-B kinase-β (IKK-β) serine phosphorylation induced by LPS was inhibited by Nox2 silencing. We examined whether Nox2-dependent, LPS-mediated IKK-β phosphorylation was regulated by protein phosphatase 2A (PP2A) or TGF-β associated kinase-1 (TAK1) in HPMEC. LPS increased PP2A activity in HPMEC, and inhibition of PP2A did not alter LPS-mediated ICAM-1 expression but attenuated IKK-β phosphorylation. TAK1 inhibition decreased LPS-induced ICAM-1 expression in HPMEC, and Nox2 silencing attenuated LPS-mediated TAK1 phosphorylation (Thr184/187). We demonstrate that Nox2 regulates LPS-mediated endothelial activation in pulmonary endothelial cells by modulating phosphorylation of key kinases in the TLR signaling cascade. Our data support a novel mechanism by which Nox-dependent signaling regulates proinflammatory signaling in pulmonary endothelial cells. Inhibition of vascular Nox may potentially limit lung injury and alveolar remodeling caused by infections in BPD. PMID:23333803

  4. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity

    PubMed Central

    Cazzaniga, Alessandra; Castiglioni, Sara; Maier, Jeanette A. M.

    2014-01-01

    Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. PMID:25210716

  5. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    SciTech Connect

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

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

    PubMed Central

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a

  7. Mesenchymal Stem Cells Transmigrate Between and Directly Through TNF-α-activated Endothelial Cells

    PubMed Central

    Teo, Grace S.L.; Ankrum, James A.; Martinelli, Roberta; Boetto, Sarah E.; Simms, Kayla; Sciuto, Tracey E.; Dvorak, Ann M.; Karp, Jeffrey M.; Carman, Christopher V.

    2012-01-01

    SUMMARY Systemically administered adult mesenchymal stem cells (MSC), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high-resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow-derived MSC preferentially adhere to and migrate across TNF-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein coupled receptor (GPCR) signaling-dependent manner. As several studies have suggested, we observed that a fraction of MSC integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Para (between endothelial cells)- and trans (directly through individual endothelial cells)-cellular diapedesis through discrete pores and gaps in the endothelial monolayer, in association with VCAM-1-enriched ‘transmigratory cups’. Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited non-apoptotic membrane blebbing activity, that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte-like and novel mechanisms. PMID:22887987

  8. Increased Capillary Endothelial Cell Protease Activity in Response to Angiogenic Stimuli in vitro

    NASA Astrophysics Data System (ADS)

    Gross, Janet L.; Moscatelli, David; Rifkin, Daniel B.

    1983-05-01

    Bovine capillary endothelial (BCE) cells produce increased amounts of the proteases plasminogen activator (PA) and latent collagenase when cultured in the presence of the following preparations which are known to contain angiogenic activities: bovine retinal extract, mouse adipocyte conditioned medium, and human hepatoma cell lysate. These preparations stimulated both BCE cell PA and collagenase activities in a dose-dependent manner. Both activities were increased to about the same level by these preparations as by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. Mitogens that are not angiogenic, such as insulin, epidermal and fibroblast growth factors, and endothelial cell growth supplement, had no effect on BCE cell PA and collagenase activities. Two of the angiogenic preparations (retinal extract and mouse adipocyte-conditioned medium) had no effect on PA activity in endothelial cells derived from bovine aortae (BAE cells). The angiogenic preparations had little (human hepatoma cell lysate, mouse adipocyte-conditioned medium) or no (bovine retinal extract) effect on BAE cell collagenase activities. In the bovine system, the induction of high levels of both PA and collagenase activities by angiogenic preparations is limited to capillary endothelial cells.

  9. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE.

    PubMed Central

    Rollins, B. J.; Yoshimura, T.; Leonard, E. J.; Pober, J. S.

    1990-01-01

    We have demonstrated inducible expression of the mRNA encoding the monocyte chemoattractant MCP-1, the human homolog of the JE gene, in endothelial cells within 3 hours of treatment with IL-1 beta and tumor necrosis factor. IFN-gamma also induced expression of this mRNA after 24 hours, but to a lesser extent. MCP-1/JE protein steadily accumulated in the medium of endothelial cells during a 48-hour exposure to IL-1 beta. Medium conditioned by IL-1 beta-treated endothelial cells contained monocyte chemoattractant activity that was immunoadsorbed by anti-MCP-1 antibodies. These results suggest that endothelial cells secrete a monocyte chemoattractant, MCP-1/JE, in response to inflammatory mediators, and thus may contribute to the accumulation of monocytes at sites of inflammation. Images Figure 1 Figure 2 PMID:2113354

  10. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    SciTech Connect

    Beebe, D.P.; Wood, L.L.; Moos, M. )

    1990-07-15

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between {sup 125}I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.

  11. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    SciTech Connect

    Askari, Ara A.; Thomson, Scott; Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C.; Bishop-Bailey, David

    2014-04-04

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.

  12. Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

    SciTech Connect

    Madden, M.C.; Eling, T.E.; Friedman, M.

    1987-09-01

    We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H/sub 2/O/sub 2/ is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.

  13. Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3.

    PubMed

    Tölle, M; Klöckl, L; Wiedon, A; Zidek, W; van der Giet, M; Schuchardt, M

    2016-08-01

    Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 lead to downstream activation of eNOS in

  14. Inhibition of Cellular Methyltransferases Promotes Endothelial Cell Activation by Suppressing Glutathione Peroxidase 1 Protein Expression*

    PubMed Central

    Barroso, Madalena; Florindo, Cristina; Kalwa, Hermann; Silva, Zélia; Turanov, Anton A.; Carlson, Bradley A.; de Almeida, Isabel Tavares; Blom, Henk J.; Gladyshev, Vadim N.; Hatfield, Dolph L.; Michel, Thomas; Castro, Rita; Loscalzo, Joseph; Handy, Diane E.

    2014-01-01

    S-Adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNASec to the Um34 form. The formation of methylated tRNASec facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNASec and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNASec hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype. PMID:24719327

  15. GTP cyclohydrolase I expression and enzymatic activity are present in caveolae of endothelial cells

    PubMed Central

    Peterson, Timothy E.; d’Uscio, Livius V.; Cao, Sheng; Wang, Xiao-Li; Katusic, Zvonimir S.

    2009-01-01

    Tetrahydrobiopterin is an essential cofactor required for the synthesis of nitric oxide. GTP cyclohydrolase I (GTPCH I) is the rate limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrate that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial nitric oxide synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a two-fold increase in GTPCH I activity was detected in the aortas of caveolin-1 deficient mice suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells and tetrahydrobiopterin production occurs in close proximity to endothelial nitric oxide synthase. Additionally, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1. PMID:19104007

  16. Markers of endothelial cell activation and immune activation are increased in patients with severe leptospirosis and associated with disease severity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: Previous studies concluded that haemorrhage is one of the most accurate prognostic factors of mortality in leptospirosis. Therefore, endothelial cell activation was investigated in relation to disease severity in severe leptospirosis. Methods: Prospective cohort study of severe leptospi...

  17. Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism

    PubMed Central

    Yang, Ying; Nie, Wei; Yuan, Jianmin; Zhang, Bingkun; Wang, Zhong

    2010-01-01

    Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser1179 which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models. PMID:20926919

  18. Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation

    SciTech Connect

    Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung; Kim, Hyo Shin; Lee, Sang Ki; Lee, Kwon Ho; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2008-03-28

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.

  19. Pterostilbene, an Active Constituent of Blueberries, Stimulates Nitric Oxide Production via Activation of Endothelial Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells.

    PubMed

    Park, Seong Hoon; Jeong, Sun-Oh; Chung, Hun-Teag; Pae, Hyun-Ock

    2015-09-01

    Endothelial dysfunction, a key process in development of cardiovascular diseases, is largely due to reduced nitric oxide (NO) derived from endothelial NO synthase (eNOS). Resveratrol has been reported to stimulate NO production via estrogen receptor α (ERα) activation in endothelial cells. Here, we investigated whether two natural methylated analogs of resveratrol, pterostilbene (Pts) and trans-3,5,4'-trimethoxystilbene (TMS), similarly to resveratrol, could influence endothelial NO release in human umbilical vein endothelial cells (HUVECs). In HUVECs exposed to Pts or TMS, NO production and phosphorylation of eNOS, protein kinase B (Akt), and ERα were measured by using a fluorimetric NO assay kit and Western blot analysis, respectively. Dimethylated Pts, but not trimethylated TMS, stimulated dose-dependent NO production via eNOS phosphorylation. Pts also stimulated dose-dependent phosphorylation of Akt, but not of ERα. NO production and eNOS phosphorylation in response to Pts were significantly abolished by the phosphoinositide 3-kinase (PI3K)/Akt inhibitor LY294002, but not by the ERα antagonist ICI182780. Our results suggest that Pts, but not TMS, is capable of inducing eNOS phosphorylation and the subsequent NO release, presumably, by activating PI3K/Akt pathway. The potential efficacy of Pts, an active constituent of blueberries, may aid in the prevention of cardiovascular diseases characterized by endothelial dysfunction. PMID:26008990

  20. Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines.

    PubMed

    Shahabuddin, S; Ponath, P; Schleimer, R P

    2000-04-01

    Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines. PMID:10725746

  1. Antiphospholipid antibodies prolong the activation of endothelial cells induced by necrotic trophoblastic debris: implications for the pathogenesis of preeclampsia.

    PubMed

    Chen, Q; Guo, F; Hensby-Bennett, S; Stone, P; Chamley, L

    2012-10-01

    The symptoms of preeclampsia are preceded by endothelial cell activation/dysfunction which is induced by a placental trigger(s) but maternal risk factor(s) also contribute to the pathogenesis of preeclampsia. In this work we have investigated the interactions of a maternal risk factor, antiphospholipid antibodies, and a placental trigger, necrotic trophoblastic debris, on the activation of endothelial cells. Trophoblastic debris, from placental explants, was induced to become necrotic by freeze-thawing then exposed to endothelial cells for 24 h. After washing away residual trophoblastic debris antiphospholipid antibodies or a control antibody were added to the cultures then replaced with fresh medium in the presence or absence of antibodies. Endothelial cell activation was quantified by examining cell-surface ICAM-1 expression and monocyte adhesion. Endothelial cells exposed to necrotic trophoblastic debris for 24 h became activated but the activation was lost 24 h after removal of the debris. Antiphospholipid antibodies alone did not active untreated endothelial cells, but did prolong the activation of endothelial cells which had been activated by pre-treatment with necrotic trophoblastic debris. When exposed to antiphospholipid antibodies the endothelial cells remained activated despite removal of the trophoblastic debris. In contrast, a control antibody did not prolong endothelial cell activation. Our data suggest that in women with antiphospholipid antibodies, activation of endothelial cells induced by necrotic trophoblastic debris could be maintained even if the endothelial cells were only intermittently exposed to necrotic debris. This might in part explain why antiphospholipid antibodies are such a strong maternal risk factor for preeclampsia. PMID:22902008

  2. Activated T Cell Trans-Endothelial Migration Relies on Myosin-IIA Contractility for Squeezing the Cell Nucleus through Endothelial Cell Barriers

    PubMed Central

    Jacobelli, Jordan; Estin Matthews, Miriam; Chen, Stephanie; Krummel, Matthew F.

    2013-01-01

    Following activation, T cells are released from lymph nodes to traffic via the blood to effector sites. The re-entry of these activated T cells into tissues represents a critical step for them to carry out local effector functions. Here we have assessed defects in effector T cells that are acutely depleted in Myosin-IIA (MyoIIA) and show a T cell intrinsic requirement for this motor to facilitate the diapedesis step of extravasation. We show that MyoIIA accumulates at the rear of T cells undergoing trans-endothelial migration. T cells can extend protrusions and project a substantial portion of their cytoplasm through the endothelial wall in the absence of MyoIIA. However, this motor protein plays a crucial role in allowing T cells to complete the movement of their relatively rigid nucleus through the endothelial junctions. In vivo, this defect manifests as poor entry into lymph nodes, tumors and into the spinal cord, during tissue-specific autoimmunity, but not the spleen. This suggests that therapeutic targeting of this molecule may allow for differential attenuation of tissue-specific inflammatory responses. PMID:24069389

  3. When the endothelium scores an own goal: endothelial cells actively augment metastatic extravasation through endothelial-mesenchymal transition.

    PubMed

    Gasparics, Ákos; Rosivall, László; Krizbai, István A; Sebe, Attila

    2016-05-01

    Endothelial-mesenchymal transition (EndMT) is an important mechanism during organ development and in certain pathological conditions. For example, EndMT contributes to myofibroblast formation during organ fibrosis, and it has been identified as an important source of cancer-associated fibroblasts, facilitating tumor progression. Recently, EndMT was proposed to modulate endothelial function during intravasation and extravasation of metastatic tumor cells. Evidence suggests that endothelial cells are not passive actors during transendothelial migration (TEM) of cancer cells, as there are profound changes in endothelial junctional protein expression, signaling, permeability, and contractility. This review describes these alterations in endothelial characteristics during TEM of metastatic tumor cells and discusses them in the context of EndMT. EndMT could play an important role during metastatic intravasation and extravasation, a novel hypothesis that may lead to new therapeutic approaches to tackle metastatic disease. PMID:26993222

  4. Cancer cell-associated fatty acid synthase activates endothelial cells and promotes angiogenesis in colorectal cancer.

    PubMed

    Zaytseva, Yekaterina Y; Elliott, Victoria A; Rychahou, Piotr; Mustain, W Conan; Kim, Ji Tae; Valentino, Joseph; Gao, Tianyan; O'Connor, Kathleen L; Neltner, Janna M; Lee, Eun Y; Weiss, Heidi L; Evers, B Mark

    2014-06-01

    Upregulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation are unknown. Since angiogenesis is crucial for metastasis, we investigated the role of FASN in the neovascularization of CRC. The effect of FASN on tumor vasculature was studied in orthotopic CRCs, the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models using immunohistochemistry, immunofluorescent staining and confocal microscopy. Cell secretion was evaluated by ELISA and antibody arrays. Proliferation, migration and tubulogenesis of endothelial cells (ECs) were assessed in CRC-EC coculture models. In this study, we found that stable knockdown of FASN decreased microvessel density in HT29 and HCT116 orthotopic CRCs and resulted in 'normalization' of tumor vasculature in both orthotopic and CAM models. Furthermore, FASN regulated secretion of pro- and antiangiogenic factors, including vascular endothelial growth factor-A (VEGF-A). Mechanisms associated with the antiangiogenic activity noted with knockdown of FASN included: downregulation of VEGF(189), upregulation of antiangiogenic isoform VEGF(165b) and a decrease in expression and activity of matrix metalloproteinase-9. Furthermore, conditioned medium from FASN knockdown CRC cells inhibited activation of vascular endothelial growth factor receptor-2 and its downstream signaling and decreased proliferation, migration and tubulogenesis of ECs as compared with control medium. Together, these results suggest that cancer cell-associated FASN regulates tumor vasculature through alteration of the profile of secreted angiogenic factors and regulation of their bioavailability. Inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC. PMID:24510238

  5. Cancer cell-associated fatty acid synthase activates endothelial cells and promotes angiogenesis in colorectal cancer

    PubMed Central

    Evers, B.Mark

    2014-01-01

    Upregulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation are unknown. Since angiogenesis is crucial for metastasis, we investigated the role of FASN in the neovascularization of CRC. The effect of FASN on tumor vasculature was studied in orthotopic CRCs, the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models using immunohistochemistry, immunofluorescent staining and confocal microscopy. Cell secretion was evaluated by ELISA and antibody arrays. Proliferation, migration and tubulogenesis of endothelial cells (ECs) were assessed in CRC–EC coculture models. In this study, we found that stable knockdown of FASN decreased microvessel density in HT29 and HCT116 orthotopic CRCs and resulted in ‘normalization’ of tumor vasculature in both orthotopic and CAM models. Furthermore, FASN regulated secretion of pro- and antiangiogenic factors, including vascular endothelial growth factor-A (VEGF-A). Mechanisms associated with the antiangiogenic activity noted with knockdown of FASN included: downregulation of VEGF189, upregulation of antiangiogenic isoform VEGF165b and a decrease in expression and activity of matrix metalloproteinase-9. Furthermore, conditioned medium from FASN knockdown CRC cells inhibited activation of vascular endothelial growth factor receptor-2 and its downstream signaling and decreased proliferation, migration and tubulogenesis of ECs as compared with control medium. Together, these results suggest that cancer cell-associated FASN regulates tumor vasculature through alteration of the profile of secreted angiogenic factors and regulation of their bioavailability. Inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC. PMID:24510238

  6. Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation

    PubMed Central

    Zhou, Tao; You, Wen-ting; Ma, Zeng-chun; Liang, Qian-de; Tan, Hong-ling; Xiao, Cheng-rong; Tang, Xiang-lin; Zhang, Bo-li; Wang, Yu-guang; Gao, Yue

    2016-01-01

    Aim: Pregnane X receptor (PXR) is a nuclear receptor that regulates a number of genes encoding drug metabolism enzymes and transporters and plays a key role in xeno- and endobiotic detoxification. Ginkgolide B has shown to increase the activity of PXR. Here we examined whether ginkgolide B activated PXR and attenuated xenobiotic-induced injuries in endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with ginkgolide B. The expression of PXR, CYP3A4, MDR1, VCAM-1, E-selectin and caspase-3 were quantified with qRT-PCR and Western blot analysis. Cell apoptosis was analyzed with flow cytometry. Fluorescently labeled human acute monocytic leukemia cells (THP-1 cells) were used to examine cell adhesion. Results: Ginkgolide B (30–300 μmol/L) did not change the mRNA and protein levels of PXR in the cells, but dose-dependently increased nuclear translocation of PXR protein. Ginkgolide B increased the expression of CYP3A4 and MDR1 in the cells, which was partially reversed by pretreatment with the selective PXR signaling antagonist sulforaphane, or transfection with PXR siRNA. Functionally, ginkgolide B dose-dependently attenuated doxorubicin- or staurosporine-induced apoptosis, which was reversed by transfection with PXR siRNA. Moreover, ginkgolide B suppressed TNF-α-induced THP-1 cell adhesion and TNF-α-induced expression of vascular adhesion molecule 1 (VCAM-1) and E-selectin in the cells, which was also reversed by transfection with PXR siRNA. Conclusion: Ginkgolide B exerts anti-apoptotic and anti-inflammatory effects on endothelial cells via PXR activation, suggesting that a PXR-mediated endothelial detoxification program may be important for protecting endothelial cells from xeno- and endobiotic-induced injuries. PMID:26775663

  7. Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

    PubMed

    Fleming, Ingrid; Fisslthaler, Beate; Dixit, Madhulika; Busse, Rudi

    2005-09-15

    The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway. PMID:16118242

  8. Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells

    PubMed Central

    Reinhard, Nathalie R.; van Helden, Suzanne F.; Anthony, Eloise C.; Yin, Taofei; Wu, Yi I.; Goedhart, Joachim; Gadella, Theodorus W. J.; Hordijk, Peter L.

    2016-01-01

    Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells. PMID:27147504

  9. Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

    PubMed

    Reinhard, Nathalie R; van Helden, Suzanne F; Anthony, Eloise C; Yin, Taofei; Wu, Yi I; Goedhart, Joachim; Gadella, Theodorus W J; Hordijk, Peter L

    2016-01-01

    Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells. PMID:27147504

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

    PubMed

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

    2016-10-01

    Endothelial cell activation resulting from biomaterial contact or biomaterial-induced blood activation may in turn also affect hemostasis and inflammatory processes in the blood. Current in vitro hemocompatibility assays typically ignore these modulating effects of the endothelium. This study describes a co-incubation system of human whole blood, biomaterial and endothelial cells (ECs) that was developed to overcome this limitation. First, human endothelial cells were characterized in terms of their expression of coagulation- and inflammation-relevant markers in response to various activators. Subsequently, their capacity to regulate hemostasis as well as complement and granulocyte activation was monitored in a hemocompatibility assay. After blood contact, quiescent ECs exhibited anticoagulant and anti-inflammatory properties. When they were co-incubated with surfaces exhibiting pro-coagulant or pro-inflammatory characteristics, the ECs down-regulated coagulation but not complement or leukocyte activation. Analysis of intracellular levels of the endothelial activation markers E-selectin and tissue factor showed that co-incubation with model surfaces and blood significantly increased the activation state of ECs. Finally, the coagulation- and inflammation-modulating properties of the ECs were tested after blood/biomaterial exposure. Pre-activation of ECs by biomaterials in the blood induced a pro-coagulant and pro-inflammatory state of the ECs, wherein the pro-coagulant response was higher for biomaterial/blood pre-activated ECs than for TNF-α-pre-activated cells. This work provides evidence that biomaterials, even without directly contacting the endothelium, affect the endothelial activation state with and have consequences for plasmatic and cellular reactions in the blood. PMID:27472163

  11. Endothelial cell activation by antiphospholipid antibodies is modulated by Krüppel-like transcription factors

    PubMed Central

    Allen, Kristi L.; Hamik, Anne; Jain, Mukesh K.

    2011-01-01

    Antiphospholipid syndrome is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLAs). The majority of APLAs are directed against phospholipid-binding proteins, particularly β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner through a pathway that involves NF-κB. Krüppel-like factors (KLFs) play a critical role in regulating the endothelial response to inflammatory stimuli. We hypothesized that activation of endothelial cells by APLA/anti-β2GPI antibodies might be associated with decreased expression of KLFs, which in turn might facilitate cellular activation mediated through NF-κB. Our experimental results confirmed this hypothesis, demonstrating markedly decreased expression of KLF2 and KLF4 after incubation of cells with APLA/anti-β2GPI antibodies. Restoration of KLF2 or KLF4 levels inhibited NF-κB transcriptional activity and blocked APLA/anti-β2GPI–mediated endothelial activation despite NF-κB p65 phosphorylation. Chromatin immunoprecipitation analysis demonstrated that inhibition of NF-κB transcriptional activity by KLFs reflects sequestration of the cotranscriptional activator CBP/p300, making this cofactor unavailable to NF-κB. These findings suggest that the endothelial response to APLA/anti-β2GPI antibodies reflects competition between KLFs and NF-κB for their common cofactor, CBP/p300. Taken together, these observations are the first to implicate the KLFs as novel participants in the endothelial proinflammatory response to APLA/anti-β2GPI antibodies. PMID:21482710

  12. GAP JUNCTION COMMUNICATION MEDIATES TRANSFORMING GROWTH FACTOR-BETA ACTIVATION AND ENDOTHELIAL-INDUCED MURAL CELL DIFFERENTIATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-beta (TGF-beta) activation. We investigated whether gap junction channels are formed between endothelial cells and recrui...

  13. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    SciTech Connect

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-04-15

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O{sub 2}{sup {center_dot}}{sup -} generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67{sup phox} siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91{sup phox} knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47{sup phox}, p67{sup phox} and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67{sup phox} siRNA. Exposure of MPMVEC obtained from gp91{sup phox} knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly

  14. Androgen Modulates Functions of Endothelial Progenitor Cells through Activated Egr1 Signaling

    PubMed Central

    Ye, Yizhou; Li, Xizhe; Zhang, You; Shen, Zhenya; Yang, Junjie

    2016-01-01

    Researches show that androgens have important effects on migration of endothelial cells and endothelial protection in coronary heart disease. Endothelial progenitor cells (EPCs) as a progenitor cell type that can differentiate into endothelial cells, have a critical role in angiogenesis and endothelial protection. The relationship between androgen and the functions of EPCs has animated much interest and controversy. In this study, we investigated the angiogenic and migratory functions of EPCs after treatment by dihydrotestosterone (DHT) and the molecular mechanisms as well. We found that DHT treatment enhanced the incorporation of EPCs into tubular structures formed by HUVECs and the migratory activity of EPCs in the transwell assay dose dependently. Moreover, microarray analysis was performed to explore how DHT changes the gene expression profiles of EPCs. We found 346 differentially expressed genes in androgen-treated EPCs. Angiogenesis-related genes like Egr-1, Vcan, Efnb2, and Cdk2ap1 were identified to be regulated upon DHT treatment. Furthermore, the enhanced angiogenic and migratory abilities of EPCs after DHT treatment were inhibited by Egr1-siRNA transfection. In conclusion, our findings suggest that DHT markedly enhances the vessel forming ability and migration capacity of EPCs. Egr1 signaling may be a possible pathway in this process. PMID:26697079

  15. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  16. Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

    PubMed

    Kumar Vr, Santhosh; Darisipudi, Murthy N; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P; Mulay, Shrikant R; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D; Lindenmeyer, Maja T; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido; Anders, Hans-Joachim

    2016-06-01

    Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases. PMID:26567242

  17. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    PubMed

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression. PMID:16651461

  18. Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Whitson, P. A.

    1993-01-01

    Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.

  19. Recombinant Treponema pallidum Protein Tp0965 Activates Endothelial Cells and Increases the Permeability of Endothelial Cell Monolayer

    PubMed Central

    Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

    2014-01-01

    The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis. PMID:25514584

  20. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    PubMed

    Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

    2014-01-01

    The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis. PMID:25514584

  1. Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia.

    PubMed

    McDonald, Braedon; Jenne, Craig N; Zhuo, Lisheng; Kimata, Koji; Kubes, Paul

    2013-12-01

    A key pathological feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4(-/-)Tie2(tlr4)), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan-associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver. PMID:24113769

  2. Maggot debridement therapy promotes diabetic foot wound healing by up-regulating endothelial cell activity.

    PubMed

    Sun, Xinjuan; Chen, Jin'an; Zhang, Jie; Wang, Wei; Sun, Jinshan; Wang, Aiping

    2016-03-01

    To determine the role of maggot debridement therapy (MDT) on diabetic foot wound healing, we compared growth related factors in wounds before and after treatment. Furthermore, we utilized human umbilical vein endothelial cells (HUVECs) to explore responses to maggot excretions/secretions on markers of angiogenesis and proliferation. The results showed that there was neo-granulation and angiogenesis in diabetic foot wounds after MDT. Moreover, significant elevation in CD34 and CD68 levels was also observed in treated wounds. In vitro, ES increased HUVEC proliferation, improved tube formation, and increased expression of vascular endothelial growth factor receptor 2 in a dose dependent manner. These results demonstrate that MDT and maggot ES can promote diabetic foot wound healing by up-regulating endothelial cell activity. PMID:26782021

  3. Ionizing Radiation Shifts the PAI-1/ID-1 Balance and Activates Notch Signaling in Endothelial Cells

    SciTech Connect

    Scharpfenecker, Marion; Kruse, Jacqueline; Sprong, Debbie; Russell, Nicola S.; Dijke, Peter ten; Stewart, Fiona A.

    2009-02-01

    Purpose: Transforming growth factor-{beta} (TGF-{beta}) and Notch signaling pathways are important regulators of vascular homeostasis and vessel remodeling; mutations in these pathways can lead to vascular disorders. Similar vascular phenotypes develop in the normal tissues of cancer patients as a long-term effect of radiotherapy. Irradiation most severely affects the capillaries, which become leaky and dilated and might eventually rupture. To investigate the mechanism of such capillary damage, we studied the effect of TGF-{beta} and Notch signaling in microvascular endothelial cells. Methods and Materials: Human microvascular endothelial cells were irradiated with 5 or 10 Gy and activation of TGF-{beta} and Notch signaling pathways was assessed by biochemical methods and a cell migration assay. Results: Ionizing radiation induced Smad2 phosphorylation and nuclear translocation and increased mRNA and protein expression of the activin-like kinase 5 (ALK5) target gene plasminogen activator inhibitor-1 (PAI-1). At the same time, we observed diminished Smad1/5/8 activation and downregulation of the ALK1 downstream target, inhibitor of DNA binding-1 (ID-1). We also measured an upregulation of the Notch ligand Jagged-1 and the target gene Hey1. Decreased inhibitor of DNA binding-1 levels coincided with a reduced ability of the cells to migrate. Conclusion: Ionizing radiation shifts the balance from ALK1 to ALK5 signaling and activates the Notch pathway in endothelial cells. This combination of anti-angiogenic signals contributes to reduced cell migration after irradiation.

  4. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  5. Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells.

    PubMed

    Kwok, Willson; Lee, Sang Ho; Culberson, Cathy; Korneszczuk, Katarzyna; Clemens, Mark G

    2009-11-01

    Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs. PMID:20501440

  6. Borane-protected phosphines are redox-active radioprotective agents for endothelial cells.

    PubMed

    Crowe, Megan E; Lieven, Christopher J; Thompson, Alex F; Sheibani, Nader; Levin, Leonard A

    2015-12-01

    Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine-borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a (137)Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1 µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1 µM vs. 4 mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity. PMID:26188467

  7. Borane-protected phosphines are redox-active radioprotective agents for endothelial cells

    PubMed Central

    Crowe, Megan E.; Lieven, Christopher J.; Thompson, Alex F.; Sheibani, Nader; Levin, Leonard A.

    2015-01-01

    Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine–borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a 137Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1 µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1 µM vs. 4 mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity. PMID:26188467

  8. Formyl Peptide Receptor Activation Elicits Endothelial Cell Contraction and Vascular Leakage.

    PubMed

    Wenceslau, Camilla F; McCarthy, Cameron G; Webb, R Clinton

    2016-01-01

    The major pathophysiological characteristic of systemic inflammatory response syndrome (SIRS) and sepsis is the loss of control of vascular tone and endothelial barrier dysfunction. These changes are attributed to pro-inflammatory mediators. It has been proposed that in patients and rats without infection, cell components from damaged tissue are the primary instigators of vascular damage. Mitochondria share several characteristics with bacteria, and when fragments of mitochondria are released into the circulation after injury, they are recognized by the innate immune system. N-Formyl peptides are common molecular signatures of bacteria and mitochondria and are known to play a role in the initiation of inflammation by activating the formyl peptide receptor (FPR). We have demonstrated that infusion of mitochondrial N-formyl peptides (F-MIT) leads to sepsis-like symptoms, including vascular leakage. We have also observed that F-MIT, via FPR activation, elicits changes in cytoskeleton-regulating proteins in endothelial cells. Therefore, we hypothesize that these FPR-mediated changes in cytoskeleton can cause endothelial cell contraction and, consequently vascular leakage. Here, we propose that endothelial FPR is a key contributor to impaired barrier function in SIRS and sepsis patients following trauma. PMID:27532003

  9. Formyl Peptide Receptor Activation Elicits Endothelial Cell Contraction and Vascular Leakage

    PubMed Central

    Wenceslau, Camilla F.; McCarthy, Cameron G.; Webb, R. Clinton

    2016-01-01

    The major pathophysiological characteristic of systemic inflammatory response syndrome (SIRS) and sepsis is the loss of control of vascular tone and endothelial barrier dysfunction. These changes are attributed to pro-inflammatory mediators. It has been proposed that in patients and rats without infection, cell components from damaged tissue are the primary instigators of vascular damage. Mitochondria share several characteristics with bacteria, and when fragments of mitochondria are released into the circulation after injury, they are recognized by the innate immune system. N-Formyl peptides are common molecular signatures of bacteria and mitochondria and are known to play a role in the initiation of inflammation by activating the formyl peptide receptor (FPR). We have demonstrated that infusion of mitochondrial N-formyl peptides (F-MIT) leads to sepsis-like symptoms, including vascular leakage. We have also observed that F-MIT, via FPR activation, elicits changes in cytoskeleton-regulating proteins in endothelial cells. Therefore, we hypothesize that these FPR-mediated changes in cytoskeleton can cause endothelial cell contraction and, consequently vascular leakage. Here, we propose that endothelial FPR is a key contributor to impaired barrier function in SIRS and sepsis patients following trauma. PMID:27532003

  10. Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions.

    PubMed

    Bao, Xiaoping; Lian, Xiaojun; Palecek, Sean P

    2016-01-01

    Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined, growth factor- and serum-free system by temporal modulation of Wnt/β-catenin signaling via small molecules. We demonstrate a 10-day, two-stage process that recapitulates endothelial cell development, in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. PMID:27590162

  11. TIMP-2 Modulates VEGFR-2 Phosphorylation and Enhances Phosphodiesterase Activity in Endothelial Cells

    PubMed Central

    Lee, Seo-Jin; Tsang, Patricia; Diaz, Tere; Wei, Bei-yang; Stetler-Stevenson, William George

    2010-01-01

    In the present study we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. In order to focus on metalloproteinase-independent mechanisms, we utilized the TIMP-2 analog known as Ala+TIMP-2 that is deficient in matrix metalloproteinase (MMP) inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 following VEGF-A stimulation and disrupts the downstream activation of PLC-γ, Ca+2 flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation and downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity. PMID:20084057

  12. Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

    PubMed

    Burns, E H; Marciel, A M; Musser, J M

    1996-11-01

    Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection. PMID:8890235

  13. Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

    PubMed Central

    Burns, E H; Marciel, A M; Musser, J M

    1996-01-01

    Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection. PMID:8890235

  14. Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

    PubMed Central

    Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

    2016-01-01

    Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

  15. Collagen-binding VEGF mimetic peptide: Structure, matrix interaction, and endothelial cell activation

    NASA Astrophysics Data System (ADS)

    Chan, Tania R.

    Long term survival of artificial tissue constructs depends greatly on proper vascularization. In nature, differentiation of endothelial cells and formation of vasculature are directed by dynamic spatio-temporal cues in the extracellular matrix that are difficult to reproduce in vitro. In this dissertation, we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF), which can be used to encode spatially controlled angiogenic signals in collagen-based scaffolds. The peptide, QKCMP, contains a collagen mimetic domain (CMP) that binds to type I collagen by a unique triple helix hybridization mechanism and a VEGF mimetic domain (QK) with pro-angiogenic activity. We demonstrate QKCMP's ability to hybridize with native and heat denatured collagens through a series of binding studies on collagen and gelatin substrates. Circular dichroism experiments show that the peptide retains the triple helical structure vital for collagen binding, and surface plasmon resonance study confirms the molecular interaction between the peptide and collagen strands. Cell culture studies demonstrate QKCMP's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. We also show that the peptide can be used to spatially modify collagen-based substrates to promote localized endothelial cell activation and network formation. To probe the biological events that govern these angiogenic cellular responses, we investigated the cell signaling pathways activated by collagen-bound QKCMP and determined short and long-term endothelial cell response profiles for p38, ERK1/2, and Akt signal transduction cascades. Finally, we present our efforts to translate the peptide's in vitro bioactivity to an in vivo burn injury animal model. When implanted at the wound site, QKCMP functionalized biodegradable hydrogels induce enhanced neovascularization in the granulation tissue. The results show QKCMP

  16. Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

    PubMed Central

    Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

    2013-01-01

    Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by

  17. Eugenol dilates mesenteric arteries and reduces systemic BP by activating endothelial cell TRPV4 channels

    PubMed Central

    Peixoto-Neves, Dieniffer; Wang, Qian; Leal-Cardoso, Jose H; Rossoni, Luciana V; Jaggar, Jonathan H

    2015-01-01

    Background and Purpose Eugenol, a vanilloid molecule found in some dietary plants, relaxes vasculature in part via an endothelium-dependent process; however, the mechanisms involved are unclear. Here, we investigated the endothelial cell-mediated mechanism by which eugenol modulates rat mesenteric artery contractility and systemic BP. Experimental Approach The isometric tension of rat mesenteric arteries (size 200–300 μm) was measured using wire myography; non-selective cation currents (ICat) were recorded in endothelial cells using patch clamp electrophysiology. Mean arterial pressure (MAP) and heart rate (HR) were determined in anaesthetized rats. Key Results Eugenol relaxed endothelium-intact arteries in a concentration-dependent manner and this effect was attenuated by endothelium denudation. L-NAME, a NOS inhibitor, a combination of TRAM-34 and apamin, selective blockers of intermediate and small conductance Ca2+-activated K+ channels, respectively, and HC-067047, a TRPV4 channel inhibitor, but not indomethacin, a COX inhibitor, reduced eugenol-induced relaxation in endothelium-intact arteries. Eugenol activated HC-067047-sensitive ICat in mesenteric artery endothelial cells. Short interfering RNA (siRNA)-mediated TRPV4 knockdown abolished eugenol-induced ICat activation. An i.v. injection of eugenol caused an immediate, transient reduction in both MAP and HR, which was followed by prolonged, sustained hypotension in anaesthetized rats. This sustained hypotension was blocked by HC-067047. Conclusions and Implications Eugenol activates TRPV4 channels in mesenteric artery endothelial cells, leading to vasorelaxation, and reduces systemic BP in vivo. Eugenol may be therapeutically useful as an antihypertensive agent and is a viable molecular candidate from which to develop second-generation TRPV4 channel activators that reduce BP. PMID:25832173

  18. The antiangiogenic compound aeroplysinin-1 induces apoptosis in endothelial cells by activating the mitochondrial pathway.

    PubMed

    Martínez-Poveda, Beatriz; Rodríguez-Nieto, Salvador; García-Caballero, Melissa; Medina, Miguel-Ángel; Quesada, Ana R

    2012-09-01

    Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo. In this work, we provide evidence of a selective induction of apoptosis by aeroplysinin-1 in endothelial cells. Studies on the nuclear morphology of treated cells revealed that aeroplysinin-1 induces chromatin condensation and nuclear fragmentation, and it increases the percentage of cells with sub-diploid DNA content in endothelial, but not in HCT-116, human colon carcinoma and HT-1080 human fibrosarcoma cells. Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism. Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound. The observation that aeroplysinin-1 prevents the phosphorylation of Bad relates to the mitochondria-mediated induction of apoptosis by this compound. PMID:23118719

  19. Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

    SciTech Connect

    Hernandez-Montes, Eva; Pollard, Susan E.; Vauzour, David; Jofre-Montseny, Laia; Rota, Cristina; Rimbach, Gerald; Weinberg, Peter D.; Spencer, Jeremy P.E. . E-mail: j.p.e.spencer@reading.ac.uk

    2006-08-04

    Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of {gamma}-glutamylcysteine synthetase-heavy subunit ({gamma}-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.

  20. [AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism].

    PubMed

    Bai, Hong-Bo; Wang, Yun; Zhang, Yu-Hua; Zhang, Yuan

    2016-02-25

    The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost

  1. The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

    PubMed Central

    Zhang, Jing-Chuan; Doñate, Fernando; Qi, Xiaoping; Ziats, Nicholas P.; Juarez, Jose C.; Mazar, Andrew P.; Pang, Yuan-Ping; McCrae, Keith R.

    2002-01-01

    Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin. PMID:12196635

  2. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Wyroba, Elzbieta; Papiewska-Pajak, Izabela; Cierniewski, Czeslaw S

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells. PMID:23474086

  3. Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase.

