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Sample records for adult human endothelial

  1. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  2. Use of tritiated thymidine as a marker to compare the effects of matrix proteins on adult human vascular endothelial cell attachment: implications for seeding of vascular prostheses

    Energy Technology Data Exchange (ETDEWEB)

    Hasson, J.E.; Wiebe, D.H.; Sharefkin, J.B.; D' Amore, P.A.; Abbott, W.M.

    1986-11-01

    We have developed a technique to measure attachment of adult human vascular endothelial cells to test surfaces with tritiated thymidine used as a marker. With this technique, we measured attachment of adult human vascular endothelial cells to a series of extracellular matrix proteins, including fibronectin-coated (10 micrograms/cm/sup 2/), laminin-coated (10 micrograms/cm/sup 2/), and collagen-coated (1% gelatin) surfaces because of the role of these proteins in promoting cell attachment and growth. For a typical experiment, in the presence of serum, initial attachment (at 1 hour) was greatest on fibronectin-coated (63%) and gelatin-coated (60%) tissue culture plastic (polystyrene) and was least on laminin-coated (28%) or untreated polystyrene (18%). The data suggest that fibronectin, either alone, or with a more complex combination of extracellular components may need to be present on prosthetic surfaces to produce maximal cell attachment and subsequent growth to confluence in vivo. The described method of measuring attachment is independent of surface properties, ensures complete recovery of cells, and will allow systematic exploration of those properties that best support human endothelial cell attachment to vascular prosthetic surfaces.

  3. Comparison of Fibronectin and Collagen in Supporting the Isolation and Expansion of Endothelial Progenitor Cells from Human Adult Peripheral Blood.

    Directory of Open Access Journals (Sweden)

    Elena Colombo

    Full Text Available Endothelial colony-forming cells (ECFCs, are circulating endothelial progenitor cells increasingly studied in various diseases because of their potential for clinical translation. Experimental procedures for their ex vivo culture still lack standardization. In particular two different extracellular matrix proteins, either fibronectin or collagen, are commonly used by different Authors for coating plastic plates, both allowing to obtain cells that have all the features of ECFCs. However, possible differences in the impact of each substrate on ECFCs have not been analysed, so far. Therefore, in this study we investigated whether fibronectin and collagen may differentially affect ECFC cultures.ECFCs were isolated and cultured from peripheral blood mononuclear cells of healthy donors. The impact of fibronectin compared with collagen as the only variable of the experimental procedure was analysed separately in the phase of isolation of ECFC colonies and in the following phase of cell expansion. In the isolation phase, although similar frequencies of colonies were obtained on the two substrates, ECFC colonies appeared some days earlier when mononuclear cells were seeded on fibronectin rather than collagen. In the expansion phase, ECFCs cultured on collagen showed a longer lifespan and higher cell yields compared with ECFCs cultured on fibronectin, possibly related to the higher levels of IL-6 and IL-8 measured in their supernatants. ECFCs cultured on both substrates showed similar immunophenotype and ability for in vitro tube formation.Overall, the results of this study indicate that, although both fibronectin and collagen efficiently sustain ECFC cultures, each of them brings some advantages within individual steps of the entire process. We suggest that colony isolation performed on fibronectin followed by cell expansion performed on collagen may represent a novel and the most efficient strategy to obtain ECFCs from adult peripheral blood samples.

  4. Endothelial potential of human embryonic stem cells

    OpenAIRE

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

    2007-01-01

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

  5. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Samaras, Susan E. [Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN (United States); Chen, Billy [Molecular Medicine Program, Department of Medicine, Boston University School of Medicine, Boston, MA (United States); Koch, Stephen R. [Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN (United States); Sawyer, Douglas B.; Lim, Chee Chew [Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN (United States); Davidson, Jeffrey M., E-mail: jeff.davidson@vanderbilt.edu [Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN (United States); Research Service, Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. Black-Right-Pointing-Pointer Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. Black-Right-Pointing-Pointer Differential degradation appears related to nuclear vs. sarcolemmal localization. Black-Right-Pointing-Pointer Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

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

    Directory of Open Access Journals (Sweden)

    Maria Magdalena eMontt-Guevara

    2015-07-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    OpenAIRE

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

    2015-01-01

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

  9. Radioprotection of human endothelial cells with amifostine

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-11-01

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

  10. Higher Levels of Adiponectin in Vascular Endothelial Cells are Associated with Greater Brachial Artery Flow-mediated Dilation in Older Adults

    OpenAIRE

    Yoo, Jeung-Ki; Hwang, Moon-Hyon; Luttrell, Meredith J.; Kim, Han-Kyul; Meade, Thomas H.; English, Mark; Segal, Mark S.; Christou, Demetra D.

    2015-01-01

    Adiponectin, an adipocyte-derived protein, exerts anti-atherosclerotic effects on the vascular endothelium. Recently adiponectin protein has been reported in murine vascular endothelial cells, however, whether adiponectin is present in human vascular endothelial cells remains unexplored. We sought to examine 1) adiponectin protein in vascular endothelial cells collected from older adults free of overt cardiovascular disease; 2) the relation between endothelial cell adiponectin and in vivo vas...

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

    Science.gov (United States)

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

    2015-10-01

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

  12. Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

    OpenAIRE

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

    2011-01-01

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

  13. Fetoplacental Vascular Endothelial Dysfunction as an Early Phenomenon in the Programming of Human Adult Diseases in Subjects Born from Gestational Diabetes Mellitus or Obesity in Pregnancy

    Directory of Open Access Journals (Sweden)

    Andrea Leiva

    2011-01-01

    Full Text Available Gestational diabetes mellitus (GDM and obesity in pregnancy (OP are pathological conditions associated with placenta vascular dysfunction coursing with metabolic changes at the fetoplacental microvascular and macrovascular endothelium. These alterations are seen as abnormal expression and activity of the cationic amino acid transporters and endothelial nitric oxide synthase isoform, that is, the “endothelial L-arginine/nitric oxide signalling pathway.” Several studies suggest that the endogenous nucleoside adenosine along with insulin, and potentially arginases, are factors involved in GDM-, but much less information regards their role in OP-associated placental vascular alterations. There is convincing evidence that GDM and OP prone placental endothelium to an “altered metabolic state” leading to fetal programming evidenced at birth, a phenomenon associated with future development of chronic diseases. In this paper it is suggested that this pathological state could be considered as a metabolic marker that could predict occurrence of diseases in adulthood, such as cardiovascular disease, obesity, diabetes mellitus (including gestational diabetes, and metabolic syndrome.