    PubMed

    Bevan, Heather S; Slater, Sadie C; Clarke, Hayley; Cahill, Paul A; Mathieson, Peter W; Welsh, Gavin I; Satchell, Simon C

    2011-10-01

    Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall. PMID:21775480

  4. Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

    PubMed

    Zhang, J C; Claffey, K; Sakthivel, R; Darzynkiewicz, Z; Shaw, D E; Leal, J; Wang, Y C; Lu, F M; McCrae, K R

    2000-12-01

    We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule. PMID:11099478

  5. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    PubMed Central

    Askari, Ara A.; Thomson, Scott; Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C.; Bishop-Bailey, David

    2014-01-01

    The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity. PMID:24631907

  6. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    SciTech Connect

    Onuma, Hirohisa; Inukai, Kouichi Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  7. Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

    PubMed

    Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

    2016-04-15

    Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts. PMID:26851257

  8. Regulation of NOD-like receptors and inflammasome activation in cerebral endothelial cells.

    PubMed

    Nagyőszi, Péter; Nyúl-Tóth, Ádám; Fazakas, Csilla; Wilhelm, Imola; Kozma, Mihály; Molnár, Judit; Haskó, János; Krizbai, István A

    2015-11-01

    Cerebral endothelial cells (CECs) forming the blood-brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen- and damage-associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD-like receptors (NLRs) - including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX - in human brain endothelial cells. Inflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and IL-1β had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase-cleaved interleukins IL-1β and IL-33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL-1β secretion in a caspase- and ERK1/2 kinase-dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood-brain barrier in neuroimmune and neuroinflammatory processes. PMID:26083549

  9. Endothelial cell activation promotes foam cell formation by monocytes following transendothelial migration in an in vitro model

    PubMed Central

    Westhorpe, Clare L. V.; Dufour, Eric M.; Maisa, Anna; Jaworowski, Anthony; Crowe, Suzanne M.; Muller, William A.

    2012-01-01

    Foam cells are a pathological feature present at all stages of atherosclerosis. Foam cells develop from monocytes that enter the nascent atheroma and subsequently ingest modified low density lipoproteins (LDL). The regulation of this process has previously been studied in vitro using cultured macrophages fed modified LDL. We used our existing in vitro model of transendothelial migration (TEM) to study this process in a more physiologically relevant setting. In our model, monocytes undergo TEM across a primary endothelial monolayer into an underlying three-dimensional collagen matrix in the presence of 20% human serum. Foam cells were detected by Oil Red O staining for intracellular lipid droplets. We demonstrate that sub-endothelial monocytes can develop into foam cells within 48 hours of TEM across TNF-α activated endothelium, in the absence of additional lipids. Our data indicate a role for both monocyte-endothelial interactions and soluble factors in the regulation of foam cell development, including oxidation of LDL in situ from lipid present in culture medium following TNF-α stimulation of the endothelial cells. Our study provides a simple model for investigating foam cell development in vitro that mimics cell migration in vivo, and demonstrates the critical role of inflammation in regulating early atherogenic events. PMID:22609311

  10. Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

    NASA Astrophysics Data System (ADS)

    Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

    2014-04-01

    To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

  11. TNFα-Damaged-HUVECs Microparticles Modify Endothelial Progenitor Cell Functional Activity

    PubMed Central

    Luna, Carlos; Carmona, Andrés; Alique, Matilde; Carracedo, Julia; Ramirez, Rafael

    2015-01-01

    Endothelial progenitor cells (EPCs) have an important role in the maintenance of vascular integrity and homeostasis. While there are many studies that explain EPCs mechanisms action, there are few studies that demonstrate how they interact with other emerging physiological elements such as Endothelial Microparticles (EMPs). EMPs are membranous structures with a size between 100 and 1000 nm that act as molecular information transporter in biological systems and are known as an important elements in develop different pathologies; moreover a lot of works explains that are novel biomarkers. To elucidate these interactions, we proposed an in vitro model of endothelial damage mediated by TNFalpha, in which damaged EMPs and EPCs are in contact to assess EPCs functional effects. We have observed that damaged EMPs can modulate several EPCs classic factors as colony forming units (CFUs), contribution to repair a physically damaged endothelium (wound healing), binding to mature endothelium, and co-adjuvants to the formation of new vessels in vitro (angiogenesis). All of these in a dose-dependent manner. Damaged EMPs at a concentration of 103 MPs/ml have an activating effect of these capabilities, while at concentrations of 105 MPs/ml these effects are attenuated or reduced. This in vitro model helps explain that in diseases where there is an imbalance between these two elements (EPCs and damaged EMPs), the key cellular elements in the regeneration and maintenance of vascular homeostasis (EPCs) are not fully functional, and could explain, at least in part, endothelial dysfunction associated in various pathologies. PMID:26733886

  12. Endothelial RIG-I activation impairs endothelial function

    SciTech Connect

    Asdonk, Tobias; Nickenig, Georg; Zimmer, Sebastian

    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.

  13. KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    PubMed Central

    Wang, Yinan; Yang, Chuanhe; Gu, Qingqing; Sims, Michelle; Gu, Weiwang; Pfeffer, Lawrence M.; Yue, Junming

    2015-01-01

    The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in regulating cell proliferation, migration and differentiation in a variety of human cells and is one of four factors required for the induction of pluripotent stem cell reprogramming. However, its role has not been addressed in ocular neovascular diseases. This study investigated the role of KLF4 in angiogenesis and underlying molecular mechanisms in human retinal microvascular endothelial cells (HRMECs). The functional role of KLF4 in HRMECs was determined following lentiviral vector mediated inducible expression and shRNA knockdown of KLF4. Inducible expression of KLF4 promotes cell proliferation, migration and tube formation. In contrast, silencing KLF4 inhibits cell proliferation, migration, tube formation and induces apoptosis in HRMECs. KLF4 promotes angiogenesis by transcriptionally activating VEGF expression, thus activating the VEGF signaling pathway in HRMECs. PMID:26075898

  14. T-kininogen inhibits kinin-mediated activation of ERK in endothelial cells.

    PubMed

    Leiva-Salcedo, Elias; Perez, Viviana; Acuña-Castillo, Claudio; Walter, Robin; Sierra, Felipe

    2002-01-01

    Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo. PMID:12415746

  15. Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells.

    PubMed

    Nagarajan, Shunmugan; Raj, Rajendran Kadarkarai; Saravanakumar, Venkatesan; Balaguru, Uma Maheswari; Behera, Jyotirmaya; Rajendran, Vinoth Kumar; Shathya, Yogarajan; Ali, B Mohammed Jaffar; Sumantran, Venil; Chatterjee, Suvro

    2016-01-01

    Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2', 7'-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks. PMID:27345770

  16. Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells

    PubMed Central

    Nagarajan, Shunmugan; Raj, Rajendran Kadarkarai; Saravanakumar, Venkatesan; Balaguru, Uma Maheswari; Behera, Jyotirmaya; Rajendran, Vinoth Kumar; Shathya, Yogarajan; Ali, B. Mohammed Jaffar; Sumantran, Venil; Chatterjee, Suvro

    2016-01-01

    Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2′, 7′-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks. PMID:27345770

  17. R-Ras protein inhibits autophosphorylation of vascular endothelial growth factor receptor 2 in endothelial cells and suppresses receptor activation in tumor vasculature.

    PubMed

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-03-27

    Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis. PMID:25645912

  18. Human umbilical cord blood mononuclear cells activate the survival protein Akt in cardiac myocytes and endothelial cells that limits apoptosis and necrosis during hypoxia.

    PubMed

    Henning, Robert J; Dennis, Steve; Sawmiller, Darrell; Hunter, Lorynn; Sanberg, Paul; Miller, Leslie

    2012-06-01

    We have previously reported that human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic, mesenchymal, and endothelial stem cells, can significantly reduce acute myocardial infarction size. To determine the mechanism whereby HUCBC increase myocyte and vascular endothelial cell survival, we treated cardiac myocytes and coronary artery endothelial cells in separate experiments with HUCBC plus culture media or culture media alone and subjected the cells to 24 h of hypoxia or normoxia. We then determined in myocytes and endothelial cells activation of the cell survival protein Akt by Western blots. We also determined in these cells apoptosis by annexin V staining and necrosis by propidium iodide staining. Thereafter, we inhibited with API, a specific and sensitive Akt inhibitor, Akt activation in myocytes and endothelial cells cultured with HUCBC during hypoxia and determined cell apoptosis and necrosis. In cells cultured without HUCBC, hypoxia only slightly activated Akt. Moreover, hypoxia increased myocyte apoptosis by ≥ 226% and necrosis by 58% in comparison with myocytes in normoxia. Hypoxic treatment of endothelial cells without HUCBC increased apoptosis by 94% and necrosis by 59%. In contrast, hypoxia did not significantly affect HUCBC. Moreover, in myocyte + HUCBC cultures in hypoxia, HUCBC induced a ≥ 135% increase in myocyte phospho-Akt. Akt activation decreased myocyte apoptosis by 76% and necrosis by 35%. In endothelial cells, HUCBC increased phospho-Akt by 116%. HUCBC also decreased endothelial cell apoptosis by 58% and necrosis by 42%. Inhibition of Akt with API in myocytes and endothelial cells cultured with HUCBC during hypoxia nearly totally prevented the HUCBC-induced decrease in apoptosis and necrosis. We conclude that HUCBC can significantly decrease hypoxia-induced myocyte and endothelial cell apoptosis and necrosis by activating Akt in these cells and in this manner HUCBC can limit myocardial ischemia and injury. PMID

  19. 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.

    PubMed

    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. PMID:27333954

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

    PubMed

    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. PMID:24752501

  1. Anti-atherosclerotic activity of platycodin D derived from roots of Platycodon grandiflorum in human endothelial cells.

    PubMed

    Wu, Jingtao; Yang, Guiwen; Zhu, Wenxing; Wen, Wujun; Zhang, Fumiao; Yuan, Jinduo; An, Liguo

    2012-01-01

    This study examined the effects of platycodin D (PD), a triterpene saponin from the the root of Platycodon grandiflorum A.DC on human umbilical vein endothelial cells (HUVECs) in vitro, which were pre-treated with PD (0.01, 0.15, 0.25 mg/mL), respectively, and treated with 50 mg/L oxidized low-density lipoprotein (oxLDL). The levels of nitric oxide (NO) and malonaldehyde (MAD) in the culture medium, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) mRNA expression in endothelium cells and the adhesion of monocytes to endothelial cells were measured. The results showed that PD increased NO concentration and decreased MDA level induced by oxLDL in the medium of endothelial cells. Moreover, PD significantly inhibited the oxLDL-induced increase in monocyte adhesion to endothelial cells as well as decreasing mRNA expression levels of VCAM-1 and ICAM-1 on these cells. Based on these results, it is suggested that PD is a promising anti-atherosclerotic activity, which is at least in part the result of its increasing NO concentration, reducing the oxLDL-induced cell adhesion molecule expression in endothelial cells and the endothelial adhesion to monocytes. PMID:22863916

  2. STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity

    PubMed Central

    Demaria, Olivier; De Gassart, Aude; Coso, Sanja; Gestermann, Nicolas; Di Domizio, Jeremy; Flatz, Lukas; Gaide, Olivier; Michielin, Olivier; Hwu, Patrick; Petrova, Tatiana V.; Martinon, Fabio; Modlin, Robert L.; Speiser, Daniel E.; Gilliet, Michel

    2015-01-01

    Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma. PMID:26607445

  3. Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

    PubMed Central

    Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

    2016-01-01

    Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

  4. Angiogenesis Interactome and Time Course Microarray Data Reveal the Distinct Activation Patterns in Endothelial Cells

    PubMed Central

    Chu, Liang-Hui; Lee, Esak; Bader, Joel S.; Popel, Aleksander S.

    2014-01-01

    Angiogenesis involves stimulation of endothelial cells (EC) by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the “angiome”) could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A). We used the Short Time-series Expression Miner (STEM) to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME) show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC) and human microvascular EC (MEC). The results show that VEGFR1–VEGFR2 levels are more closely coupled than VEGFR1–VEGFR3 or VEGFR2–VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle. PMID:25329517

  5. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

    PubMed Central

    Wojta, J.; Holzer, M.; Hufnagl, P.; Christ, G.; Hoover, R. L.; Binder, B. R.

    1991-01-01

    The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 37 degrees C for 24 hours as evidenced by continuously increased amounts of PAI-1 released into the conditioned media. t-PA release, however, decreased during the 24-hour period at 37 degrees C after exposure to hyperthermia. No difference in PAI-1 antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 37 degrees C could be found. Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells. Images Figure 6 PMID:1928306

  6. Asymmetric dimethylarginine inhibits HSP90 activity in Pulmonary Arterial Endothelial Cells: Role of Mitochondrial Dysfunction

    PubMed Central

    Sud, Neetu; Wells, Sandra M.; Wiseman, Dean A.; Wilham, Jason; Black, Stephen M.

    2013-01-01

    Increased ADMA levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus, the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that that ADMA increased NOS uncoupling both in recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Further, we found that this eNOS uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of HSP90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states. PMID:18385287

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

    PubMed Central

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

    2016-01-01

    Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised. PMID:27052191

  8. Fatty acids and glucose increase neutral endopeptidase activity in human microvascular endothelial cells.

    PubMed

    Muangman, Pornprom; Spenny, Michelle L; Tamura, Richard N; Gibran, Nicole S

    2003-06-01

    Neutral endopeptidase (NEP), a membrane-bound metallopeptidase enzyme that degrades neuropeptides, bradykinin, atrial natriuretic factor, enkephalins, and endothelin may regulate response to injury. We have previously demonstrated increased NEP localization and enzyme activity in diabetic wounds and skin compared with normal controls. We hypothesized that hyperlipidemia and hyperglycemia associated with type 2 diabetes mellitus may induce excessive NEP activity and thereby diminish normal response to injury. Human microvascular endothelial cells were treated with five different fatty acids (40 microM) with varying degrees of saturation, including oleic acid, linoleic acid, palmitic acid, stearic acid, and linolenic acid and/or glucose (40 mM) for 48 h. The effect of the antioxidative agents vitamin E and C on NEP enzyme activation was determined by treating the cultured cells with alpha-tocopherol succinate and/or L-ascorbic acid. Cell membrane preparations were assayed for NEP activity by incubation with glutaryl-Ala-Ala-Phe-4-methoxy-beta naphthylamide to generate a fluorescent degradation product methoxy 2 naphthylamine. High glucose or fatty acid concentration upregulated NEP activity. The highest NEP activity was observed with combined elevated glucose, linoleic acid, and oleic acid (P < 0.05). Antioxidant vitamin E and C treatment significantly reduced NEP enzyme activity after fatty acid exposure (P < 0.05). Thus, hyperglycemia and hyperlipidemia associated with type 2 diabetes mellitus may increase endothelial cell NEP activity and thereby decrease early pro-inflammatory responses. The modulator effect of vitamin E and C on NEP membrane enzyme activity after exposure to fatty acid stimulation suggests that lipid oxidation may activate NEP. PMID:12785004

  9. Asbestos-induced endothelial cell activation and injury. Demonstration of fiber phagocytosis and oxidant-dependent toxicity.

    PubMed

    Garcia, J G; Gray, L D; Dodson, R F; Callahan, K S

    1988-10-01

    Vascular endothelial cell injury is important in the development of a variety of chronic interstitial lung disorders. However, the involvement of such injury in the inflammatory response associated with the inhalation of asbestos fibers is unclear and the mechanism of asbestos fiber cytotoxicity remains unknown. In the present study, human umbilical vein endothelial cells were challenged with amosite asbestos and several parameters of cellular function were examined. Electron microscopic examination revealed that endothelial cell exposure to asbestos resulted in active phagocytosis of these particulates. Biochemical evidence of dose-dependent asbestos-mediated endothelial cell activation was indicated by increased metabolism of arachidonic acid. For example, amosite asbestos (500 micrograms/ml) produced a ninefold increase in prostacyclin (PGI2) levels over those levels in non-exposed cells. Incubation of human endothelial cells with asbestos fibers induced specific 51Cr release in both a dose- and time-dependent fashion indicative of cellular injury. Injury induced by amosite asbestos was not significantly attenuated by treatment of the endothelial cell monolayer with either the iron chelator deferoxamine, which prevents hydroxyl radical (.OH) formation, or by the superoxide anion (O2-) scavenger, superoxide dismutase. However, significant dose-dependent protection was observed with the hydrogen peroxide (H2O2) scavenger, catalase. Chelation of elemental iron present within amosite asbestos fibers by deferoxamine produced a 33% reduction in asbestos cytotoxicity, suggesting a potential role for hydroxyl radical-mediated injury via the iron-catalyzed Haber-Weiss reaction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3202471

  10. The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells*

    PubMed Central

    Jiang, He; Salmon, Richard M.; Upton, Paul D.; Wei, Zhenquan; Lawera, Aleksandra; Davenport, Anthony P.; Morrell, Nicholas W.; Li, Wei

    2016-01-01

    BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling. PMID:26631724

  11. Hypoxia increases membranal and secreted HLA-DR in endothelial cells, rendering them T-cell activators.

    PubMed

    Lahat, Nitza; Bitterman, Haim; Weiss-Cerem, Lea; Rahat, Michal A

    2011-10-01

    Transplantation involves preoperative ischemic periods that contribute to endothelial cell (EC) dysfunction and T-cell activation, leading to graft rejection. As hypoxia is a major constituent of ischemia, we evaluated its effect on the ability of ECs to express HLA-DR, which is required for presentation of antigens to T cells, and by itself serves as an important target for allogeneic T cells. Primary human umbilical vein ECs (HUVEC) and the human endothelial cell line EaHy926 were incubated in normoxia or hypoxia (PO(2) < 0.3%). Hypoxia increased the membranal expression (by 4-6 fold, P < 0.01) and secretion (by sixfold, P < 0.05) of HLA-DR protein, without influencing the accumulation of its mRNA. Alternative splicing, attenuated trafficking, or shedding from the plasma membrane were not observed, but the lysosomal inhibitor bafilomycin A1 reduced HLA-DR secretion. Hypoxia-induced endothelial HLA-DR elevated and diminished the secretion of IL-2 and IL-10, respectively, from co-cultured allogeneic CD4(+) T cells in a HLA-DR-dependent manner, as demonstrated by the use of monoclonal anti-HLA-DR. Our results indicate a yet not fully understood post-translational mechanism(s), which elevate both membranal and soluble HLA-DR expression. This elevation is involved in allogeneic T-cell activation, highlighting the pivotal role of ECs in ischemia/hypoxia-associated injury and graft rejection. PMID:21806687

  12. Tumor cell-secreted angiogenin induces angiogenic activity of endothelial cells by suppressing miR-542-3p.

    PubMed

    He, Ting; Qi, Feifei; Jia, Lin; Wang, Shan; Wang, Chunying; Song, Nan; Fu, Yan; Li, Lin; Luo, Yongzhang

    2015-11-01

    Therapeutic strategies for targeting angiogenesis have been proven as successful treatments for divergent cancers. We previously discovered an anti-angiogenic miR-542-3p, which directly targeted the key angiogenesis-promoting protein Angiopoietin-2 to inhibit tumor angiogenesis in breast cancer models. In this study, to further investigate the mechanism of miR-542-3p induced angiogenic inhibition, we screened for tumor cell derived factors which were responsible for miR-542-3p alteration in endothelial cells. We found that tumor cell-derived angiogenin downregulated miR-542-3p in endothelial cells. Overexpression of angiogenin in tumor cells facilitated angiogenic activation in both in vitro and in vivo models via inhibition of miR-542-3p. Furthermore, our results showed that angiogenin could suppress CEBPB and POU2F1, which were transcription factors for miR-542-3p, suggesting a novel tumor cell-endothelial cell signal pathway. In addition, the level of angiogenin in primary breast carcinomas correlated with clinical progression. Serum levels of angiogenin were associated with metastatic development of breast cancer patients. Together, these findings reveal a novel regulatory pathway whereby tumor-derived angiogenin directly activates angiogenesis through inhibition of miR-542-3p, suggesting that angiogenin may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression. PMID:26272182

  13. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli

    SciTech Connect

    Boncela, Joanna; Przygodzka, Patrycja; Wyroba, Elzbieta; Papiewska-Pajak, Izabela; Cierniewski, Czeslaw S.

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43 kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells. - Highlights: ► LPS stimulates generation of secretory vesicles containing SerpinB2. ► SerpinB2 concentrates in TGN46 positive vesicles close to the plasma membrane. ► Brefeldin A inhibits secretion of SerpinB2. ► The secreted SerpinB2 was predominantly in its nonglycosylated 43 kDa.

  14. R-Ras inhibits VEGF-induced p38MAPK activation and HSP27 phosphorylation in endothelial cells

    PubMed Central

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2016-01-01

    R-Ras is a Ras family small GTPase highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes, and smooth muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. R-Ras attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and phosphorylation of downstream heat shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion, and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNAi increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  15. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

    PubMed

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-01-01

    R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  16. Endosulfan activates the extrinsic coagulation pathway by inducing endothelial cell injury in rats.

    PubMed

    Zhang, Lianshuang; Wei, Jialiu; Guo, Fangzi; Duan, Junchao; Li, Yanbo; Shi, Zhixiong; Yang, Yumei; Zhou, Xianqing; Sun, Zhiwei

    2015-10-01

    Endosulfan, a persistent organic pollutant, is widely used in agriculture as a pesticide. The aim of the present study was to evaluate the blood toxicity of different doses of endosulfan in Wistar rats. The experimental sample was composed of four groups, a control group that did not receive endosulfan and three endosulfan-exposed groups that respectively received 1, 5, or 10 mg/kg/day (doses below LD50), of endosulfan for 21 days. The results showed that endosulfan significantly decreased the prothrombin time (PT) and upregulated the activated coagulation factors VIIa, Xa, and XIIIa; thrombin-antithrombin complex (TAT); and P-selectin. Plasma levels of tissue factor (TF) and malondialdehyde (MDA) were increased in the endosulfan groups. The activated partial thromboplastin time (APTT) and the level of activated coagulation factor IXa showed no obvious changes. Immunohistochemical results showed increased expression of von Willebrand factor (vWF) and the inflammatory cytokine interleukin (IL)-1β in the groups exposed to endosulfan. The pathology and electron microscopy results showed impaired vascular tissue accompanied by the exfoliation of endothelial cells and mitochondrial damage in the endosulfan-exposed groups. In summary, our results suggest that endosulfan damages endothelial cells via oxidative stress and the inflammatory response, leading to the release of TF and vWF into the blood. The TF and vWF in the blood may activate extrinsic coagulation factors and platelets, thus triggering the extrinsic coagulation pathway. There were no obvious effects on the intrinsic coagulation pathway. PMID:26028348

  17. REDV Peptide Conjugated Nanoparticles/pZNF580 Complexes for Actively Targeting Human Vascular Endothelial Cells.

    PubMed

    Shi, Changcan; Li, Qian; Zhang, Wencheng; Feng, Yakai; Ren, Xiangkui

    2015-09-16

    Herein, we demonstrate that the REDV peptide modified nanoparticles (NPs) can serve as a kind of active targeting gene carrier to condensate pZNF580 for specific promotion of the proliferation of endothelial cells (ECs). First, we synthesized a series of biodegradable amphiphilic copolymers by ring-opening polymerization reaction and graft modification with REDV peptide. Second, we prepared active targeting NPs via self-assembly of the amphiphilic copolymers using nanoprecipitation technology. After condensation with negatively charged pZNF580, the REDV peptide modified NPs/pZNF580 complexes were formed finally. Due to the binding affinity toward ECs of the specific peptide, these REDV peptide modified NPs/pZNF580 complexes could be recognized and adhered specifically by ECs in the coculture system of ECs and human artery smooth muscle cells (SMCs) in vitro. After expression of ZNF580, as the key protein to promote the proliferation of ECs, the relative ZNF580 protein level increased from 15.7% to 34.8%. The specificity in actively targeting ECs of the REDV peptide conjugated NPs/pZNF580 complexes was still retained in the coculture system. These findings in the present study could facilitate the development of actively targeting gene carriers for the endothelialization of artificial blood vessels. PMID:26373583

  18. Angiogenin stimulates endothelial cell prostacyclin secretion by activation of phospholipase A2.

    PubMed Central

    Bicknell, R; Vallee, B L

    1989-01-01

    Angiogenin stimulates capillary and umbilical vein endothelial cell prostacyclin secretion but not that of prostaglandins of the E series. The response was quantitated by radioimmunoassay and by [3H]arachidonate labeling followed by analysis of the secreted prostaglandins. The stimulated secretion lasts for several minutes and is optimal at 2-4 min. The dose-response (peak at 1-10 ng/ml) is similar to that previously observed for activation of endothelial cell phospholipase C. Stimulated secretion was blocked by pretreatment with the inhibitors of prostacyclin synthesis, indomethacin and tranylcypromine, and also the specific inhibitor of phospholipase A2, quinacrine, as well as pertussis toxin and the diglyceryl and monoglyceryl lipase inhibitor RHC 80267. Stimulated secretion was also abolished in cells that were either pretreated for 48 hr with phorbol ester to down-regulate protein kinase C or incubated with the protein kinase inhibitor H7. Hydrolysis of phosphatidylinositol by phospholipase A2 appears to be the source of angiogenin-mobilized arachidonate; angiogenin-induced hydrolysis of phosphatidylcholine was not detected. Activation of phospholipase A2 occurs in the absence of an angiogenin-induced calcium flux. The results are discussed in terms of mechanisms of agonist-induced intracellular arachidonate mobilization and relevance to angiogenesis. PMID:2646638

  19. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1.

    PubMed

    Lin, Chiou-Feng; Chiu, Shu-Chen; Hsiao, Yu-Ling; Wan, Shu-Wen; Lei, Huan-Yao; Shiau, Ai-Li; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Chen, Shun-Hua; Liu, Ching-Chuan; Lin, Yee-Shin

    2005-01-01

    Vascular dysfunction is a hallmark associated with disease onset in dengue hemorrhagic fever and dengue shock syndrome. In addition to direct viral damage, immune responses to dengue virus (DV) infection may also underlie the pathogenesis of disease. We have proposed a mechanism of molecular mimicry in which Abs directed against DV nonstructural protein 1 (NS1) cross-react with endothelial cells and induce damage. In this study, we demonstrated the inflammatory endothelial cell activation induced by anti-DV NS1 via the transcription factor NF-kappaB-regulated pathway. Protein phosphorylation and NF-kappaB activation were observed after anti-DV NS1 stimulation in a human microvascular endothelial cell line-1. The cytokine and chemokine production, including IL-6, IL-8, and MCP-1, but not RANTES, in endothelial cells increased after treatment with anti-DV NS1 Abs. The expression of IL-6, IL-8, and MCP-1 was blocked by the preabsorption of anti-DV NS1 with DV NS1 or by the inhibition of NF-kappaB activation. Furthermore, the increases in both ICAM-1 expression and the ability of human PBMC to adhere to endothelial cells were also observed, and these effects were inhibited by pretreatment with anti-ICAM-1 or anti-MCP-1 Abs. Therefore, in addition to endothelial cell apoptosis, as previously reported, inflammatory activation occurs in endothelial cells after stimulation by anti-DV NS1 Abs. These results suggest the involvement of anti-DV NS1 Abs in the vasculopathy of DV infection. PMID:15611263

  20. Cell Penetrating Peptide-Mediated Caveolae-Dependent Activation of Lung Endothelial Nitric Oxide Synthase.

    PubMed

    Hutchinson, Tarun E; Hu, Hanbo; Patel, Jawaharlal M

    2016-01-01

    Cell penetrating peptides can be used as therapeutic agents via modulation of selective cell functions. Nitric oxide (NO) generated by vascular endothelial NO synthase (eNOS) plays a critical role in the NO/ cyclic guanosine 5'-monophosphate (cGMP)-mediated pulmonary vascular function. Here we examined whether internalization of a fifteen amino acid (KRFNSISCSSWRRKR) synthetic peptide (P3) enhances the catalytic activity of eNOS via caveolae/eNOS dissociation leading to NO release and increased cGMP production in pulmonary artery endothelial cells (EC). ECs were treated with varying concentrations of P3 and used to monitor internalization, isolation of caveolae-enriched fraction, the catalytic activity of eNOS, NO/cGMP production, and intracellular Ca(2+) release. Confocal images show timedependent internalization of P3 in EC. Treatment of EC with P3, but not scrambled P3, increased the catalytic activity of eNOS in a dose-dependent manner without change in eNOS expression or phosphorylation. Treatment of EC with P3 stimulated intracellular Ca(2+) release, increased the catalytic activity of phospatidylinsositide 3 kinase (PI3K) and resulted in eNOS/caveolae-1 (Cav-1) dissociation leading to translocation of eNOS to intracellular compartment in EC. P3- mediated activation of eNOS was abolished by intracellular Ca(2+) chelator 1,2-bis(2-aminophenooxy)ethane-N,N,N',N'- tertraacetic acid-AM (BAPTA-AM), PI3K inhibition, or by siRNA-mediated Cav-1 suppression. These results demonstrate that exogenous peptide consisting of cationic amino acids can internalize and enhance the catalytic activity of eNOS via modulation of caveolar signaling and intracellular Ca(2+) release in EC. PMID:26655728

  1. Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation

    PubMed Central

    Vijayan, Vijith; Hiller, Oliver; Figueiredo, Constanca; Aljabri, Abid; Blasczyk, Rainer; Theilmeier, Gregor; Becker, Jan Ulrich; Larmann, Jan; Immenschuh, Stephan

    2015-01-01

    Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation. PMID:26690352

  2. Equid herpesvirus 1 infection of endothelial cells requires activation of putative adhesion molecules: an in vitro model

    PubMed Central

    SMITH, D; HAMBLIN, A; EDINGTON, N

    2002-01-01

    Antisera to activated equine endothelial cells, which detected surface molecules of 116 kD, 97 kD, 42 kD and 38 kD, were made to investigate the role of endothelial adhesion molecules in equid herpes virus 1 infection. These putative adhesion molecules could be induced by 17-β oestradiol, chorionic gonadotrophin, or IL-2, as well as by LPS and PWM. In an in vitro flow system, using equine veins or arteries, equid herpesvirus 1 in leucocytes was only transferred to infect endothelial cells if both leucocytes and endothelial cells expressed these surface molecules. Blocking of the membrane molecules with polyclonal antibodies prevented transfer of virus to the endothelial cells, indicating that the adhesion molecules had a key role in effecting transfer of virus. These in vitro observations give particular insight into the reports that in the natural course of infection in horses infection of endothelial cells is restricted to certain tissues, and in a wider context the results illustrate the complexity of factors that may direct tissue tropism. PMID:12165084

  3. Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-{beta}

    SciTech Connect

    Zhao, Haige; Hiroi, Toyoko; Hansen, Baranda S.; Rade, Jeffrey J.

    2009-11-27

    Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A{sub 2}, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A{sub 2} synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-{kappa}{beta} signaling pathway.

  4. Analysis of active components in Salvia miltiorrhiza injection based on vascular endothelial cell protection.

    PubMed

    Shen, Jie; Yang, Kai; Sun, Caihua; Zheng, Minxia

    2014-09-01

    Correlation analysis based on chromatograms and pharmacological activities is essential for understanding the effective components in complex herbal medicines. In this report, HPLC and measurement of antioxidant properties were used to describe the active ingredients of Salvia miltiorrhiza injection (SMI). HPLC results showed that tanshinol, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, protocatechuic acid and their metabolites in rat serum may contribute to the efficacy of SMI. Assessment of antioxidant properties indicated that differences in the composition of serum powder of SMI caused differences in vascular endothelial cell protection. When bivariate correlation was carried out it was found that salvianolic acid B, tanshinol and protocatechuic aldehyde were active components of SMI because they were correlated to antioxidant properties. PMID:25296678

  5. PAR-1 activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

    PubMed Central

    Bae, Jong-Sup; Rezaie, Alireza R.

    2008-01-01

    Summary We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs, however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-α-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-β-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders. PMID:18612544

  6. Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

    PubMed Central

    Nilius, B; Prenen, J; Szücs, G; Wei, L; Tanzi, F; Voets, T; Droogmans, G

    1997-01-01

    1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i

  7. Endotoxin induces fibrosis in vascular endothelial cells through a mechanism dependent on transient receptor protein melastatin 7 activity.

    PubMed

    Echeverría, Cesar; Montorfano, Ignacio; Hermosilla, Tamara; Armisén, Ricardo; Velásquez, Luis A; Cabello-Verrugio, Claudio; Varela, Diego; Simon, Felipe

    2014-01-01

    The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial-to-mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis. PMID:24710004

  8. Endotoxin Induces Fibrosis in Vascular Endothelial Cells through a Mechanism Dependent on Transient Receptor Protein Melastatin 7 Activity

    PubMed Central

    Echeverría, Cesar; Montorfano, Ignacio; Hermosilla, Tamara; Armisén, Ricardo; Velásquez, Luis A.; Cabello-Verrugio, Claudio; Varela, Diego; Simon, Felipe

    2014-01-01

    The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial­to­mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis. PMID:24710004

  9. Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells

    SciTech Connect

    He Yingbo; Chang Guodong; Zhan Shunli; Song Xiaomin; Wang Xiaofeng; Luo Yongzhang

    2008-06-06

    The level of circulating tissue factor (TF) is up-regulated in human angiogenesis-related malignancies. However, whether circulating TF has angiogenic activities has not been determined. Soluble TF (sTF) is the main domain of circulating TF. Here, using cell migration, wound healing, and tubule formation assays, human recombinant sTF was found to significantly promote the migration and differentiation of endothelial cells. The stress fiber formation and rearrangement induced by sTF observed through immunofluorescence microscope may be responsible for the stimulatory migration effect of sTF. Nevertheless, sTF had no effects on endothelial cell proliferation. Interestingly, sTF can be internalized by endothelial cells, which implies a novel mechanism for sTF in angiogenesis. These results suggest that sTF has unique angiogenic activities and may serve as a potential therapeutic target to treat diseases associated with angiogenesis such as cancer and rheumatoid arthritis.

  10. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells.

    PubMed

    Goutzourelas, Nikolaos; Stagos, Dimitrios; Spanidis, Ypatios; Liosi, Maria; Apostolou, Anna; Priftis, Alexandros; Haroutounian, Serko; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

    2015-10-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  11. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells

    PubMed Central

    GOUTZOURELAS, NIKOLAOS; STAGOS, DIMITRIOS; SPANIDIS, YPATIOS; LIOSI, MARIA; APOSTOLOU, ANNA; PRIFTIS, ALEXANDROS; HAROUTOUNIAN, SERKO; SPANDIDOS, DEMETRIOS A.; TSATSAKIS, ARISTIDIS M.; KOURETAS, DEMETRIOS

    2015-01-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  12. House dust mite extracts activate cultured human dermal endothelial cells to express adhesion molecules and secrete cytokines.

    PubMed

    Arlian, Larry G; Elder, B Laurel; Morgan, Marjorie S

    2009-05-01

    The human skin contacts molecules from house dust mites that are ubiquitous in many environments. These mite-derived molecules may penetrate the skin epidermis and dermis and contact microvascular endothelial cells and influence their function. The purpose of this study was to determine the response of normal human dermal microvascular endothelial cells to extracts of the dust mites, Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei with and without endotoxin (lipopolysaccharide). Endothelial cells were stimulated with mite extracts and the expression of surface molecules and the secretion of cytokines were measured in the absence and presence of polymyxin B to bind endotoxin. All three mite extracts stimulated endothelial cells to express intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin and to secrete interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP-1), and granulocyte/macrophage colony stimulating factor (GM-CSF). Euroglyphus maynei-induced expression of all the cell surface molecules was not inhibited when the endotoxin activity in the mite extract was inhibited. In contrast, endothelial cells challenged with D. farinae or D. pteronyssinus extract depleted of endotoxin activity expressed only constitutive levels of ICAM-1, VCAM-1, and E-selectin. D. farinae and E. maynei extracts depleted of endotoxin activity still induced secretion of IL-8 and MCP-1 but at reduced levels. Only constitutive amounts of IL-6, G-CSF, and GM-CSF were secreted in response to any of the endotoxin-depleted mite extracts. Extracts of D. farinae, D. pteronyssinus, and E. maynei contain both endotoxins and other molecules that can stimulate expression of cell adhesion molecules and chemokine receptors and the secretion of cytokines by normal human microvascular endothelial cells. PMID:19496432

  13. Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation.

    PubMed

    Li, Yong D; Block, Edward R; Patel, Jawaharlal M

    2002-10-01

    Signaling events involving angiotensin IV (ANG IV)-mediated pulmonary artery endothelial cell (PAEC) proliferation were examined. ANG IV significantly increased upstream phosphatidylinositide (PI) 3-kinase (PI3K), PI-dependent kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), and protein kinase B-alpha/Akt (PKB-alpha) activities, as well as downstream p70 ribosomal S6 kinase (p70S6K) activities and/or phosphorylation of these proteins. ANG IV also significantly increased 5-bromo-2'-deoxy-uridine incorporation into newly synthesized DNA in a concentration- and time-dependent manner. Pretreatment of cells with wortmannin and LY-294002, inhibitors of PI3K, or rapamycin, an inhibitor of the mammalian target of rapamycin kinase and p70S6K, diminished the ANG IV-mediated activation of PDK-1 and PKB-alpha as well as phosphorylation of p70S6K. Although an inhibitor of mitogen-activated protein kinase kinase, PD-98059, but not rapamycin, blocked ANG IV-induced phosphorylation of ERK1/2, both PD-98059 and rapamycin independently caused partial reduction in ANG IV-mediated cell proliferation. However, simultaneous treatment with PD-98059 and rapamycin resulted in total inhibition of ANG IV-induced cell proliferation. These results demonstrate that ANG IV-induced DNA synthesis is regulated in a coordinated fashion involving multiple signaling modules in PAEC. PMID:12225947

  14. Loss of Interleukin-21 Receptor Activation in Hypoxic Endothelial Cells Impairs Perfusion Recovery after Hindlimb Ischemia

    PubMed Central

    Wang, Tao; Cunningham, Alexis; Dokun, Ayotunde O; Hazarika, Surovi; Houston, Kevin; Chen, Lingdan; Lye, R. John; Spolski, Rosanne; Leonard, Warren J.; Annex, Brian H.

    2016-01-01

    Objective Surgical hindlimb ischemia (HLI) in mice has become a valuable preclinical model to study peripheral arterial disease (PAD). We previously identified that the different phenotypical outcomes following HLI across inbred mouse strains is related a region on the short arm of mouse chromosome 7. The gene coding the interleukin-21 receptor (IL-21R) lies at the peak of association in this region. Approach and Results With quantitative RT-PCR, we found that a mouse strain with a greater ability to up-regulate IL-21R following HLI had better perfusion recovery than a strain with no up-regulation after HLI. Immunofluorescent staining of ischemic hind-limb tissue showed IL-21R expression on endothelial cells (EC) from these C57BL/6 mice. An EC-enriched fraction isolated from ischemic hind-limb muscle showed higher Il-21R levels than an EC-enriched fraction from non-ischemic limbs. In-vitro, human umbilical vein EC (HUVEC) showed elevated IL-21R expression after hypoxia and serum starvation. Under these conditions, IL-21 treatment increased cell viability, decreased cell apoptosis, and augmented tube formation. In-vivo, either knockout Il21r or blocking IL-21 signaling by treating with IL-21R-Fc (fusion protein that blocks IL-21 binding to its receptor) in C57BL/6 mice resulted in less perfusion recovery after HLI. Both in-vitro and in-vivo modulation of the IL-21/IL-21R axis under hypoxic conditions resulted in increasedSTAT3 phosphorylation and a subsequent increase in the BCL-2/BAX ratio. Conclusion Our data indicate that IL-21R up-regulation and ligand activation in hypoxic endothelial cells may help perfusion recovery by limiting/preventing apoptosis and/or favoring cell survival and angiogenesis through the STAT3 pathway. PMID:25838422

  15. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

  16. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  17. Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells

    PubMed Central

    Janssens, Dominique; Delaive, Edouard; Houbion, Andrée; Eliaers, François; Remacle, José; Michiels, Carine

    2000-01-01

    Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases. PMID:10928952

  18. Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells

    PubMed Central

    Ferraro, Francesco; Mafalda Lopes da, Silva; Grimes, William; Lee, Hwee Kuan; Ketteler, Robin; Kriston-Vizi, Janos; Cutler, Daniel F.

    2016-01-01

    Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall. PMID:27576551

  19. Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells.

    PubMed

    Ferraro, Francesco; Mafalda Lopes da, Silva; Grimes, William; Lee, Hwee Kuan; Ketteler, Robin; Kriston-Vizi, Janos; Cutler, Daniel F

    2016-01-01

    Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall. PMID:27576551

  20. A Natural Small Molecule Harmine Inhibits Angiogenesis and Suppresses Tumour Growth through Activation of p53 in Endothelial Cells

    PubMed Central

    Dai, Fujun; Chen, Yihua; Song, Yajuan; Huang, Li; Zhai, Dong; Dong, Yanmin; Lai, Li; Zhang, Tao; Li, Dali; Pang, Xiufeng; Liu, Mingyao; Yi, Zhengfang

    2012-01-01

    Activation of p53 effectively inhibits tumor angiogenesis that is necessary for tumor growth and metastasis. Reactivation of the p53 by small molecules has emerged as a promising new strategy for cancer therapy. Several classes of small-molecules that activate the p53 pathway have been discovered using various approaches. Here, we identified harmine (β-carboline alkaloid) as a novel activator of p53 signaling involved in inhibition of angiogenesis and tumor growth. Harmine induced p53 phosphorylation and disrupted the p53-MDM2 interaction. Harmine also prevented p53 degradation in the presence of cycloheximide and activated nuclear accumulation of p53 followed by increasing its transcriptional activity in endothelial cells. Moreover, harmine not only induced endothelial cell cycle arrest and apoptosis, but also suppressed endothelial cell migration and tube formation as well as induction of neovascularity in a mouse corneal micropocket assay. Finally, harmine inhibited tumor growth by reducing tumor angiogenesis, as demonstrated by a xenograft tumor model. Our results suggested a novel mechanism and bioactivity of harmine, which inhibited tumor growth by activating the p53 signaling pathway and blocking angiogenesis in endothelial cells. PMID:23300602

  1. Troglitazone inhibits endothelial cell proliferation through suppression of casein kinase 2 activity

    SciTech Connect

    Lee, Kuy-Sook; Park, Jin-Hee; Lee, Seahyoung; Lim, Hyun-Joung; Jang, Yangsoo; Park, Hyun-Young . E-mail: hypark65@nih.go.kr

    2006-07-21

    Troglitazone, an agonist of peroxisome proliferator activated receptor{gamma} (PPAR{gamma}), has been reported to inhibit endothelial cell proliferation by suppressing Akt activation. Recently, it has been also proposed that phosphatase and tensin homolog deleted from chromosome 10 (PTEN) plays an important role in such effect of troglitazone. However, the mechanism of how troglitazone regulates PTEN remains to be elucidated. We therefore investigated the effects of troglitazone on casein kinase 2 (CK2), which is known to negatively regulate PTEN activity. Troglitazone significantly inhibited serum-induced proliferation of HUVEC in a concentration dependent manner. Serum-induced Akt and its downstream signaling pathway activation was attenuated by troglitazone (10 {mu}M) pretreatment. The phosphorylation of PTEN, which was directly related to Akt activation, was decreased with troglitazone pretreatment and was inversely proportional to CK2 activity. DRB, a CK2 inhibitor, also showed effects similar to that of troglitazone on Akt and its downstream signaling molecules. In conclusion, our results suggest that troglitazone inhibits proliferation of HUVECs through suppression of CK2 activity rendering PTEN to remain activated, and this effect of troglitazone in HUVECs seems to be PPAR{gamma} independent.

  2. Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis.

    PubMed

    Wu, Xiao-Ming; Gao, Yan-Bin; Cui, Fang-Qiang; Zhang, Na

    2016-01-01

    The interaction between glomerular endothelial cells (GECs) and glomerular mesangial cells (GMCs) is an essential aspect of diabetic nephropathy (DN). Therefore, understanding how GECs communicate with GMCs in the diabetic environment is crucial for the development of new targets for the prevention and treatment of DN. Exosomes, nanometer-sized extracellular membrane vesicles secreted by various cell types, play important roles in cell-to-cell communication via the transfer of mRNA, microRNA and protein. In this study, we demonstrate that high glucose (HG)-treated GECs secrete a higher number of exosomes highly enriched in TGF-β1 mRNA compared with normal glucose (NG)-treated GECs. Exosomes released by HG-treated GECs can promote α-smooth muscle actin (α-SMA) expression, proliferation and extracellular matrix protein overproduction in GMCs through the TGF-β1/Smad3 signaling pathway. Thus, we provide new insights into the pathogenesis of DN that involves intercellular transfer of TGF-β1 mRNA in the GEC-to-GMC direction via exosomes. PMID:27010029

  3. Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis

    PubMed Central

    Wu, Xiao-ming; Gao, Yan-bin; Cui, Fang-qiang; Zhang, Na

    2016-01-01

    ABSTRACT The interaction between glomerular endothelial cells (GECs) and glomerular mesangial cells (GMCs) is an essential aspect of diabetic nephropathy (DN). Therefore, understanding how GECs communicate with GMCs in the diabetic environment is crucial for the development of new targets for the prevention and treatment of DN. Exosomes, nanometer-sized extracellular membrane vesicles secreted by various cell types, play important roles in cell-to-cell communication via the transfer of mRNA, microRNA and protein. In this study, we demonstrate that high glucose (HG)-treated GECs secrete a higher number of exosomes highly enriched in TGF-β1 mRNA compared with normal glucose (NG)-treated GECs. Exosomes released by HG-treated GECs can promote α-smooth muscle actin (α-SMA) expression, proliferation and extracellular matrix protein overproduction in GMCs through the TGF-β1/Smad3 signaling pathway. Thus, we provide new insights into the pathogenesis of DN that involves intercellular transfer of TGF-β1 mRNA in the GEC-to-GMC direction via exosomes. PMID:27010029

  4. Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

    NASA Astrophysics Data System (ADS)

    Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

    The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed

  5. Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells.

    PubMed

    Lee, T H; Rhim, T; Kim, S S

    1998-10-30

    Recently, O'Reilly et al. (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277-285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simple in vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 microg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos. PMID:9786880

  6. Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells.

    PubMed

    Halamoda Kenzaoui, Blanka; Chapuis Bernasconi, Catherine; Guney-Ayra, Seher; Juillerat-Jeanneret, Lucienne

    2012-02-01

    Different types of NPs (nanoparticles) are currently under development for diagnostic and therapeutic applications in the biomedical field, yet our knowledge about their possible effects and fate in living cells is still limited. In the present study, we examined the cellular response of human brain-derived endothelial cells to NPs of different size and structure: uncoated and oleic acid-coated iron oxide NPs (8-9 nm core), fluorescent 25 and 50 nm silica NPs, TiO2 NPs (21 nm mean core diameter) and PLGA [poly(lactic-co-glycolic acid)]-PEO [poly(ethylene oxide)] polymeric NPs (150 nm). We evaluated their uptake by the cells, and their localization, generation of oxidative stress and DNA-damaging effects in exposed cells. We show that NPs are internalized by human brain-derived endothelial cells; however, the extent of their intracellular uptake is dependent on the characteristics of the NPs. After their uptake by human brain-derived endothelial cells NPs are transported into the lysosomes of these cells, where they enhance the activation of lysosomal proteases. In brain-derived endothelial cells, NPs induce the production of an oxidative stress after exposure to iron oxide and TiO2 NPs, which is correlated with an increase in DNA strand breaks and defensive mechanisms that ultimately induce an autophagy process in the cells. PMID:22026563

  7. Platelet activating factors alters calcium homeostasis in cultured vascular endothelial cells

    SciTech Connect

    Brock, T.A.; Gimbrone, M.A. Jr.

    1986-06-01

    Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine; PAF), a potent in vivo mediator of allergic and inflammatory reactions, induced a rapid (onset less than 30 s), concentration-dependent (threshold approximately 10(-11) M, half-maximal approximately 10(-10) M, maximal approximately 10(-8)-10(-7) M) efflux of /sup 45/Ca/sup 2 +/ from preloaded cultured bovine aortic endothelial cells (BAEC). In contrast, deacetylated and other PAF analogues were essentially ineffective. PAF (10(-7) M) was also shown to increase cytosolic free calcium (49 +/- 5%) in suspensions of quin 2 (calcium-sensitive fluorescent dye)-loaded BAEC. PAF-stimulated /sup 45/Ca/sup 2 +/ efflux was not blocked by aspirin treatment (100 or 500 microM, 30 min). In the absence of external calcium, PAF was still highly effective in stimulating unidirectional /sup 45/Ca/sup 2 +/ efflux, thus suggesting that PAF mobilized a sequestered pool of intracellular calcium. CV-3988, a PAF antagonist, inhibited PAF-stimulated /sup 45/Ca/sup 2 +/ efflux in a dose-dependent manner. Pretreatment of BAEC with PAF (10(-8) M, 15 min), but not with other PAF analogues, resulted in a decrease in subsequent PAF-stimulated /sup 45/Ca/sup 2 +/ efflux, thus suggesting an agonist-specific desensitization. PAF also stimulated a 30% net decrease in the equilibrium /sup 45/Ca/sup 2 +/ content of BAEC within 1 min, which gradually recovered to prestimulus levels in 10-15 min. PAF-stimulated /sup 45/Ca/sup 2 +/ efflux was also observed in endothelial cells cultured from human umbilical vein and baboon cephalic vein but not from cultured human dermal fibroblasts or bovine aortic smooth muscle. These studies provide direct evidence for agonist- and cell-specific effects of PAF on vascular endothelium.

  8. Shear stress activation of SREBP1 in endothelial cells is mediated by integrins.

    PubMed

    Liu, Yi; Chen, Benjamin P-C; Lu, Min; Zhu, Yi; Stemerman, Michael B; Chien, Shu; Shyy, John Y-J

    2002-01-01

    We investigated the effect of shear stress on the sterol regulatory element-binding protein 1 (SREBP1) in vascular endothelial cells (ECs) and the mechanotransduction mechanism involved. Application of a shear stress (12 dyn/cm(2)) caused the proteolytic cleavage of SREBP1 and the ensuing translocation of its transcription factor domain into the nucleus. As a result, shear stress increased the mRNAs encoding the low density lipoprotein receptor (LDLR), as well as the binding of (125)I-LDL. Using a step flow channel, we showed that SREBP1 activation in ECs under laminar flow is transient, but disturbed flow causes sustained activation. In studying the shear stress-elicited molecular signaling that activates SREBP1, we found that blocking the beta(1)-integrin with the AIIB2 blocking-type monoclonal antibody inhibited SREBP1 activation induced by shear stress. EC attachment to fibronectin or the activation of beta(1)-integrin in the suspended ECs by the TS2/16 monoclonal antibody was sufficient for SREBP1 activation. Furthermore, transient transfection assays showed that dominant-negative mutants of focal adhesion kinase and c-Src attenuated the shear stress-increased LDLR promoter activity. These results demonstrate that integrin signaling plays a critical role in the modulation of SREBP in ECs in response to shear stress. PMID:11788464

  9. Hyperoxia enhanced the production of plasminogen activator by cultured pulmonary artery endothelial cells

    SciTech Connect

    Phillips, P.G.; Ryan, T.J.; Birnby, L.; Tsan, M.F. New York State Health Lab., Albany )

    1990-02-26

    Pulmonary artery endothelial cell (EC) monolayers exposed to hyperoxia show increased permeability to albumin and marked alterations in actin filament distribution. The mechanism for these cytoskeletal changes is unknown. The authors now report that cells exposed to hyperoxia (95% O{sub 2}) produce significant quantities of urokinase-type plasminogen activators (u-PA). Zymographic analysis revealed that plasminogen-dependent caseinolytic activity in conditioned media from O{sub 2}-exposed cells was several fold higher than controls, especially at 48 hr. Cell-associated lytic activity was markedly increased at 48 and 72 hr when oxidative effects on the cytoskeleton were most apparent. Amidolytic assays confirmed these findings. (1) Conditioned media, CTA U/ml (mean {+-} SEM): Control 0.73{+-}0.05 vs 48 hr O{sub 2} 2.09{+-}0.50, (2) Cell-associated activity (preparations enriched for adhesion plaques), CTA mU/10{sup 6} cells: Control 0.17{+-}0.06; O{sub 2}: 24 hr 0.18{+-}0.07; 48 hr 0.46{+-}0.08; 72 hr 0.48{+-}0.08; O{sub 2} 48 hr or 72 hr vs Control. Immunocytochemical analysis demonstrated breakdown and restructuring of fibronectin and collagen components of the extracellular matrix at 48 and 72 hours. They conclude that hyperoxia enhanced both PA production and dismantling of matrix by EC. These changes may in part be responsible for some of the actin filament alterations which occur in conjunction with O{sub 2}-induced permeability increases.

  10. A vascular injury model using focal heat-induced activation of endothelial cells

    PubMed Central

    Sylman, J.L.; Artzer, D.T.; Rana, K.; Neeves, K.B.

    2015-01-01

    Endothelial cells (EC) both inhibit and promote platelet function depending on their activation state. Quiescent EC inhibit platelet activation by constitutive secretion of platelet inhibitors. Activated EC promote platelet adhesion by secretion of von Willebrand factor (vWF). EC also secrete an extracellular matrix that support platelet adhesion when exposed following vascular injury. Previous studies of EC-platelet interactions under flow activate entire monolayers of cells by chemical activation. In this study, EC cultured in microfluidic channels were focally activated by heat from an underlying microelectrode. Based on finite element modeling, microelectrodes induced peak temperature increases of 10–40 °C above 37 °C after applying 5–9 V for 30 s resulting in three zones: (1) A quiescent zone corresponded to peak temperatures of less than 15 °C characterized by no EC activation or platelet accumulation. (2) An activation zone corresponding to an increase of 16–22 °C yielded EC that were viable, secreted elevated levels of vWF, and were P-selectin positive. Platelets accumulated in the retracted spaces between EC in the activation zone at a wall shear rate of 150 s−1. Experiments with blocking antibodies show that platelets adhere via GPIbα-vWF and α6β1-laminin interactions. (3) A kill zone corresponded to peak temperatures of greater than 23 °C where EC were not viable and did not support platelet adhesion. These data define heating conditions for the activation of EC, causing the secretion of vWF and the exposure of a subendothelial matrix that support platelet adhesion and aggregation. This model provides for spatially defined zones of EC activation that could be a useful tool for measuring the relative roles of anti- and prothrombotic roles of EC at the site of vascular injury. PMID:26087748

  11. Instant membrane resealing in nlrp3 inflammmasome activation of endothelial cells.

    PubMed

    Chen, Yang; Yuan, Ming; Xia, Min; Wang, Lei; Zhang, Yang; Li, Pin-Lan

    2016-01-01

    The present study explored the molecular mechanisms by which instant cell membrane resealing (CMR) controls the activation of NOD-like receptor pyrin domain containing 3 (Nlrp3) inflammasomes. Using wavelength-switching fluorescent microscopy with PI and fura-2 as indicators, we monitored instant CMR simultaneously with (Ca(2+))i in mouse microvascular endothelial cell (MVECs). LCWE or saponin wad found to produce membrane injury, which was resealed in a Ca(2+)-dependent manner, but abolished by FasL, a membrane raft (MR) clustering stimulator. Even in the presence of Ca(2+), FasL prolonged the CMR time as shown by an earlier onset of PI influx (48±12 sec vs. 17±3 min. of control). These effects of FasL were substantially blocked by an MR disruptor, methyl-beta-cyclodextrin (MCD). The failure of CMR upon FasL activated Nlrp3 inflammasomes, which was blocked by MCD, a membrane resealing compound, VA64 or siRNA of an MR-facilitating enzyme, acid sphingomyelinase. This inflammasome activation was due to increased lysosomal permeability and cathepsin B release. It is concluded that an MR-associated CMR protects ECs from Nlrp3 inflammasome activation induced by membrane injury. PMID:26709796

  12. NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

    SciTech Connect

    Pecorelli, Alessandra; Bocci, Velio; Acquaviva, Alessandra; Belmonte, Giuseppe; Gardi, Concetta; Virgili, Fabio; Ciccoli, Lucia; Valacchi, Giuseppe

    2013-02-15

    During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O{sub 3} per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H{sub 2}O{sub 2}) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H{sub 2}O{sub 2} are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment.

  13. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser

    PubMed Central

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc’h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3–57.3) of CECs with low mortality (0.5%, range (0.55–1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for

  14. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser.

    PubMed

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc'h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future

  15. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  16. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  17. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

    PubMed Central

    Romer, L H; McLean, N; Turner, C E; Burridge, K

    1994-01-01

    Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes. Images PMID:8049526

  18. A proton-activated, outwardly rectifying chloride channel in human umbilical vein endothelial cells

    SciTech Connect

    Ma Zhiyong; Zhang Wei; Chen Liang; Wang Rong; Kan Xiaohong; Sun Guizhen; Liu Chunxi; Li Li Zhang Yun

    2008-07-04

    Extracellular acidic pH-activated chloride channel I{sub Cl,acid}, has been characterized in HEK 293 cells and mammalian cardiac myocytes. This study was designed to characterize I{sub Cl,acid} in human umbilical vein endothelial cells(HUVECs). The activation and deactivation of the current rapidly and repeatedly follows the change of the extracellular solution at pH 4.3, with the threshold pH 5.3. In addition, at very positive potentials, the current displays a time-dependent facilitation. pH-response relationship for I{sub Cl,acid} revealed that EC{sub 50} is pH 4.764 with a threshold pH value of pH 5.3 and nH of 14.545. The current can be blocked by the Cl{sup -} channel inhibitor DIDS (100 {mu}M). In summary, for the first time we report the presence of proton-activated, outwardly rectifying chloride channel in HUVECs. Because an acidic environment can develop in local myocardium under pathological conditions such as myocardial ischemia, I{sub Cl,acid} would play a role in regulation of EC function under these pathological conditions.

  19. Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: A novel antiangiogenic therapy

    SciTech Connect

    Kamisasanuki, Taro; Tokushige, Saori; Terasaki, Hiroto; Khai, Ngin Cin; Wang, Yuqing; Sakamoto, Taiji; Kosai, Ken-ichiro

    2011-09-16

    Highlights: {yields} CD9 plays stimulus-independent roles in angiogenesis in vitro and in vivo. {yields} Targeting CD9 expression is effective in an angiogenic disease model. {yields} Targeting CD9 expression predominantly affects activated endothelial cells. {yields} CD9 is involved in endothelial cell proliferation, but not survival. {yields} CD9 is part of angiogenic machinery in endothelial cells during angiogenesis. -- Abstract: The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus

  20. Endothelin-1 Impairs Nitric Oxide Signaling in Endothelial Cells Through a Protein Kinase Cδ-Dependent Activation of STAT3 and Decreased Endothelial Nitric Oxide Synthase Expression

    PubMed Central

    Sud, Neetu

    2009-01-01

    In an ovine model of persistent pulmonary hypertension of the newborn (PPHN), endothelin-1 (ET-1) expression is increased, while endothelial nitric oxide synthase (eNOS) expression is decreased. However, the molecular mechanisms by which ET-1 attenuates eNOS expression in endothelial cells are not completely understood. Thus, the goal of this study was to determine if the overexpression of ET-1 decreases eNOS expression in pulmonary arterial endothelial cells isolated from fetal lambs. To increase the ET-1 expression, cells were transfected with a plasmid coding for Prepro-ET-1, a precursor of ET-1. After overexpression of Prepro-ET-1, ET-1 levels in the culture medium were significantly increased (control = 805.3 ± 69.8; Prepro-ET-1 overexpression = 1351 ± 127.9). eNOS promoter activity, protein levels, and NO generation were all significantly decreased by the overexpression of Prepro-ET-1. The decrease in transcription correlated with increased activity of protein kinase Cδ (PKCδ) and STAT3. Further, DNA binding activity of STAT3 was also increased by Prepro-ET-1 overexpression. The increase in STAT3 activity and decrease in eNOS promoter activity were inhibited by the overexpression of dominant negative mutants of PKCδ or STAT3. Further, a 2 bp mutation in the STAT3 binding site in the eNOS promoter inhibited STAT3 binding and led to enhanced promoter activity in the presence of Prepro-ET-1 overexpression. In conclusion, ET-1 secretion is increased by Prepro-ET-1 overexpression. This results in activation of PKCδ, which phosphorylates STAT3, increasing its binding to the eNOS promoter. This in turn decreases eNOS promoter activity, protein levels, and NO production. Thus, ET-1 can reduce eNOS expression and NO generation in fetal pulmonary artery endothelial cells through PKCδ-mediated activation of STAT3. PMID:19754268

  1. Pore size assessment during corneal endothelial cells permeabilization by femtosecond laser activated carbon nanoparticles

    NASA Astrophysics Data System (ADS)

    Jumelle, C.; Mauclair, C.; Houzet, J.; Bernard, A.; He, Z.; Piselli, S.; Perrache, C.; Egaud, G.; Baubeau, E.; Gain, P.; Thuret, G.

    2015-07-01

    Corneal therapeutic molecules delivery represents a promising solution to maintain human corneal endothelial cells (HCECs) viability, but the difficulty is transport across cell membrane. A new delivery method published recently consists in ephemerally permeabilizing cell membranes using a photo-acoustic reaction produced by carbon nanoparticles (CNPs) and femtosecond laser (FsL). The aim of this work is to investigate the size of pores formed at cell membrane by this technique. To induce cell permeabilization, HCECs were put in contact with CNPs and irradiated with a 500 μm diameter Ti:Sa FsL focalized spot. Four sizes of marker molecules were delivered into HCECs to investigate pore sizes: calcein (1.2 nm), FITC-Dextran 4kDa (2.8 nm) and FITC-Dextran 70kDa (12 nm) and FITC-Dextran 2MDa (50 nm). Delivery of each molecule was assessed by flow cytometry, a technique able to measure their presence into cells. We showed that the delivery rate was dependent of their size. Calcein was delivered in 56.1±8.2% of HCECs, FITC-Dextran 4kDa in 42.2±3.5%, FITC-Dextran 70 kDa in 21.5±2.7% and finally FITC-Dextran 2MDa in 12.9±2.0%. It means that a large number of pores in the size ranging from 1.2 to 2.8 nm were formed. However, 12 nm and larger pores were almost half more infrequent. Pore sizes formed at cell membrane by the technique of cell permeabilization by FsL activated CNPs was investigated. The results indicated that the pore sizes are large enough for the efficient delivery of small, medium and big therapeutics molecules on HCECs by this technique.

  2. Nicotine improves the functional activity of late endothelial progenitor cells via nicotinic acetylcholine receptors.

    PubMed

    Yu, Min; Liu, Qian; Sun, Jing; Yi, Kaihong; Wu, Libiao; Tan, Xuerui

    2011-08-01

    The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10 nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs. PMID:21774635

  3. Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1.

    PubMed

    Zanotti, L; Angioni, R; Calì, B; Soldani, C; Ploia, C; Moalli, F; Gargesha, M; D'Amico, G; Elliman, S; Tedeschi, G; Maffioli, E; Negri, A; Zacchigna, S; Sarukhan, A; Stein, J V; Viola, A

    2016-05-01

    Mesenchymal stem cells (MSC) represent a promising therapeutic approach in many diseases in view of their potent immunomodulatory properties, which are only partially understood. Here, we show that the endothelium is a specific and key target of MSC during immunity and inflammation. In mice, MSC inhibit activation and proliferation of endothelial cells in remote inflamed lymph nodes (LNs), affect elongation and arborization of high endothelial venules (HEVs) and inhibit T-cell homing. The proteomic analysis of the MSC secretome identified the tissue inhibitor of metalloproteinase-1 (TIMP-1) as a potential effector molecule responsible for the anti-angiogenic properties of MSC. Both in vitro and in vivo, TIMP-1 activity is responsible for the anti-angiogenic effects of MSC, and increasing TIMP-1 concentrations delivered by an Adeno Associated Virus (AAV) vector recapitulates the effects of MSC transplantation on draining LNs. Thus, this study discovers a new and highly efficient general mechanism through which MSC tune down immunity and inflammation, identifies TIMP-1 as a novel biomarker of MSC-based therapy and opens the gate to new therapeutic approaches of inflammatory diseases. PMID:26898191

  4. Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1

    PubMed Central

    Zanotti, L; Angioni, R; Calì, B; Soldani, C; Ploia, C; Moalli, F; Gargesha, M; D'Amico, G; Elliman, S; Tedeschi, G; Maffioli, E; Negri, A; Zacchigna, S; Sarukhan, A; Stein, J V; Viola, A

    2016-01-01

    Mesenchymal stem cells (MSC) represent a promising therapeutic approach in many diseases in view of their potent immunomodulatory properties, which are only partially understood. Here, we show that the endothelium is a specific and key target of MSC during immunity and inflammation. In mice, MSC inhibit activation and proliferation of endothelial cells in remote inflamed lymph nodes (LNs), affect elongation and arborization of high endothelial venules (HEVs) and inhibit T-cell homing. The proteomic analysis of the MSC secretome identified the tissue inhibitor of metalloproteinase-1 (TIMP-1) as a potential effector molecule responsible for the anti-angiogenic properties of MSC. Both in vitro and in vivo, TIMP-1 activity is responsible for the anti-angiogenic effects of MSC, and increasing TIMP-1 concentrations delivered by an Adeno Associated Virus (AAV) vector recapitulates the effects of MSC transplantation on draining LNs. Thus, this study discovers a new and highly efficient general mechanism through which MSC tune down immunity and inflammation, identifies TIMP-1 as a novel biomarker of MSC-based therapy and opens the gate to new therapeutic approaches of inflammatory diseases. PMID:26898191

  5. Normal human IgG prevents endothelial cell activation induced by TNFα and oxidized low-density lipoprotein atherogenic stimuli

    PubMed Central

    RONDA, N; BERNINI, F; GIACOSA, R; GATTI, R; BALDINI, N; BUZIO, C; ORLANDINI, G

    2003-01-01

    Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-α (TNFα) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFα induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)′2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies. PMID:12869027

  6. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles.

    PubMed

    Freitag, Tanja C; Maisner, Andrea

    2016-03-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  7. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles

    PubMed Central

    Freitag, Tanja C.

    2015-01-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  8. Ferroportin and Exocytoplasmic Ferroxidase Activity Are Required for Brain Microvascular Endothelial Cell Iron Efflux*

    PubMed Central

    McCarthy, Ryan C.; Kosman, Daniel J.

    2013-01-01

    The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of 59Fe efflux. Both hBMVEC Fpn protein and 59Fe efflux activity are restored upon incubation with 6.6 nm soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrin- or non-transferrin-bound 59Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp. PMID:23640881

  9. Hepatocyte growth factor suppresses hypoxia/reoxygenation-induced XO activation in cardiac microvascular endothelial cells.

    PubMed

    Zhang, Yingqian; Hu, Shunying; Chen, Yundai

    2015-07-01

    Hypoxia/reoxygenation (H/R) is one of the cellular stresses in pathological conditions, such as myocardial infarction, stroke and organ transplantation. Oxidative stress caused by reactive oxygen species (ROS) is a crucial element of H/R injury in vascular endothelial cells (ECs). Xanthine oxidase (XO) has been recognized to contribute to H/R injury. Of note, xanthine oxidoreductase is synthesized as xanthine dehydrogenase (XDH) and needs to be converted to XO to become a source of superoxide. Hepatocyte growth factor (HGF) has been found to protect ECs against H/R injury. The relation, however, between HGF and XO in ECs under H/R conditions remains to be determined. Primary cultured rat cardiac microvascular endothelial cells (CMECs) were exposed to 4 h of hypoxia and followed by 1 h of reoxygenation. Generation of ROS and cytosolic Ca2+ concentration was measured by flow cytometry qualification of DCFHDA and fluo-3 AM staining cells, respectively. XDH mRNA was qualified by qRT-PCR analysis. XO activity was determined by colorimetric assay and XO protein levels were determined by Western blot. Cell apoptosis was assessed by caspase-3 activity and Annexin V/PI staining. After H/R, cellular ROS production significantly increased. Both XO activity and XO protein increased after H/R. Cellular ROS elevation was inhibited by allopurinol (a potent XO inhibitor), indicting XO accounting for the generation of ROS after H/R. In addition, XDH mRNA increased after H/R, indicating a de novo XDH synthesis, which needs to be converted to XO to become a source of superoxide. Pretreatment of HGF inhibited the elevation of XO activity and XO protein level after H/R; however, HGF has no effect on the increase of XDH mRNA. We also find an increase of the cytosolic Ca2+ in CMECs after H/R. BAPTA-AM, a cell-permeable Ca2+ chelator, prevented the increase of XO activity and XO protein levels, implicating the elevated cytosolic Ca2+ concentration involvement in XO conversion and XO

  10. Human Pulmonary Endothelial Cells in Culture

    PubMed Central

    Johnson, Alice R.

    1980-01-01

    Endothelial cells were cultured from various different human vessels, including aortas, pulmonary, ovarian, and umbilical arteries, and pulmonary, ovarian, and umbilical veins. The cultured cells were identified as endothelial cells by the presence of Factor VIII antigen and antiotensin I converting enzyme (kininase II). They retained these markers for at least five passages in culture, and some cells had them for seven passages or more. Endothelial cells from the various vessels were compared with respect to their ability to metabolize angiotensins I and II and bradykinin. Cells from arteries had three to five times the angiotensin I converting enzyme activity as cells from veins. The activity of angiotensinase A (aspartyl aminopeptidase) had a similar distribution, and cells from arteries were consistently more active than cells from veins. Cultures of endothelial cells from pulmonary and umbilical vessels formed prostacyclin in response to mechanical stimulation. Media from cell monolayers that were subjected to a change of medium and gentle agitation inhibited aggregation of human platelets. This inhibitory activity was generated within 2-5 min, and it was not formed by cells that were treated with indomethacin or tranylcypromine. Addition of prostaglandin (PG)H2 to indomethacin-treated cells restored the ability to form the inhibitor, but cells treated with tranylcypromine were not responsive to PGH2. In experiments where [14C]arachidonic acid was added to the cells before stimulation, the major metabolite identified by thin-layer chromatography was 6-keto PGF1α. Thus, it appears that pulmonary endothelial cells, as well as umbilical cord cells, can form prostacyclin. In experiments comparing the ability of arterial and venous cells to form prostacyclin, arterial cells were more active than venous cells. These studies of cells from various human vessels suggest that the vascular origin of cultured endothelial cells determines how they metabolize vasoactive

  11. Mechanisms of endothelial cell-dependent leukocyte adhesion stimulated by platelet-activating factor.

    PubMed

    Ding, Z; Li, S; Wu, Z

    1992-04-01

    Platelet-activating factor (PAF) stimulates leukocyte-endothelial cell (EC) adhesion through its effects either on leukocytes or on ECs. ECs may be injured, synthesize, or express new adhesive proteins to increase leukocyte adhesion. Intermediary mediators produced by activated ECs are also likely involved in promoting leukocyte adhesion. Our experiments demonstrated that PAF induced no obvious damage to bovine pulmonary artery ECs evaluated by lactic dehydrogenase release rate, angiotensin-converting enzyme activity, and cellular malondialdehyde content. Treatment of EC monolayers with 10(-9) M PAF increased polymorphonuclear leukocyte (PMN) adhesion. Increasing PAF concentration did not induce more PMN adherence. PAF elicited both a rapid and prolonged increment of PMN adherence to EC monolayers. The rapid adherence was greatly attenuated by pretreatment of ECs with PAF receptor antagonist SRI 63-441 but was not affected by pretreatment of PMNs with SRI 63-441, suggesting that PAF increases PMN adherence rapidly through its effects on specific receptors on ECs. Increased PMN adherence lasted if PAF treatment of ECs was sustained for 3 or 6 h. Pretreatment of ECs with actinomycin D, a protein synthesis inhibitor, significantly decreased PAF-induced sustained PMN adherence, but the inhibition is incomplete, suggesting that other mechanisms than protein synthesis also participated in the prolonged PMN adherence. We concluded from the results that PAF may induce both rapid and prolonged PMN adhesion to ECs. The effects are receptor mediated. The prolonged PMN adhesion is partly the result of protein synthesis. PMID:1592489

  12. Omega-3 fatty acid oxidation products prevent vascular endothelial cell activation by coplanar polychlorinated biphenyls

    SciTech Connect

    Majkova, Zuzana; Layne, Joseph; Sunkara, Manjula; Morris, Andrew J.; Toborek, Michal; Hennig, Bernhard

    2011-02-15

    Coplanar polychlorinated biphenyls (PCBs) may facilitate development of atherosclerosis by stimulating pro-inflammatory pathways in the vascular endothelium. Nutrition, including fish oil-derived long-chain omega-3 fatty acids, such as docosahexaenoic acid (DHA, 22:6{omega}-3), can reduce inflammation and thus the risk of atherosclerosis. We tested the hypothesis that cyclopentenone metabolites produced by oxidation of DHA can protect against PCB-induced endothelial cell dysfunction. Oxidized DHA (oxDHA) was prepared by incubation of the fatty acid with the free radical generator 2,2-azo-bis(2-amidinopropane) dihydrochloride (AAPH). Cellular pretreatment with oxDHA prevented production of superoxide induced by PCB77, and subsequent activation of nuclear factor-{kappa}B (NF-{kappa}B). A{sub 4}/J{sub 4}-neuroprostanes (NPs) were identified and quantitated using HPLC ESI tandem mass spectrometry. Levels of these NPs were markedly increased after DHA oxidation with AAPH. The protective actions of oxDHA were reversed by treatment with sodium borohydride (NaBH{sub 4}), which concurrently abrogated A{sub 4}/J{sub 4}-NP formation. Up-regulation of monocyte chemoattractant protein-1 (MCP-1) by PCB77 was markedly reduced by oxDHA, but not by un-oxidized DHA. These protective effects were proportional to the abundance of A{sub 4}/J{sub 4} NPs in the oxidized DHA sample. Treatment of cells with oxidized eicosapentaenoic acid (EPA, 20:5{omega}-3) also reduced MCP-1 expression, but less than oxDHA. Treatment with DHA-derived cyclopentenones also increased DNA binding of NF-E2-related factor-2 (Nrf2) and downstream expression of NAD(P)H:quinone oxidoreductase (NQO1), similarly to the Nrf-2 activator sulforaphane. Furthermore, sulforaphane prevented PCB77-induced MCP-1 expression, suggesting that activation of Nrf-2 mediates the observed protection against PCB77 toxicity. Our data implicate A{sub 4}/J{sub 4}-NPs as mediators of omega-3 fatty acid-mediated protection against the

  13. Omega-3 fatty acid oxidation products prevent vascular endothelial cell activation by coplanar polychlorinated biphenyls

    PubMed Central

    Majkova, Zuzana; Layne, Joseph; Sunkara, Manjula; Morris, Andrew J.; Toborek, Michal; Hennig, Bernhard

    2011-01-01

    Coplanar polychlorinated biphenyls (PCBs) may facilitate development of atherosclerosis by stimulating pro-inflammatory pathways in the vascular endothelium. Nutrition, including fish oil-derived long-chain omega-3 fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can reduce inflammation and thus the risk of atherosclerosis. We tested the hypothesis that cyclopentenone metabolites produced by oxidation of DHA can protect against PCB-induced endothelial cell dysfunction. Oxidized DHA (oxDHA) was prepared by incubation of the fatty acid with the free radical generator 2,2-azo-bis(2-amidinopropane) dihydrochloride (AAPH). Cellular pretreatment with oxDHA prevented production of superoxide induced by PCB77, and subsequent activation of nuclear factor-κB (NF-κB). A4/J4-neuroprostanes (NPs) were identified and quantitated using HPLC ESI tandem mass spectrometry. Levels of these NPs were markedly increased after DHA oxidation with AAPH.. The protective actions of oxDHA were reversed by treatment with sodium borohydride (NaBH4), which concurrently abrogated A4/J4-NP formation. Up-regulation of monocyte chemoattractant protein-1 (MCP-1)by PCB77 was markedly reduced by oxDHA, but not by un-oxidized DHA. These protective effects were proportional to the abundance of A4/J4NPs in the oxidized DHA sample. Treatment of cells with oxidized eicosapentaenoic acid (EPA, 20:5ω-3) also reduced MCP-1 expression, but less than oxDHA. Treatment with DHA-derived cyclopentenones also increased DNA binding of NF-E2-related factor-2 (Nrf2)and downstream expression of NAD(P)H:quinone oxidoreductase (NQO1), similarly to the Nrf-2 activator sulforaphane. Furthermore, sulforaphane prevented PCB77-induced MCP-1 expression, suggesting that activation of Nrf-2 mediates the observed protection against PCB77 toxicity. Our data implicate A4/J4-NPs as mediators of omega-3 fatty acid-mediated protection against the endothelial toxicity of coplanar PCBs. PMID:21130106

  14. Effects of the contraceptive skin patch and subdermal contraceptive implant on markers of endothelial cell activation and inflammation.

    PubMed

    Hernandez-Juarez, Jesus; Sanchez-Serrano, Juan Carlos; Moreno-Hernandez, Manuel; Alvarado-Moreno, Jose Antonio; Hernandez-Lopez, Jose Rubicel; Isordia-Salas, Irma; Majluf-Cruz, Abraham

    2015-07-01

    Changes in blood coagulation factors may partially explain the association between hormonal contraceptives and thrombosis. Therefore, the likely effects of the contraceptive skin patch and subdermal contraceptive implant on levels of inflammatory markers and endothelial activation were analyzed. This was an observational, prospective, longitudinal, nonrandomized study composed of 80 women between 18 and 35 years of age who made the decision to use the contraceptive skin patch or subdermal contraceptive implant. vascular cell adhesion molecule-1 (VCAM-1), endothelial cell leukocyte adhesion molecule-1 (ELAM-1), von Willebrand factor (VWF), and plasminogen activator inhibitor type 1(PAI-1) as well as high-sensitivity C-reactive protein (hsCRP) were assayed before and after 4 months of use of the contraceptive method. VCAM-1, VWF, and PAI-1 remained unchanged in the contraceptive skin patch group; however, a significant increase in hsCRP (0.29-0.50 mg/dL; P =.012) and a significant decrease in ELAM-1 (44-25 ng/mL; P =.022) were observed. A significant diminution in VCAM-1 (463-362 ng/mL; P =.022) was also found in the subdermal contraceptive implant group. Our results strongly suggest that these contraceptive methods do not induce endothelial activation after 4 months of use. Increase in hsCRP levels was unrelated to changes in markers of endothelial activation. PMID:25655356

  15. Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells.