  14. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

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

    2002-04-01

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

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

    OpenAIRE

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

    1993-01-01

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

  16. Paclitaxel Induces Thrombomodulin Downregulation in Human Aortic Endothelial Cells

    OpenAIRE

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

    2011-01-01

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

  17. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

    OpenAIRE

    Patsch, Christoph; Challet-Meylan, Ludivine; Eva C Thoma; Urich, Eduard; Heckel, Tobias; O’Sullivan, John F.; Grainger, Stephanie J.; Kapp, Friedrich G.; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H. C.; He, Wei; Pan, Wei; Prummer, Michael

    2015-01-01

    The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of ei...

  18. Relation of C-Reactive Protein to Endothelial Fibrinolytic Function in Healthy Adults

    OpenAIRE

    Weil, Brian R.; Greiner, Jared J.; Stauffer, Brian L.; DeSouza, Christopher A.

    2011-01-01

    Elevated plasma concentrations of C-reactive protein (CRP) independently predict future atherothrombotic events in healthy, asymptomatic adults. CRP may promote atherothrombosis by altering fibrinolytic balance; however, the influence of elevated plasma CRP concentrations on endothelial fibrinolysis in healthy adults is unclear. We tested the hypothesis that endothelial release of tissue-type plasminogen activator (t-PA) is impaired in adults with elevated plasma CRP concentrations independen...

  19. "All-laser" endothelial corneal transplant in human patients

    Science.gov (United States)

    Rossi, Francesca; Menabuoni, Luca; Malandrini, Alex; Canovetti, Annalisa; Lenzetti, Ivo; Pini, Roberto

    2012-03-01

    Femtosecond laser sculpturing of corneal tissue is commonly used for the preparation of endothelial flaps. Diode laser welding of ocular tissues is a procedure that enables minimally invasive suturing of tissues. The combination of these laser based techniques results in a new approach to minimally invasive ophthalmic surgery, such as in endothelial corneal transplant (or endothelial keratoplasty - EK). In this work we present the "all laser" EK performed in human subjects. 24 pseudophakic patients with bullous keratopathy underwent EK: the femtosecond laser was used to prepare the 100 ìm thick and 8.5 mm diameter donor Descemet endothelial flap. After staining the stromal layer of the donor flap with a liquid ICG solution, the donor flap was inserted in the recipient eye by the use of the Busin injector. Then, the endothelial layer was laser-welded to the recipient eye (10 laser spots around the periphery of the flap), in order to reduce the risk of postoperative dislocation of the transplanted flap. A transplanted flap engraftment was observed in all the treated eyes. The staining procedure used to perform laser welding also enabled to evidence the stromal side of the donor flap, so as the flap was always placed in the right side position. The endothelial cells counts in both the laserwelded flaps and in a control group were in good agreement. The proposed technique is easy to perform and enables the reduction of postoperative endothelial flap dislocations.

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

  2. Human endothelial senescence can be induced by TNF-α

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    TNF-α is one of the most important proinfiammatory cytokines in mediating multiple physio-pathological functions during immunological responses. Vascular endothelial cells, when stimulated by TNF-α2 can increase the expression of multiple cytokines and cellular adhesion molecules and, in turn, actively promote the inflammatory responses by recruiting and activating of leukocytes to the inflammatory site. In addition to endothelial death induced by TNF-α2 we found for the first time that TNF-α can also induce the human endothelial cells senescence. The induced senescent endothelial cells will display SA-β-Gal staining and they were arrested in G0-G1 phase. We found that Aψm would always be up-regulated in response to TNF-α stimulation at early time but when the cells become senescent, A ψmshows a tendency to decrease. It may reflect the sthenic function of mitochondria at early time in response to TNF-αstimulation and decay when the endothelial cells were induced senescent. ROS fluctuates at early time and also decreases when the endothelial cells become senescent. Our results show that the change of mitochondrial function may be related to the senescent process.``

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

    NARCIS (Netherlands)

    Tinken, T.M.; Thijssen, D.H.J.; Hopkins, N.; Dawson, E.A.; Cable, N.T.; Green, D.J.

    2010-01-01

    Although episodic changes in shear stress have been proposed as the mechanism responsible for the effects of exercise training on the vasculature, this hypothesis has not been directly addressed in humans. We examined brachial artery flow-mediated dilation, an index of NO-mediated endothelial functi

  4. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    1978-04-01

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

  6. Endothelial dysfunction is associated with left ventricular mass (assessed using MRI) in an adult population (MESA)

    OpenAIRE

    Yeboah, J; Crouse, JR; Bluemke, DA; Lima, JAC; Polak, JF; Burke, GL; Herrington, DM

    2010-01-01

    Brachial flow-mediated dilation (FMD) is a measure of endothelial nitric oxide bioavailability. Endothelial nitric oxide controls vascular tone and is likely to modify the ventricular muscle coupling mechanism. The association between left ventricular mass and FMD is not well understood. We assessed the association between left ventricular mass index (LVMI) and FMD in participants of the Multi-Ethnic Study of Atherosclerosis (MESA). MESA is a population-based study of 6814 adults free of clin...

  7. Coffee polyphenols improve peripheral endothelial function after glucose loading in healthy male adults.

    Science.gov (United States)

    Ochiai, Ryuji; Sugiura, Yoko; Shioya, Yasushi; Otsuka, Kazuhiro; Katsuragi, Yoshihisa; Hashiguchi, Teruto

    2014-02-01

    Brewed coffee is a widely consumed beverage, and many studies have examined its effects on human health. We investigated the vascular effects of coffee polyphenols (CPPs), hypothesizing that a single ingestion of CPP during glucose loading would improve endothelial function. To test this hypothesis, we conducted a randomized acute clinical intervention study with crossover design and measured reactive hyperemia index (RHI) to assess the acute effects of a 75-g glucose load with CPP in healthy, nondiabetic adult men. Blood glucose and insulin levels were elevated after glucose loading with and without CPP, with no significant differences between treatments. The RHI did not significantly decrease after glucose loading without CPP. With CPP, however, RHI significantly (P < .05) increased over baseline after glucose loading. The difference between treatments was statistically significant (P < .05). No significant changes were observed in an oxidative stress marker after glucose loading with or without CPP. These findings suggest that a single ingestion of CPP improves peripheral endothelial function after glucose loading in healthy subjects.