    PubMed

    Kása, Anita; Czikora, István; Verin, Alexander D; Gergely, Pál; Csortos, Csilla

    2013-09-01

    Reversible Ser/Thr phosphorylation of cytoskeletal and adherent junction (AJ) proteins has a critical role in the regulation of endothelial cell (EC) barrier function. We have demonstrated earlier that protein phosphatase 2A (PP2A) activity is important in EC barrier integrity. In the present work, macro- and microvascular EC were examined and we provided further evidence on the significance of PP2A in the maintenance of EC cytoskeleton and barrier function with special focus on the Bα (regulatory) subunit of PP2A. Immunofluorescent staining revealed that the inhibition of PP2A results in changes in the organization of EC cytoskeleton as microtubule dissolution and actin re-arrangement were detected. Depletion of Bα regulatory subunit of PP2A had similar effect on the cytoskeleton structure of the cells. Furthermore, transendothelial electric resistance measurements demonstrated significantly slower barrier recovery of Bα depleted EC after thrombin treatment. AJ proteins, VE-cadherin and β-catenin, were detected along with Bα in pull-down assay. Also, the inhibition of PP2A (by okadaic acid or fostriecin) or depletion of Bα caused β-catenin translocation from the membrane to the cytoplasm in parallel with its phosphorylation on Ser552. In conclusion, our data suggest that the A/Bα/C holoenzyme form of PP2A is essential in EC barrier integrity both in micro- and macrovascular EC. PMID:23721711

  16. Cyclic AMP enhances agonist-induced Ca2+ entry into endothelial cells by activation of potassium channels and membrane hyperpolarization.

    PubMed Central

    Graier, W F; Kukovetz, W R; Groschner, K

    1993-01-01

    The mechanism underlying cyclic AMP (cAMP)-mediated amplification of agonist-induced Ca2+ responses in endothelial cells was investigated in pig endothelial cells. Forskolin, adenosine and isoprenaline, as well as the membrane-permeant cAMP analogue dibutyryl cAMP, enhanced bradykinin-induced rises in intracellular free Ca2+ as well as bradykinin-induced Mn2+ entry. These agents were also found to hyperpolarize endothelial cells without increasing intracellular Ca2+ by itself, i.e. in the absence of bradykinin. Both amplification of bradykinin effects and the hyperpolarizing action was blocked by the protein kinase inhibitor H-8. The involvement of K+ channels in the hyperpolarizing effects of forskolin was consequently studied in perforated outside-out vesicles. Two different types of K+ channels were recorded, one of which had a large conductance (170 pS) and was activated by forskolin. We suggest that stimulation of endothelial adenylate cyclase results in activation of large-conductance K+ channels and consequently in membrane hyperpolarization, which in turn enhances bradykinin-induced entry of Ca2+ by increasing its electrochemical gradient. PMID:8385935

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

    PubMed Central

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

    2015-01-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte–endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum

  18. Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

    SciTech Connect

    Nagane, Masaki; Yasui, Hironobu; Sakai, Yuri; Yamamori, Tohru; Niwa, Koichi; Hattori, Yuichi; Kondo, Takashi; Inanami, Osamu

    2015-01-02

    Highlights: • eNOS activity is increased in BAECs exposed to X-rays. • ATM is involved in this increased eNOS activity. • HSP90 modulates the radiation-induced activation of ATM and eNOS. - Abstract: In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90.

  19. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Li, Xin-Yuan; Hu, Qing-Qing; Ma, He-Ping

    2016-01-01

    Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (PO). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and PO. Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.

  20. Endothelial cell activation by hemodynamic shear stress derived from arteriovenous fistula for hemodialysis access.

    PubMed

    Franzoni, Marco; Cattaneo, Irene; Longaretti, Lorena; Figliuzzi, Marina; Ene-Iordache, Bogdan; Remuzzi, Andrea

    2016-01-01

    Intimal hyperplasia (IH) is the first cause of failure of an arteriovenous fistula (AVF). The aim of the present study was to investigate the effects on endothelial cells (ECs) of shear stress waveforms derived from AVF areas prone to develop IH. We used a cone-and-plate device to obtain real-time control of shear stress acting on EC cultures. We exposed human umbilical vein ECs for 48 h to different shear stimulations calculated in a side-to-end AVF model. Pulsatile unidirectional flow, representative of low-risk stenosis areas, induced alignment of ECs and actin fiber orientation with flow. Shear stress patterns of reciprocating flow, derived from high-risk stenosis areas, did not affect EC shape or cytoskeleton organization, which remained similar to static cultures. We also evaluated flow-induced EC expression of genes known to be involved in cytoskeletal remodeling and expression of cell adhesion molecules. Unidirectional flow induced a significant increase in Kruppel-like factor 2 mRNA expression, whereas it significantly reduced phospholipase D1, α4-integrin, and Ras p21 protein activator 1 mRNA expression. Reciprocating flow did not increase Kruppel-like factor 2 mRNA expression compared with static controls but significantly increased mRNA expression of phospholipase D1, α4-integrin, and Ras p21 protein activator 1. Reciprocating flow selectively increased monocyte chemoattractant protein-1 and IL-8 production. Furthermore, culture medium conditioned by ECs exposed to reciprocating flows selectively increased smooth muscle cell proliferation compared with unidirectional flow. Our results indicate that protective vascular effects induced in ECs by unidirectional pulsatile flow are not induced by reciprocating shear forces, suggesting a mechanism by which oscillating flow conditions may induce the development of IH in AVF and vascular access dysfunction. PMID:26497959

  1. Sirt 1 activator inhibits the AGE-induced apoptosis and p53 acetylation in human vascular endothelial cells.

    PubMed

    Li, Peng; Zhang, Lina; Zhou, Changyong; Lin, Nan; Liu, Aiguo

    2015-01-01

    Advanced glycation end products (AGEs) by nonenzymatic glycation reactions are extremely accumulated in the diabetic vascular cells, neurons, and glia, and are confirmed to play important role in the pathogenesis of diabetes mellitus -induced cardiovascular complications. Sirt 1, known as mammalian sirtuin, has been recognized to regulate insulin secretion and protect cells against oxidative stress, which is promoted by the accumulated AGEs in cardiovascular cells. In the present study, we treated human endothelial Eahy926 cells with AGEs, and determined the apoptosis induction, caspase activation, the Sirt 1 activity, the expression and acetylation of p53. Then we manipulated Sirt 1 activity with a Sirt 1 activator, Resveratrol (RSV), and a Sirt 1 inhibitor, sirtinol, in the AGE-BSA-treated Eahy926 cells, and then re-evaluated the apoptosis induction, caspase activation, the expression and acetylation of p53. Results demonstrated that AGEs induced apoptosis in the human endothelial Eahy926 cells, by promoting the cytochrome c release, activation of caspase 9/3. Also, the AGE-BSA treatment promoted the total p53 level and acetylated (Ac) p53, but reduced the Sirt 1 level and activity. On the other hand, the Sirt 1 inhibitor/activator not only deteriorated/ameliorated the promotion to p53 level and Ac p53, but also aggravated/inhibited the AGE-induced apoptosis and the promotion to apoptosis-associated signaling molecules. In conclusion, the present study confirmed the apoptosis promotion by AGEs in endothelial Eahy926 cells, by regulating the Sirt 1 activity and p53 signaling, it also implies the protective role of Sirt 1 activator against the AGE-induced apoptosis. PMID:26354378

  2. The anti-inflammatory drug nimesulide inhibits neutrophil adherence to and migration across monolayers of cytokine-activated endothelial cells.

    PubMed

    Dapino, P; Ottonello, L; Dallegri, F

    1994-01-01

    Neutrophil migration through the microvascular endothelium represents a fundamental event for the cell accumulation at sites of tissue injury. Owing to their capacity to modify the structural and functional characteristics of endothelial cells, inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) play a pivotal role in directing circulating neutrophils away from the bloodstream to the interstitial tissue. In order to study neutrophil transendothelial migration, human umbilical vein endothelial cells were grown to confluence on the polycarbonate filter of two-compartment migration chambers. Pretreatment of the endothelial cell monolayers with TNF alpha for 4 h resulted in rapid migration of approximately 50% of subsequently added neutrophils across the layers. In contrast, < 10% of added neutrophils penetrated untreated endothelial monolayers. Using TNF alpha-treated endothelium, neutrophil transmigration was inhibited by the methane sulfonanilide anti-inflammatory drug nimesulide. Moreover, neutrophil adherence to TNF alpha-treated endothelial monolayers, cultured in microtiter wells, was markedly reduced by nimesulide. A linear correlation between the drug-dependent inhibition of neutrophil transmigration and neutrophil adherence was found. Finally, nimesulide did not interfere with the TNF alpha ability to convert resting endothelium into a pro-adhesive and pro-locomotory cell layer. The data suggest that nimesulide reduces neutrophil transendothelial migration primarily by limiting the cell anchorage to the TNF alpha-activated endothelium. Therefore, the drug has the potential to down-regulate neutrophil extravasation and, in turn, the burden of neutrophil oxidants and proteases leading to tissue injury at sites of inflammation. PMID:7824814

  3. Intima modifier locus 2 controls endothelial cell activation and vascular permeability

    PubMed Central

    Smolock, Elaine M.; Burke, Ryan M.; Wang, Chenjing; Thomas, Tamlyn; Batchu, Sri N.; Qiu, Xing; Zettel, Martha; Fujiwara, Keigi; Berk, Bradford C.

    2014-01-01

    Carotid intima formation is a significant risk factor for cardiovascular disease. C3H/FeJ (C3H/F) and SJL/J (SJL) inbred mouse strains differ in susceptibility to immune and vascular traits. Using a congenic approach we demonstrated that the Intima modifier 2 (Im2) locus on chromosome 11 regulates leukocyte infiltration. We sought to determine whether inflammation was due to changes in circulating immune cells or activation of vascular wall cells in genetically pure Im2 (C3H/F.SJL.11.1) mice. Complete blood counts showed no differences in circulating monocytes between C3H/F and C3H/F.SJL.11.1 compared with SJL mice. Aortic vascular cell adhesion molecule-1 (VCAM-1) total protein levels were dramatically increased in SJL and C3H/F.SJL.11.1 compared with C3H/F mice. Immunostaining of aortic endothelial cells (EC) showed a significant increase in VCAM-1 expression in SJL and C3H/F.SJL.11.1 compared with C3H/F under steady flow conditions. Immunostaining of EC membranes revealed a significant decrease in EC size in SJL and C3H/F.SJL.11.1 vs. C3H/F in regions of disturbed flow. Vascular permeability was significantly higher in C3H/F.SJL.11.1 compared with C3H/F. Our results indicate that Im2 regulation of leukocyte infiltration is mediated by EC inflammation and permeability. RNA sequencing and pathway analyses comparing genes in the Im2 locus to C3H/F provide insight into candidate genes that regulate vascular wall inflammation and permeability highlighting important genetic mechanisms that control vascular intima in response to injury. PMID:24986958

  4. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    SciTech Connect

    Schwarz, Margaret A. . E-mail: m.schwarz@umdnj.edu; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

    2005-12-10

    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function.

  5. α-enolase Causes Pro-Inflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils through Plasmin Activation of Protease-Activated Receptor-2

    PubMed Central

    Bock, Ashley; Tucker, Nicole; Kelher, Marguerite R.; Khan, Samina Y.; Gonzalez, Eduardo; Wohlauer, Max; Hansen, Kirk; Dzieciatkowska, Monika; Sauaia, Angels; Banerjee, Anirban; Moore, Ernest E.; Silliman, Christopher C.

    2015-01-01

    Pro-inflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung (ALI) and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients primes PMNs and causes pro-inflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity. Methods Proteomic analyses of field plasma samples from injured vs. healthy patients was used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and ICAM-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease activated receptor-1 (PAR-1) and PAR-2 and co-precipitation of α-enolase with PAR-2 and plasminogen/plasmin. Results α-enolase increased 10.8-fold in injured patients (p<0.05). Thrombin and α-enolase significantly increased ICAM-1 surface expression on HMVECs, which was inhibited by anti-proteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-enolase co-precipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required. We conclude that α-enolase increases post-injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such pro-inflammatory endothelial activation may predispose to PMN-mediated organ injury. PMID:25944790

  6. cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

    PubMed Central

    Ginsburg, D; Zeheb, R; Yang, A Y; Rafferty, U M; Andreasen, P A; Nielsen, L; Dano, K; Lebo, R V; Gelehrter, T D

    1986-01-01

    Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7. Images PMID:3097076

  7. Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.

    PubMed

    Sipkens, Jessica A; Hahn, Nynke; van den Brand, Carlien S; Meischl, Christof; Cillessen, Saskia A G M; Smith, Desirée E C; Juffermans, Lynda J M; Musters, René J P; Roos, Dirk; Jakobs, Cornelis; Blom, Henk J; Smulders, Yvo M; Krijnen, Paul A J; Stehouwer, Coen D A; Rauwerda, Jan A; van Hinsbergh, Victor W M; Niessen, Hans W M

    2013-11-01

    Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells. PMID:22038300

  8. Experimental study on apoptosis of TNFR1 receptor pro-endothelial progenitor cells activated by high glucose induced oxidative stress

    PubMed Central

    Liu, Yong; Xei, Fei; Xu, Xiong-Fei; Zeng, Hong; He, Hu-Qiang; Zhang, Lei; Zheng, Ying-Qiang; He, Yan-Zheng

    2015-01-01

    Objective: To investigate whether high glucose in vitro activating TNFR1 and further promote rat marrow endothelial progenitor cells (EPCs) apoptosis. Methods: Rat morrow endothelial progenitor cells were cultured and identified by Confocal Microscopy; then were treated with high glucose (5.5, 15, 30, 60 mmol/L), mannitol (15, 30, 60, 90 mmol/L), high glucose + Tempol and high glucose+ MAB430. Apoptosis rate of the above cells were detected by flow cytometry. ROS and MDA level and anti-O2- were detected by colorimetric technique; the expression level of TNFR1 induced signal pathway related proteins were detected by Western blotting. Results: High glucose can induce endothelial progenitor cells apoptosis, which is mostly in the later stage (72 h-96 h) instead of the earlier stage (24 h-48 h); high glucose can also induce oxidative stress reaction and the produces ROS and MDA increase significantly in the later stage (after 72 h), but anti-O2- decrease significantly. TNF apoptosis signal pathway related protein expression level not increase in the earlier stage (before 24 h) but increase significantly in the later stage (after 72 h). Tempol and MAB430 down-regulate TNF apoptosis signal pathway related protein expression and reduce EPCs apoptosis. Conclusion: High glucose activates the TNFR1 of TPCs through oxidative stress reaction and further induces cell apoptosis. PMID:26884909

  9. Donepezil attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells through SIRT1 activation.

    PubMed

    Zhang, Tao; Tian, Feng; Wang, Jing; Zhou, Shanshan; Dong, Xueqing; Guo, Kai; Jing, Jing; Zhou, Ying; Chen, Yundai

    2015-09-01

    Cellular senescence of endothelial cells is a damage and stress response which induces pro-inflammatory, pro-atherosclerotic, and pro-thrombotic phenotypes. Donepezil is a drug used for the treatment of mild to moderate dementia of the Alzheimer's disease (AD). The aim of the present study was to investigate the attenuation of endothelial cell senescence by donepezil and to explore the mechanisms underlying the anti-aging effects of donepezil. Our results indicated that high glucose (HG) markedly decreased cell viability of human umbilical vein endothelial cells (HUVECs), and this phenomenon was reversed by treatment with donepezil. Importantly, our results displayed that the frequency of senescent (SA-ß-gal-positive) cells and the expression level of senescence genes (PAI-1 and p21) were significantly higher in the HG group compared with the normal glucose (NG) group, and these changes were blocked by treatment with donepezil. Also, our results showed that donepezil inhibits the generation of reactive oxygen species (ROS), which promotes cellular senescence. Pretreatment with nicotinamide (NAM), a sirtuin 1 (SIRT1) inhibitor, inhibited the reduction in senescence associated with donepezil. Indeed, our results indicated that donepezil increased the SIRT1 enzyme activity. Therefore, these results show that donepezil delays cellular senescence that is promoted under HG condition via activation of SIRT1. PMID:26194321

  10. Reduced Ang2 expression in aging endothelial cells.

    PubMed

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

    2016-06-01

    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. PMID:27137842

  11. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Kennedy, C; Gould, G W; Plevin, R

    1996-03-01

    1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor. PMID:8882634

  12. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

    PubMed Central

    Graham, A.; McLees, A.; Kennedy, C.; Gould, G. W.; Plevin, R.

    1996-01-01

    1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8882634

  13. Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.

    PubMed Central

    Key, N S; Vercellotti, G M; Winkelmann, J C; Moldow, C F; Goodman, J L; Esmon, N L; Esmon, C T; Jacob, H S

    1990-01-01

    Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor. Within 4 hr of infection of HUVECs, TM activity measured by thrombin-dependent protein C activation declined 21 +/- 3% (P less than 0.05) and by 18 hr, 48 +/- 5% (P less than 0.001). Similar significant TM decrements accompanied infection of bovine aortic endothelial cells. Identical TM loss was induced with HSV-2 infection but not with adenovirus infection. Decreased surface expression of TM antigen (measured by the specific binding of a polyclonal antibody to bovine TM) closely paralleled the loss of TM activity. As examined by Northern blotting, these losses apparently reflected rapid onset (within 4 hr of HSV infection) loss of mRNA for TM. In contrast, HSV infection induced a viral-dose-dependent increase in synthesis of tissue factor protein, adding to the procoagulant state. The results indicate that loss of endothelial protein-synthetic capacity is not a universal effect of HSV infection. We suggest that the procoagulant state induced by reduction in TM activity and amplified tissue factor activity accompanying HSV infection of endothelium could contribute to deposition of thrombi on atherosclerotic plaques and to the "coagulant-necrosis" state that characterizes HSV-infected mucocutaneous lesions. Images PMID:2169619

  14. Endothelial cell-derived nitric oxide enhances aerobic glycolysis in astrocytes via HIF-1α-mediated target gene activation.

    PubMed

    Brix, Britta; Mesters, Jeroen R; Pellerin, Luc; Jöhren, Olaf

    2012-07-11

    Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation. PMID:22787058

  15. The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

    PubMed Central

    Bae, Jong-Sup; Yang, Likui; Manithody, Chandrashekhara

    2007-01-01

    Recent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the γ-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin–sensitive Gi-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells. PMID:17823308

  16. AZD3582 increases heme oxygenase-1 expression and antioxidant activity in vascular endothelial and gastric mucosal cells.

    PubMed

    Berndt, Georg; Grosser, Nina; Hoogstraate, Janet; Schröder, Henning

    2005-06-01

    AZD3582 [4-(nitrooxy)-butyl-(2S)-2-(6-methoxy-2-naphthyl)-propanoate] is a COX-inhibiting nitric oxide donator (CINOD). Incubation of human endothelial cells (derived from umbilical cord) with AZD3582 (10-100muM) led to increased expression of heme oxygenase (HO)-1 mRNA and protein. Heme oxygenase-1 (HO-1) is a crucial mediator of antioxidant and tissue-protective actions. In contrast, naproxen (a non-selective NSAID) and rofecoxib (a selective inhibitor of COX-2), did not affect HO-1 expression. Pre-treating endothelial cells with AZD3582 at concentrations that were effective at inducing HO-1 also reduced NADPH-dependent production of oxygen radicals. Antioxidant activity in the endothelial cells persisted after AZD3582 had been washed out from the incubation medium. When added exogenously to the cells at low micromolar concentrations, the HO-1 metabolite, bilirubin, virtually abolished NADPH-dependent oxidative stress. AZD3582-induced blockade of free-radical formation was reversed in the presence of the HO-1 inhibitor, tin protoporphyrin-IX (SnPP). Similar results were obtained in human gastric mucosal cells (KATO-III). Our results demonstrate that HO-1 is a novel target of AZD3582. PMID:15911218

  17. Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: Antithrombin binding on cultured endothelial cells and perfused rat aorta

    SciTech Connect

    de Agostini, A.I.; Watkins, S.C.; Slayter, H.S.; Youssoufian, H.; Rosenberg, R.D. )

    1990-09-01

    We have studied the interaction of {sup 125}I-antithrombin ({sup 125}I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with {sup 125}I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.

  18. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation. PMID:25683686

  19. Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells.

    PubMed

    Kurpios-Piec, Dagmara; Grosicka-Maciąg, Emilia; Woźniak, Katarzyna; Kowalewski, Cezary; Kiernozek, Ewelina; Szumiło, Maria; Rahden-Staroń, Iwonna

    2015-02-01

    Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 µg/mL or 2 µg/mL) (0.2 µM or 0.8 µM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action. PMID:25752435

  20. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

    PubMed

    Belcher, John D; Chen, Chunsheng; Nguyen, Julia; Milbauer, Liming; Abdulla, Fuad; Alayash, Abdu I; Smith, Ann; Nath, Karl A; Hebbel, Robert P; Vercellotti, Gregory M

    2014-01-16

    Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme

  1. Anthocyanins and their gut metabolites reduce the adhesion of monocyte to TNFα-activated endothelial cells at physiologically relevant concentrations.

    PubMed

    Krga, Irena; Monfoulet, Laurent-Emmanuel; Konic-Ristic, Aleksandra; Mercier, Sylvie; Glibetic, Maria; Morand, Christine; Milenkovic, Dragan

    2016-06-01

    An increasing number of evidence suggests a protective role of dietary anthocyanins against cardiovascular diseases. Anthocyanins' extensive metabolism indicates that their metabolites could be responsible for the protective effects associated with consumption of anthocyanin-rich foods. The aim of this work was to investigate the effect of plasma anthocyanins and their metabolites on the adhesion of monocytes to TNFα-activated endothelial cells and on the expression of genes encoding cell adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were exposed to circulating anthocyanins: cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, anthocyanin degradation product: 4-hydroxybenzaldehyde, or to their gut metabolites: protocatechuic, vanillic, ferulic and hippuric acid, at physiologically-relevant concentrations (0.1-2 μM) and time of exposure. Both anthocyanins and gut metabolites decreased the adhesion of monocytes to HUVECs, with a magnitude ranging from 18.1% to 47%. The mixture of anthocyanins and that of gut metabolites also reduced monocyte adhesion. However, no significant effect on the expression of genes encoding E-selectin, ICAM1 and VCAM1 was observed, suggesting that other molecular targets are involved in the observed effect. In conclusion, this study showed the potency of anthocyanins and their gut metabolites to modulate the adhesion of monocytes to endothelial cells, the initial step in atherosclerosis development, under physiologically-relevant conditions. PMID:26873533

  2. Grape pomace extract exerts antioxidant effects through an increase in GCS levels and GST activity in muscle and endothelial cells.

    PubMed

    Goutzourelas, Nikolaos; Stagos, Dimitrios; Housmekeridou, Anastasia; Karapouliou, Christina; Kerasioti, Efthalia; Aligiannis, Nektarios; Skaltsounis, Alexios L; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

    2015-08-01

    In a previous study, we demonstrated that a grape pomace extract (GPE) exerted antioxidant activity in endothelial (EA.hy926) and muscle (C2C12) cells through an increase in glutathione (GSH) levels. In the present study, in order to elucidate the mechanisms responsible for the antioxidant activity of GPE, its effects on the expression of critical antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD)1, heme oxygenase 1 (HO-1) and gamma-glutamylcysteine synthetase (GCS) were assessed in EA.hy926 and C2C12 cells. Moreover, the effects of GPE on CAT, SOD and glutathione S-transferase (GST) enzymatic activity were evaluated. For this purpose, the C2C12 and EA.hy926 cells were treated with GPE at low and non-cytotoxic concentrations (2.5 and 10 µg/ml for the C2C12 cells; 0.068 and 0.250 µg/ml for the EA.hy926 cells) for 3, 6, 12, 18 and 24 h. Following incubation, enzymatic expression and activity were assessed. The results revealed that treatment with GPE significantly increased GCS levels and GST activity in both the C2C12 and EA.hy926 cells. However, GPE significantly decreased CAT levels and activity, but only in the muscle cells, while it had no effect on CAT levels and activity in the endothelial cells. Moreover, treatment with GPE had no effect on HO-1 and SOD expression and activity in both cell lines. Therefore, the present results provide further evidence of the crucial role of GSH systems in the antioxidant effects exerted by GPE. Thus, GPE may prove to be effective for use as a food supplement for the treatment of oxidative stress-induced pathological conditions of the cardiovascular and skeletal muscle systems, particularly those associated with low GSH levels. PMID:26082074

  3. Grape pomace extract exerts antioxidant effects through an increase in GCS levels and GST activity in muscle and endothelial cells

    PubMed Central

    GOUTZOURELAS, NIKOLAOS; STAGOS, DIMITRIOS; HOUSMEKERIDOU, ANASTASIA; KARAPOULIOU, CHRISTINA; KERASIOTI, EFTHALIA; ALIGIANNIS, NEKTARIOS; SKALTSOUNIS, ALEXIOS L; SPANDIDOS, DEMETRIOS A; TSATSAKIS, ARISTIDIS M; KOURETAS, DEMETRIOS

    2015-01-01

    In a previous study, we demonstrated that a grape pomace extract (GPE) exerted antioxidant activity in endothelial (EA.hy926) and muscle (C2C12) cells through an increase in glutathione (GSH) levels. In the present study, in order to elucidate the mechanisms responsible for the antioxidant activity of GPE, its effects on the expression of critical antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD)1, heme oxygenase 1 (HO-1) and gamma-glutamylcysteine synthetase (GCS) were assessed in EA.hy926 and C2C12 cells. Moreover, the effects of GPE on CAT, SOD and glutathione S-transferase (GST) enzymatic activity were evaluated. For this purpose, the C2C12 and EA.hy926 cells were treated with GPE at low and non-cytotoxic concentrations (2.5 and 10 µg/ml for the C2C12 cells; 0.068 and 0.250 µg/ml for the EA.hy926 cells) for 3, 6, 12, 18 and 24 h. Following incubation, enzymatic expression and activity were assessed. The results revealed that treatment with GPE significantly increased GCS levels and GST activity in both the C2C12 and EA.hy926 cells. However, GPE significantly decreased CAT levels and activity, but only in the muscle cells, while it had no effect on CAT levels and activity in the endothelial cells. Moreover, treatment with GPE had no effect on HO-1 and SOD expression and activity in both cell lines. Therefore, the present results provide further evidence of the crucial role of GSH systems in the antioxidant effects exerted by GPE. Thus, GPE may prove to be effective for use as a food supplement for the treatment of oxidative stress-induced pathological conditions of the cardiovascular and skeletal muscle systems, particularly those associated with low GSH levels. PMID:26082074

  4. In vivo hematopoietic Myc activation directs a transcriptional signature in endothelial cells within the bone marrow microenvironment.

    PubMed

    Franke, Katharina; Vilne, Baiba; Prazeres da Costa, Olivia; Rudelius, Martina; Peschel, Christian; Oostendorp, Robert A J; Keller, Ulrich

    2015-09-01

    Cancer pathogenesis involves tumor-intrinsic genomic aberrations and tumor-cell extrinsic mechanisms such as failure of immunosurveillance and structural and functional changes in the microenvironment. Using Myc as a model oncogene we established a conditional mouse bone marrow transduction/transplantation model where the conditional activation of the oncoprotein Myc expressed in the hematopoietic system could be assessed for influencing the host microenvironment. Constitutive ectopic expression of Myc resulted in rapid onset of a lethal myeloproliferative disorder with a median survival of 21 days. In contrast, brief 4-day Myc activation by means of the estrogen receptor (ER) agonist tamoxifen did not result in gross changes in the percentage/frequency of hematopoietic lineages or hematopoietic stem/ progenitor cell (HSPC) subsets, nor did Myc activation significantly change the composition of the non-hematopoietic microenvironment defined by phenotyping for CD31, ALCAM, and Sca-1 expression. Transcriptome analysis of endothelial CD45- Ter119- cells from tamoxifen-treated MycER bone marrow graft recipients revealed a gene expression signature characterized by specific changes in the Rho subfamily pathway members, in the transcription-translation-machinery and in angiogenesis. In conclusion, intra-hematopoietic Myc activation results in significant transcriptome alterations that can be attributed to oncogene-induced signals from hematopoietic cells towards the microenvironment, e. g. endothelial cells, supporting the idea that even pre-leukemic HSPC highjack components of the niche which then could protect and support the cancer-initiating population. PMID:26308666

  5. High expression levels of egfl7 correlate with low endothelial cell activation in peritumoral vessels of human breast cancer

    PubMed Central

    Pannier, Diane; Philippin-Lauridant, Géraldine; Baranzelli, Marie-Christine; Bertin, Delphine; Bogart, Emilie; Delprat, Victor; Villain, Gaëlle; Mattot, Virginie; Bonneterre, Jacques; Soncin, Fabrice

    2016-01-01

    Tumor blood vessels participate in the immune response against cancer cells and we previously used pre-clinical models to demonstrate that egfl7 (VE-statin) promotes tumor cell evasion from the immune system by repressing endothelial cell activation, preventing immune cells from entering the tumor mass. In the present study, the expression levels of egfl7 and that of ICAM-1 as a marker of endothelium activation, were evaluated in peritumoral vessels of human breast cancer samples. Breast cancer samples (174 invasive and 30 in situ) from 204 patients treated in 2005 were immunostained for CD31, ICAM-1 and stained for egfl7 using in situ hybridization. The expression levels of ICAM-1 and egfl7 were assessed in peritumoral areas using semi-quantitative scales. There was a strong and significant inverse correlation between the expression of ICAM-1 and that of egfl7 in CD31+ blood vessels. When the ICAM-1 score increased, the egfl7 score reduced significantly (P=0.004), and vice-versa (Cuzick's test for trend across ordered groups). In order to determine which gene influenced the other gene between egfl7 and ICAM-1, the expression levels of either gene were modulated in endothelial cells. Egfl7 regulated ICAM-1 expression while ICAM-1 had no effects on egfl7 expression in the same conditions. Altogether, these results provide further results that egfl7 serves a regulatory role in endothelial cell activation in relation to immune infiltration and that it is a potential therapeutic target to consider for improving anticancer immunotherapies. PMID:27446447

  6. 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

    SciTech Connect

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

    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

  7. IQ Domain GTPase-Activating Protein 1 is Involved in Shear Stress-Induced Progenitor-Derived Endothelial Cell Alignment

    PubMed Central

    Rami, Lila; Auguste, Patrick; Thebaud, Noélie B.; Bareille, Reine; Daculsi, Richard; Ripoche, Jean; Bordenave, Laurence

    2013-01-01

    Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m2) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This “switch” was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress. PMID:24278215

  8. Interstitial Fluid Flow Intensity Modulates Endothelial Sprouting in Restricted Src-Activated Cell Clusters During Capillary Morphogenesis

    PubMed Central

    Hernández Vera, Rodrigo; Genové, Elsa; Alvarez, Lery; Borrós, Salvador; Kamm, Roger; Lauffenburger, Douglas

    2009-01-01

    Development of tissues in vitro with dimensions larger than 150 to 200 μm requires the presence of a functional vascular network. Therefore, we have studied capillary morphogenesis under controlled biological and biophysical conditions with the aim of promoting vascular structures in tissue constructs. We and others have previously demonstrated that physiological values of interstitial fluid flow normal to an endothelial monolayer in combination with vascular endothelial growth factor play a critical role during capillary morphogenesis by promoting cell sprouting. In the present work, we studied the effect that a range of interstitial flow velocities (0–50 μm/min) has in promoting the amount, length, and branching of developing sprouts during capillary morphogenesis. The number of capillary-like structures developed from human umbilical vein endothelial cell monolayers across the interstitial flow values tested was not significantly affected. Instead, the length and branching degree of the sprouts presented a significant maximum at flow velocities of 10 to 20 μm/min. More-over, at these same flow values, the phosphorylation level of Src also showed its peak. We discovered that capillary morphogenesis is restricted to patches of Src-activated cells (phosphorylated Src (pSrc)) at the monolayer, suggesting that the transduction pathway in charge of sensing the mechanical stimulus induced by flow is promoting predetermined mechanically sensitive areas (pSrc) to undergo capillary morphogenesis. PMID:18636940

  9. Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier.

    PubMed

    DiStefano, Peter V; Smrcka, Alan V; Glading, Angela J

    2016-01-01

    The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability. PMID:27612188

  10. Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor

    NASA Astrophysics Data System (ADS)

    Ishikawa, Fuyuki; Miyazono, Kohei; Hellman, Ulf; Drexler, Hannes; Wernstedt, Christer; Hagiwara, Koichi; Usuki, Kensuke; Takaku, Fumimaro; Risau, Werner; Heldin, Carl-Henrik

    1989-04-01

    Cloning and sequencing of the complementary DNA for platelet-derived endothelial cell growth factor indicates that it is a novel factor distinct from previously characterized proteins. The factor, a protein with a relative molecular mass of about 45,000, stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo.

  11. HLA Class II Antibody Activation of Endothelial Cells Promotes Th17 and Disrupts Regulatory T Lymphocyte Expansion.

    PubMed

    Lion, J; Taflin, C; Cross, A R; Robledo-Sarmiento, M; Mariotto, E; Savenay, A; Carmagnat, M; Suberbielle, C; Charron, D; Haziot, A; Glotz, D; Mooney, N

    2016-05-01

    Kidney transplantation is the most successful treatment option for patients with end-stage renal disease, and chronic antibody-mediated rejection is the principal cause of allograft loss. Predictive factors for chronic rejection include high levels of HLA alloantibodies (particularly HLA class II) and activation of graft endothelial cells (ECs). The mechanistic basis for this association is unresolved. We used an experimental model of HLA-DR antibody stimulation of microvascular ECs to examine the mechanisms underlying the association between HLA class II antibodies, EC activation and allograft damage. Activation of ECs with the F(Ab')2 fragment of HLA-DR antibody led to phosphorylation of Akt, ERK and MEK and increased IL-6 production by ECs cocultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an Akt-dependent manner. We previously showed that HLA-DR-expressing ECs induce polarization of Th17 and FoxP3(bright) regulatory T cell (Treg) subsets. Preactivation of ECs with anti-HLA-DR antibody redirected EC allogenicity toward a proinflammatory response by decreasing amplification of functional Treg and by further increasing IL-6-dependent Th17 expansion. Alloimmunized patient serum containing relevant HLA-DR alloantibodies selectively bound and increased EC secretion of IL-6 in cocultures with PBMCs. These data contribute to understanding of potential mechanisms of antibody-mediated endothelial damage independent of complement activation and FcR-expressing effector cells. PMID:26614587

  12. ICAM-1-activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration.

    PubMed

    Liu, Guoquan; Place, Aaron T; Chen, Zhenlong; Brovkovych, Viktor M; Vogel, Stephen M; Muller, William A; Skidgel, Randal A; Malik, Asrar B; Minshall, Richard D

    2012-08-30

    Polymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)-dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)-mediated transendothelial migration. An important unanswered question is whether ICAM-1-activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1-dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1(-/-) and eNOS(-/-) mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr(518)Phe ICAM-1 mutant, induced SHP-2-dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr(518)Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho-ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti-PECAM-1 mAb-binding activity. These results collectively show that ICAM-1-activated Src and eNOS signaling sequentially induce PECAM-1-mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation. PMID:22806890

  13. Monocrotaline pyrrole-induced changes in angiotensin-converting enzyme activity of cultured pulmonary artery endothelial cells.

    PubMed Central

    Hoorn, C. M.; Roth, R. A.

    1993-01-01

    1. Changes in the structural and functional integrity of endothelium have been recognized as relatively early features of delayed and progressive pulmonary vascular injury caused by the pyrrolizidine alkaloid, monocrotaline (MCT). Although a number of investigators have evaluated angiotensin-converting enzyme (ACE) activity in the lungs of rats treated with MCT, the exact nature of changes in activity of this enzyme and the role they may play in MCT pneumotoxicity remain controversial. 2. We examined the direct effects of monocrotaline pyrrole (MCTP), a toxic metabolite of MCT, on cultured endothelial cell ACE activity. Post-confluent monolayers of porcine or bovine pulmonary artery endothelial cells (PECs or BECs, respectively) were treated with a single administration of MCTP at time 0; then they were examined for their ability to degrade the synthetic peptide, [3H]-benzoyl-Phe-Ala-Pro. 3. In PECs, which are relatively insensitive to the direct cytolytic effects of MCTP, monolayer ACE activity was unchanged initially but gradually decreased within 4 days after treatment with a high concentration of MCTP (150 microM). This decrease was transient, and PEC monolayer ACE activity returned to the control value by 10 days post treatment. 4. BEC monolayer ACE activity was also unchanged initially but rapidly declined within 4 days after MCTP treatment and remained depressed throughout the post treatment period. BECs were quite sensitive to the cytolytic effects of MCTP and the decline in ACE activity occurred coincident with the decrease in monolayer cellularity and appearance of marked cytotoxicity. 5. We conclude that high concentrations of MCTP decrease endothelial ACE activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8242234

  14. Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    PubMed Central

    Yoon, Yae Jin; Kim, Dae-Kyum; Yoon, Chang Min; Park, Jaesung; Kim, Yoon-Keun; Roh, Tae-Young; Gho, Yong Song

    2014-01-01

    Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference–mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases. PMID:25502753

  15. Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway

    PubMed Central

    Leffler, Nancy R.; Asch, Adam S.; Witte, Owen N.; Yang, Li V.

    2011-01-01

    Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2′,5′-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a Gi signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the Gs/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells. PMID:22110680

  16. Endothelial Cell Response to Fusobacterium nucleatum.

    PubMed

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

    2016-07-01

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

  17. Clostridium sordellii Lethal-Toxin Autoprocessing and Membrane Localization Activities Drive GTPase Glucosylation Profiles in Endothelial Cells

    PubMed Central

    Craven, Ryan

    2015-01-01

    ABSTRACT Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. One of the principle virulence factors is TcsL, a large protein toxin which glucosylates host GTPases to cause cytopathic and cytotoxic effects. TcsL has two enzymatic domains, an N-terminal glucosyltransferase domain (GTD) and an autoprocessing domain responsible for release of the GTD within the cell. The GTD can then use its N-terminal membrane localization domain (MLD) for orientation on membranes and modification of GTPases. This study describes the use of conditionally immortalized murine pulmonary microvascular endothelial cells as a model for the study of TcsL functional activities. Point mutations that disrupt the glucosyltransferase, autoprocessing, or membrane localization activities were introduced into a recombinant version of TcsL, and the activities of these mutants were compared to those of wild-type toxin. We observed that all mutants are defective or impaired in cytotoxicity but differ in their modification of Rac1 and Ras. The data suggest a model where differences in GTPase localization dictate cellular responses to intoxication and highlight the importance of autoprocessing in the function of TcsL. IMPORTANCE Clostridium sordellii is a bacterium that can infect humans and cause serious disease and death. The principle virulence factor associated with clinical symptoms is a large protein toxin known as lethal toxin. The mechanism of lethal-toxin intoxication is assumed to be similar to that of the homologous toxins from C. difficile, but very few studies have been done in the context of endothelial cells, a relevant target in C. sordellii infections. This study was designed to test the role of the lethal-toxin enzymatic activities and membrane localization in endothelial cell toxicity and host substrate modification. PMID:27303685

  18. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

  19. Engineering a Prostate-Specific Membrane Antigen–Activated Tumor Endothelial Cell Prodrug for Cancer Therapy

    PubMed Central

    Denmeade, Samuel R.; Mhaka, Annastasiah M.; Rosen, D. Marc; Brennen, W. Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; DeMarzo, Angelo M.; Wilding, George; Carducci, Michael A.; Dionne, Craig A.; Møller, Jesper V.; Nissen, Poul; Christensen, S. Brøgger; Isaacs, John T.

    2013-01-01

    Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer. PMID:22745436

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

    SciTech Connect

    Kito, Hiroaki; Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto; Ohya, Susumu; Asai, Kiyofumi; Imaizumi, Yuji

    2015-04-10

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

  1. Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation

    PubMed Central

    2016-01-01

    Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs. PMID:27382348

  2. Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation.

    PubMed

    Hu, Min; Liu, Bo

    2016-07-01

    Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs. PMID:27382348

  3. The control of vascular endothelial cell injury.

    PubMed

    Murota, S; Morita, I; Suda, N

    1990-01-01

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

  4. Low Concentrations of Uncouplers of Oxidative Phosphorylation Prevent Inflammatory Activation of Endothelial Cells by Tumor Necrosis Factor.

    PubMed

    Romaschenko, V P; Zinovkin, R A; Galkin, I I; Zakharova, V V; Panteleeva, A A; Tokarchuk, A V; Lyamzaev, K G; Pletjushkina, O Yu; Chernyak, B V; Popova, E N

    2015-05-01

    In endothelial cells, mitochondria play an important regulatory role in physiology as well as in pathophysiology related to excessive inflammation. We have studied the effect of low doses of mitochondrial uncouplers on inflammatory activation of endothelial cells using the classic uncouplers 2,4-dinitrophenol and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, as well as the mitochondria-targeted cationic uncoupler dodecyltriphenylphosphonium (C12TPP). All of these uncouplers suppressed the expression of E-selectin, adhesion molecules ICAM1 and VCAM1, as well as the adhesion of neutrophils to endothelium induced by tumor necrosis factor (TNF). The antiinflammatory action of the uncouplers was at least partially mediated by the inhibition of NFκB activation due to a decrease in phosphorylation of the inhibitory subunit IκBα. The dynamic concentration range for the inhibition of ICAM1 expression by C12TPP was three orders of magnitude higher compared to the classic uncouplers. Probably, the decrease in membrane potential inhibited the accumulation of penetrating cations into mitochondria, thus lowering the uncoupling activity and preventing further loss of mitochondrial potential. Membrane potential recovery after the removal of the uncouplers did not abolish its antiinflammatory action. Thus, mild uncoupling could induce TNF resistance in endothelial cells. We found no significant stimulation of mitochondrial biogenesis or autophagy by the uncouplers. However, we observed a decrease in the relative amount of fragmented mitochondria. The latter may significantly change the signaling properties of mitochondria. Earlier we showed that both classic and mitochondria-targeted antioxidants inhibited the TNF-induced NFκB-dependent activation of endothelium. The present data suggest that the antiinflammatory effect of mild uncoupling is related to its antioxidant action. PMID:26071781

  5. High-mobility group box 1 activates integrin-dependent homing of endothelial progenitor cells.

    PubMed

    Chavakis, Emmanouil; Hain, Andreas; Vinci, Maria; Carmona, Guillaume; Bianchi, Marco E; Vajkoczy, Peter; Zeiher, Andreas M; Chavakis, Triantafyllos; Dimmeler, Stefanie

    2007-02-01

    Endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. Integrins contribute to EPC homing. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly on cell necrosis and tissue damage, eliciting a proinflammatory response and stimulating tissue repair. In the present study, we investigated the effects of HMGB1 on EPC homing. EPCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products) and TLR2 (Toll-like receptor 2). EPC migration was stimulated by HMGB1 in a RAGE-dependent manner. In addition, the HMGB1-induced migration of EPCs on fibronectin and fibrinogen was significantly inhibited by antibodies against beta1 and beta2 integrins, respectively. Short-term prestimulation of EPCs with HMGB1 also increased EPC adhesion to endothelial cell monolayers, and this effect was blocked by antibodies to beta2 integrins or RAGE. HMGB1 increased EPC adhesion to the immobilized integrin ligands intercellular adhesion molecule-1 and fibronectin in a RAGE-dependent manner. Strikingly, HMGB1 rapidly increased integrin affinity and induced integrin polarization. Using intravital microscopy in a tumor model of neovascularization, prestimulation of EPCs with HMGB1 enhanced the initial in vivo adhesion of EPCs to microvessels and the recruitment of EPCs in the tumor tissue. In addition, prestimulation of EPCs with HMGB1 increased the homing of EPCs to ischemic muscles. In conclusion, these data represent a link between HMGB1 and integrin functions of EPCs and demonstrate that HMGB1 stimulates EPC homing to ischemic tissues. These results may provide a platform for the development of novel therapeutic approaches to improve EPC homing. PMID:17218606

  6. Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

    PubMed

    Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

    2016-11-01

    The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. PMID:27420801

  7. Role of erythropoietin in the angiogenic activity of bone marrow endothelial cells of MGUS and multiple myeloma patients

    PubMed Central

    Ferrucci, Arianna; Ria, Roberto; Ruggieri, Simona; Racanelli, Vito; Rao, Luigia; Annese, Tiziana; Nico, Beatrice; Vacca, Angelo; Ribatti, Domenico

    2016-01-01

    Increasing evidences suggest several biological roles for erythropoietin and its receptor (Epo and EpoR), unrelated to erythropoiesis, including angiogenesis. Here, we detected the expression of EpoR in bone marrow-derived endothelial cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients (MGECs and MMECs, respectively) and assessed whether Epo plays a role in MGECs- and MMECs-mediated angiogenesis. We show that EpoR is expressed by both MGECs and MMECs even though at a higher level in the first ones. Both EC types respond to rHuEpo in terms of cell proliferation, whereas other responses, including activation of JAK2/STAT5 and PI3K/Akt pathways, cell migration and capillarogenesis are enhanced by Epo in MGECs, but not in MMECs. In addition, the conditioned media of both Epo-treated cells induce a strong angiogenic response in vivo in the chorioallantoic membrane assay, comparable to that of vascular endothelial growth factor (VEGF). Overall, these data highlight the effect of Epo on MGECs- and MMECs-mediated angiogenesis: MGECs are more responsive to Epo treatment than MMECs, probably because over-angiogenic phenotype of MMECs is already activated by their autocrine/paracrine loops occurring in the “angiogenic switch” from MGUS. PMID:26919105

  8. Role of erythropoietin in the angiogenic activity of bone marrow endothelial cells of MGUS and multiple myeloma patients.

    PubMed

    Lamanuzzi, Aurelia; Saltarella, Ilaria; Ferrucci, Arianna; Ria, Roberto; Ruggieri, Simona; Racanelli, Vito; Rao, Luigia; Annese, Tiziana; Nico, Beatrice; Vacca, Angelo; Ribatti, Domenico

    2016-03-22

    Increasing evidences suggest several biological roles for erythropoietin and its receptor (Epo and EpoR), unrelated to erythropoiesis, including angiogenesis. Here, we detected the expression of EpoR in bone marrow-derived endothelial cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients (MGECs and MMECs, respectively) and assessed whether Epo plays a role in MGECs- and MMECs-mediated angiogenesis. We show that EpoR is expressed by both MGECs and MMECs even though at a higher level in the first ones. Both EC types respond to rHuEpo in terms of cell proliferation, whereas other responses, including activation of JAK2/STAT5 and PI3K/Akt pathways, cell migration and capillarogenesis are enhanced by Epo in MGECs, but not in MMECs. In addition, the conditioned media of both Epo-treated cells induce a strong angiogenic response in vivo in the chorioallantoic membrane assay, comparable to that of vascular endothelial growth factor (VEGF). Overall, these data highlight the effect of Epo on MGECs- and MMECs-mediated angiogenesis: MGECs are more responsive to Epo treatment than MMECs, probably because over-angiogenic phenotype of MMECs is already activated by their autocrine/paracrine loops occurring in the "angiogenic switch" from MGUS. PMID:26919105

  9. Acanthoic acid inhibits LPS-induced inflammatory response by activating LXRα in human umbilical vein endothelial cells.

    PubMed

    Li, Yong; Zhang, Xiao-Shi; Yu, Jin-Long

    2016-03-01

    Acanthoic acid, a pimaradiene diterpene isolated from Acanthopanax koreanum, has been reported to have anti-inflammatory activities. However, the effect of acanthoic acid on vascular inflammation has not been investigated. The aim of this study was to investigate the anti-inflammatory effects of acanthoic acid on lipopolysaccharide (LPS)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs). The production of cytokines TNF-α and IL-8 was detected by ELISA. The expression of VCAM-1, ICAM-1, E-selectin, NF-κB and LXRα were detected by Western blotting. Adhesion of monocytes to HUVECs was detected by monocytic cell adhesion assay. The results showed that acanthoic acid dose-dependently inhibited LPS-induced TNF-α and IL-8 production. Acanthoic acid also inhibited TNF-α-induced IL-8 and IL-6 production. LPS-induced endothelial cell adhesion molecules, VCAM-1 and ICAM-1 were also inhibited by acanthoic acid. Acanthoic acid inhibited LPS-induced NF-κB activation. Furthermore, acanthoic acid dose-dependently up-regulated the expression of LXRα. In addition, our results showed that the anti-inflammatory effect of acanthoic acid was attenuated by transfection with LXRα siRNA. In conclusion, the anti-inflammatory effect of acanthoic acid is due to its ability to activate LXRα. Acanthoic acid may be a therapeutic agent for inflammatory cardiovascular disease. PMID:26803523

  10. CD39/NTPDase-1 expression and activity in human umbilical vein endothelial cells are differentially regulated by leaf extracts from Rubus caesius and Rubus idaeus.

    PubMed

    Dudzinska, Dominika; Luzak, Boguslawa; Boncler, Magdalena; Rywaniak, Joanna; Sosnowska, Dorota; Podsedek, Anna; Watala, Cezary

    2014-09-01

    Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1-15 μg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 μg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells. PMID:25034034

  11. Effect of maternal anti-HPA-1a antibodies and polyclonal IVIG on the activation status of vascular endothelial cells

    PubMed Central

    RADDER, C M; BEEKHUIZEN, H; KANHAI, H H H; BRAND, A

    2004-01-01

    Maternal anti-HPA-1a antibodies can cause severe fetal and neonatal alloimmune thrombocytopenia (FNAIT), complicated by intracranial haemorrhage (ICH). Antenatal treatment with maternal intravenous immunoglobulin (IVIG) seems to protect against ICH even when thrombocytopenia persists. The aim of this study was to investigate if anti-HPA-1a antibodies and IVIG potentially affect vascular endothelial cells (ECs) in order to identify susceptibility for ICH. Human umbilical cord endothelial cells (HUVEC) were incubated with anti-HPA-1a antibodies with or without polyclonal IVIG and evaluated for EC activation. Maternal sera with anti-HPA-1a antibodies affected neither the EC expression of intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and tissue factor (TF) nor the release of van Willebrand factor (vWF) or interleukin (IL)-8 nor the integrity of ECs. Maternal sera obtained after IVIG treatment and polyclonal IVIG decrease constitutive and cytokine-induced ICAM-1 and VCAM-1 expression on ECs. The results show that maternal anti-HPA-1a antibodies cause no activation or damage of ECs in this model. The clinical relevance of the de-activating properties of IVIG on EC activation with respect to ICH deserves further investigation. PMID:15196265

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

    PubMed Central

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

    2016-01-01

    Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials. PMID:27348615

  13. Dysfunctional endothelial cells directly stimulate cancer inflammation and metastasis

    PubMed Central

    Franses, Joseph W.; Drosu, Natalia C.; Gibson, William J.; Chitalia, Vipul C.; Edelman, Elazer R.

    2013-01-01

    Although the influence of context-dependent endothelial cell regulation of vascular disease and repair is well-established, the privileged roles endothelial cells play as paracrine regulators of tumor progression has only recently become appreciated. We hypothesized that if the same endothelial physiology governs vascular and cancer biology then endothelial cell control in cancer should follow endothelial regulation of vascular health. Healthy endothelial cells promote vascular repair and inhibit tumor invasiveness and metastasis; dysfunctional endothelial cells have the opposite effects. We now ask if dysfunctionally activated endothelial cells will promote cancer cell inflammatory signaling and aggressive properties. Indeed, while factors released from quiescent ECs induce balanced inflammatory signaling, correlating with decreased proliferation and invasiveness, factors released from dysfunctional ECs robustly activated NF-κB and STAT3 signaling within cancer cells, correlating with increased in vitro invasiveness and decreased proliferation and survival. Furthermore, matrix-embedded dysfunctional endothelial cells stimulated intratumoral pro-inflammatory signaling and spontaneous metastasis, while simultaneously slowing net primary tumor growth, when implanted adjacent to Lewis lung carcinoma tumors. These studies may broaden our realization of the roles of endothelial function and dysfunction, increase understanding and control of the tumor microenvironment, and facilitate optimization of anti-angiogenic and vascular-modifying therapies in cancer and related diseases. PMID:23463345

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

    PubMed Central

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

    2013-01-01

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

  15. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

    SciTech Connect

    Semino, Carlos E. . E-mail: semino@mit.edu; Kamm, Roger D.; Lauffenburger, Douglas A.

    2006-02-01

    We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures.

  16. Inhibitory effect of interferon-gamma activated ovine umbilical vein endothelial cells on the intracellular replication of Toxoplasma gondii.

    PubMed

    Dimier, I H; Bout, D T

    1996-01-01

    Toxoplasma gondii is an obligate intracellular parasite that is a major cause of abortion and neonatal mortality in sheep. In congenital toxoplasmosis, T gondii first invades the umbilical vein endothelial cells and are then disseminated throughout the fetus. Treatment of ovine umbilical vein endothelial cells with bovine recombinant gamma-interferon (IFN-gamma) blocked the growth of T gondii. Growth of the parasite was measured by 3H-uracil incorporation 18 h after the onset of the infection and by microscopic enumeration of parallel cultures. This assay revealed that when the cells were pretreated with IFN-gamma in concentrations ranging from 0.15-1,250 U/mL, a high degree of inhibition of T gondii replication was observed with the effect being dose-dependent. Maximum activation was achieved by incubating with 625 U/mL IFN-gamma and no activity was present at 0.15 U/mL. This technique could be of relevance as a first line of defense against congenital ovine Toxoplasma infection. Inhibition of T gondii replication is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. L-Arginine-dependent production of reactive nitrogen and oxygen intermediates was not responsible for the inhibition of T gondii replication. Supplements of five amino acids were able to overcome the inhibition partially but significantly. The mechanism of the inhibition remains to be elucidated. PMID:8822620

  17. Platelet endothelial cell adhesion molecule-1 and mechanotransduction in vascular endothelial cells.

    PubMed

    Fujiwara, K

    2006-04-01

    Endothelial cells are known to respond to mechanical forces such as fluid shear stress and cyclic stretch, but elucidating the mechanism for mechanosensing has been difficult. Experimental data indicate that there are probably several sensing mechanisms. We have recently proposed a novel mechanoresponse mechanism that involves platelet endothelial cell adhesion molecule-1 (PECAM-1). When endothelial cells are stimulated by fluid shear stress, PECAM-1 is tyrosine phosphorylated and activates the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signalling cascade. The same signalling events occurred when we applied pulling force directly on PECAM-1 on the endothelial cell surface using magnetic beads coated with antibodies against the external domain of PECAM-1. These results appear to indicate that PECAM-1 is a mechanotransduction molecule. To our knowledge, this is the first mammalian molecule that is shown to respond to mechanical force directly exerted to it. PMID:16594905

  18. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

    PubMed

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Angel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A; Víctor, Víctor M; Esplugues, Juan V; Rojas, José M; Sánchez-Madrid, Francisco; Serrador, Juan M

    2008-07-29

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  19. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

    PubMed Central

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Ángel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A.; Víctor, Víctor M.; Esplugues, Juan V.; Rojas, José M.; Sánchez-Madrid, Francisco; Serrador, Juan M.

    2008-01-01

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  20. Network formation through active migration of human vascular endothelial cells in a multilayered skeletal myoblast sheet.

    PubMed

    Nagamori, Eiji; Ngo, Trung Xuan; Takezawa, Yasunori; Saito, Atsuhiro; Sawa, Yoshiki; Shimizu, Tatsuya; Okano, Teruo; Taya, Masahito; Kino-oka, Masahiro

    2013-01-01

    Autologous transplantation of myoblast sheet has attracted attention as a new technique for curing myocardial infarction. Myoblast sheet has the ability to secret cytokines that improve heart function via the facilitation of angiogenesis on affected part. To mimic the in vivo angiogenesis in the myoblast sheet after transplantation, a five-layered cell sheet of human skeletal muscle myoblasts (HSMMs) was overlaid on human umbilical vein endothelial cells (HUVECs) which enables evaluation of dynamic HUVEC behavior. HUVECs existing initially at the bottom of the sheet changed to be a stretched shape and migrated upward compared with the surrounding HSMMs in the sheet. Prolonged incubation resulted in network formation of HUVECs in the middle of the sheet, although non-networked HUVECs continued to migrate to the top of the sheet, which meant the spatial habitation of HUVECs in the cell sheet. Image processing was performed to determine the variation in the extent of network formation at different HUVEC densities. It was found that the extent of formed network depended on the frequency of encounters among HUVECs in the middle of the sheet. The present system, which can evaluate network formation, is considered to be a promising in vitro angiogenesis model. PMID:23117213

  1. Cell-cell interaction promotes rat marrow stromal cell differentiation into endothelial cell via activation of TACE/TNF-alpha signaling

    PubMed Central

    Xu, Jian; Liu, Xinfeng; Chen, Jieli; Zacharek, Alex; Cui, Xu; Savant-Bhonsale, Smita; Chopp, Michael; Liu, Zhenguo

    2010-01-01

    Objective Marrow stromal cells (MSCs) are capable of differentiating into various cell types including endothelial cells. Microenvironment is important in cell fate determination. Tumor necrosis factor alpha converting enzyme (TACE), a well characterized “sheddase”, participates in the differentiation process of multiple lineages by the proteolytic release of membrane-bound proteins such as tumor necrosis factor alpha (TNF-alpha). Methods and Results We investigated the endothelial differentiation of MSCs under two co-culture conditions: 1) direct MSCs-rat brain microvascular endothelial cells (rBMECs) contact co-culture; and 2) indirect co-culture of MSCs and rBMECs. Also, we examined the role of TACE/TNF-alpha signaling in the process of differentiation under direct co-culture condition. We found that endothelial differentiation of MSCs was substantially enhanced in MSCs-rBMECs direct contact co-culture, but not in indirect transwell co-culture condition. Transcript levels of TACE and TNF-alpha as well as TACE protein expression were significantly upregulated in direct, but not in indirect co-culture condition. Addition of human recombinant TACE promoted gene expression of endothelial specific markers including vWF, CD31, VE-cadherin, Flk-1 and Flt-1 in the differentiating MSCs. Furthermore, inhibition of TACE with TAPI-2 or inhibition of TNF-alpha with Etanercept attenuated endothelial differentiation of MSCs in the direct co-culture condition. Conclusions We demonstrated for the first time that direct MSCs-rBMECs interaction stimulated the endothelial differentiation of MSCs via TACE/TNF alpha signaling. PMID:19796498

  2. Irradiation-Induced Up-Regulation of HLA-E on Macrovascular Endothelial Cells Confers Protection against Killing by Activated Natural Killer Cells

    PubMed Central

    Riederer, Isabelle; Sievert, Wolfgang; Eissner, Günther; Molls, Michael; Multhoff, Gabriele

    2010-01-01

    Background Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. Methodology/Principal Findings Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. Conclusion/Significance These data emphasize that an irradiation

  3. Down-regulation of histamine-induced endothelial cell activation as potential anti-atherosclerotic activity of peptides from Spirulina maxima.

    PubMed

    Vo, Thanh-Sang; Kim, Se-Kwon

    2013-10-01

    Histamine, a potent inflammatory mediator, has been known to cause the pathogenesis of atherosclerosis. In this sense, two bioactive peptides P1 (LDAVNR; 686Da) and P2 (MMLDF; 655Da) purified from gastric enzymatic hydrolysate of Spirulina maxima were examined for their protective effects against early atherosclerotic responses induced by histamine in EA.hy926 endothelial cells. Interestingly, both P1 and P2 exhibited inhibitory activities on the production and expression of IL-6 and MCP-1. Furthermore, P1 and P2 inhibited the production of adhesion molecules including P-selectin and E-selectin, and thus reducing in vitro cell adhesion of monocyte onto endothelial cells. In addition, the production of intracellular reactive oxygen species was observed to reduce in the presence of P1 or P2. Notably, the inhibitory activities of P1 and P2 were found due to down-regulating Egr-1 expression via histamine receptor and PKCδ-dependent MAPKs activation pathway. These results suggest that peptides P1 and P2 from S. maxima are effective to suppress histamine-induced endothelial cell activation that may contribute to the prevention of early atherosclerosis. PMID:23856417

  4. KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling*

    PubMed Central

    DiStefano, Peter V.; Kuebel, Julia M.; Sarelius, Ingrid H.; Glading, Angela J.

    2014-01-01

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1. PMID:25320085

  5. The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Xiong, Lingxin; Xie, Jingshu; Song, Chenxue; Liu, Jinping; Zheng, Jingtong; Liu, Chuangui; Zhang, Xiaotian; Li, Pingya; Wang, Fang

    2015-01-01

    Epidemiological studies have verified the critical role that antioxidative stress plays in protecting vascular endothelial cells. The aims of the present study were to investigate the antioxidative activities and differential regulation of nuclear erythroid-related factor 2- (Nrf2-) mediated gene expression by Xueshuan Xinmaining Tablet (XXT), a traditional Chinese medicine with the effect of treating cardiovascular diseases. The antioxidative activities of XXT were investigated using quantitative real-time PCR (qPCR), a PCR array, and western blotting. Our results indicated that XXT exhibited potent antioxidative activities by suppressing the levels of hydrogen peroxide- (H2O2-) induced reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs). We were also conscious of strong Nrf2-mediated antioxidant induction. XXT enhanced the expressions of Keap1, Nrf2, and Nrf2-mediated genes, such as glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H: quinine oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), and glutathione peroxidase (GPX) in HUVECs. In summary, XXT strongly activated Nrf2 and its downstream regulated genes, which may contribute to the antioxidative and vascular endothelial cell protective activities of XXT. PMID:26681964

  6. A Case of Abnormal Lymphatic-Like Differentiation and Endothelial Progenitor Cell Activation in Neovascularization Associated with Hemi-Retinal Vein Occlusion

    PubMed Central

    Loukovaara, Sirpa; Gucciardo, Erika; Repo, Pauliina; Lohi, Jouko; Salven, Petri; Lehti, Kaisa

    2015-01-01

    Purpose Pathological vascular differentiation in retinal vein occlusion (RVO)-related neovessel formation remains poorly characterized. The role of intraocular lymphatic-like differentiation or endothelial progenitor cell activity has not been studied in this disease. Methods Vitrectomy was performed in an eye with hemi-RVO; the neovessel membrane located at the optic nerve head was removed and subjected to immunohistochemistry. Characterization of the neovascular tissue was performed using hematoxylin and eosin, α-smooth muscle actin, and the pan-endothelial cell (EC) adhesion molecule CD31. The expression of lymphatic EC markers was studied by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), podoplanin (PDPN), and prospero-related homeobox protein 1 (Prox-1). Potential vascular stem/progenitor cells were identified by active cellular proliferation (Ki67) and expression of the stem cell marker CD117. Results The specimen contained blood vessels lined by ECs and surrounded by pericytes. Immunoreactivity for LYVE-1 and Prox-1 was detected, with Prox-1 being more widely expressed in the active Ki67-positive lumen-lining cells. PDPN expression was instead found in the cells residing in the extravascular tissue. Expression of the stem cell markers CD117 and Ki67 suggested vascular endothelial progenitor cell activity. Conclusions Intraocular lymphatic-like differentiation coupled with progenitor cell activation may be involved in the pathology of neovessel formation in ischemia-induced human hemi-RVO. PMID:26327908

  7. Endothelial progenitor cells: identity defined?

    PubMed Central

    Timmermans, Frank; Plum, Jean; Yöder, Mervin C; Ingram, David A; Vandekerckhove, Bart; Case, Jamie

    2009-01-01

    Abstract In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ, a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133+ cells or CD45+ haematopoietic cells. PMID:19067770

  8. Effects of carbon dioxide level (PCO sub 2 ) on the fibrinolytic activity (FA) of pulmonary artery endothelial cells (PAEC)

    SciTech Connect

    Langleben, D.; Moroz, L.A.; Danes, D. )

    1990-02-26

    Recovery from pulmonary thromboembolism depends on the rapidity and completeness of clot lysis. This involves endogenous fibrinolytic mechanisms, particularly the balance between plasminogen activators and inhibitors produced by endothelial cells. Hypocapnia is common in pulmonary embolism, however it is not known if endothelial fibrinolytic function is affected by PCO{sub 2}. The authors therefore measured the FA in medium (MCDB-131, 0.5% albumin) conditioned for 20 hours in-vitro by exposure to confluent cultures of bovine proximal PAEC. During conditioning, cells were exposed to 5% CO{sub 2} in air (PCO{sub 2} - 36-40mm Hg, CONTROL), or various PCO{sub 2} levels (30-55 mmHg, in air). FA of conditioned medium was determined by {sup 125}I-fibrin solid phase assay, with addition of plasminogen (10 ug/ml). With PCO{sub 2} levels {le} 35 mmHg, FA in the conditioned medium was 5 to 18% higher than CONTROL FA. When PCO{sub 2} was {ge} 45 mmHg, FA decreased 5 to 60% as compared to CONTROL FA. There was a significant negative linear relationship between PCO{sub 2} and FA. Thus, PCO{sub 2} level can affect PAEC mediated plasminogen activation. This finding may be relevant to in-vivo clearance of clots from pulmonary arteries.

  9. Microarray profiling of L1-overexpressing endothelial cells reveals STAT3 activation via IL-6/IL-6Rα axis

    PubMed Central

    Magrini, Elena; Cavallaro, Ugo; Bianchi, Fabrizio

    2015-01-01

    We recently identified a novel role for the L1 transmembrane glycoprotein (also known as L1CAM or CD171) in the regulation of tumor angiogenesis and vessels stabilization. L1 overexpression in cultured endothelial cells of the lung (luECs) exerted a pleiotropic effect in that it regulated proliferation, migration, tubulogenesis, vascular permeability, and endothelial-to-mesenchymal transition (EndMT). In addition, we provided strong evidence that antibody-mediated targeting of L1 may be an effective strategy for vessel normalization with the potential to increase efficacy of chemotherapeutic agents. High-throughput microarray expression profile revealed that L1 modulates the expression of hundreds of genes mainly involved in cell cycle regulation, DNA replication, cellular assembly, migration, development and organization. By using a ‘pathway-oriented’ analysis strategy we were able to identify a network of 105 genes modulated by L1 through the predicted activation of five transcription factors: STAT1, STAT2, STAT3, IRF7, and ATF4. Indeed, L1 overexpression resulted in the strong induction of STAT3 phosphorylation which was abolished by antibody-mediated neutralization of IL-6Rα. These results indicated that L1 promoted STAT3 activation via the IL-6/IL-6Rα axis. PMID:26484199

  10. Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2

    PubMed Central

    Wu, Florence TH; Lee, Christina R; Bogdanovic, Elena; Prodeus, Aaron; Gariépy, Jean; Kerbel, Robert S

    2015-01-01

    Angiopoietin-1 (Ang1) activation of Tie2 receptors on endothelial cells (ECs) reduces adhesion by tumor cells (TCs) and limits junctional permeability to TC diapedesis. We hypothesized that systemic therapy with Vasculotide (VT)—a purported Ang1 mimetic, Tie2 agonist—can reduce the extravasation of potentially metastatic circulating TCs by similarly stabilizing the host vasculature. In vitro, VT and Ang1 treatments impeded endothelial hypermeability and the transendothelial migration of MDA-MB-231•LM2-4 (breast), HT29 (colon), or SN12 (renal) cancer cells to varying degrees. In mice, VT treatment inhibited the transit of TCs through the pulmonary endothelium, but not the hepatic or lymphatic endothelium. In the in vivo LM2-4 model, VT monotherapy had no effect on primary tumors, but significantly delayed distant metastatic dissemination to the lungs. In the post-surgical adjuvant treatment setting, VT therapeutically complemented sunitinib therapy, an anti-angiogenic tyrosine kinase inhibitor which limited the local growth of residual disease. Unexpectedly, detailed investigations into the putative mechanism of action of VT revealed no evidence of Tie2 agonism or Tie2 binding; alternative mechanisms have yet to be determined. PMID:25851538

  11. Hypoxia activates 15-PGDH and its metabolite 15-KETE to promote pulmonary artery endothelial cells proliferation via ERK1/2 signalling

    PubMed Central

    Ma, Cui; Liu, Yun; Wang, Yanyan; Zhang, Chen; Yao, Hongmin; Ma, Jun; Zhang, Lei; Zhang, Dandan; Shen, Tingting; Zhu, Daling

    2014-01-01

    BACKGROUND AND PURPOSE Dysfunction and injury of endothelial cells in the pulmonary artery play critical roles in the hypertension induced by chronic hypoxia. One consequence of hypoxia is increased activity of 15-hydroxyprostaglandin dehydrogenase (PGDH). Here, we have explored, in detail, the effects of hypoxia on the proliferation of pulmonary artery endothelial cells. EXPERIMENTAL APPROACH We used bromodeoxyuridine incorporation, cell-cycle analysis, immunohistochemistry and Western blot analysis to study the effects of hypoxia, induced 15-PGDH) activity and its product, 15-keto-6Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15-KETE), on endothelial cell proliferation. Scratch-wound and tube formation assays were also used to study migration of endothelial cells. KEY RESULTS 15-KETE increased DNA synthesis and enhanced the transition from the G0/G1 phase to the S phase in hypoxia. Inhibition of 15-PGDH or siRNA for 15-PGDH reversed these effects. 15-KETE also activated the ERK1/2 signalling pathway. 15-KETE-induced cell migration and tube formation were reversed by blocking ERK1/2, but not the p38 MAPK pathway. CONCLUSIONS AND IMPLICATIONS Hypoxia-induced endothelial proliferation and migration, an important underlying mechanism contributing to hypoxic pulmonary vascular remodelling, appears to be mediated by 15-PGDH and 15-KETE, via the ERK1/2 signalling pathway. PMID:24467360

  12. Advanced glycation endproducts induce apoptosis of endothelial progenitor cells by activating receptor RAGE and NADPH oxidase/JNK signaling axis

    PubMed Central

    Chen, Jianfei; Jing, Jun; Yu, Shiyong; Song, Minbao; Tan, Hu; Cui, Bin; Huang, Lan

    2016-01-01

    Elevated levels of advanced glycation endproducts (AGEs) is an important risk factor for atherosclerosis. Dysfunction of endothelial progenitor cells (EPCs), which is essential for re-endothelialization and neovascularization, is a hallmark of atherosclerosis. However, it remains unclear whether and how AGEs acts on EPCs to promote pathogenesis of atherosclerosis. In this study, EPCs were exposed to different concentrations of AGEs. The expression of NADPH and Rac1 was measured to investigate the involvement of NADPH oxidase pathway. ROS was examined to indicate the level of oxidative stress in EPCs. Total JNK and p-JNK were determined by Western blotting. Cell apoptosis was evaluated by both TUNEL staining and flow cytometry. Cell proliferation was measured by 3H thymidine uptake. The results showed that treatment of EPCs with AGEs increased the levels of ROS in EPCs. Mechanistically, AGEs increased the activity of NADPH oxidase and the expression of Rac1, a major component of NADPH. Importantly, treatment of EPCs with AGEs activated the JNK signaling pathway, which was closely associated with cell apoptosis and inhibition of proliferation. Our results suggest that the RAGE activation by AGEs in EPCs upregulates intracellular ROS levels, which contributes to increased activity of NADPH oxidase and expression of Rac1, thus promoting cellular apoptosis and inhibiting proliferation. Mechanistically, AGEs binding to the receptor RAGE in EPCs is associated with hyperactivity of JNK signaling pathway, which is downstream of ROS. Our findings suggest that dysregulation of the AGEs/RAGE axis in EPCs may promote atherosclerosis and identify the NADPH/ROS/JNK signaling axis as a potential target for therapeutic intervention. PMID:27347324

  13. αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

    PubMed Central

    Rusnati, Marco; Tanghetti, Elena; Dell’Era, Patrizia; Gualandris, Anna; Presta, Marco

    1997-01-01

    Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3 antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells. PMID:9398667

  14. Real-time monitoring of cell mechanical changes induced by endothelial cell activation and their subsequent binding with leukemic cell lines.

    PubMed

    Tan, Liang; Lin, Peiling; Pezeshkian, Bahareh; Rehman, Abdul; Madlambayan, Gerard; Zeng, Xiangqun

    2014-06-15

    Endothelial cell (EC) activation and their subsequent binding with different cells have various mechanical consequences that, if monitored real time, can serve as a functional biomarker of many pathophysiological response mechanisms. This work presents an innovative and facile strategy to conduct such monitoring using quartz crystal microbalance (QCM), thereby relating the shifts in its frequency and motional resistance to morphological changes upon cell-cell and cell-substrate interactions. By activating ECs with TNF-α and then characterizing their binding with HL-60 and KG-1 leukemia cells, we are able to induce the mechanical changes in ECs especially in the region of cell-substrate contact which resulted in dynamically coupled mass and viscoelastic changes representing the extent of both activation and binding. The activated ECs suffered a decrease of cellular contact area, leading to positive frequency shift and decreased motional resistance. The binding of leukemia cells onto pre-activated ECs exerted a mechanical force to regain the cell surface contact which resulted in the obvious QCM responses opposite to that of activation, and proportional to the number of cells added, in spite of the fact that these added cells are extremely outside the extinction boundary of the shear wave generated by QCM. Different cell lines demonstrate different attachment behavior, which was detected by the QCM. Despite these variations are quite subtle, yet the sensitivity of the technique for dynamic changes at the interface makes them detectable. Moreover, the reproducibility of the generated data determined at each step by deviation measurements (<10%) in response plot was very high despite the high possible heterogeneity in cell populations. The results are explained on the basis of simple theoretical and physical models, although, the development of a more quantitative and precise model is underway in our laboratory. PMID:24487102

  15. Real-Time Monitoring of Cell Mechanical Changes Induced by Endothelial Cell Activation and their Subsequent Binding with Leukemic Cell Lines

    PubMed Central

    Tan, Liang; Lin, Peiling; Pezeshkian, Bahareh; Rehman, Abdul; Madlambayan, Gerard; Zeng, Xiangqun

    2014-01-01

    Endothelial cell (EC) activation and their subsequent binding with different cells have various mechanical consequences that, if monitored real time, can serve as a functional biomarker of many pathophysiological response mechanisms. This work presents an innovative and facile strategy to conduct such monitoring using quartz crystal microbalance (QCM), thereby relating the shifts in its frequency and motional resistance to morphological changes upon cell-cell and cell-substrate interactions. By activating ECs with TNF-α and then characterizing their binding with HL-60 and KG-1 leukemia cells, we are able to induce the mechanical changes in ECs especially in the region of cell-substrate contact which resulted in dynamically coupled mass and viscoelastic changes representing the extent of both activation and binding. The activated ECs suffered a decrease of cellular contact area, leading to positive frequency shift and decreased motional resistance. The binding of leukemia cells onto pre-activated ECs exerted a mechanical force to regain the cell surface contact which resulted in the obvious QCM responses opposite to that of activation, and proportional to the number of cells added, in spite of the fact that these added cells are extremely outside the extinction depth of the shear wave generated by QCM. Different cell lines demonstrate different attachment behavior, which was detected by the QCM. Despite these variations are quite subtle, yet the sensitivity of the technique for dynamic changes at the interface makes them detectable. Moreover, the reproducibility of the generated data determined at each step by deviation measurements (<10%) in response plot was very high despite the high possible heterogeneity in cell populations. The results are explained on the basis of simple theoretical and physical models, although, the development of a more quantitative and precise model is underway in our laboratory. PMID:24487102

  16. REGULATION OF ENDOTHELIAL CELL ACTIVATION AND ANGIOGENESIS BY INJECTABLE PEPTIDE NANOFIBERS

    PubMed Central

    Cho, Hongkwan; Balaji, Swathi; Sheikh, Abdul Q.; Hurley, Jennifer R.; Tian, Ye F.; Collier, Joel H.; Crombleholme, Timothy M.; Narmoneva, Daria A.

    2011-01-01

    RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. Results were validated in vivo using mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, increased β3 integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of β3 and MEK inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. Addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of β3 integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize bio-compatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffolds materials. PMID:21925628

  17. [Protective effect of combined administration of active ingredients of Danhong on cerebral micro-vascular endothelial cell injured by hypoxia].