  8. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-04-11

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

  9. Endothelial and lipoprotein lipases in human and mouse placenta

    DEFF Research Database (Denmark)

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

    2005-01-01

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

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

    OpenAIRE

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

    1996-01-01

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

  11. Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations.

    Science.gov (United States)

    Antonov, A S; Nikolaeva, M A; Klueva, T S; Romanov YuA; Babaev, V R; Bystrevskaya, V B; Perov, N A; Repin, V S; Smirnov, V N

    1986-01-01

    Endothelial cells (EC) were harvested by 0.1% collagenase treatment for adult human thoracic aortas obtained 1-3 h after sudden death. At least 35-70% of EC were removed from the intimal surface of aorta, 90-95% of them being viable. Plating efficiency was 70-80%. Monolayer formation was achieved at a seeding density of 5-8 X 10(2) cells/mm2. The cells were identified as endothelium by the presence of Factor VIII antigen, Weigel-Palade bodies and typically endothelial morphology at confluence. Unlike endothelial cultures derived from human umbilical veins and infant aortas, primary cultures obtained from human adult aortas contain multinuclear EC with Factor VIII antigen and Weibel-Palade bodies. The number of multinuclear EC in cultures isolated from aortas affected by atherosclerosis was 2-fold higher (P less than 0.05) than in cultures obtained from grossly normal aortas taken from donors of the same age. EC with numerous lipid inclusions revealed by oil-red-O staining were present in all the EC primary cultures derived from aortas affected by atherosclerosis. No oil-red-O-positive cells were detected among the EC cultured from infant aorta, aorta of young donors, and umbilical vein. An electron microscopic examination of EC from atherosclerotic aorta in culture and in situ failed to reveal any ultrastructural peculiarities distinguishing multinuclear EC from the mononuclear EC. PMID:3004520

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

    Science.gov (United States)

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tang Rining

    2012-09-01

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

  14. Cell pattern in adult human corneal endothelium.

    Directory of Open Access Journals (Sweden)

    Carlos H Wörner

    Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

  15. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Bruno Lorusso

    2015-01-01

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

  16. Endothelial and lipoprotein lipases in human and mouse placenta

    DEFF Research Database (Denmark)

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

    2005-01-01

    Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin......-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher Na......Cl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL...

  17. Endothelial Function and the Regulation of Muscle Protein Anabolism in Older Adults

    OpenAIRE

    Timmerman, Kyle L.; Volpi, Elena

    2012-01-01

    Sarcopenia, the loss of skeletal muscle mass and function with aging, is a major contributor to frailty and morbidity in older adults. Recent evidence has emerged suggesting that endothelial dysfunction and insulin resistance of muscle protein metabolism may significantly contribute to the development of sarcopenia. In this article we review: 1) recent studies and theories on the regulation of skeletal muscle protein balance in older adults; 2) the link between insulin-resistance of muscle pr...

  18. Coffee polyphenol consumption improves postprandial hyperglycemia associated with impaired vascular endothelial function in healthy male adults.

    Science.gov (United States)

    Jokura, Hiroko; Watanabe, Isamu; Umeda, Mika; Hase, Tadashi; Shimotoyodome, Akira

    2015-10-01

    Epidemiological studies indicate that habitual coffee consumption lowers the risk of diabetes and cardiovascular diseases. Postprandial hyperglycemia is a direct and independent risk factor for cardiovascular diseases. We previously demonstrated that coffee polyphenol ingestion increased secretion of Glucagon-like peptide 1 (GLP-1), which has been shown to exhibit anti-diabetic and cardiovascular effects. We hypothesized coffee polyphenol consumption may improve postprandial hyperglycemia and vascular endothelial function by increasing GLP-1 release and/or reducing oxidative stress. To examine this hypothesis, we conducted a randomized, acute, crossover, intervention study in healthy male adults, measuring blood parameters and flow-mediated dilation (FMD) after ingestion of a meal with or without coffee polyphenol extract (CPE). Nineteen subjects consumed a test meal with either a placebo- or CPE-containing beverage. Blood biomarkers and FMD were measured at fasting and up to 180 minutes postprandially. The CPE beverage led to a significantly lower peak postprandial increase in blood glucose and diacron-reactive oxygen metabolite, and significantly higher postprandial FMD than the placebo beverage. Postprandial blood GLP-1 increase tended to be higher after ingestion of the CPE beverage, compared with placebo. Subclass analysis revealed that the CPE beverage significantly improved postprandial blood GLP-1 response and reduced blood glucose increase in the subjects with a lower insulinogenic index. Correlation analysis showed postprandial FMD was negatively associated with blood glucose increase after ingestion of the CPE beverage. In conclusion, these results suggest that coffee polyphenol consumption improves postprandial hyperglycemia and vascular endothelial function, which is associated with increased GLP-1 secretion and decreased oxidative stress in healthy humans.

  19. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

    Science.gov (United States)

    Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

    2005-05-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

  20. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  1. Modeling human endothelial cell transformation in vascular neoplasias

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    Victoria W. Wen

    2013-09-01

    Full Text Available Endothelial cell (EC-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, which responds poorly to current treatments and has a very high mortality rate. The development of treatments that are more effective for these disorders will be expedited by insight into the processes that promote abnormal proliferation and malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by the rarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a central role in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This review describes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes that underpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through the introduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed in relation to their relevance as models of vascular neoplasia.