    PubMed

    Zhou, Hui-fen; He, Yu; Zhang, Yu-yan; Yang, Jie-hong; Zhao, Tao; Fu, Wei; Zhou, Peng; Wan, Hai-tong

    2014-11-01

    To study the protective effect of combined administration of active ingredients of Danhong on cultured primary mice's brain microvascular endothelial cells (rBMECs) injured by hypoxia. Primary mice's brain micro-vascular endothelial cells were cultured to establish the 4 h hypoxia model. Meanwhile, active ingredients (protocatechuic aldehyde, salvianolic acid B, hydroxysafflor yellow A and tanshinol) of Danhong were administered in rBMECs. The non-toxic dosage was determined by MTT. The leakage of lactate dehydrogenase(LDH), cell superoxide dismutase (SOD) activity and MDA level were detected by the colorimetric method. The expressions of ICAM-1, MMP-9, P53 mRNA were detected by RT-PCR method. Changes in rBMECs cell cycle and early apoptosis were detected by flow cytometry. Danhong's active ingredients and prescriptions 1, 2, 3, 7, 8, 9 could be combined to significantly restrain LDH in hypoxic cells supernatant. Prescriptions 1, 2, 3, 7, 8, 9 could significantly enhance SOD activity in anoxic cells; Prescriptions 1, 2, 3, 8, 9 could significantly decrease the MDA level; Prescriptions 1, 2, 6, 7, 9 could significantly inhibit the early rB-MECs apoptosis induced by hypoxia. After hypoxia, the up-regulated P53 mRNA expression could cause retardation in G, phase and promote cell apoptosis. This proved that the regulatory function of P53 gene lay in monitoring of calibration points in G, phase. Prescriptions 1, 2, 5, 6, 7, 8, 9 could significantly down-regulate the P53 mRNA expression; Prescriptions 1, 4, 7, 8, 9 could significantly down-regulate the ICAM-1 mRNA expression; Prescriptions 1, 3, 6, 9 could significantly down-regulate the MMP-9 mRNA expression. The combined administration of Danhong's active ingredients showed a significant protective effect on primary cultured rBMECs injury induced by hypoxia Its mechanism may be related to the enhancement of cellular antioxidant capacity and the inhibition of inflammatory response and cell apoptosis. This study could

  18. Air Pollution Upregulates Endothelial Cell Procoagulant Activity Via Ultrafine Particle-Induced Oxidant Signaling and Tissue Factor Expression

    EPA Science Inventory

    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 mecha...

  19. Down-regulation of tumor endothelial marker 8 suppresses cell proliferation mediated by ERK1/2 activity

    PubMed Central

    Cao, Chuangjie; Wang, Zhuo; Huang, Leilei; Bai, Lihong; Wang, Yuefeng; Liang, Yingjie; Dou, Chengyun; Wang, Liantang

    2016-01-01

    Tumor endothelial marker 8 (TEM8) was recently suggested as a putative anti-tumor target in several types of human cancer based on its selective overexpression in tumor versus normal endothelial cells. The objective of this study was to detect the potential functions of TEM8 in osteosarcoma. Overall, TEM8 was mainly located in cytoplasm and was up-regulated in osteosarcoma compared to benign bone lesions and adjacent non tumor tissue (ANT). High TEM8 expression group had a significant lower overall survival rate than that in the low TEM8 expression group. TEM8 knock-down by siRNA or shRNA results in significant reduction of osteosarcoma cell growth and proliferation both in vitro and in vivo. Ablation of TEM8 led to increasing of p21 and p27 and suppression of cyclin D1 mediated by Erk1/2 activity. These findings suggest that down-regulation of TEM8 play an important role in the inhibition of tumorigenesis and development of osteosarcoma. PMID:26996335

  20. A selective estrogen receptor modulator inhibits TNF-alpha-induced apoptosis by activating ERK1/2 signaling pathway in vascular endothelial cells.

    PubMed

    Yu, Jing; Eto, Masato; Akishita, Masahiro; Okabe, Tetsuro; Ouchi, Yasuyoshi

    2009-07-01

    Tumor necrosis factor (TNF-alpha) is a pleiotropic cytokine exerting both inflammatory and cell death activity and is thought to play a role in the pathogenesis of atherosclerosis. The present study was designed to examine whether the raloxifene analogue, LY117018 could inhibit TNF-alpha-induced apoptosis in vascular endothelial cells and to clarify the involved mechanisms. Apoptosis of endothelial cells was determined by DNA fragmentation assay and the activation of caspase-3. LY117018 significantly inhibited TNF-alpha-induced caspase-3 activation and cell DNA fragmentation levels in bovine carotid artery endothelial cells. The inhibitory effect of LY117018 was abolished by an estrogen receptor antagonist ICI 182,780. p38 MAPK, JNK, ERK1/2 and Akt have been shown to act as apoptotic or anti-apoptotic signals. TNF-alpha stimulated the phosphorylation levels of p38 MAPK, JNK, ERK1/2 and Akt in vascular endothelial cells. TNF-alpha-induced apoptosis was significantly decreased by SB203580, a p38 MAPK inhibitor or SP600125, a JNK inhibitor, but was enhanced by an ERK1/2 pathway inhibitor, PD98059 or a PI3-kinase/Akt pathway inhibitor, wortmannin. The anti-apoptotic effect of LY117018 was abrogated only by PD98059 but was not affected by the inhibitors for p38 MAPK, JNK, or Akt. LY117018 stimulated the further increase in phosphorylation of ERK1/2 in TNF-alpha treated endothelial cells but it did not affect phosphorylation levels of p38 MAPK, JNK or Akt. These results suggest that LY 110718 prevents caspase-3 dependent apoptosis induced by TNF-alpha in vascular endothelial cells through activation of the estrogen receptors and the ERK1/2 signaling pathway. PMID:19275968

  1. Protein Never in Mitosis Gene A Interacting-1 regulates calpain activity and the degradation of cyclooxygenase-2 in endothelial cells

    PubMed Central

    Liu, Tongzheng; Schneider, Ryan A; Shah, Vaibhav; Huang, Yongcheng; Likhotvorik, Rostislav I; Keshvara, Lakhu; Hoyt, Dale G

    2009-01-01

    Background The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates turnover of inducible nitric oxide synthase (iNOS) in murine aortic endothelial cells (MAEC) stimulated with E. coli endotoxin (LPS) and interferon-γ (IFN). Degradation of iNOS was reduced by a calpain inhibitor, suggesting that PIN1 may affect induction of other calpain-sensitive inflammatory proteins, such as cyclooxygenase (COX)-2, in MAEC. Methods MAEC, transduced with lentivirus encoding an inactive control short hairpin (sh) RNA or one targeting PIN1 that reduced PIN1 by 85%, were used. Cells were treated with LPS/IFN, calpain inhibitors (carbobenzoxy-valinyl-phenylalaninal (zVF), PD150606), cycloheximide and COX inhibitors to determine the effect of PIN1 depletion on COX-2 and calpain. Results LPS or IFN alone did not induce COX-2. However, treatment with 10 μg LPS plus 20 ng IFN per ml induced COX-2 protein 10-fold in Control shRNA MAEC. Induction was significantly greater (47-fold) in PIN1 shRNA cells. COX-2-dependent prostaglandin E2 production increased 3-fold in KD MAEC, but did not increase in Control cells. The additional increase in COX-2 protein due to PIN1 depletion was post-transcriptional, as induction of COX-2 mRNA by LPS/IFN was the same in cells containing or lacking PIN1. Instead, the loss of COX-2 protein, after treatment with cycloheximide to block protein synthesis, was reduced in cells lacking PIN1 in comparison with Control cells, indicating that degradation of the enzyme was reduced. zVF and PD150606 each enhanced the induction of COX-2 by LPS/IFN. zVF also slowed the loss of COX-2 after treatment with cycloheximide, and COX-2 was degraded by exogenous μ-calpain in vitro. In contrast to iNOS, physical interaction between COX-2 and PIN1 was not detected, suggesting that effects of PIN1 on calpain, rather than COX-2 itself, affect COX-2 degradation. While cathepsin activity was unaltered, depletion of PIN1 reduced calpain activity

  2. Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Nezu, A.; Shinoda, H.; Minami, M.

    1996-01-01

    1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified. PMID:8789382

  3. Oxidized High-Density Lipoprotein Impairs Endothelial Progenitor Cells' Function by Activation of CD36-MAPK-TSP-1 Pathways

    PubMed Central

    Wu, Jianxiang; He, Zhiqing; Gao, Xiang; Wu, Feng; Ding, Ru; Ren, Yusheng; Jiang, Qijun; Fan, Min

    2015-01-01

    Abstract Aims: High-density lipoprotein (HDL) levels inversely correlate with cardiovascular events due to the protective effects on vascular wall and stem cells, which are susceptible to oxidative modifications and then lead to potential pro-atherosclerotic effects. We proposed that oxidized HDL (ox-HDL) might lead to endothelial progenitor cells (EPCs) dysfunction and investigated underlying mechanisms. Results: ox-HDL was shown to increase apoptosis and intracellular reactive oxygen species levels, but to reduce migration, angiogenesis, and cholesterol efflux of EPCs in a dose-dependent manner. p38 mitogen-activated protein kinase (MAPK) and NF-κB were activated after ox-HDL stimulation, which also upregulated thrombospondin-1 (TSP-1) expression without affecting vascular endothelial growth factor. Effects caused by ox-HDL could be significantly attenuated by pretreatment with short hairpin RNA-mediated CD36 knockdown or probucol. Data of in vivo experiments and the inverse correlation of ox-HDL and circulating EPC numbers among patients with coronary artery diseases (CAD) or CAD and type 2 diabetes also supported it. Meanwhile, HDL separated from such patients could significantly increase cultured EPC's caspase 3 activity, further supporting our proposal. Innovation: This is the most complete study to date of how ox-HDL would impair EPCs function, which was involved with activation of CD36-p38 MAPK-TSP-1 pathways and proved by not only the inverse relationship between ox-HDL and circulating EPCs in clinic but also pro-apoptotic effects of HDL separated from patients' serum. Conclusion: Activation of CD36-p38 MAPK-TSP-1 pathways contributes to the pathological effects of ox-HDL on EPCs' dysfunction, which might be one of the potential etiological factors responsible for the disturbed neovascularization in chronic ischemic disease. Antioxid. Redox Signal. 22, 308–324. PMID:25313537

  4. The volatile oil of Nardostachyos Radix et Rhizoma induces endothelial nitric oxide synthase activity in HUVEC cells.

    PubMed

    Maiwulanjiang, Maitinuer; Bi, Cathy W C; Lee, Pinky S C; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M; Xiong, Aizhen; Li, Ning; Dong, Tina T X; Aisa, Haji A; Tsim, Karl W K

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production. PMID:25643147

  5. The Volatile Oil of Nardostachyos Radix et Rhizoma Induces Endothelial Nitric Oxide Synthase Activity in HUVEC Cells

    PubMed Central

    Maiwulanjiang, Maitinuer; Bi, Cathy W. C.; Lee, Pinky S. C.; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M.; Xiong, Aizhen; Li, Ning; Dong, Tina T. X.; Aisa, Haji A.; Tsim, Karl W. K.

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca2+ level, and BAPTA-AM, a Ca2+ chelator, reduced the Ca2+ surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production. PMID:25643147

  6. Activation of adult rat CNS endothelial cells by opioid-induced toll-like receptor 4 (TLR4) signaling induces proinflammatory, biochemical, morphological, and behavioral sequelae

    PubMed Central

    Grace, Peter M.; Ramos, Khara M.; Rodgers, Krista M.; Wang, Xiaohui; Hutchinson, Mark R.; Lewis, Makenzie T.; Morgan, Kelly N.; Kroll, Juliet L.; Taylor, Frederick R.; Strand, Keith A.; Zhang, Yingning; Berkelhammer, Debra; Huey, Madeline G.; Greene, Lisa I.; Cochran, Thomas A.; Yin, Hang; Barth, Daniel S.; Johnson, Kirk W.; Rice, Kenner; Maier, Steven F.; Watkins, Linda R.

    2014-01-01

    CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at TLR4, presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (-)-morphine or its mu-opioid receptor (MOR) inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated NFκB, increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 from these cells. (-)-Morphine-induced upregulation of TNFα mRNA and prostaglandin E2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequalae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by

  7. Synthesis of 13-β-elemene ester derivatives and evaluation of their antioxidant activity in human umbilical vein endothelial cells.

    PubMed

    Chen, Ji-Chao; Duan, Wen-Li; Bai, Ren-Ren; Yao, He-Quan; Wu, Xiao-Ming; Shang, Jing; Xu, Jin-Yi

    2015-08-01

    In the present study, a series of 13-β-elemene ester derivatives were designed and prepared, and their antioxidant activity was investigated in the H2O2-treated human umbilical vein endothelial cells (HUVECs). Among the test compounds, the dimer compounds 5v and 5w exhibited the most potent antioxidant activity with significant ROS suppression being observed. Both compounds markedly inhibited the H2O2-induced changes in various biochemical substances, such as superoxide dismutase (SOD), malonyldialdehyde (MDA), nitric oxide (NO), and lactic dehydrogenase (LDH), which were superior to that of the positive control vitamin E. Further more, they did not produce any obvious cytotoxicity, but increased the viability of HUVECs injured by H2O2 in a dose-dependent manner. Additionally, compound 5w, designed as a prodrug-like compound, showed improved stability relative to compound 4 in vitro. PMID:26253495

  8. miR-143 Activation Regulates Smooth Muscle and Endothelial Cell Crosstalk in Pulmonary Arterial Hypertension

    PubMed Central

    Stevens, Hannah; Lu, Ruifang; Caudrillier, Axelle; McBride, Martin; McClure, John D; Grant, Jenny; Thomas, Matthew; Frid, Maria; Stenmark, Kurt; White, Kevin; Seto, Anita G.; Morrell, Nicholas W.; Bradshaw, Angela C; MacLean, Margaret R.; Baker, Andrew H.

    2015-01-01

    Rationale The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH. Methods and Results We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-β (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143−/− and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology. PMID:26311719

  9. A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

    PubMed Central

    Allen, Kristi L.; Fonseca, Fabio V.; Betapudi, Venkaiah; Willard, Belinda; Zhang, Jainwei

    2012-01-01

    Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β2GPI, and activate endothelial cells (ECs) in a β2GPI-dependent manner after binding of β2GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β2GPI Ab–mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88–dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β2GPI Ab–mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β2GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β2GPI–associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome. PMID:22106343

  10. A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies.

    PubMed

    Allen, Kristi L; Fonseca, Fabio V; Betapudi, Venkaiah; Willard, Belinda; Zhang, Jainwei; McCrae, Keith R

    2012-01-19

    Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β(2)GPI, and activate endothelial cells (ECs) in a β(2)GPI-dependent manner after binding of β(2)GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β(2)GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β(2)GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β(2)GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β(2)GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome. PMID:22106343

  11. TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

    PubMed

    Sartain, Sarah E; Turner, Nancy A; Moake, Joel L

    2016-01-15

    Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1β or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1β had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure. PMID:26673143

  12. Myricitrin attenuates endothelial cell apoptosis to prevent atherosclerosis: An insight into PI3K/Akt activation and STAT3 signaling pathways.

    PubMed

    Qin, Meng; Luo, Yun; Meng, Xiang-bao; Wang, Min; Wang, Hong-wei; Song, Shi-yu; Ye, Jing-xue; Pan, Rui-le; Yao, Fan; Wu, Ping; Sun, Gui-bo; Sun, Xiao-bo

    2015-07-01

    Blood vessel endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) has been implicated in the pathogenesis of atherosclerosis and vasculopathy. The ox-LDL-elicited reactive oxygen species (ROS) release has been assumed to serve a critical function in endothelial damage. Myricitrin (from Myrica cerifera) is a natural antioxidant that has strong anti-oxidative, anti-inflammatory, and anti-nociceptive activities. However, the protective effect of myricitrin on ROS-induced endothelial cell injury and its related molecular mechanisms have never been investigated. This study demonstrates that myricitrin can inhibit ox-LDL-induced endothelial apoptosis and prevent plaque formation at an early stage in an atherosclerotic mouse model. The administration of myricitrin in vivo decreases the thickness of the vascular wall in the aortic arch of ApoE-/- mice. In vitro study shows that ox-LDL-induced human umbilical vein endothelial cell apoptosis can be reduced upon receiving myricitrin pre-treatment. Treatment with myricitrin significantly attenuated ox-LDL-induced endothelial cell apoptosis by inhibiting LOX-1 expression and by increasing the activation of the STAT3 and PI3K/Akt/eNOS signaling pathways. At the same time, our result demonstrates that myricitrin treatment optimizes the balance of pro/anti-apoptosis proteins, including Bax, Bad, XIAP, cIAP-2, and survivin. Our study suggests that myricitrin treatment can effectively protect cells from ox-LDL-induced endothelial cell apoptosis, which results in reduced atherosclerotic plaque formation. This result indicates that myricitrin can be used as a drug candidate for the treatment of cardiovascular diseases. PMID:25849952

  13. Effects of bleomycin A5 on caspase-3, P53, bcl-2 expression and telomerase activity in vascular endothelial cells

    PubMed Central

    Huang, Yi-Deng; Li, Ping; Tong, Xiao; He, Yue; Zhuo, Yang; Xia, Si-Wen; Luo, Xing-Hua

    2015-01-01

    Objective: The aim of this study was to investigate the molecular mechanism of bleomycin A5 in inducing the apoptosis of human umbilical vein endothelial cells (ECV304). Materials and Methods: ECV304 cells were cultured and passaged, and then were divided into control group and three treatment groups. The later three groups were treated with 15, 75, and 150 μg/ml bleomycin A5 for 24 hours, respectively. The expressions of caspase-3, p53, and bcl-2 in ECV304 cells were detected by flow cytometry, and the activity of telomerase was determined using telomere repeat amplification protocol (TRAP)-silver staining method. Results: After treatment with different concentrations of bleomycin A5, the expression of caspase-3 in ECV304 cells was increased. It was significantly decreased with the increase of bleomycin A5 concentration, but the difference between 75 μg/ml and 150 μg/ml groups was not significant. Bleomycin A5 could significantly increase the expression of p53, with concentration dependence. It had no obvious effect on bcl-2 expression. There was high expression of telomerase in control group. After treatment with different concentration of bleomycin A5, the telomerase activity was significantly decreased. Conclusion: Bleomycin A5 can increase caspase-3 and p53 levels and inhibit telomerase activity to induce ECV304 apoptosis. PMID:25821312

  14. Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

    PubMed

    Luzak, Boguslawa; Boncler, Magdalena; Rywaniak, Joanna; Dudzinska, Dominika; Rozalski, Marek; Krajewska, Urszula; Balcerczak, Ewa; Podsedek, Anna; Redzynia, Malgorzata; Watala, Cezary

    2014-12-01

    The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 μg/ml, and up to 33 % for 15 μg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 μg/ml and 48 ± 6 % for 15 μg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 μg/ml and 18 ± 7 % for 15 μg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 μg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium. PMID:25407137

  15. Endothelial Cells Derived From Nuclear Reprogramming

    PubMed Central

    Wong, Wing Tak; Huang, Ngan F.; Botham, Crystal M.; Sayed, Nazish; Cooke, John P.

    2012-01-01

    The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell culture have been developed and refined. More recently, methods to derive endothelial cells from pluripotent cells have extended the scientific range of human endothelial cell studies. Pluripotent stem cells may be generated, expanded and then differentiated into endothelial cells for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific endothelial cells to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of endothelial cells from stem cells; their characterization by genetic, histological and functional studies; and their translational applications. PMID:23104878

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

    SciTech Connect

    Weber, Martina; Baker, Meredith B.; Moore, Jeffrey P.; Searles, Charles D.

    2010-03-19

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

  17. Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway.

    PubMed

    Mai, Jietang; Nanayakkara, Gayani; Lopez-Pastrana, Jahaira; Li, Xinyuan; Li, Ya-Feng; Wang, Xin; Song, Ai; Virtue, Anthony; Shao, Ying; Shan, Huimin; Liu, Fang; Autieri, Michael V; Kunapuli, Satya P; Iwakura, Yoichiro; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-Feng

    2016-03-01

    Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4(+) T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)(-/-)/IL-17A(-/-) mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway. PMID:26733204

  18. Brain endothelial miR-146a negatively modulates T-cell adhesion through repressing multiple targets to inhibit NF-κB activation.

    PubMed

    Wu, Dongsheng; Cerutti, Camilla; Lopez-Ramirez, Miguel A; Pryce, Gareth; King-Robson, Josh; Simpson, Julie E; van der Pol, Susanne Ma; Hirst, Mark C; de Vries, Helga E; Sharrack, Basil; Baker, David; Male, David K; Michael, Gregory J; Romero, Ignacio A

    2015-03-01

    Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation. PMID:25515214

  19. Brain endothelial miR-146a negatively modulates T-cell adhesion through repressing multiple targets to inhibit NF-κB activation

    PubMed Central

    Wu, Dongsheng; Cerutti, Camilla; Lopez-Ramirez, Miguel A; Pryce, Gareth; King-Robson, Josh; Simpson, Julie E; van der Pol, Susanne MA; Hirst, Mark C; de Vries, Helga E; Sharrack, Basil; Baker, David; Male, David K; Michael, Gregory J; Romero, Ignacio A

    2015-01-01

    Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation. PMID:25515214

  20. Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells

    PubMed Central

    Han, Jung Min; Kim, Sunghoon; Celada, Antonio; Ribas de Pouplana, Lluís

    2011-01-01

    Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity. PMID:22140588

  1. Role of plasminogen activator inhibitor in the reciprocal regulation of bovine aortic endothelial and smooth muscle cell migration by TGF-beta 1.

    PubMed Central

    Petzelbauer, E.; Springhorn, J. P.; Tucker, A. M.; Madri, J. A.

    1996-01-01

    Vascular endothelial and smooth muscle cells exhibit reciprocal migratory responses after transforming growth factor (TGF)-beta 1 treatment. Endothelial cells exhibit a decreased migratory rate and smooth muscle cells exhibit an increased migratory rate. Previous studies have demonstrated increases in extracellular matrix and integrin synthesis and expression in response to TGF-beta 1. In this report, we illustrate the roles of plasminogen activator inhibitor in modulating the migratory rates in these two cell types. Endothelial cells appear to require a proteolytic phenotype for rapid migration, whereas vascular smooth muscle cells appear to require an anti-proteolytic phenotype. Modulation of proteinase/anti-proteinase activity ratios was accomplished via TGF-beta 1 induction, addition of exogenous plasminogen activator inhibitor, addition of anti-catalytic antibodies directed against urokinase plasminogen activator, overexpression of plasminogen activator inhibitor utilizing stable transfectants, and the use of vitronectin as a substratum. The reciprocal migratory behaviors exhibited by these two vascular cell types in response to TGF-beta 1 is discussed in the context that these two vascular cell types utilize distinct adhesive and signaling pathways in their interactions with extracellular matrix components and responsiveness to proteolytic activity. Images Figure 1 Figure 2 Figure 3 PMID:8780396

  2. Involvement of marrow-derived endothelial cells in vascularization.

    PubMed

    Larrivée, B; Karsan, A

    2007-01-01

    Until recently, the adult neovasculature was thought to arise only through angiogenesis, the mechanism by which new blood vessels form from preexisting vessels through endothelial cell migration and proliferation. However, recent studies have provided evidence that postnatal neovasculature can also arise though vasculogenesis, a process by which endothelial progenitor cells are recruited and differentiate into mature endothelial cells to form new blood vessels. Evidence for the existence of endothelial progenitors has come from studies demonstrating the ability of bone marrow-derived cells to incorporate into adult vasculature. However, the exact nature of endothelial progenitor cells remains controversial. Because of the lack of definitive markers of endothelial progenitors, the in vivo contribution of progenitor cells to physiological and pathological neovascularization remains unclear. Early studies reported that endothelial progenitor cells actively integrate into the adult vasculature and are critical in the development of many types of vascular-dependent disorders such as neoplastic progression. Moreover, it has been suggested that endothelial progenitor cells can be used as a therapeutic strategy aimed at promoting vascular growth in a variety of ischemic diseases. However, increasing numbers of studies have reported no clear contribution of endothelial progenitors in physiological or pathological angiogenesis. In this chapter, we discuss the origin of the endothelial progenitor cell in the embryo and adult, and we discuss the cell's link to the primitive hematopoietic stem cell. We also review the potential significance of endothelial progenitor cells in the formation of a postnatal vascular network and discuss the factors that may account for the current lack of consensus of the scientific community on this important issue. PMID:17554506

  3. Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

    PubMed Central

    Archipoff, G.; Beretz, A.; Bartha, K.; Brisson, C.; de la Salle, C.; Froget-Léon, C.; Klein-Soyer, C.; Cazenave, J. P.

    1993-01-01

    1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium. Images Figure 5 Figure 6 PMID:7684300

  4. Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

    PubMed Central

    Kim, Seung Joon; Wan, Qiaoqiao; Cho, Eunhye; Han, Bumsoo; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Na, Sungsoo

    2014-01-01

    Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs. PMID:24393843

  5. DIFFERENTIAL TRANSCRIPTION FACTOR ACTIVATION AD GENE EXPRESSION PROFILES IN HUMAN VASCULAR ENDOTHELIAL CELLS ON EXPOSURE TO RESIDUAL OIL FLY ASH (ROFA) AND VANADIUM

    EPA Science Inventory


    Differential transcription factor activation and gene expression profiles in human vascular endothelial cells on exposure to residual oil fly ash (ROFA) and vanadium.
    Srikanth S. Nadadur and Daniel L. Costa, US EPA, ORD, NHEERL (ETD, Pulmonary Toxicology Branch), Research ...

  6. Endothelial cells in dengue hemorrhagic fever.

    PubMed

    Srikiatkhachorn, Anon; Kelley, James F

    2014-09-01

    Therapies to prevent or reverse endothelial dysfunction and vascular leak found in dengue hemorrhagic fever (DHF) have not been identified. In this review we summarize dengue viruses and the spectrum of human disease and highlight evidence of endothelial cell dysfunction in DHF based on studies in patients and mouse and tissue culture models. Evidence suggests that both virus antigen and host immune response, can cause endothelial cell dysfunction and weaken endothelial barrier integrity. We suggest possible therapeutic interventions and highlight how therapies targeting altered endothelial function might be evaluated in animal models and in patients with DHF. PMID:25025934

  7. Endothelial cell micropatterning: Methods, effects, and applications

    PubMed Central

    Anderson, Deirdre E.J.; Hinds, Monica T.

    2012-01-01

    The effects of flow on endothelial cells have been widely examined for the ability of fluid shear stress to alter cell morphology and function; however, the effects of endothelial cell morphology without flow have only recently been observed. An increase in lithographic techniques in cell culture spurred a corresponding increase in research aiming to confine cell morphology. These studies lead to a better understanding of how morphology and cytoskeletal configuration affect the structure and function of the cells. This review examines endothelial cell micropatterning research by exploring both the many alternative methods used to alter endothelial cell morphology and the resulting changes in cellular shape and phenotype. Micropatterning induced changes in endothelial cell proliferation, apoptosis, cytoskeletal organization, mechanical properties, and cell functionality. Finally, the ways these cellular manipulation techniques have been applied to biomedical engineering research, including angiogenesis, cell migration, and tissue engineering, is discussed. PMID:21761242

  8. Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.

    PubMed

    Zhou, Jing; Lee, Pei-Ling; Tsai, Chien-Sung; Lee, Chih-I; Yang, Tung-Lin; Chuang, Han-Sheng; Lin, Wei-Wen; Lin, Ting-Er; Lim, Seh Hong; Wei, Shu-Yi; Chen, Yuh-Lien; Chien, Shu; Chiu, Jeng-Jiann

    2012-05-15

    Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, but the mechanism of force-specific activation of their signaling to modulate cellular function remains unclear. We have demonstrated that bone morphogenetic protein receptor (BMPR)-specific Smad1/5 can be force-specifically activated by oscillatory shear stress (OSS) in ECs to cause cell cycle progression. Smad1/5 is highly activated in ECs of atherosclerotic lesions in diseased human coronary arteries from patients with end-stage heart failure undergoing heart transplantation and from apolipoprotein E-deficient mice. Application of OSS (0.5 ± 4 dyn/cm(2)) causes the sustained activation of Smad1/5 in ECs through activations of mammalian target of rapamycin and p70S6 kinase, leading to up-regulation of cyclin A and down-regulations of p21(CIP1) and p27(KIP1) and, hence, EC cycle progression. En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the inner, but not the outer, curvature of aortic arch, nor the straight segment of thoracic aorta [corrected]. Immunohistochemical and en face examinations of the experimentally stenosed abdominal aorta in rats show high levels of phospho-Smad1/5 in ECs at poststenotic sites, where OSS occurs. These OSS activations of EC Smad1/5 in vitro and in vivo are not inhibited by the BMP-specific antagonist Noggin and, hence, are independent of BMP ligand. Transfecting ECs with Smad1/5-specific small interfering RNAs inhibits the OSS-induced EC cycle progression. Our findings demonstrate the force-specificity of the activation of Smad1/5 and its contribution to cell cycle progression in ECs induced by disturbed flow. PMID:22550179

  9. Isolation of Murine Embryonic Hemogenic Endothelial Cells.

    PubMed

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

    2016-01-01

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

  10. Isolation of Murine Embryonic Hemogenic Endothelial Cells

    PubMed Central

    Marcelo, Kathrina L.; Hirschi, Karen K.

    2016-01-01

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

  11. Progenitor endothelial cell involvement in Alzheimer's disease

    SciTech Connect

    Budinger, Thomas F.

    2003-05-01

    There is compelling evidence that endothelial cells of the brain and periphery are dysfunctional in Alzheimer's Disease. There is evidence for a fundamental defect in, or abnormal aging of, endothelial progenitor cells in atherosclerosis. The possibility that endothelial cell defects are a primary cause for Alzheimer's Disease or other dementias can be researched by molecular and cell biology studies as well as cell trafficking studies using recently demonstrated molecular imaging methods. The evidence for abnormal endothelial function and the methods to explore this hypothesis are presented.

  12. Acidic microenvironments induce lymphangiogenesis and IL-8 production via TRPV1 activation in human lymphatic endothelial cells.

    PubMed

    Nakanishi, Masako; Morita, Yoshihiro; Hata, Kenji; Muragaki, Yasuteru

    2016-07-15

    Local acidosis is one of the characteristic features of the cancer microenvironment. Many reports indicate that acidosis accelerates the proliferation and invasiveness of cancer cells. However, whether acidic conditions affect lymphatic metastasis is currently unknown. In the present study, we focused on the effects of acidosis on lymphatic endothelial cells (LECs) to assess the relationship between acidic microenvironments and lymph node metastasis. We demonstrated that normal human LECs express various acid receptors by immunohistochemistry and reverse transcriptase-polymerase chain reaction (PCR). Acidic stimulation with low pH medium induced morphological changes in LECs to a spindle shape, and significantly promoted cellular growth and tube formation. Moreover, real-time PCR revealed that acidic conditions increased the mRNA expression of interleukin (IL)-8. Acidic stimulation increased IL-8 production in LECs, whereas a selective transient receptor potential vanilloid subtype 1 (TRPV1) antagonist, 5'-iodoresiniferatoxin, decreased IL-8 production. IL-8 accelerated the proliferation of LECs, and inhibition of IL-8 diminished tube formation and cell migration. In addition, phosphorylation of nuclear factor (NF)-κB was induced by acidic conditions, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that acidic microenvironments in tumors induce lymphangiogenesis via TRPV1 activation in LECs, which in turn may promote lymphatic metastasis. PMID:27312995

  13. Heparin Binds Endothelial Cell Growth Factor, the Principal Endothelial Cell Mitogen in Bovine Brain

    NASA Astrophysics Data System (ADS)

    Maciag, Thomas; Mehlman, Tevie; Friesel, Robert; Schreiber, Alain B.

    1984-08-01

    Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

  14. Activation of endothelial β-catenin signaling induces heart failure

    PubMed Central

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  15. Activation of endothelial β-catenin signaling induces heart failure.

    PubMed

    Nakagawa, Akito; Naito, Atsuhiko T; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P; Adams, Ralf H; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  16. Endothelial cells enhance migration of meniscus cells

    PubMed Central

    Yuan, Xiaoning; Eng, George M.; Arkonac, Derya E.; Chao, Pen-hsiu Grace; Vunjak-Novakovic, Gordana

    2014-01-01

    Objective To study the interactions between vascular endothelial cells and meniscal fibrochondrocytes from the inner avascular and outer vascular regions of the meniscus, and identify angiogenic factors that enhance cell migration and integrative repair. Methods Bovine meniscal fibrochondrocytes (bMFCs) from the inner and outer regions of meniscus were cultured for seven days with and without human umbilical vein endothelial cells (HUVECs) in a micropatterned three-dimensional hydrogel system for cell migration. Angiogenic factors secreted by HUVECs were probed for their role in paracrine mechanisms governing bMFC migration, and applied to a full-thickness defect model of meniscal repair in explants from the inner and outer regions over four weeks. Results Endothelial cells enhanced migration of inner and outer bMFCs in the micropatterned system via endothelin-1 (ET-1) signaling. Supplementation of ET-1 significantly enhanced integration strength of full-thickness defects in inner and outer explants, and cell migration at the macro-scale, compared to controls without ET-1 treatment. Conclusion We report for the first time that bMFCs from both the avascular and vascular regions respond to the presence of endothelial cells with increased migration. Paracrine signaling by endothelial cells regulates the bMFCs differentially by region, but we identify ET-1 as an angiogenic factor that stimulates migration of inner and outer cells at the micro-scale, and integrative repair of inner and outer explants at the macro-scale. These findings reveal the regional interactions between vasculature and MFCs, and suggest ET-1 as a potential new treatment modality for avascular meniscal injuries, in order to prevent the development of osteoarthritis. PMID:25307081

  17. Replication of human endothelial cells in culture.

    PubMed

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

    1973-08-01

    Investigative studies dealing with the properties and functions of endothelial cells have been hampered because there has been little or no success in the isolation, growth, and passage of individual cells in large numbers. We have developed a system whereby pure cultures of endothelial cells derived from umbilical veins can be subcultured for at least five serial passages. Many facets of endothelial function and interaction can be evaluated with the use of this new adaptive system of isolation and culture. PMID:4718112

  18. Senescent endothelial cells: Potential modulators of immunosenescence and ageing.

    PubMed

    Pantsulaia, Ia; Ciszewski, Wojciech Michal; Niewiarowska, Jolanta

    2016-08-01

    Recent studies have demonstrated that the accumulation of senescent endothelial cells may be the primary cause of cardiovascular diseases. Because of their multifunctional properties, endothelial cells actively take part in stimulating the immune system and inflammation. In addition, ageing is characterized by the progressive deterioration of immune cells and a decline in the activation of the immune response. This results in a loss of the primary function of the immune system, which is eliminating damaged/senescent cells and neutralizing potential sources of harmful inflammatory reactions. In this review, we discuss cellular senescence and the senescence-associated secretory phenotype (SASP) of endothelial cells and summarize the link between endothelial cells and immunosenescence. We describe the possibility that age-related changes in Toll-like receptors (TLRs) and microRNAs can affect the phenotypes of senescent endothelial cells and immune cells via a negative feedback loop aimed at restraining the excessive pro-inflammatory response. This review also addresses the following questions: how do senescent endothelial cells influence ageing or age-related changes in the inflammatory burden; what is the connection between ECs and immunosenescence, and what are the crucial hypothetical pathways linking endothelial cells and the immune system during ageing. PMID:27235855

  19. Interleukin-1beta-induced type IIA secreted phospholipase A2 gene expression and extracellular activity in rat vascular endothelial cells.

    PubMed

    Schwemmer, M; Aho, H; Michel, J B

    2001-06-01

    Two phospholipase A2 (PLA2) isoforms, secretory and cytosolic, have been implicated in inflammation. Secretory type IIA PLA2 (sPLA2-IIA), which hydrolyzes fatty acids bound at the sn-2 position of glycerophospholipids, has been detected universally in a variety of mammalian tissues and cells. The expression of the sPLA2-IIA gene and its extracellular activity were shown to be regulated by different factors such as hypoxia, cytokines and phorbol esters. In the present study, we examined the effects of interleukin-1beta (IL-1beta) on the expression of the 14kDa sPLA2-IIA, determined using reverse transcription polymerase chain reaction and radiometric Escherichia coli enzyme assay in primary cultures of rat endothelial cells and in two different rat endothelial cell lines (SVAREC and RBE4). These experiments revealed that IL-1beta induces sPLA2-IIa gene expression and secretion of the enzyme in endothelial cells in a dose- and time-dependent manner. The cAMP-elevator forskolin did not augment the cytokine-induced elevation of sPLA2-IIa enzyme activity but significantly increased the IL-1beta-stimulated sPLA2-IIa mRNA contents in endothelial cells. PMID:11469536

  20. Cell-Based Screening Identifies the Active Ingredients from Traditional Chinese Medicine Formula Shixiao San as the Inhibitors of Atherosclerotic Endothelial Dysfunction

    PubMed Central

    Wang, Xiaofan; Zhang, Ruowen; Gu, Liqiang; Zhang, Yuanyuan; Zhao, Xu; Bi, Kaishun; Chen, Xiaohui

    2015-01-01

    In this study, we performed a phenotypic screening in human endothelial cells exposed to oxidized low density lipoprotein (an in vitro model of atherosclerotic endothelial dysfunction) to identify the effective compounds in Shixiao San. After investigating the suitability and reliability of the cell-based screening method using atorvastatin as the positive control drug, this method was applied in screening Shixiao San and its extracts. The treatment of n-butanol fraction on endothelial cells exhibited stronger healing effects against oxidized low density lipoprotein-induced insult when compared with other fractions. Cell viability, the level of nitric oxide, endothelial nitric oxide synthase and endothelin-1 were measured, respectively. The assays revealed n-butanol fraction significantly elevated the survival ratio of impaired cells in culture. In parallel, n-butanol fraction exhibited the highest inhibition of inflammation. The generation of prostaglandin-2 and adhesion molecule (soluble intercellular adhesion molecule-1) was obviously declined. Furthermore, n-butanol fraction suppressed the production of reactive oxygen species and malondialdehyde, and restored the activity of superoxide dismutase. Compounds identification of the n-butanol fraction was carried out by ultra high liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. The active ingredients including quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside revealed the ability of anti-atherosclerosis after exposing on endothelial cells. The current work illustrated the pharmacology effect of Shixiao San and clearly indicated the major active components in Shixiao San. More importantly, the proposed cell-based screening method might be particularly suitable for fast evaluating the anti-atherosclerosis efficacy of Traditional Chinese Medicines and screening out the interesting

  1. Platelet-activating factor increases VE-cadherin tyrosine phosphorylation in mouse endothelial cells and its association with the PtdIns3'-kinase

    PubMed Central

    Hudry-Clergeon, Hélène; Stengel, Dominique; Ninio, Ewa; Vilgrain, Isabelle

    2005-01-01

    Platelet-activating-Factor (PAF), a potent inflammatory mediator, is involved in endothelial permeability. This study was designed to characterize PAF receptor (PAF-R) expression and its specific contribution to the modifications of adherens junctions in mouse endothelial cells. We demonstrated that PAF-R was expressed in mouse endothelial cells and was functionally active in stimulating p42/p44 MAPK and phosphatidylinositol 3-kinase (PtdIns3′-kinase)/Akt activities. Treatment of cells with PAF induced a rapid, time- and dose-dependent (10−7 to 10−10M) increase in tyrosine phosphorylation of a subset of proteins ranging from 90 kDa to 220 kDa, including the VE-cadherin, the latter effect being prevented by the tyrosine kinase inhibitors, herbimycin A and bis-tyrphostin. Furthermore, we demonstrated that PAF promoted formation of multimeric aggregates of VE-cadherin with PtdIns3′-kinase which was also inhibited by herbimycin and bis-tyrphostin. Finally, we showed by immunostaining of endothelial cells VE-cadherin, that PAF dissociated adherens junctions. The present data provide the first evidence that the treatment of endothelial cells with PAF promoted activation of tyrosine kinases and the VE-cadherin tyrosine phosphorylation and PtdIns3′-kinase association, that ultimately lead to the dissociation of adherens junctions. Physical association between PtdIns3′-kinase, serving as a docking protein, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of PAF-induced cellular activation. PMID:15791001

  2. Low concentration of 4-hydroxy hexenal increases heme oxygenase-1 expression through activation of Nrf2 and antioxidative activity in vascular endothelial cells

    SciTech Connect

    Ishikado, Atsushi; Nishio, Yoshihiko; Morino, Katsutaro; Ugi, Satoshi; Kondo, Hajime; Makino, Taketoshi; Kashiwagi, Atsunori; Maegawa, Hiroshi

    2010-11-05

    Research highlights: {yields} Low doses of 4-HHE and 4-HNE induce HO-1 expression in vascular endothelial cells. {yields} 4-HHE and 4-HNE increase the intranuclear expression and DNA binding of Nrf2. {yields} 4-HHE and 4-HNE-induced HO-1 expression depends on the activation of Nrf2. {yields} Pretreatment with 4-HHE and 4-HNE prevents oxidative stress-induced cytotoxicity. -- Abstract: Large-scale clinical studies have shown that n-3 polyunsaturated fatty acids (n-3 PUFAs) such as eicosapentaenoic and docosahexaenoic acids reduce cardiovascular events without improving classical risk factors for atherosclerosis. Recent studies have proposed that direct actions of n-3 PUFAs themselves, or of their enzymatic metabolites, have antioxidative and anti-inflammatory effects on vascular cells. Although a recent study showed that plasma 4-hydroxy hexenal (4-HHE), a peroxidation product of n-3 PUFA, increased after supplementation of docosahexaenoic acid, the antiatherogenic effects of 4-HHE in vascular cells remain unclear. In the present study, we tested the hypothesis that 4-HHE induces the antioxidative enzyme heme oxygenase-1 (HO-1) through activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulatory transcriptional factor, and prevents oxidative stress-induced cytotoxicity in vascular endothelial cells. This mechanism could partly explain the cardioprotective effects of n-3 PUFAs. Human umbilical vein endothelial cells were stimulated with 1-10 {mu}M 4-HHE or 4-hydroxy nonenal (4-HNE), a peroxidation product of n-6 PUFAs. Both 4-HHE and 4-HNE dose-dependently increased HO-1 mRNA and protein expression, and intranuclear expression and DNA binding of Nrf2 at 5 {mu}M. Small interfering RNA for Nrf2 significantly reduced 4-HHE- or 4-HNE-induced HO-1 mRNA and protein expression. Furthermore, pretreatment with 4-HHE or 4-HNE prevented tert-butyl hydroperoxide-induced cytotoxicity. In conclusion, 4-HHE, a peroxidation product of n-3 PUFAs, stimulated

  3. [Transplantation of corneal endothelial cells].

    PubMed

    Amano, Shiro

    2002-12-01

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

  4. 2,2',4,6,6'-pentachlorobiphenyl (PCB 104) induces apoptosis of human microvascular endothelial cells through the caspase-dependent activation of CREB.

    PubMed

    Lee, Yong Woo; Park, Hyen Joo; Son, Kwang Won; Hennig, Bernhard; Robertson, Larry W; Toborek, Michal

    2003-05-15

    It has been proposed that endothelial integrity can play an active regulatory role in the extravasation of tumor cells during cancer metastasis. Since polychlorinated biphenyls (PCBs) have been shown to cause endothelial cell activation or injury and to lead to various diseases that involve dysfunction of the vascular endothelium, the present study was designed to determine the cellular and molecular signaling mechanisms of PCB-induced apoptosis in human microvascular endothelial cells (HMEC-1). A significant and marked decrease in cell viability was observed in HMEC-1 treated with 2,2',4,6,6'-pentachlorobiphenyl (PCB 104) in a time- and dose-dependent manner. Exposure of HMEC-1 to PCB 104 also dramatically induced internucleosomal DNA fragmentation. However, the caspase inhibitor zVAD-fmk significantly reversed the PCB 104-induced DNA fragmentation in HMEC-1, suggesting that endothelial cell death induced by PCB 104 exposure is, at least in part, due to caspase-dependent apoptotic pathways. To elucidate the molecular signaling mechanisms of PCB 104-induced apoptotic cell death in human microvascular endothelial cells, the present study focused on the effects of acute exposure of PCB 104 on the activation of several transcription factors, such as cAMP responsive element-binding protein (CREB), activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and signal transducers and activators of transcription (STAT1), which have been known to play a pivotal role in the molecular signaling cascades for the induction of apoptosis. A series of electrophoretic mobility shift assay showed that PCB 104 specifically increased only CREB DNA-binding activity in a dose-dependent manner. AP-1, NF-kappaB, and STAT1, however, were not activated. In addition, zVAD-fmk significantly and dose-dependently blocked the CREB activation enhanced by PCB 104 exposure. These results suggest that PCB-induced death of human microvascular endothelial cells is mediated, at least in part, via

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

    PubMed

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

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro- and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy. PMID:23044355

  6. Carbon Monoxide Induces Heme Oxygenase-1 to Modulate STAT3 Activation in Endothelial Cells via S-Glutathionylation

    PubMed Central

    Yang, Yan-Chang; Huang, Yu-Ting; Hsieh, Chia-Wen; Yang, Po-Min; Wung, Being-Sun

    2014-01-01

    IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs. PMID:25072782

  7. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation

    PubMed Central

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

  8. Quantitation of Endothelial Cell Adhesiveness In Vitro

    PubMed Central

    Lowe, Donna J.; Raj, Kenneth

    2015-01-01

    One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive. PMID:26132714

  9. FAK and p38-MAP Kinase-Dependent Activation of Apoptosis and Caspase-3 in Retinal Endothelial Cells by α1(IV)NC1

    PubMed Central

    Boosani, Chandra S.; Nalabothula, Narasimharao; Munugalavadla, Veerendra; Cosgrove, Dominic; Keshamouni, Venkateshwar G.; Sheibani, Nader; Sudhakar, Akulapalli

    2009-01-01

    Purpose To determine the impact of the antiangiogenic factor α1(IV)NC1 on vascular endothelial growth factor mediated proangiogenic activity in mouse retinal endothelial cell (MLEC). Methods Primary culture of mouse retinal endothelial cells were established as previously described and used to determine the effects of α1(IV)NC1 on proangiogenic activity of VEGF. Cell proliferation was evaluated using [H3] thymidine incorporation and 3,(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide colorimetric assays. Cell migration was determined using modified Boyden chamber and scratch wound assays and tube formation was assessed on Matrigel. The intracellular signaling events Bcl-2/Bcl-xL and caspase-3/poly (ADP-ribose) polymerase (PARP) activities were evaluated in cells stimulated with VEGF and plated on type IV collagen coated dishes. Apoptosis was assessed by measuring different caspases activity as well as quantitative fluorescence analysis using fluorescence-activated cell sorting assay. Subcutaneously injected VEGF induced in-vivo neovascularization was studied using Matrigel plug assay. Results VEGF induced sub-confluent MREC proliferation, migration, and tube formation was significantly inhibited by α1(IV)NC1 at 1.0µM (P<0.001). α1(IV)NC1 induced MREC apoptosis mediating through by inhibition of Bcl-2 and Bcl-xL expressions and activation of caspase-3/PARP through FAK/p38-MAPK signaling. In addition, α1(IV)NC1 dose dependently inhibited VEGF-mediated neovascularization in-vivo. Conclusions α1(IV)NC1 inhibited VEGF-mediated angiogenesis by promoting apoptosis, caspase-3/PARP activation and negatively impacting FAK/p38-MAPK phosphorylation, Bcl-2 and Bcl-xL expressions leading to MREC death. The endothelial specific inhibitory actions of recombinant α1(IV)NC1 may be of benefit in the treatment of a variety of eye diseases with a neovascular component. PMID:19443723

  10. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    PubMed

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing. PMID:26612622

  11. Seasonal Changes in Testes Vascularisation in the Domestic Cat (Felis domesticus): Evaluation of Microvasculature, Angiogenic Activity, and Endothelial Cell Expression

    PubMed Central

    Alexandre-Pires, Graça; Mateus, Luísa; Martins, Catarina; Ferreira-Dias, Graça

    2012-01-01

    Some male seasonal breeders undergo testicular growth and regression throughout the year. The objective of this study was to understand the effect of seasonality on: (i) microvasculature of cat testes; (ii) angiogenic activity in testicular tissue in vitro; and (iii) testicular endothelial cells expression throughout the year. Testicular vascular areas increased in March and April, June and July, being the highest in November and December. Testes tissue differently stimulated in vitro angiogenic activity, according to seasonality, being more evident in February, and November and December. Even though CD143 expression was higher in December, smaller peaks were present in April and July. As changes in angiogenesis may play a role on testes vascular growth and regression during the breeding and non-breeding seasons, data suggest that testicular vascularisation in cats is increased in three photoperiod windows of time, November/December, March/April and June/July. This increase in testicular vascularisation might be related to higher seasonal sexual activity in cats, which is in agreement with the fact that most queens give birth at the beginning of the year, between May and July, and in September. PMID:22567311

  12. Chemical constituents of Hericium erinaceum associated with the inhibitory activity against cellular senescence in human umbilical vascular endothelial cells.

    PubMed

    Noh, Hyung Jun; Yang, Hyo Hyun; Kim, Geum Soog; Lee, Seung Eun; Lee, Dae Young; Choi, Je Hun; Kim, Seung Yu; Lee, Eun Suk; Ji, Seung Heon; Kang, Ki Sung; Park, Hye-Jin; Kim, Jae-Ryong; Kim, Ki Hyun

    2015-12-01

    Hericium erinaceum is an edible and medicinal mushroom widely used in Korea, Japan, and China. On the search for biologically active compounds supporting the medicinal usage, the MeOH extract of the fruiting bodies of H. erinaceum was investigated for its chemical constituents. Six compounds were isolated and identified as hericenone D (1), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (2), erinacerin B (3), hericenone E (4), hericenone F (5) and isohericerin (6) by comparing their spectroscopic data with previously reported values. The inhibitory effects on adriamycin-induced cellular senescence in human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) of the isolates (1-6) were studied. Among the isolated compounds, ergosterol peroxide (2) reduced senescence associated β-galactosidase (SA-β-gal) activity increased in HUVECs treated with adriamycin. According to experimental data obtained, the active compound may inspire the development of a new pharmacologically useful substance to be used in the treatment and prevention of age-related diseases. PMID:25676326

  13. Endothelial Cell Integrin Laminin Receptor Expression in Multiple Sclerosis Lesions

    PubMed Central

    Sobel, Raymond A.; Hinojoza, Julian R.; Maeda, Atsuko; Chen, Michael

    1998-01-01

    Laminin, a major glycoprotein component of vessel basement membranes, is recognized by β1- and β3-integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions, integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions, endothelial cell VLA-6 and β1 subunits were decreased compared to controls whereas αv subunit and VLA-1 were increased. In chronic inactive lesions β1, VLA-6 and αv were the same as controls but VLA-1 remained increased. α3 subunit was constant in all samples. By immunoelectron microscopy VLA-1, VLA-6, β1, and laminin were distributed throughout endothelial cells; αv was adjacent to and on luminal surfaces; αv and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell β1/VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and blood-brain barrier breakdown. αv and VLA-1 on intercellular junctions may participate in re-establishing vessel integrity after leukocyte migration. Luminal surface αv also likely binds intraluminal ligands and cells. In chronic inactive plaques persistently elevated endothelial cell VLA-1 correlates with longstanding endothelial cell and blood-brain barrier dysfunction. PMID:9708801

  14. Effect of ascorbate on plasminogen activator inhibitor-1 expression and release from platelets and endothelial cells in an in-vitro model of sepsis.

    PubMed

    Swarbreck, Scott B; Secor, Dan; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel

    2015-06-01

    The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis. PMID:25730478

  15. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed Central

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-01-01

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  16. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-04-15

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  17. Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action

    PubMed Central

    Lombardo, Giusy; Dentelli, Patrizia; Togliatto, Gabriele; Rosso, Arturo; Gili, Maddalena; Gallo, Sara; Deregibus, Maria Chiara; Camussi, Giovanni; Brizzi, Maria Felice

    2016-01-01

    Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices. PMID:27157262

  18. The P2Y2 Receptor Interacts with VE-Cadherin and VEGF Receptor-2 to Regulate Rac1 Activity in Endothelial Cells

    PubMed Central

    Liao, Zhongji; Cao, Chen; Wang, Jianjie; Huxley, Virginia H.; Baker, Olga; Weisman, Gary A.

    2015-01-01

    Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1. PMID:25657827

  19. The vascular disrupting activity of OXi8006 in endothelial cells and its phosphate prodrug OXi8007 in breast tumor xenografts.

    PubMed

    Strecker, Tracy E; Odutola, Samuel O; Lopez, Ramona; Cooper, Morgan S; Tidmore, Justin K; Charlton-Sevcik, Amanda K; Li, Li; MacDonough, Matthew T; Hadimani, Mallinath B; Ghatak, Anjan; Liu, Li; Chaplin, David J; Mason, Ralph P; Pinney, Kevin G; Trawick, Mary Lynn

    2015-12-01

    This study describes the vascular disrupting ability and the mechanism of action of the indole-based tubulin-binding compound, OXi8006, and its water-soluble phosphate prodrug OXi8007. Treatment of rapidly proliferating human umbilical vein endothelial cells (HUVECs), used as a model for the tumor vasculature, with OXi8006 or OXi8007, caused potent microtubule disruption followed by extensive reorganization of the cytoskeletal network. The mechanism of action involved an increase in focal adhesion formation associated with an increase in phosphorylation of both non-muscle myosin light chain and focal adhesion kinase. These effects were dramatically diminished by an inhibitor of RhoA kinase, a downstream effector of RhoA. Cell cycle blockade at G2/M and cytotoxicity toward rapidly proliferating HUVECs were also observed. Capillary-like networks of HUVECs were disrupted by the action of both OXi8006 and OXi8007. The prodrug OXi8007 exhibited potent and rapid dose-dependent antivascular activity assessed by dynamic bioluminescence imaging (BLI) in an MDA-MB-231-luc breast cancer xenograft mouse model. By 6 hours post treatment, over 93% of the BLI signal was abolished with only a slight recovery at 24 hours. These findings were confirmed by histology. The results from this study demonstrate that OXi8007 is a potent vascular disrupting agent acting through an anti-microtubule mechanism involving RhoA. PMID:26325604

  20. Effects of a novel cyclic RGD peptidomimetic on cell proliferation, migration and angiogenic activity in human endothelial cells

    PubMed Central

    2014-01-01

    Background Cyclic RGD peptidomimetics containing a bifunctional diketopiperazine scaffold are a novel class of high-affinity ligands for the integrins αVβ3 and αVβ5. Since integrins are a promising target for the modulation of normal and pathological angiogenesis, the present study aimed at characterizing the ability of the RGD peptidomimetic cyclo[DKP-RGD] 1 proliferation, migration and network formation in human umbilical vein endothelial cells (HUVEC). Methods Cell viability was assessed by flow cytometry and annexin V (ANX)/propidium iodide (PI) staining. Cell proliferation was evaluated by the ELISA measurement of bromodeoxyuridine (BrdU) incorporation. Network formation by HUVEC cultured in Matrigel-coated plates was evaluated by optical microscopy and image analysis. Integrin subunit mRNA expression was assessed by real time-PCR and Akt phosphorylation by western blot analysis. Results Cyclo[DKP-RGD] 1 does not affect cell viability and proliferation either in resting conditions or in the presence of the pro-angiogenic growth factors VEGF, EGF, FGF, and IGF-I. Addition of cyclo[DKP-RGD] 1 however significantly decreased network formation induced by pro-angiogenic growth factors or by IL-8. Cyclo[DKP-RGD] 1 did not affect mRNA levels of αV, β3 or β5 integrin subunits, however it significantly reduced the phosphorylation of Akt. Conclusions Cyclo[DKP-RGD] 1 can be a potential modulator of angiogenesis induced by different growth factors, possibly devoid of the adverse effects of cytotoxic RGD peptidomimetic analogues. PMID:25053992

  1. Suppressive effects of anti-inflammatory agents on human endothelial cell activation and induction of heat shock proteins.

    PubMed Central

    Amberger, A.; Hala, M.; Saurwein-Teissl, M.; Metzler, B.; Grubeck-Loebenstein, B.; Xu, Q.; Wick, G.

    1999-01-01

    BACKGROUND: Studies from our laboratory have shown that the earliest stages of atherosclerosis may be mediated by an autoimmune reaction against heat shock protein 60 (Hsp60). The interactions of Hsp60-specific T cells with arterial endothelial cells (EC) require expression of both Hsp60 and certain adhesion molecules shown to be induced simultaneously in EC by mechanical and other types of stress. Recently, it was shown that suppression of T cell-mediated immune responses by cyclosporin A (CyA) enhanced atherosclerotic lesion formation in mice. In contrast, aspirin was found to lower the risk of myocardial infarction in men. These conflicting observations may be due to different effects of anti-inflammatory agents on adhesion molecule and Hsp expression in EC, respectively. MATERIAL AND METHODS: In the present study, we analyzed the effects of CyA, aspirin, and indomethacin on T cell proliferation using a proliferation assay. To explore the expression of adhesion molecules, monocyte chemoattractant protein-1 (MCP-1), and Hsp60 in human umbilical vein endothelial cells (HUVECs), Northern blot analyses were used. To examine the activation status of the transcription factors nuclear factor kappaB (NF-kappaB) and heat shock factor-1 (HSF-1), electrophoretic mobility shift assays were performed. RESULTS: With the exception of indomethacin, the used immunosuppressive and anti-inflammatory agents significantly inhibited T cell proliferation in response to influenza virus antigen in a dose-dependent manner. Interestingly, CyA and indomethacin did not suppress tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression on HUVECs, whereas aspirin had an inhibitory effect. These observations correlated with the modulation of NF-kappaB activity in EC. All agents tested induced expression of Hsp60 6 hr after application. In addition, aspirin and indomethacin, but not CyA, induced Hsp70 expression in HUVECs that correlated with induction of HSF-1 activity

  2. The Association of Vitamin D Status with Dyslipidaemia and Biomarkers of Endothelial Cell Activation in Older Australians

    PubMed Central

    Alyami, Ali M.; Lam, Virginie; Soares, Mario J.; Zhao, Yun; Sherriff, Jillian L.; Mamo, John C.; James, Anthony P.; Coombes, Fiona

    2016-01-01

    Background/Aims: Vitamin D has been investigated for many non-skeletal effects. The objective of this study was to determine whether circulating lipids, systemic inflammation, and biomarkers of endothelial cell activation varied with the vitamin D status of older Australians. Methods: One hundred and one participants were proportionately and randomly sampled across tertiles of 25 hydroxy vitamin D (25(OH)D) from a larger cohort of free living older adults (T1 median = 97; T2 median = 74.5; T3 median = 56.8 nmol/L). Overnight fasting blood samples were assayed for 25(OH)D, parathyroid hormone (PTH), insulin, triacylglycerol (TAG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C). Markers of systemic inflammation (high sensitivity C-reactive protein (hsCRP), tumour necrosis factor-α (TNF-α)) and endothelial activation (hepatocyte growth factor (HGF), P-selectin and soluble vascular cell adhesion molecule (sVCAM), soluble intracellular adhesion molecule (sICAM)) were determined. A general linear model multivariate analysis with a backward elimination procedure was performed. Results: Eighty-three participants (48 women, 35 men), aged 65 ± 7.7 years, BMI 28 ± 4.5 kg/m2, with complete data were analyzed. The final parsimonious model controlled for age, gender, BMI, and McAuley’s index, but excluded season, medications, and PTH. There were significant differences across 25(OH)D tertiles in TC (T1 < T3, p = 0.003; T2 < T3, p = 0.001), LDL-C (T1 < T3, p = 0.005; T2 < T3, p = 0.001), TAG (T2 < T3, p = 0.026), HGF (T1 > T3, p = 0.009) and sVCAM (T1 > T3, P = 0.04). Conclusions: Higher vitamin D status may protect the endothelium through reduced dyslipidaemia and increased HGF. PMID:27483306

  3. Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 by Orientia tsutsugamushi in Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1

    PubMed Central

    Cho, Nam-Hyuk; Seong, Seung-Yong; Huh, Myung-Sook; Kim, Na-Hyun; Choi, Myung-sik; Kim, Ik-sang

    2002-01-01

    Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1. Orientia tsutsugamushi causes vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated the cis-acting and trans-acting elements involved in O. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected with O. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected with O. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the

  4. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

  5. Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis.

    PubMed

    Tanaka, Aya; Itoh, Fumiko; Nishiyama, Koichi; Takezawa, Toshiaki; Kurihara, Hiroki; Itoh, Susumu; Kato, Mitsuyasu

    2010-05-20

    E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect. PMID:20231428

  6. Irisin Induces Angiogenesis in Human Umbilical Vein Endothelial Cells In Vitro and in Zebrafish Embryos In Vivo via Activation of the ERK Signaling Pathway

    PubMed Central

    Wu, Fei; Song, Haibo; Zhang, Yuan; Zhang, Yuzhu; Mu, Qian; Jiang, Miao; Wang, Fang; Zhang, Wen; Li, Liang; Li, Huanjie; Wang, Yunshan; Zhang, Mingxiang; Li, Shiwu; Yang, Lijun; Meng, Yan; Tang, Dongqi

    2015-01-01

    As a link between exercise and metabolism, irisin is assumed to be involved in increased total body energy expenditure, reduced body weight, and increased insulin sensitivity. Although our recent evidence supported the contribution of irisin to vascular endothelial cell (ECs) proliferation and apoptosis, further research of irisin involvement in the angiogenesis of ECs was not conclusive. In the current study, it was found that irisin promoted Human Umbilical Vein Endothelial Cell (HUVEC) angiogenesis via increasing migration and tube formation, and attenuated chemically-induced intersegmental vessel (ISV) angiogenic impairment in transgenic TG (fli1: GFP) zebrafish. It was further demonstrated that expression of matrix metalloproteinase (MMP) 2 and 9 were also up-regulated in endothelial cells. We also found that irisin activated extracellular signal–related kinase (ERK) signaling pathways. Inhibition of ERK signaling by using U0126 decreased the pro-migration and pro-angiogenic effect of irisin on HUVEC. Also, U0126 inhibited the elevated expression of MMP-2 and MMP-9 when they were treated with irisin. In summary, these findings provided direct evidence that irisin may play a pivotal role in maintaining endothelium homeostasis by promoting endothelial cell angiogenesis via the ERK signaling pathway. PMID:26241478

  7. Vitamin D protects human endothelial cells from H₂O₂ oxidant injury through the Mek/Erk-Sirt1 axis activation.

    PubMed

    Polidoro, Lorella; Properzi, G; Marampon, F; Gravina, G L; Festuccia, C; Di Cesare, E; Scarsella, L; Ciccarelli, C; Zani, B M; Ferri, C

    2013-04-01

    Endothelium homeostasis alterations govern the pathogenesis of cardiovascular diseases. Several studies show that vitamins anti-oxidant proprieties rescue the endothelial functions adversely affected by oxidative stress in several diseases. We investigated the vitamin D anti-oxidant potential in human endothelial cells exposed to H2O2 oxidative stress. Vitamin D protected endothelial cells against H2O2 oxidative stress counteracting the superoxide anion generation, the apoptosis and blocking the extrinsic caspase cascade by positively controlling phospho-active ERKs level. MEKs/ERKs inhibitor U0126 reverted the vitamin D anti-oxidant effects. Characterizing the vitamin D downstream effector, we found that vitamin D up-regulated SirT-1 and reverted the SirT-1 down-regulation induced by H2O2. ERKs activation by vitamin D strictly correlated with SirT-1 protein accumulation since both MEKs/ERKs inhibition and ERK1/2 silencing decreased SIRT-1. SirT-1 inhibition by Sirtinol reverted the vitamin D anti-oxidant effects. Thus, vitamin D significantly reduced the endothelial malfunction and damage caused by oxidative stress, through the activation of MEKs/ERKs/SirT-1 axis. PMID:23247634

  8. Signaling hierarchy regulating human endothelial cell development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  9. Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

    PubMed

    Heyninck, Karen; Sabbe, Linde; Chirumamilla, Chandra Sekhar; Szarc Vel Szic, Katarzyna; Vander Veken, Pieter; Lemmens, Kristien J A; Lahtela-Kakkonen, Maija; Naulaerts, Stefan; Op de Beeck, Ken; Laukens, Kris; Van Camp, Guy; Weseler, Antje R; Bast, Aalt; Haenen, Guido R M M; Haegeman, Guy; Vanden Berghe, Wim

    2016-06-01

    Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA. PMID:27045103

  10. ERK-dependent activation of Sp1 is required for low-power laser irradiation-induced vascular endothelial cell proliferation

    NASA Astrophysics Data System (ADS)

    Feng, Jie; Xing, Da

    2012-12-01

    Here, we report that low-power laser irradiation (LPLI) activates ERK/Sp1 pathway to upregulate VEGF expression and promote vascular endothelial cell proliferation. We demonstrate for the first time that LPLI enhances DNA-binding activity and transactivation activity of Sp1 on VEGF promoter. Additionally, ERK translocates from cytoplasm to nucleus following LPLI. Moreover, activated ERK phosphorylates Sp1 and results in increased EKR-Sp1 interaction. Selective inhibition of Sp1 or ERK suppresses the effect of LPLI on the promotion of cell cycle progression and proliferation. These findings provide a novel link between LPLI and angiogenesis, supplying potential therapy strategies for angiogenesis with LPLI.

  11. Poly(lactic-co-glycolic) Acid/Nanohydroxyapatite Scaffold Containing Chitosan Microspheres with Adrenomedullin Delivery for Modulation Activity of Osteoblasts and Vascular Endothelial Cells

    PubMed Central

    Li, Chunyan; Chen, Yingxin; Dong, Shujun; Chen, Xuesi; Zhou, Yanmin

    2013-01-01

    Adrenomedullin (ADM) is a bioactive regulatory peptide that affects migration and proliferation of diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. This study investigated the effects of sustained release of ADM on the modulation activity of osteoblasts and vascular endothelial cells in vitro. Chitosan microspheres (CMs) were developed for ADM delivery. Poly(lactic-co-glycolic) acid and nano-hydroxyapatite were used to prepare scaffolds containing microspheres with ADM. The CMs showed rough surface morphology and high porosity, and they were well-distributed. The scaffolds exhibited relatively uniform pore sizes with interconnected pores. The addition of CMs improved the mechanical properties of the scaffolds without affecting their high porosity. In vitro degradation tests indicated that the addition of CMs increased the water absorption of the scaffolds and inhibited pH decline of phosphate-buffered saline medium. The expression levels of osteogenic-related and angiogenic-related genes were determined in MG63 cells and in human umbilical vein endothelial cells cultured on the scaffolds, respectively. The expression levels of osteogenic-related and angiogenic-related proteins were also detected by western blot analysis. Their expression levels in cells were improved on the ADM delivery scaffolds at a certain time point. The in vitro evaluation suggests that the microsphere-scaffold system is suitable as a model for bone tissue engineering. PMID:23841075

  12. Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration

    PubMed Central

    White, Charles R; Haidekker, Mark A; Stevens, Hazel Y; Frangos, John A

    2004-01-01

    Hand–arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynaud's phenomenon. Chronic exposure to hand-held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low-volume fluid oscillation. Rapid low-volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal-regulated kinase (ERK1/2) phosphorylation and endothelin-1 (ET-1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET-1 release compared to steady flow, and endothelin-1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium. PMID:14724194

  13. Production of soluble Neprilysin by endothelial cells

    SciTech Connect

    Kuruppu, Sanjaya; Rajapakse, Niwanthi W.; Minond, Dmitriy; Smith, A. Ian

    2014-04-04

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

  14. Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

    PubMed Central

    Maier, J A; Statuto, M; Ragnotti, G

    1994-01-01

    We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function. Images PMID:8114717

  15. Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho.

    PubMed

    Gratzinger, Dita; Canosa, Sandra; Engelhardt, Britta; Madri, Joseph A

    2003-08-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoglobulin family vascular adhesion molecule, is involved in endothelial cell migration and angiogenesis (1, 2). We found that endothelial cells lacking PECAM-1 exhibit increased single cell motility and extension formation but poor wound healing migration, reminiscent of cells in which Rho activity has been suppressed by overexpressing a GTPase-activating protein (3). The ability of PECAM-1 to restore wound healing migration to PECAM-1-deficient cells was independent of its extracellular domain or signaling via its immunoreceptor tyrosine-based inhibitory motif. PECAM-1-deficient endothelial cells had a selective defect in RhoGTP loading, and inhibition of Rho activity mimicked the PECAM-1-deficient phenotype of increased chemokinetic single cell motility at the expense of coordinated wound healing migration. The wound healing advantage of PECAM-1-positive endothelial cells was not only Rho mediated but pertussis toxin inhibitable, characteristic of migration mediated by heterotrimeric G-protein-linked seven-transmembrane receptor signaling such as signaling in response to the serum sphingolipid sphingosine-1-phosphate (S1P) (4, 5). Indeed, we found that the wound healing defect of PECAM-1 null endothelial cells is minimized in sphingolipid-depleted media; moreover, PECAM-1 null endothelial cells fail to increase their migration in response to S1P. We have also found that PECAM-1 localizes to rafts and that in its absence heterotrimeric G-protein components are differentially recruited to rafts, providing a potential mechanism for PECAM-1-mediated coordination of S1P signaling. PECAM-1 may thus support the effective S1P/RhoGTP signaling required for wound healing endothelial migration by allowing for the spatially directed, coordinated activation of Galpha signaling pathways. PMID:12890700

  16. Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration.

    PubMed

    Rotrosen, D; Edwards, J E; Gibson, T R; Moore, J C; Cohen, A H; Green, I

    1985-12-01

    To elucidate the pathogenesis of hematogenous Candida infections, we developed an in vitro model of Candida adherence to and penetration of human endothelial cells. We enhanced or inhibited adherence in order to probe mechanisms of attachment. Adherence of Candida albicans showed a linear relation to Candida inoculum (range, 10(2)-10(5) cfu, r = .99, P less than .01) and exceeded that of less virulent Candida species and that of Saccharomyces cerevisiae (P less than .01). Candida immune serum blocked attachment (greater than 95% inhibition; P less than .001), however, this activity was abolished by immunoprecipitation of immune serum with C. albicans mannan (P less than .001) and was unaffected by immunoprecipitation with S. cerevisiae mannan or by adsorption with particulate chitin. Adherence was diminished by exposing C. albicans to heat (greater than 99% inhibition; P less than .01), UV light (98% inhibition; P less than .01), or sodium periodate (greater than 72% inhibition; P less than .01). An extract from heat-exposed C. albicans blocked adherence (greater than 51% inhibition; P less than .001). Transmission electron microscopy demonstrated that viable or killed Candida organisms were attached to endothelial cells, were enveloped by membrane processes from the endothelial cell surface, and were incorporated into the endothelial cells within phagosomes. Cytochalasin B blocked incorporation without blocking surface attachment. PMID:3905987

  17. African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

    PubMed Central

    Vallée, Isabelle; Tait, Stephen W. G.; Powell, Penelope P.

    2001-01-01

    African swine fever (ASF) is an asymptomatic infection of warthogs and bushpigs, which has become an emergent disease of domestic pigs, characterized by hemorrhage, lymphopenia, and disseminated intravascular coagulation. It is caused by a large icosohedral double-stranded DNA virus, African swine fever virus (ASFV), with infection of macrophages well characterized in vitro and in vivo. This study shows that virulent isolates of ASFV also infect primary cultures of porcine aortic endothelial cells and bushpig endothelial cells (BPECs) in vitro. Kinetics of early and late gene expression, viral factory formation, replication, and secretion were similar in endothelial cells and macrophages. However, ASFV-infected endothelial cells died by apoptosis, detected morphologically by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and nuclear condensation and biochemically by poly(ADP-ribose) polymerase (PARP) cleavage at 4 h postinfection (hpi). Immediate-early proinflammatory responses were inhibited, characterized by a lack of E-selectin surface expression and interleukin 6 (IL-6) and IL-8 mRNA synthesis. Moreover, ASFV actively downregulated interferon-induced major histocompatibility complex class I surface expression, a strategy by which viruses evade the immune system. Significantly, Western blot analysis showed that the 65-kDa subunit of the transcription factor NF-κB, a central regulator of the early response to viral infection, decreased by 8 hpi and disappeared by 18 hpi. Both disappearance of NF-κB p65 and cleavage of PARP were reversed by the caspase inhibitor z-VAD-fmk. Interestingly, surface expression and mRNA transcription of tissue factor, an important initiator of the coagulation cascade, increased 4 h after ASFV infection. These data suggest a central role for vascular endothelial cells in the hemorrhagic pathogenesis of the disease. Since BPECs infected with ASFV also undergo apoptosis, resistance of the natural host must involve

  18. Epidermal growth factor and perlecan fragments produced by apoptotic endothelial cells co-ordinately activate ERK1/2-dependent antiapoptotic pathways in mesenchymal stem cells.

    PubMed

    Soulez, Mathilde; Sirois, Isabelle; Brassard, Nathalie; Raymond, Marc-André; Nicodème, Frédéric; Noiseux, Nicolas; Durocher, Yves; Pshezhetsky, Alexei V; Hébert, Marie-Josée

    2010-04-01

    Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum-free medium conditioned by apoptotic EC increases extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl-xL protein levels and poly (ADP-ribose) polymerase cleavage) of human MSC. A C-terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with beta1-integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering beta1-integrin and EGF receptor-dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response. PMID:20201065

  19. Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells

    PubMed Central

    Das, Jharna R; Gutkind, J. Silvio; Ray, Patricio E

    2016-01-01

    Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms. PMID:27097314

  20. Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells.

    PubMed

    Das, Jharna R; Gutkind, J Silvio; Ray, Patricio E

    2016-01-01

    Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms. PMID:27097314

  1. Fibrinogen induces endothelial cell permeability

    PubMed Central

    Tyagi, Neetu; Roberts, Andrew M.; Dean, William L.; Tyagi, Suresh C.

    2010-01-01

    Many cardiovascular and cerebrovascular disorders are accompanied by an increased blood content of fibrinogen (Fg), a high molecular weight plasma adhesion protein. Fg is a biomarker of inflammation and its degradation products have been associated with microvascular leakage. We tested the hypothesis that at pathologically high levels, Fg increases endothelial cell (EC) permeability through extracellular signal regulated kinase (ERK) signaling and by inducing F-actin formation. In cultured ECs, Fg binding to intercellular adhesion molecule-1 and to α5β1 integrin, caused phosphorylation of ERK. Subsequently, F-actin formation increased and coincided with formation of gaps between ECs, which corresponded with increased permeability of ECs to albumin. Our data suggest that formation of F-actin and gaps may be the mechanism for increased albumin leakage through the EC monolayer. The present study indicates that elevated un-degraded Fg may be a factor causing microvascular permeability that typically accompanies cardiovascular and cerebrovascular disorders. PMID:17849175

  2. Resveratrol analog piceatannol restores the palmitic acid-induced impairment of insulin signaling and production of endothelial nitric oxide via activation of anti-inflammatory and antioxidative heme oxygenase-1 in human endothelial cells.