  2. Daily egg consumption in hyperlipidemic adults - Effects on endothelial function and cardiovascular risk

    Directory of Open Access Journals (Sweden)

    Gonzalez-Simon Anjelica L

    2010-07-01

    Full Text Available Abstract Background Limiting consumption of eggs, which are high in cholesterol, is generally recommended to reduce risk of cardiovascular disease. However, recent evidence suggests that dietary cholesterol has limited influence on serum cholesterol or cardiac risk. Objective To assess the effects of egg consumption on endothelial function and serum lipids in hyperlipidemic adults. Methods Randomized, placebo-controlled crossover trial of 40 hyperlipidemic adults (24 women, 16 men; average age = 59.9 ± 9.6 years; weight = 76.3 ± 21.8 kilograms; total cholesterol = 244 ± 24 mg/dL. In the acute phase, participants were randomly assigned to one of the two sequences of a single dose of three medium hardboiled eggs and a sausage/cheese breakfast sandwich. In the sustained phase, participants were then randomly assigned to one of the two sequences of two medium hardboiled eggs and 1/2 cup of egg substitute daily for six weeks. Each treatment assignment was separated by a four-week washout period. Outcome measures of interest were endothelial function measured as flow mediated dilatation (FMD and lipid panel. Results Single dose egg consumption had no effects on endothelial function as compared to sausage/cheese (0.4 ± 1.9 vs. 0.4 ± 2.4%; p = 0.99. Daily consumption of egg substitute for 6 weeks significantly improved endothelial function as compared to egg (1.0 ± 1.2% vs. -0.1 ± 1.5%; p p p = 0.01. Study results (positive or negative are expressed in terms of change relative to baseline. Conclusions Egg consumption was found to be non-detrimental to endothelial function and serum lipids in hyperlipidemic adults, while egg substitute consumption was beneficial.

  3. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

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

    OpenAIRE

    Geelen, S; Bhattacharyya, C; Tuomanen, E

    1992-01-01

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

  5. Human endothelial senescence induced by IL-1α in vitro

    Institute of Scientific and Technical Information of China (English)

    YAO Aiyu; ZHOU Jianjun; LIU Yabing; FENG Meifu; ZHOU Rouli

    2004-01-01

    Interleukin 1(IL-1) is an important proinflammatory cytokine that causes pleiotropic effects. Vascular endothelial cells stimulated by IL-1α can lead to the inflammatory response. Reactive oxygen species (ROS) are also generated at the site of inflammation and serve as an important factor against foreign invader. Here we report that long-term stimulation of human vein endothelial cells with IL-1α can accelerate their senescence associated with β-galactosidase activity. The flow cytometric analyses showed that most of the induced cells entered G0-G1 phase. DNA damage was more severe in senescent cells by comet assay. The induced cells by IL-1α had higher levels of ROS and malonyldialdehyde (MDA), lower activity of antioxidant enzymes and lower capacity of total antioxidant systems than control, which led to cell damage and cell degeneration, that is to say, which contributed to cellular senescence. Our results gave a direct proof to a new hypothesis-"the inflammation hypothesis of aging" on cellular level, and also provided a basis for the study on anti-aging and aging-related diseases.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Ulisse Garbin

    2008-01-01

    Full Text Available The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L for 24 hours and oxidative stress was induced by the addition of oxidized (ox-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin; proteins linked to inflammation (IL-6 and TNFalpha, thrombotic state (tissue factor, PAI-1 and uPA, hypertension such as endothelin-1 (ET-1, and vascular remodeling such as metalloproteinases (MMP-2, MMP-9 and protease inhibitor (TIMP-1. The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

  8. Glycoconjugates and Related Molecules in Human Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Norihiko Sasaki

    2013-01-01

    Full Text Available Vascular endothelial cells (ECs form the inner lining of blood vessels. They are critically involved in many physiological functions, including control of vasomotor tone, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and immunity. It is considered that impairment of EC functions leads to the development of vascular diseases. The carbohydrate antigens carried by glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans mainly present on the cell surface serve not only as marker molecules but also as functional molecules. Recent studies have revealed that the carbohydrate composition of the EC surface is critical for these cells to perform their physiological functions. In this paper, we consider the expression and functional roles of endogenous glycoconjugates and related molecules (galectins and glycan-degrading enzymes in human ECs.

  9. Human growth hormone stimulates proliferation of human retinal microvascular endothelial cells in vitro

    International Nuclear Information System (INIS)

    Growth hormone (GH) has been implicated in the pathogenesis of proliferative diabetic retinopathy. The authors sought to determine whether this could be mediated by an effect of GH on proliferation of endothelial cells, and, for this purpose, established long-term cultures of human retinal microvascular endothelial cells (hREC) from normal postmortem human eyes. High-purity hREC preparations were selected for experiments, based on immunogluorescence with acetylated low density lipoprotein (LDL) and anti-factor VIII-related antigen. Growth requirements for these cells were complex, including serum for maintenance at slow growth rates and additional mitogens for more rapid proliferation. Exposure of hREC to physiologic doses of human GH (hGH) resulted in 100% greater cell number vs. control but could be elicited only in the presence of serum. When differing serum conditions were compared, hGH stimulated [3H]thymidine incorporation up to 1.6- to 2.2-fold under each condition and increased DNA content significantly in the presence of human, horse, and fetal calf serum. In summary, hREC respond to physiologic concentrations of hGH in vitro with enhanced proliferation. This specific effect of GH on retinal microvascular endothelial cells supports the hypothesis of role for GH in endothelial cell biology

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

    OpenAIRE

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

    1999-01-01

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

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

    Science.gov (United States)

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

    1993-06-01

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

  12. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    Science.gov (United States)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  13. Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules

    OpenAIRE

    1991-01-01

    Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their ...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Ugele, B; Lange, F

    2001-01-01

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

  16. INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co-culture or direct contact co-culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

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

    Directory of Open Access Journals (Sweden)

    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  18. ASSOCIATION BETWEEN INSULIN RESISTANCE AND NITRIC OXIDE IN HUMAN RETINAL MICROVASCULAR ENDOTHELIAL CELLS IN VITRO

    OpenAIRE

    Bushra, Sumbul

    2015-01-01

    Diabetic retinopathy (DR) a major consequence of diabetes is considered the leading cause of vision loss and blindness worldwide among working adults. Endothelial dysfunction expediting imbalance in vascular homeostasis, is one of the primary manifestation leading to the pathogenesis of DR. NO a major vasodilator involved in the regulation of vascular homeostasis is reported to be released by insulin dependent PI3K/ Akt signaling pathway. Endothelial dysfunction impairs ocular ...

  19. Production of factor VIII by human liver sinusoidal endothelial cells transplanted in immunodeficient uPA mice.

    Directory of Open Access Journals (Sweden)

    Marina E Fomin

    Full Text Available Liver sinusoidal endothelial cells (LSECs form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII. Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA transgene (uPA-NOG mice. Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.