    PubMed

    Jeong, Sun-Oh; Son, Yong; Lee, Ju Hwan; Cheong, Yong-Kwan; Park, Seong Hoon; Chung, Hun-Taeg; Pae, Hyun-Ock

    2015-07-01

    Growing evidence suggests that the elevation of free fatty acids, including palmitic acid (PA), are associated with inflammation and oxidative stress, which may be involved in endothelial dysfunction, characterized by the reduced bioavailability of nitric oxide (NO) synthesized from endothelial NO synthase (eNOS). Heme oxygenase-1 (HO-1) is important in the preservation of NO bioavailability. Piceatannol (Pic), with similar chemical structure to resveratrol, is suggested to possess similar protective effects as resveratrol. In the present study, human umbilical vein endothelial cells (HUVECs), stimulated with PA, were used to examine the endothelial protective effects of Pic. Pic increased the expression of HO-1 via nuclear factor erythroid-2-related factor-2 activation in the HUVECs, and decreased the PA-induced secretions of interleukin-6 and tumor necrosis factor-α, and the formation of reactive oxygen species ROS via inhibition of NF-κB activation. Notably, following inhibition of HO-1 activity by tin protoporphryin-IX, Pic did not prevent cytokine secretion, ROS formation, and NF-κB activation in the PA-stimulated HUVECs. PA attenuated insulin-mediated insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, leading to decreased glucose uptake, and phosphorylation of eNOS, leading to a reduction in the production of NO. Pic effectively mitigated the inhibitory effects of PA on the insulin-mediated phosphorylation of IRS-1 and eNOS, which was not observed following inhibition of HO‑1 activity. The results of the present study suggested that Pic may have the potential to prevent PA-induced impairment of insulin signaling and eNOS function, by inducing the expression of the anti-inflammatory and antioxidant, HO-1. PMID:25815690

  3. Paradoxic effects of metformin on endothelial cells and angiogenesis.

    PubMed

    Dallaglio, Katiuscia; Bruno, Antonino; Cantelmo, Anna R; Esposito, Alessia I; Ruggiero, Luca; Orecchioni, Stefania; Calleri, Angelica; Bertolini, Francesco; Pfeffer, Ulrich; Noonan, Douglas M; Albini, Adriana

    2014-05-01

    The biguanide metformin is used in type 2 diabetes management and has gained significant attention as a potential cancer preventive agent. Angioprevention represents a mechanism of chemoprevention, yet conflicting data concerning the antiangiogenic action of metformin have emerged. Here, we clarify some of the contradictory effects of metformin on endothelial cells and angiogenesis, using in vitro and in vivo assays combined with transcriptomic and protein array approaches. Metformin inhibits formation of capillary-like networks by endothelial cells; this effect is partially dependent on the energy sensor adenosine-monophosphate-activated protein kinase (AMPK) as shown by small interfering RNA knockdown. Gene expression profiling of human umbilical vein endothelial cells revealed a paradoxical modulation of several angiogenesis-associated genes and proteins by metformin, with short-term induction of vascular endothelial growth factor (VEGF), cyclooxygenase 2 and CXC chemokine receptor 4 at the messenger RNA level and downregulation of ADAMTS1. Antibody array analysis shows an essentially opposite regulation of numerous angiogenesis-associated proteins in endothelial and breast cancer cells including interleukin-8, angiogenin and TIMP-1, as well as selective regulation of angiopioetin-1, -2, endoglin and others. Endothelial cell production of the cytochrome P450 member CYP1B1 is upregulated by tumor cell supernatants in an AMPK-dependent manner, metformin blocks this effect. Metformin inhibits VEGF-dependent activation of extracellular signal-regulated kinase 1/2, and the inhibition of AMPK activity abrogates this event. Metformin hinders angiogenesis in matrigel pellets in vivo, prevents the microvessel density increase observed in obese mice on a high-fat diet, downregulating the number of white adipose tissue endothelial precursor cells. Our data show that metformin has an antiangiogenic activity in vitro and in vivo associated with a contradictory short

  4. Transcriptional activation of endothelial cells by TGFβ coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury.

    PubMed

    Benton, Richard L; Maddie, Melissa A; Dincman, Toros A; Hagg, Theo; Whittemore, Scott R

    2009-01-01

    Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI. PMID:19663807

  5. Transcriptional activation of endothelial cells by TGFβ coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury

    PubMed Central

    Benton, Richard L; Maddie, Melissa A; Dincman, Toros A; Hagg, Theo; Whittemore, Scott R

    2009-01-01

    Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI. PMID:19663807

  6. Apicobasal polarity of brain endothelial cells.

    PubMed

    Worzfeld, Thomas; Schwaninger, Markus

    2016-02-01

    Normal brain homeostasis depends on the integrity of the blood-brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood-brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. PMID:26661193

  7. Increased endothelial cell permeability in endoglin-deficient cells.

    PubMed

    Jerkic, Mirjana; Letarte, Michelle

    2015-09-01

    Endoglin (ENG) is a TGF-β superfamily coreceptor essential for vascular endothelium integrity. ENG mutations lead to a vascular dysplasia associated with frequent hemorrhages in multiple organs, whereas ENG null mouse embryos die at midgestation with impaired heart development and leaky vasculature. ENG interacts with several proteins involved in cell adhesion, and we postulated that it regulates vascular permeability. The current study assessed the permeability of ENG homozygous null (Eng(-/-)), heterozygous (Eng(+/-)), and normal (Eng(+/+)) mouse embryonic endothelial cell (EC) lines. Permeability, measured by passage of fluorescent dextran through EC monolayers, was increased 2.9- and 1.7-fold for Eng(-/-) and Eng(+/-) ECs, respectively, compared to control ECs and was not increased by TGF-β1 or VEGF. Prolonged starvation increased Eng(-/-) EC permeability by 3.7-fold with no effect on control ECs; neutrophils transmigrated faster through Eng(-/-) than Eng(+/+) monolayers. Using a pull-down assay, we demonstrate that Ras homolog gene family (Rho) A is constitutively active in Eng(-/-) and Eng(+/-) ECs. We show that the endothelial barrier destabilizing factor thrombospondin-1 and its receptor-like protein tyrosine phosphatase are increased, whereas stabilizing factors VEGF receptor 2, vascular endothelial-cadherin, p21-activated kinase, and Ras-related C3 botulinum toxin substrate 2 are decreased in Eng(-/-) cells. Our findings indicate that ENG deficiency leads to EC hyperpermeability through constitutive activation of RhoA and destabilization of endothelial barrier function. PMID:25972355

  8. Activation of ERK pathway is required for 15-HETE-induced angiogenesis in human umbilical vascular endothelial cells.

    PubMed

    Wang, Shuang; Cao, Weiwei; Xing, Hao; Chen, Ying Li; Li, Qian; Shen, Tingting; Jiang, Chun; Zhu, Daling

    2016-06-01

    Angiogenesis plays a critical role in the progression of cardiovascular disease, retinal ischemia, or tumorigenesis. The imbalance of endothelial cell proliferation and apoptosis disturbs the establishment of the vasculogenesis, which is affected by several arachidonic acid metabolites. 15-Hydroxyeicosatetraenoic acid (15-HETE) is one of the metabolites. However, the underlying mechanisms of angiogenesis induced by 15-HETE in human umbilical vascular endothelial cells (HUVECs) are still poorly understood. Since extracellular signal-regulated kinase (ERK) is a critical regulator of cell proliferation, there may be a crosstalk between 15-HETE-regulating angiogenic process and ERK-proliferative effect in HUVECs. To test this hypothesis, we study the effect of 15-HETE on cell proliferation, angiogenesis, and apoptosis using cell viability measurement, cell cycle analysis, western blot, scratch-wound, tube formation assay, and nuclear morphology determination. We found that 15-HETE promoted HUVEC angiogenesis, which were mediated by ERK. Moreover, 15-HETE-induced proliferation and cell cycle transition from the G0/G1 phase to the G2/M + S phase. All these effects were reversed after blocking ERK with PD98059 (an ERK inhibitor). In addition, HUVEC apoptosis was relieved by 15-HETE through the ERK pathway. Thus, ERK is necessary for the effects of 15-HETE in the regulation of HUVEC angiogenesis, which may be a novel potential target for the treatment of angiogenesis-related diseases. PMID:26460784

  9. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    NASA Astrophysics Data System (ADS)

    Shen, Qin; Goderie, Susan K.; Jin, Li; Karanth, Nithin; Sun, Yu; Abramova, Natalia; Vincent, Peter; Pumiglia, Kevin; Temple, Sally

    2004-05-01

    Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

  10. Immunological functions of liver sinusoidal endothelial cells.

    PubMed

    Knolle, Percy A; Wohlleber, Dirk

    2016-05-01

    Liver sinusoidal endothelial cells (LSECs) line the liver sinusoids and separate passenger leukocytes in the sinusoidal lumen from hepatocytes. LSECs further act as a platform for adhesion of various liver-resident immune cell populations such as Kupffer cells, innate lymphoid cells or liver dendritic cells. In addition to having an extraordinary scavenger function, LSECs possess potent immune functions, serving as sentinel cells to detect microbial infection through pattern recognition receptor activation and as antigen (cross)-presenting cells. LSECs cross-prime naive CD8 T cells, causing their rapid differentiation into memory T cells that relocate to secondary lymphoid tissues and provide protection when they re-encounter the antigen during microbial infection. Cross-presentation of viral antigens by LSECs derived from infected hepatocytes triggers local activation of effector CD8 T cells and thereby assures hepatic immune surveillance. The immune function of LSECs complements conventional immune-activating mechanisms to accommodate optimal immune surveillance against infectious microorganisms while preserving the integrity of the liver as a metabolic organ. PMID:27041636

  11. Pregnancy-associated plasma protein-A promotes TF procoagulant activity in human endothelial cells by Akt-NF-κB axis.

    PubMed

    Cirillo, Plinio; Conte, Stefano; Pellegrino, Grazia; Ziviello, Francesca; Barra, Giusi; De Palma, Raffaele; Leonardi, Antonio; Trimarco, Bruno

    2016-08-01

    Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a controversial role in pathophysiology of cardiovascular disease. It seems involved in progression of atherosclerosis and is widely represented in atherosclerotic plaque. PAPP-A plasma levels are elevated in patients with acute coronary syndromes (ACS), thus it has been suggested that it might be a prognostic marker for developing major cardiovascular events. However, the pathophysiological link(s) between PAPP-A and ACS are still unknown. Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates whether PAPP-A, at concentrations measurable in ACS patients, might induce TF expression in human endothelial cells in culture (HUVEC). In HUVEC, PAPP-A induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by FACS analysis and pro-coagulant activity assay. PAPP-A induced TF expression through the activation of Akt/NF-κB axis, as demonstrated by luciferase assay and by suppression of TF-mRNA transcription as well as of TF expression/activity by Akt and NF-κB inhibitors. These data indicate that PAPP-A promotes TF expression in human endothelial cells and support the hypothesis that this proteinase, besides being involved in progression of atherosclerosis, does not represent an independent risk factor for adverse cardiovascular events, but it rather might play an "active" role in the pathophysiology of ACS as an effector molecule able to induce a pro-thrombotic phenotype in endothelial cells. PMID:27007282

  12. Coupling factor 6 downregulates platelet endothelial cell adhesion molecule-1 via c-Src activation and acts as a proatherogenic molecule.

    PubMed

    Kumagai, Akiko; Osanai, Tomohiro; Katoh, Chisato; Tanaka, Makoto; Tomita, Hirofumi; Morimoto, Takeshi; Murakami, Reiichi; Magota, Koji; Okumura, Ken

    2008-09-01

    Coupling factor 6 (CF6), a component of ATP synthase, suppresses the generation of prostacyclin and nitric oxide (NO). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is involved in shear-induced NO production. To investigate the linkage between the actions of CF6 and PECAM-1, we examined the effects of CF6 on PECAM-1 expression and shear-mediated NO release, comparatively with those of angiotensin II (AngII). Treatment of human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells (HAEC) with CF6 at 10(-7)M or AngII at 10(-7)M for 24h suppressed PECAM-1 gene and protein expression. CF6 or AngII activated c-Src at 15 min in HUVEC, and blockade of c-Src with PP1, its specific inhibitor, restored them. Efrapeptin, an inhibitor of ATPase, attenuated CF6-induced suppression of PECAM-1 gene expression by blockade of acidification, whereas superoxide dismutase or apocinin, an inhibitor of NADPH oxidase, blocked AngII-induced suppression of PECAM-1. Exposure of the cells to shear stress at 25 dynes/cm(2) for 30 min enhanced phosphorylation of eNOS at Ser(1177) and NO release. Pretreatment with CF6 or AngII for 24h attenuated them in HUVEC and HAEC. These suggest that CF6 downregulates PECAM-1 expression via c-Src activation and attenuates shear-induced NO release presumably by suppressing eNOS phosphorylation. PMID:18243211

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

    PubMed

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

    2015-04-01

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

  14. Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1

    PubMed Central

    Gjyshi, Olsi; Flaherty, Stephanie; Veettil, Mohanan Valiya; Johnson, Karen E.; Chandran, Bala

    2014-01-01

    ABSTRACT Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase Cζ (PKCζ) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (γGCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63–ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV

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

    SciTech Connect

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

    2013-03-29

    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

  16. Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment.

    PubMed

    Seng, Wen Lin; Eng, Kurt; Lee, Jenny; McGrath, Patricia

    2004-01-01

    We have recently generated a monoclonal antibody (mAb), Phy-V002, which specifically labels activated vascular endothelial cells (EC) in zebrafish. Here, we show that this mAb labels activated EC in newly formed vessels in vivo without staining mature vessels or other tissues. Using this mAb, drug effects on in vivo EC migration and vessel formation were visually assessed by whole-mount immunochemical staining in the transparent embryo. In addition, we have developed a quantitative microplate-based ELISA that measures EC proliferation in vivo after drug treatment. We have validated the quantitative in vivo ELISA using several antiangiogenic small molecules with different mechanisms of action which were added directly to the fish water. Some of these drugs, including: 2-methoxyestradiol, flavopiridol, paclitaxel, and genistein, are currently in clinical trials. We also injected large molecule drugs, including 3TSR and TSR2+KRFK, recombinant human antiangiogenic peptides of thrombospondin-1, a natural protein. To demonstrate that proangiogenic effects can also be assessed in zebrafish, we assessed effects of penicillamine and simvastatin, two proangiogenic compounds shown to stimulate vessel formation in rodents. Using whole-mount immunochemical staining with Phy-V002, inhibition of EC migration and inhibition or stimulation of vessel formation were visually observed for each compound. Next, using the quantitative in vivo angiogenesis ELISA, we generated dose-response curves for each compound. Compared to conventional assays, advantages of using zebrafish to assess drug effects on angiogenesis include: (1) a short assay time; (2) easy animal maintenance; (3) use of small quantities of drug; (4) single dosing; (5) a quantitative assay format; and (6) use of statistically significant number of animals per test. PMID:15609079

  17. Blood cells and endothelial barrier function.

    PubMed

    Rodrigues, Stephen F; Granger, D Neil

    2015-01-01

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

  18. Blood cells and endothelial barrier function

    PubMed Central

    Rodrigues, Stephen F; Granger, D Neil

    2015-01-01

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

  19. Chalcone inhibits the activation of NF-kappaB and STAT3 in endothelial cells via endogenous electrophile.

    PubMed

    Liu, Yen-Chin; Hsieh, Chia-Wen; Wu, Chun-Ching; Wung, Being-Sun

    2007-03-20

    Chalcone, an alpha,beta-unsaturated flavonoid, possesses anti-inflammatory properties. In our present study, we have demonstrated chalcone inhibited IL-6- and LPS-induced ICAM-1 gene expression. In adhesion assay, chalcone reduced the LPS-induced adhesion of THP-1 cells to endothelial cells (ECs). Chalcone was found to abrogate the activation of STAT3 and NF-kappaB in a dose- and time-dependent manner, in IL-6- and LPS-treated ECs. Other flavonoids, quercetin and cyanidin, which lack alpha,beta-unsaturated carbonyl group, showed weaker or no inhibitory effect on both IL-6-induced STAT3 phosphorylation and LPS-induced p65 translocation. However, the electrophilic compounds curcumin and crotonaldehyde, which also contain an alpha,beta-unsaturated carbonyl moiety, mimic the inhibitory effects of chalcone with different efficiencies. In addition, N-acetyl-L-cysteine (NAC) could reverse the inhibition of STAT3 phosphorylation when preincubated with chalcone. The use of buthionine sulfoximine (BSO) to decrease intracellular GSH levels further enhanced the effects of chalcone. On the other hand, in ECs treated with BSO only no abrogation of IL-6-induced STAT3 phosphorylation was observed. We also found that chalcone could reduce the GSH level in vitro. Furthermore, the cellular GSH levels were rapidly reduced after 25 microM chalcone treatment. Following 6 h exposure, however, chalcone treatment rescued the GSH levels in ECs, coincident with the inhibition of STAT3 and NF-kappaB activation. In contrast, chalcone induced expression of thioredoxin reductase and heme-oxygenase genes after prolonged treatment. Furthermore, chalcone upregulated the levels of the transcription factor Nrf2 in nuclear extracts and increased antioxidant response element (ARE)-luciferase activity and thioredoxin reductase promoter activity. Hence, our present findings indicate that chalcone suppresses both IL-6- and LPS-induced signaling pathways through the thiol-dependent intracellular redox

  20. Telmisartan inhibits advanced glycation end products (AGEs)-elicited endothelial cell injury by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gammaactivation.

    PubMed

    Yamagishi, Sho-ichi; Matsui, Takanori; Nakamura, Kazuo; Takeuchi, Masayoshi; Inoue, Hiroyoshi

    2008-01-01

    Advanced glycation end products (AGEs)-their receptor (RAGE) axis plays a central role in the pathogenesis of diabetic microangiopathy. Since the pathophysiological crosstalk between the AGEs-RAGE system and angiotensin II has also been associated with diabetic microangiopathy, we examined here whether and how telmisartan, a unique angiotensin II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity, could inhibit the AGEs-elicited endothelial cell injury by suppressing RAGE expression in vitro. Telmisartan suppressed RAGE expression at both mRNA and protein levels in human cultured microvascular endothelial cells (ECs), which were prevented by GW9662, an inhibitor of PPAR-gamma. Further, telmisartan was found to inhibit up-regulation of mRNA levels for monocyte chemoattractant protein-1, intercellular adhesion molecule-1 and vascular endothelial growth factor in AGEs-exposed ECs. These results suggest that telmisartan inhibits the AGEs-elicited EC injury by down-regulating RAGE expression via PPAR-gamma activation. Our present study provides a unique beneficial aspect of telmisartan. Specifically, it could work as an anti-inflammatory agent against AGEs via PPAR-gamma activation and may play a protective role against diabetic microangiopathy. PMID:18855759

  1. Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte–endothelial cell coculture model

    PubMed Central

    Liu, Xin; Sun, Jiao

    2014-01-01

    Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte–endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease. PMID:24648726

  2. Endothelial Cell Stimulation by Candida albicans

    PubMed Central

    Phan, Quynh T.; Filler, Scott G.

    2013-01-01

    The opportunistic fungal pathogen, Candida albicans, enters the bloodstream and causes hematogenously disseminated infection in hospitalized patients. During the initiation of a hematogenously disseminated infection, endothelial cells are one of the first host cells to come in contact with C. albicans. Endothelial cells can significantly influence the local host response to C. albicans by expressing leukocyte adhesion molecules and pro-inflammatory cytokines. Thus, it is of interest to investigate the response of endothelial cells to C. albicans in vitro. We describe the use of real-time PCR and enzyme immunoassays to measure the effects of C. albicans on the endothelial cell production of E-selectin and tumor necrosis factor α in vitro. PMID:19089392

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

    EPA Science Inventory

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

  4. Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin–vascular endothelial growth factor pathway

    SciTech Connect

    Wang, Zhishan; Humphries, Brock; Xiao, Hua; Jiang, Yiguo; Yang, Chengfeng

    2013-08-15

    Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchial epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro

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

    PubMed

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

    2011-08-01

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

  6. Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

    NASA Technical Reports Server (NTRS)

    Ramachandran, Anup; Moellering, Douglas; Go, Young-Mi; Shiva, Sruti; Levonen, Anna-Liisa; Jo, Hanjoong; Patel, Rakesh P.; Parthasarathy, Sampath; Darley-Usmar, Victor M.

    2002-01-01

    Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

  7. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

    PubMed

    Berk, B C; Corson, M A; Peterson, T E; Tseng, H

    1995-12-01

    Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow. PMID:8666584

  8. Borrelia burgdorferi outer membrane protein A induces nuclear translocation of nuclear factor-kappa B and inflammatory activation in human endothelial cells.

    PubMed

    Wooten, R M; Modur, V R; McIntyre, T M; Weis, J J

    1996-11-15

    Lyme disease is caused by infection with Borrelia burgdorferi, and is characterized by bacterial persistence and inflammation in a number of host tissues. B. burgdorferi outer surface lipoproteins possess cytokine stimulatory properties that may be responsible for localized inflammation. B. burgdorferi presence is correlated with severity of disease, and the pathology of many tissues, particularly the arthritic joint, is consistent with localized cytokine production. Spirochete invasion of tissues requires interaction with and penetration of vascular endothelium, suggesting endothelial cells may participate in the inflammation of Lyme disease. In this study, outer surface protein A (OspA), a model B. burgdorferi lipoprotein, was found to be a potent stimulant of nuclear factor-kappa B (NF-kappa B) nuclear translocation in human endothelial cells, resulting in nuclear levels similar to those seen in response to known inflammatory mediators. Only the lipid-modified OspA had activity, and activity was not due to contamination with LPS. Nuclear NF-kappa B was detectable within 15 min, suggesting that OspA directly mediates NF-kappa B nuclear translocation. OspA also rapidly up-regulated endothelial cell production of several proteins whose transcription is dependent on NF-kappa B: the cytokine IL-6; the chemokine IL-8; and the adhesion molecules E-selectin, VCAM-1, and ICAM-1. The adhesion molecules were functional, as demonstrated by enhanced binding of neutrophils to OspA-stimulated endothelial monolayers. These data suggest that OspA may initiate synthesis of many proteins essential for localized inflammation via the direct activation of NF-kappa B-dependent transcription. These observations suggest that the interaction of B. burgdorferi lipoproteins with the endothelium may directly induce the inflammation responsible for the symptoms of Lyme disease. PMID:8906837

  9. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    SciTech Connect

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-12-01

    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.

  10. Gold nanoparticles induce heme oxygenase-1 expression through Nrf2 activation and Bach1 export in human vascular endothelial cells

    PubMed Central

    Lai, Tsung-Hsuan; Shieh, Jiunn-Min; Tsou, Chih-Jen; Wu, Wen-Bin

    2015-01-01

    It has been reported that increased levels and activity of the heme oxygenase-1 (HO-1) protein ameliorate tissue injuries. In the present study, we investigated the effects and mechanisms of action of gold nanoparticles (AuNPs) on HO-1 protein expression in human vascular endothelial cells (ECs). The AuNPs induced HO-1 protein and mRNA expression in a concentration- and time-dependent manner. The induction was reduced by the thiol-containing antioxidants, including N-acetylcysteine and glutathione, but not by the non-thiol-containing antioxidants and inhibitors that block the enzymes for intracellular reactive oxygen species generation. The AuNPs enhanced Nrf2 protein levels but did not affect Nrf2 mRNA expression. In response to the AuNP treatment, the cytosolic Nrf2 translocated to the nucleus, and, concomitantly, Bach1 exited the nucleus and its tyrosine phosphorylation increased. The chromatin immunoprecipitation assay revealed that the translocated Nrf2 bound to the antioxidant-response element located in the E2 enhancer region of the HO-1 gene promoter and acted as a transcription factor. Although N-acetylcysteine inhibited the AuNP-induced Nrf2 nuclear translocation, the AuNPs did not promote intracellular reactive oxygen species production or endoplasmic reticulum stress in the ECs. Knockdown of Nrf2 expression by RNA interference significantly inhibited AuNP-induced HO-1 expression at the protein and mRNA levels. In summary, AuNPs enhance the levels and nuclear translocation of the Nrf2 protein and Bach1 export/tyrosine phosphorylation, leading to Nrf2 binding to the HO-1 E2 enhancer promoter region to drive HO-1 expression in ECs. This study, together with our parallel findings, demonstrates that AuNPs can act as an HO-1 inducer, which may partially contribute to their anti-inflammatory bioactivity in human vascular ECs. PMID:26445536

  11. Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

    PubMed Central

    Xie, Xueping; Zhao, Ruozhi; Shen, Garry X.

    2012-01-01

    Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC. PMID:23443099

  12. Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

    PubMed Central

    Pili, R; Corda, S; Passaniti, A; Ziegelstein, R C; Heldman, A W; Capogrossi, M C

    1993-01-01

    The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion. Images PMID:8254056

  13. Endothelial cell transfusion ameliorates endothelial dysfunction in 5/6 nephrectomized rats

    PubMed Central

    Pacurari, Maricica; Xing, Dongqi; Hilgers, Rob H. P.; Guo, Yuan Yuan; Yang, Zhengqin

    2013-01-01

    Endothelial dysfunction is prevalent in chronic kidney disease. This study tested the hypothesis that transfusion of rat aortic endothelial cells (ECs) ameliorates endothelial dysfunction in a rat model of chronic kidney disease. Male Sprague-Dawley rats underwent sham surgery or 5/6 nephrectomy (Nx). Five weeks after Nx, EC (1.5 × 106 cells/rat) or vehicle were transfused intravenously. One week later, vascular reactivity of mesenteric artery was assessed on a wire myograph. Sensitivity of endothelium-dependent relaxation to acetylcholine and maximum vasodilation were impaired by Nx and improved by EC transfusion. Using selective pharmacological nitric oxide synthase isoform inhibitors, we demonstrated that the negative effect of Nx on endothelial function and rescue by EC transfusion are, at least in part, endothelial nitric oxide synthase mediated. Plasma asymmetric dimethylarginine was increased by Nx and decreased by EC transfusion, whereas mRNA expression of dimethylarginine dimethylaminohydrolases 1 (DDAH1) was decreased by Nx and restored by EC transfusion. Immunohistochemical staining confirmed that local expression of DDAH1 is decreased by Nx and increased by EC transfusion. In conclusion, EC transfusion attenuates Nx-induced endothelium-dependent vascular dysfunction by regulating DDAH1 expression and enhancing endothelial nitric oxide synthase activity. These results suggest that EC-based therapy could provide a novel therapeutic strategy to improve vascular function in chronic kidney disease. PMID:23955716

  14. Evaluation of Endothelial Cell Adhesion Molecules and Anti-C1q Antibody in Discriminating between Active and Non-Active Systemic Lupus Erythematosus

    PubMed Central

    Mahayidin, Hasni; Yahya, Nurul Khaiza; Wan Ghazali, Wan Syamimee; Mohd Ismail, Asmahan; Wan Ab Hamid, Wan Zuraida

    2016-01-01

    Background Detecting the active state of systemic lupus erythematosus (SLE) is important but challenging. This study aimed to determine the diagnostic accuracy of serum endothelial cell adhesion molecules (ICAM-1 and VCAM-1) and anti-C1q antibody in discriminating between active and non-active SLE. Methods Using SELENA-SLE disease activity index (SLEDAI), 95 SLE patients (45 active and 50 non-active) were assessed. A score above five was considered indicative of active SLE. The blood samples were tested for serum ICAM-1, VCAM-1 and anti-C1q antibody using enzyme-linked immunosorbent assay (ELISA). Results The levels of serum VCAM-1 and anti-C1q antibody were significantly higher in active SLE patients. Both VCAM-1 and anti-C1q were able to discriminate between active and non-active SLE (p-value < 0.001 and 0.005, respectively). From the receiver operating characteristic curves (ROCs) constructed, the optimal cut-off values for VCAM-1 and anti-C1q antibody in discriminating between active and non-active SLE were 30.5 ng/mL (69.0% sensitivity, 60.0% specificity, PPV 58.5%, NPV 66.7%) and 7.86 U/mL (75.6% sensitivity, 80% specificity, PPV 77.3%, NPV 78.4%), respectively. However, serum ICAM-1 level was unable to discriminate between the two groups (p-value = 0.193). Conclusion Anti-C1q antibody demonstrated the best diagnostic accuracy in discriminating between active and non-active SLE patients. PMID:27418866

  15. Apoptotic Cells Initiate Endothelial Cell Sprouting via Electrostatic Signaling

    PubMed Central

    Weihua, Zhang; Tsan, Rachel; Schroit, Alan J.; Fidler, Isaiah J.

    2006-01-01

    Angiogenesis, the development of new blood vessels from preexisting vessels, is crucial to tissue growth, repair, and maintenance. This process begins with the formation of endothelial cell sprouts followed by the proliferation and migration of neighboring endothelial cells along the pre-formed extensions. The initiating event and mechanism of sprouting is not known. We demonstrate that the phenotypic expression of negative-charged membrane surface in apoptotic cells initiates the formation of directional endothelial cell sprouts that extend toward the dying cells by a mechanism that involves endothelial cell membrane hyperpolarization and cytoskeleton reorganization but is independent of diffusible molecules. PMID:16357162

  16. Fibroblast nemosis induces angiogenic responses of endothelial cells

    SciTech Connect

    Enzerink, Anna; Rantanen, Ville; Vaheri, Antti

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  17. Cellular and molecular biology of aging endothelial cells.

    PubMed

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  18. Endothelial Cells Enhance Tumor Cell Invasion through a Crosstalk Mediated by CXC Chemokine Signaling1

    PubMed Central

    Warner, Kristy A; Miyazawa, Marta; Cordeiro, Mabel M R; Love, William J; Pinsky, Matthew S; Neiva, Kathleen G; Spalding, Aaron C; Nör, Jacques E

    2008-01-01

    Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells. PMID:18283335

  19. Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo: protective role of IL-10.

    PubMed

    Eissner, G; Lindner, H; Behrends, U; Kölch, W; Hieke, A; Klauke, I; Bornkamm, G W; Holler, E

    1996-09-27

    Previous work from our group has contributed to demonstrate the role of conditioning related release of proinflammatory cytokines in induction of acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). In the present report we show that ionizing radiation (IR) in a clinical relevant dose upregulates intercellular adhesion molecule 1 (ICAM-1) on cultured human microvascular endothelial cells (HMEC). Bacterial endotoxin (lipopolysaccharide, LPS) in a concentration corresponding to serum levels seen during clinical endotoxemia, is capable of further enhancing ICAM-1 expression on irradiated cells. Adhesion assays with freshly isolated peripheral blood mononuclear cells (PBMC) revealed that increased ICAM-1 on IR-treated endothelial cells led to an increased adhesion of PBMC. Again, this effect could be superinduced by LPS. Recombinant human interleukin 10 (IL-10), an antagonistic cytokine known to function as an LPS antagonist, was able to counteract the LPS-mediated enhancement of IR-triggered ICAM-1 induction and PBMC adhesion. In contrast, IL-10 could not inhibit irradiation caused effects. IL-10 seemed to interfere with the translocation of preformed intracellular ICAM-1 to the cell membrane. To investigate whether this superinductive function of IR and LPS on endothelial cells is of clinical relevance, mice were treated with total body irradiation (TBI) and inoculated with a single dose of LPS. Immunohistochemical analyses of murine tissues demonstrated that LPS superinduces IR-triggered ICAM-1 also in vivo. These findings may be of clinical importance as they suggest that the endothelium is activated after radiotherapy or TBI used for conditioning in bone marrow transplantation. The activated endothelium in turn may facilitate the accumulation of effector cells at sites of inflammation. PMID:8824483

  20. Carbamoylating Activity Associated with the Activation of the Antitumor Agent Laromustine Inhibits Angiogenesis by Inducing ASK1-Dependent Endothelial Cell Death

    PubMed Central

    Praggastis, Alexandra; Li, Yonghao; Zhou, Huanjiao Jenny; He, Yun; Ghazvinian, Roxanne; Cincotta, Dylan J.; Rice, Kevin P.; Min, Wang

    2014-01-01

    The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis. PMID:25068797

  1. Isolation and culture of pulmonary endothelial cells.

    PubMed

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan. PMID:6090112

  2. Ulinastatin attenuates pulmonary endothelial glycocalyx damage and inhibits endothelial heparanase activity in LPS-induced ARDS.

    PubMed

    Wang, Lipeng; Huang, Xiao; Kong, Guiqing; Xu, Haixiao; Li, Jiankui; Hao, Dong; Wang, Tao; Han, Shasha; Han, Chunlei; Sun, Yeying; Liu, Xiangyong; Wang, Xiaozhi

    2016-09-16

    Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure characterized by major pathologic mechanisms of increased microvascular permeability and inflammation. The glycocalyx lines on the endothelial surface, which determines the vascular permeability, and heparanase play pivotal roles in the degradation of heparan sulfate (HS). HS is the major component of the glycocalyx. The aim of this study is to examine the effects of Ulinastatin (UTI) on vascular permeability and pulmonary endothelial glycocalyx dysfunction induced by lipopolysaccharide (LPS). In our study, C57BL/6 mice and human umbilical vein endothelial cells were stimulated with LPS to induce injury models. After 6 h of LPS stimulation, pulmonary pathological changes, pulmonary edema, and vascular permeability were notably attenuated by UTI. UTI inhibited LPS-induced endothelial glycocalyx destruction and significantly decreased the production of HS as determined by ELISA and immunofluorescence. UTI also reduced the active form of heparanase (50 kDa) expression and heparanase activity. Moreover, lysosome pH was investigated because heparanase (65 kDa) can be reduced easily in its active form at 50 kDa in a low pH environment within lysosome. Results showed that UTI could inhibit LPS-induced pH elevation in lysosome. In conclusion, UTI protects pulmonary endothelial glycocalyx integrity and inhibits heparanase activity during LPS-induced ARDS. PMID:27498004

  3. Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells.

    PubMed

    Thangjam, Gagan S; Birmpas, Charalampos; Barabutis, Nektarios; Gregory, Betsy W; Clemens, Mary Ann; Newton, Joseph R; Fulton, David; Catravas, John D

    2016-05-15

    The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation. PMID:27036868

  4. Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

    PubMed

    Cheng, Yi-Fang; Young, Guang-Huar; Lin, Jiun-Tsai; Jang, Hyun-Hwa; Chen, Chin-Chen; Nong, Jing-Yi; Chen, Po-Ku; Kuo, Cheng-Yi; Kao, Shao-Hsuan; Liang, Yao-Jen; Chen, Han-Min

    2015-01-01

    The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK. PMID:26544976

  5. Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

    SciTech Connect

    Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.; Kaehler, Christian M. . E-mail: C.M.Kaehler@uibk.ac.at

    2006-09-10

    Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.

  6. Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells.

    PubMed

    Sakata, Kimimasa; Kondo, Takashi; Mizuno, Natsumi; Shoji, Miki; Yasui, Hironobu; Yamamori, Tohru; Inanami, Osamu; Yokoo, Hiroki; Yoshimura, Naoki; Hattori, Yuichi

    2015-07-01

    Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤ 20 Gy) and time-dependent (6-72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-L-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other. PMID:25869503

  7. Pyripyropenes, fungal sesquiterpenes conjugated with alpha-pyrone and pyridine moieties, exhibits anti-angiogenic activity against human umbilical vein endothelial cells.

    PubMed

    Hayashi, Asami; Arai, Masayoshi; Fujita, Mayumi; Kobayashi, Motomasa

    2009-07-01

    In the course of our search for anti-angiogenic substances, pyripyropenes A (1), B (2), and D (3) were re-discovered as selective anti-proliferative substances against human umbilical vein endothelial cells (HUVECs) from a marine-derived fungus of Aspergillus sp. Pyripyropenes showed potent anti-proliferative activity against HUVECs with IC(50) values of the range of 0.1-1.8 muM, which were cytostatic at 0.05 to 20 muM. The selective index was more than 55-fold in comparison with those of several tumor cell lines. Compound 1 inhibited vascular endothelial growth factor (VEGF)-induced migration and tubular formation of HUVECs, while 1 showed no effect on the VEGF-induced phosphorylations of extracellular signal-regulated kinase (ERK)1/2, p38, and Akt. Pyripyropenes were originally isolated as an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT-2). While, the expression level of ACATs between HUVECs and other tumor cell lines did not correspond to the selective index of the anti-proliferative activity of compound 1. Moreover, ACATs inhibitor, 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), showed growth inhibitory activity with only poor selectivity (2.4-fold) between HUVECs and human epidermoid carcinoma KB3-1 cells. PMID:19571395

  8. Infection of human monocyte-derived dendritic cells by ANDES Hantavirus enhances pro-inflammatory state, the secretion of active MMP-9 and indirectly enhances endothelial permeability

    PubMed Central

    2011-01-01

    Background Andes virus (ANDV), a rodent-borne Hantavirus, is the major etiological agent of Hantavirus cardiopulmonary syndrome (HCPS) in South America, which is mainly characterized by a vascular leakage with high rate of fatal outcomes for infected patients. Currently, neither specific therapy nor vaccines are available against this pathogen. ANDV infects both dendritic and epithelial cells, but in despite that the severity of the disease directly correlates with the viral RNA load, considerable evidence suggests that immune mechanisms rather than direct viral cytopathology are responsible for plasma leakage in HCPS. Here, we assessed the possible effect of soluble factors, induced in viral-activated DCs, on endothelial permeability. Activated immune cells, including DC, secrete gelatinolytic matrix metalloproteases (gMMP-2 and -9) that modulate the vascular permeability for their trafficking. Methods A clinical ANDES isolate was used to infect DC derived from primary PBMC. Maturation and pro-inflammatory phenotypes of ANDES-infected DC were assessed by studying the expression of receptors, cytokines and active gMMP-9, as well as some of their functional status. The ANDES-infected DC supernatants were assessed for their capacity to enhance a monolayer endothelial permeability using primary human vascular endothelial cells (HUVEC). Results Here, we show that in vitro primary DCs infected by a clinical isolate of ANDV shed virus RNA and proteins, suggesting a competent viral replication in these cells. Moreover, this infection induces an enhanced expression of soluble pro-inflammatory factors, including TNF-α and the active gMMP-9, as well as a decreased expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. These viral activated cells are less sensitive to apoptosis. Moreover, supernatants from ANDV-infected DCs were able to indirectly enhance the permeability of a monolayer of primary HUVEC. Conclusions Primary human DCs, that are primarily

  9. Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

    PubMed Central

    Félétou, Michel

    2009-01-01

    The three subtypes of calcium-activated potassium channels (KCa) of large, intermediate and small conductance (BKCa, IKCa and SKCa) are present in the vascular wall. In healthy arteries, BKCa channels are preferentially expressed in vascular smooth muscle cells, while IKCa and SKCa are preferentially located in endothelial cells. The activation of endothelial IKCa and SKCa contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na+/K+-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H2O2) hyperpolarize and relax the underlying smooth muscle cells by activating BKCa. In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BKCa. Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle KCa could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IKCa may prevent restenosis and that of BKCa channels sepsis-dependent hypotension. PMID:19187341

  10. Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration

    PubMed Central

    Kurtagic, Elma; Rich, Celeste B.; Buczek-Thomas, Jo Ann; Nugent, Matthew A.

    2015-01-01

    Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment. PMID:26672607

  11. Characterization of the threshold for NAD(P)H:quinone oxidoreductase activity in intact sulforaphane-treated pulmonary arterial endothelial cells.

    PubMed

    Bongard, Robert D; Krenz, Gary S; Gastonguay, Adam J; Williams, Carol L; Lindemer, Brian J; Merker, Marilyn P

    2011-04-15

    Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5μM, 24h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP(+) in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP(+) ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the K(m) for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold. PMID:21238579

  12. Protein kinase C activation is involved in ultraviolet B irradiation-induced endothelial cell ICAM-1 up-regulation and lymphocyte-endothelium interaction in vitro.

    PubMed

    Funk, J O; Holler, E; Kohlhuber, F; Ueffing, M; Bornkamm, G W; Kind, P; Eissner, G

    1996-10-01

    Lymphocyte-endothelium interactions are pivotal steps in mediating inflammatory responses. The authors have analysed the influence of ultraviolet B (UVB) irradiation on intercellular adhesion molecule (ICAM)-1 expression on cells of the human microvascular endothelial cell line (HMEC)-1 and the intracellular signalling pathways involved. Flow cytometry revealed dose-dependent ICAM-1 up-regulation with maximum induced expression 24h after sublethal UVB irradiation of 10 mJ/cm2. While anti-tumour necrosis factor (TNF)-alpha antibodies or recombinant human interleukin (IL)-10 did not influence this response, anti-interferon (IFN)-gamma antibodies blocked the UVB-induced ICAM-1 up-regulation. Significant induction of intracellular/membrane-bound IFN-gamma was measured as early as 6 h post-UVB. Since previous work has shown a differential role of protein kinase C (PKC) in cytokine induced ICAM-1 expression, the effect of a selective bisindolylmaleimide-derived PKC-inhibitor (GF109203X) was studied. Ultraviolet B-induced ICAM-1 up-regulation was effectively blocked by the PKC-inhibitor, whereas a PKA-inhibitor was ineffective. Moreover, immunofluorescence analysis showed a radiation-induced membrane translocation of PKC-alpha, indicative of enzyme activation, in HMEC-1 cells already 30 min post-UVB. The functional relevance of the UVB-induced ICAM-1 expression and involvement of PKC in this process was demonstrated in an adhesion assay with peripheral blood mononuclear cells. In conclusion, UVB-induced ICAM-1 expression on human endothelial cells involves PKC-dependent pathways and can be prevented by a PKC-inhibitor. The use of PKC-inhibitors as additive modulators in immune reactions may bear clinical potential. The mechanisms of IFN-gamma induction in endothelial cells by UVB deserve further investigation. PMID:8845028

  13. Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of Astragalus membranaceus using Bioassay-guided Fractionation

    PubMed Central

    Lai, Patrick Kwok-Kin; Chan, Judy Yuet-Wa; Kwok, Hin-Fai; Cheng, Ling; Yu, Hua; Lau, Ching-Po; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

    2014-01-01

    The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 3H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P < 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), 3H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro. PMID:25379465