  20. SIRT3 Deficiency Induces Endothelial Insulin Resistance and Blunts Endothelial-Dependent Vasorelaxation in Mice and Human with Obesity

    OpenAIRE

    Lu Yang; Julei Zhang; Wenjuan Xing; Xing Zhang; Jie Xu; Haifeng Zhang; Li Chen; Xiaona Ning; Gang Ji; Jia Li; Qingchuan Zhao; Feng Gao

    2016-01-01

    Recent evidence implicates the critical role of Sirtuin 3 (SIRT3) in the development of many metabolic diseases, but the contribution of SIRT3 to vascular homeostasis remains largely unknown. The aim of this study was to investigate the role of SIRT3 in endothelial insulin resistance and vascular dysfunction in obesity. We found an impaired insulin-induced mesenteric vasorelaxation and concomitant reduced vascular SIRT3 expression in morbid obese human subjects compared with the non-obese sub...

  1. Natural antioxidant dihydroxybenzyl alcohol blocks ritonavir-induced endothelial dysfunction in porcine pulmonary arteries and human endothelial cells

    OpenAIRE

    Weakley, Sarah M.; Jiang, Jun; Lü, Jianming; Wang, Xinwen; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

    2011-01-01

    Summary Background Patients with HIV have an increased incidence of pulmonary artery hypertension. This study was designed to determine if the naturally occurring antioxidant dihydroxybenzyl alcohol (DHBA) could counteract the deleterious effects of ritonavir (RTV), an HIV-protease inhibitor known to impair endothelial function and increase oxidative stress. Material/Methods Antioxidant assays were performed on DHBA in a cell free system. Glutathione (GSH) levels were measured in human pulmon...

  2. DNA damage in human endothelial cells after irradiation in anoxia

    International Nuclear Information System (INIS)

    Endothelial cells and fibroblasts have been reported to respond differently to oxidative stress. Both the effects of high oxygen tension and radiation involve the action of free radicals. DNA damage (single strand breaks, SSB, and double strand breaks, DSB) was assayed in human umbilical cord vein (HUV) cells and in Chinese hamster fibroblasts (V79) after irradiation under oxic or anoxic conditions. The cells were exposed to single doses in the range of 2-18 Gy of γ-radiation from 60Co. Significantly more DNA damage was induced in the V79 cells than in the HUV cells. As a consequence, a higher oxygen enhancement ratio was obtained for the HUV cells (6.3) as compared to the V79 cells (2.8). The repair of SSB was slower in the HUV cells than in the V79 cells, irrespective of oxic state. For the higher doses, the damage remaining at 60 min after anoxic irradiation, i.e. DSB, was only detected in the V79 cells. (orig.)

  3. A 220-kilodalton glycoprotein in yeast extract inhibits Staphylococcus aureus adherence to human endothelial cells.

    OpenAIRE

    Elliott, D.A.; Hatcher, V B; Lowy, F D

    1991-01-01

    A 220-kDa glycoprotein from yeast extract causes a twofold decrease in S. aureus adherence to human endothelial cells in vitro. Medium constituents can have a significant effect on bacterial adherence interactions.

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

    Institute of Scientific and Technical Information of China (English)

    田利民

    2014-01-01

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

  5. Exposure to Hyperoxia Decreases the Expression of Vascular Endothelial Growth Factor and Its Receptors in Adult Rat Lungs

    OpenAIRE

    Klekamp, Jessica G.; Jarzecka, Kasia; Perkett, Elizabeth A.

    1999-01-01

    Exposure to high levels of inspired oxygen leads to respiratory failure and death in many animal models. Endothelial cell death is an early finding, before the onset of respiratory failure. Vascular endothelial growth factor (VEGF) is highly expressed in the lungs of adult animals. In the present study, adult Sprague-Dawley rats were exposed to >95% FiO2 for 24 or 48 hours. Northern blot analysis revealed a marked reduction in VEGF mRNA abundance by 24 hours, which decreased to less than 50% ...

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

    Science.gov (United States)

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

    2015-01-01

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

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

    OpenAIRE

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

    2010-01-01

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

  8. Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Angela Orecchia

    Full Text Available Histone deacetylases (HDAC are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC, a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNFα and interleukin (IL-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.

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

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I;

    2006-01-01

    directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... the umbilical cords. Methods Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Main outcome measures Fold changes in gene expression levels between treated and control cultures were calculated from the microarray...

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

    Science.gov (United States)

    Srisen, Kaemisa; Röhrl, Clemens; Meisslitzer-Ruppitsch, Claudia; Ranftler, Carmen; Ellinger, Adolf; Pavelka, Margit; Neumüller, Josef

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kaemisa Srisen

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

  12. Relaxin increases human endothelial progenitor cell NO and migration and vasculogenesis in mice

    OpenAIRE

    Segal, Mark S.; Sautina, Laura; Li, Shiyu; Diao, YanPeng; Agoulnik, Alexander I.; Kielczewski, Jennifer; McGuane, Jonathan T.; Grant, Maria B.; Conrad, Kirk P.

    2012-01-01

    The ovarian peptide hormone, relaxin, circulates during pregnancy, contributing to profound maternal vasodilation through endothelial and nitric oxide (NO)–dependent mechanisms. Circulating numbers of bone marrow–derived endothelial cells (BMDECs), which facilitate angiogenesis and contribute to repair of vascular endothelium, increase during pregnancy. Thus, we hypothesized that relaxin enhances BMDEC NO production, circulating numbers, and function. Recombinant human relaxin-2 (rhRLX) stimu...

  13. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells

    OpenAIRE

    1982-01-01

    Immunofluorescence staining of cultured human umbilical vein endothelial cells has shown the presence of von Willebrand protein in the perinuclear region, in small rodlike structures through the cytoplasm, and on filaments of the extracellular matrix. Nonendothelial cells showed no staining with anti-von Willebrand protein antiserum. At the light microscope level, immunoperoxidase treatment of endothelial cells revealed the same pattern and antibody specificity as the fluorescence staining. T...

  14. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion.

    OpenAIRE

    Klein, L M; Lavker, R M; Matis, W L; Murphy, G F

    1989-01-01

    To understand better the role of mast cell secretory products in the genesis of inflammation, a system was developed for in vitro degranulation of human mast cells in skin organ cultures. Within 2 hr after morphine sulfate-induced degranulation, endothelial cells lining microvessels adjacent to affected mast cells expressed an activation antigen important for endothelial-leukocyte adhesion. Identical results were obtained when other mast cell secretagogues (anti-IgE, compound 48/80, and calci...

  15. Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema

    OpenAIRE

    Mackay, Laura S; Dodd, Sara; Dougall, Iain G; Tomlinson, Wendy; Lordan, James; Fisher, Andrew J.; Corris, Paul A

    2013-01-01

    Background Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding...

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

    OpenAIRE

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

    1987-01-01

    The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, exp...

  17. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

    Directory of Open Access Journals (Sweden)

    Vishnubalaji Radhakrishnan

    2012-01-01

    Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

  18. Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability.

    Science.gov (United States)

    Ong, Siew Pei; Ng, Mah Lee; Chu, Justin Jang Hann

    2013-12-01

    Infection with dengue virus (DV) can result in dengue hemorrhagic fever and dengue shock syndrome, where patients suffer from bleeding and plasma leakage involving endothelial cells. Angiopoietins (Ang) 1 and 2 are important angiogenic factors that affect endothelial barrier integrity. In this study, DV was observed to induce endothelial leakage at multiplicity of infection of 10 in primary human umbilical vein endothelial cells (HUVEC) with interendothelial gap formation. Immunostaining of vascular endothelial cadherin (VE-cadherin) and zona occludin 1 (ZO-1) showed the absence of these endothelial junctional proteins at the cell-cell contact zones between adjacent cells. In addition, Ang1 that is required for protecting against endothelial hyperpermeability was found to be down-regulated during DV infection. Treatment with increasing concentrations of recombinant Ang1 was shown to prevent DV-induced endothelial hyperpermeability in a dose-dependent manner by preventing the down-regulation of VE-cadherin and ZO-1 at cell membrane. In contrast, the expression of Ang2, the natural antagonist of Ang1, was observed to be up-regulated during DV infection. Recombinant Ang2 added to HUVEC at non-toxic concentrations showed decreased in transendothelial electrical resistance reading and the down-regulation of VE-cadherin and ZO-1. These findings suggest that DV reduces the expression of Ang1 and enhances the expression of Ang2 in endothelial cells and that this imbalance of Ang 1 and Ang 2 may play a contributing role to the increased permeability of human primary endothelial cells during DV infection. PMID:23989887

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

    Directory of Open Access Journals (Sweden)

    Jennifer A Greene

    Full Text Available CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina.

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

    Science.gov (United States)

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

    2013-09-01

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

  1. The effect of 193 nm excimer laser radiation on the human corneal endothelial cell density

    Energy Technology Data Exchange (ETDEWEB)

    Isager, P.; Hjortdal, J.Oe.; Ehlers, N. [Aarhus Univ. Hospital, Dept. of Ophthalmology, Aarhus (Denmark)

    1996-06-01

    The effect of 193 nm excimer laser radiation on human corneal endothelial cell density was examined. Fifty-five eyes from 35 patients underwent photorefractive keratectomy for myopia. Photomicrographs of the endothelium were taken a short time before the operation and on an average of 7 months postoperatively with a specular microscope. The average endothelial cell densities were preoperatively 3375 {+-} 266 cells/mm{sup 2} (means {+-} SD) and postoperatively 3348 {+-} 287 cells/mm{sup 2}, corresponding to a fall of 27 cells/mm{sup 2} (N = 55). This fall in endothelial cell density was not statistically significant. A significant correlation between the change in cell density and age of the patient was found, with older patients losing more cells (N = 35, 2p < 0.05). The magnification of the specular microscope was found to change with corneal thickness. The importance of correcting the endothelial cell densities for corneal thickness is discussed. (au) 14 refs.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits with the tis......Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits...... with the tissue of origin. Microvasculature was localized in situ by immunohistochemistry in breast samples. From this tissue, collagen-rich stroma and adipose tissue were dissected mechanically and further disaggregated to release microvessel organoids. BRENCs were cultured from these organoids in endothelial......-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis....

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

    Directory of Open Access Journals (Sweden)

    Azizah Ugusman

    2014-01-01

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

  4. Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft

    Institute of Scientific and Technical Information of China (English)

    WEN Shao-jun; ZHAO Li-min; WANG Shen-guo; LI Jing-xing; CHEN Hua-ying; LIU Jie-lin; LIU Ya; LUO Yi; Roo Changizi

    2007-01-01

    Background Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1α (6-keto-PGF1α).Methods Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1α in the culturing solutions were examined by radioimmunology to measure endothelial function.Results Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234±29) pg/ml and (428+98) pg/ml respectively in Group C and (196+30) pg/ml and (346±120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P<0.05 ).Conclusions Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.

  5. Unstimulated platelets evoke calcium responses in human umbilical vein endothelial cells

    NARCIS (Netherlands)

    van IJzendoorn, S C; van Gool, R G; Reutelingsperger, C P; Heemskerk, J W

    1996-01-01

    Interactions between human platelets and human umbilical vein endothelial cells (HUVEC) were studied by monitoring changes in cytosolic [Ca2+]i in both cell types. Confluent monolayers of Fura-2-loaded HUVEC, grown on gelatin-coated coverslips, responded to repeated addition of a suspension of unsti

  6. Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells

    OpenAIRE

    A. Gentile; G. Toietta; V. Pazzano; V. Tsiopoulos; A. Giglio; F. Crea; Pompilio, G; Capogrossi, M.; G. Di Rocco

    2011-01-01

    Recent studies have underscored a role for the epicardium as a source of multipotent cells. Here, we investigate the myogenic potential of adult human epicardium-derived cells (EPDCs) and analyze their ability to undergo skeletal myogenesis when cultured with differentiating primary myoblasts. Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative. We demonstrate that EPDCs spontaneously fuse with pre-existing myo...

  7. Urotensin II induces interleukin 8 expression in human umbilical vein endothelial cells.

    Directory of Open Access Journals (Sweden)

    Chung-Yi Lee

    Full Text Available BACKGROUND: Urotensin II (U-II, an 11-amino acid peptide, exerts a wide range of actions in cardiovascular systems. Interleukin-8 (IL-8 is secreted by endothelial cells, thereby enhancing endothelial cell survival, proliferation, and angiogenesis. However, the interrelationship between U-II and IL-8 as well as the detailed intracellular mechanism of U-II in vascular endothelial cells remain unclear. The aim of this study was to investigate the effect of U-II on IL-8 expression and to explore its intracellular mechanism in human umbilical vein endothelial cells. METHODS/PRINCIPAL FINDINGS: Primary human umbilical vein endothelial cells were used. Expression of IL-8 was determined by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase reporter assay. Western blot analyses and experiments with specific inhibitors were performed to reveal the downstream signaling pathways as concerned. U-II increased the mRNA/protein levels of IL-8 in human umbilical vein endothelial cells. The U-II effects were significantly inhibited by its receptor antagonist [Orn(5]-URP. Western blot analyses and experiments with specific inhibitors indicated the involvement of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase in U-II-induced IL-8 expression. Luciferase reporter assay further revealed that U-II induces the transcriptional activity of IL-8. The site-directed mutagenesis indicated that the mutation of AP-1 and NF-kB binding sites reduced U-II-increased IL-8 promoter activities. Proliferation of human umbilical vein endothelial cells induced by U-II could be inhibited significantly by IL-8 RNA interference. CONCLUSION/SIGNIFICANCE: The results show that U-II induces IL-8 expression in human umbilical vein endothelial cells via p38 mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways and IL-8 is involved in the U

  8. GABAB Receptors Expressed in Human Aortic Endothelial Cells Mediate Intracellular Calcium Concentration Regulation and Endothelial Nitric Oxide Synthase Translocation

    Directory of Open Access Journals (Sweden)

    Xu-Ping Wang

    2014-01-01

    Full Text Available GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i in a number of cells (e.g., retina, airway epithelium and smooth muscle, but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA, in cultured human aortic endothelial cells (HAECs, and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

  9. Gene Expression Analysis of Human Vascular Endothelial Cells Treated by Ouabain in Pathological Concentration

    Institute of Scientific and Technical Information of China (English)

    任延平; 吕卓人

    2004-01-01

    Objectives To study the gene expression of human vascular endothelial cells (HUVEC) treated by ouabain in pathological concentration. Methods The response of endothelial cells to ouabain of 1.8 nmol/L was explored with a complementary DNA microarray representing 8 464 different human genes. Results The results of mRNA profiles analysis indicated that 129 of the genes were differently expressed, 26 were upregulated. Conclusions The pathological role of ouabain on HUVEC may be involved in the controlling of DNA transcription、protein translation、 metabolism and signal transduction.

  10. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Institute of Scientific and Technical Information of China (English)

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG

    2004-01-01

    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

  11. Comparison between human fetal and adult skin

    OpenAIRE

    Coolen, N.A.; Schouten, K.C.; Middelkoop, E.; Ulrich, M.

    2009-01-01

    Healing of early-gestation fetal wounds results in scarless healing. Since the capacity for regeneration is probably inherent to the fetal skin itself, knowledge of the fetal skin composition may contribute to the understanding of fetal wound healing. The aim of this study was to analyze the expression profiles of different epidermal and dermal components in the human fetal and adult skin. In the human fetal skin (ranging from 13 to 22 weeks’ gestation) and adult skin biopsies, the expression...

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

    Directory of Open Access Journals (Sweden)

    Meng Deng

    2015-01-01

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

  13. Induction of heme oxygenase-1 by whisky congeners in human endothelial cells.

    Science.gov (United States)

    Suzuki, Keiko; Nemoto, Asuka; Tanaka, Izumi; Koshimizu, Seiichi; Suwa, Yoshihide; Ishihara, Hiroshi

    2010-08-01

    It is expected that the production of the cytoprotective heme oxygenase-1 (HO-1) protein in endothelial cells would reduce severity of vascular injuries, while phenolic compounds are known to induce HO-1 mRNA and protein in various cells. We investigated the activation of HO-1 by whisky, which contains various phenolic substances. The congeners of whisky stored from 4 to 18 y in oak barrels were shown to induce an increase of HO-1 protein in human umbilical vein endothelial cells, while those of freshly distilled whisky spirit exhibited no activity. To determine the compounds with potent HO-1-inducing activity among the whisky congeners, several chemicals that had been reported to exist in whisky or oak barrels were screened, and coniferyl aldehyde and sinapyl aldehyde showed the activity. Thus, compounds that emerged in whisky during barrel storage induced cytoprotective protein, HO-1, in human endothelial cells.

  14. Definitive proof of endothelialization of a Dacron arterial prosthesis in a human being.

    Science.gov (United States)

    Wu, M H; Shi, Q; Wechezak, A R; Clowes, A W; Gordon, I L; Sauvage, L R

    1995-05-01

    A 10 mm woven Dacron axillofemoral bypass graft was removed from a 65-year-old patient during redo surgery after an implant period of 26 months, because of a large seroma that surrounded the entire length of the graft. Tissue blocks were taken from representative areas along the entire length of the graft surface and evaluated by light microscopy with hematoxylin and eosin and Masson trichrome staining, scanning electron microscopy, transmission electron microscopy, and immunocytochemical staining. Paraffin-embedded sections were stained with smooth muscle cell alpha-actin, which demonstrated smooth muscle cells in the pseudointima, and Ham 56 stain to identify macrophages. Endothelial factor VIII/von Willebrand factor and Ulex europaeus agglutinin identified human endothelial cells on the flow surface, in areas far removed from the anastomoses to the native vessels. This is the first definitive proof in a human of endothelialization of a synthetic arterial graft beyond the pannus ingrowth zone. PMID:7769746

  15. Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Bingong; ZENG Qiutang; WANG Hongxiang; MAO Xiaobo

    2006-01-01

    To isolate and culture adipose stromal cells (ASCs), and study the effect of cytokines secreted by ASCs on endothelial cells, human adipose tissue was digested with collagenase type Ⅰ solution and ASCs were derived by culture. The cells surface phenotype was examined by flow cytometry. ELISA was used to detect the secretion of VEGF, HGF, SDF-1 α and RT-PCR was employed to detect the expression of their mRNA. Then the ASC medium was utilized to culture human umbilical vein endothelial cells ECV304. Cells were counted by hemacytometer to determine the proliferation and Annexin V/PI was employed for the examination of the apoptosis rate of ECV304. ASCs were derived by culture and expressed CD34, CD105 while they did not express CD31 or CD45. ASCs secreted cytokines such as VEGF, HGF and SDF-1 α so the ASC medium could stimulate proliferation and counteract apoptosis of endothelial cells (P<0.05). Bcl-2 mRNA was also found to be up-regulated in the endothelial cells. It is concluded that ASCs can secrete cytokines and has significant effect on the proliferation of endothelial cells and apoptosis.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  17. Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, L.J.; Michel, T.; Weremowicz, S.; Morton, C.C. (Brigham and Women' s Hospital, Boston, MA (United States))

    1994-01-15

    Endothelial NOS activity is a major determinant of vascular tone and blood pressure, and in several important (and sometimes hereditary) disease states, such as hypertension, diabetes, and atherosclerosis, the endothelial NO signaling system appears to be abnormal. To explore the relationship of the endothelial NOS activity, the authors isolated the human gene encoding the endothelial NOS. Genomic clones containing the 5[prime] end of this gene were identified in a human genomic library by applying a polymerase chain reaction (PCR)-based approach. Identification of the human gene for endothelial NOS (NOS3) was confirmed by nucleotide sequence analysis of the first coding exon, which was found to be identical to its cognate cDNA. The NOS3 gene spans at least 20 kb and appears to contain multiple introns. The transcription start site and promoter region of the NOS3 gene were identified by primer extension and ribonuclease protection assays. Sequencing of the putative promoter revealed consensus sequences for the shear stress-response element, as well as cytokine-responsive cis regulatory sequences, both possible important to the roles played by NOS3 in the normal and the diseased cardiovascular system. The authors also mapped the chromosomal location of the NOS3 gene. First, a chromosomal panel of human-rodent somatic cell hybrids was screened using PCR with oligonucleotide primers derived from the NOS3 genomic clone. The specificity of the amplified PCR product was confirmed by human and hamster genomic DNA controls, as well as by Southern blot analysis, using the NOS3 cDNA as probe. Definitive chromosomal assignment of the NOS3 gene to human chromosome 7 was based upon 0% discordancy; fluorescence in situ hybridization sublocalized the NOS3 gene to 7q36. The identification and characterization of the NOS3 gene may lead to further insights into heritable disease states associated with this gene product. 41 refs., 3 figs., 1 tab.

  18. RNA interference inhibits expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    CAI Chun-mei; SUN Bao-chen; LIU Xu-yang; WANG Jin-jin; LI Jun-fa; HAN Song; WANG Ning-li; LU Qing-jun

    2005-01-01

    @@ Choroidal neovascularization (CNV), a major cause of vision loss, is the result of the increased vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. It is important to inhibit the expression of VEGF protein in RPE cells.

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

    OpenAIRE

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

    2007-01-01

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

  20. Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

    Science.gov (United States)

    Krishnamurthy, Sudha; Warner, Kristy A; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B; Helman, Joseph I; Taichman, Russell S; Bellile, Emily L; McCauley, Laurie K; Polverini, Peter J; Prince, Mark E; Wicha, Max S; Nör, Jacques E

    2014-11-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling. PMID:25078284

  1. Investigation of the cytotoxicity of CCVD carbon nanotubes towards human umbilical vein endothelial cells

    OpenAIRE

    Flahaut, Emmanuel; Durrieu, Marie-Christine; Remy-Zolghadri, Murielle; Bareille, Reine; Baquey, Charles

    2006-01-01

    The cytotoxicity of different samples of carbon nanotubes synthesised by catalytic chemical vapour deposition was investigated towards human umbilical vein endothelial cells, using two cytotoxicity standard assays (neutral red assay for the cell viability and MTT assay—tetrazolinium salt—for the cell metabolic activity). No cytotoxicity was found for any sample.

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

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K;

    1990-01-01

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

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

    NARCIS (Netherlands)

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

    1999-01-01

    OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 1

  4. The Acute Effects of Grape Polyphenols Supplementation on Endothelial Function in Adults: Meta-Analyses of Controlled Trials

    OpenAIRE

    Shao-Hua Li; Hong-Bo Tian; Hong-Jin Zhao; Liang-Hua Chen; Lian-Qun Cui

    2013-01-01

    BACKGROUND: The acute effects of grape polyphenols on endothelial function in adults are inconsistent. Here, we performed meta-analyses to determine these acute effects as measured by flow-mediated dilation (FMD). METHODS: Trials were searched in PubMed, Embase and the Cochrane Library database. Summary estimates of weighted mean differences (WMDs) and 95% CIs were obtained by using random-effects models. Meta-regression and subgroup analyses were performed to identify the source of heterogen...

  5. Iptakalim rescues human pulmonary artery endothelial cells from hypoxia-induced nitric oxide system dysfunction

    OpenAIRE

    Zong, Feng; Zuo, Xiang-Rong; Wang, Qiang; ZHANG, SHI-JIANG; Xie, Wei-Ping; Wang, Hong

    2011-01-01

    The aim of this study was to assess whether hypoxia inhibits endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production, and whether iptakalim may rescue human pulmonary artery endothelial cells (HPAECs) from hypoxia-induced NO system dysfunction. HPAECs were cultured under hypoxic conditions in the absence or presence of 0.1, 10 and 1,000 μM iptakalim or the combination of 10 μM iptakalim and 1, 10 and 100 μM glibenclamide for 24 h, and the eNOS activity and NO levels...

  6. Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

    OpenAIRE

    Maruyama, I.; Salem, H H; Majerus, P W

    1984-01-01

    In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, and (b) human coagulation Factor Va. We have recently reported that the activity of Factor Va is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor Va and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activat...

  7. Kaempferia parviflora ethanolic extract promoted nitric oxide production in human umbilical vein endothelial cells.

    Science.gov (United States)

    Wattanapitayakul, Suvara K; Suwatronnakorn, Maneewan; Chularojmontri, Linda; Herunsalee, Angkana; Niumsakul, Somchit; Charuchongkolwongse, Suphan; Chansuvanich, Nuchattra

    2007-04-01

    The rhizomes of Kaempferia parviflora (KP) (Zingiberaceae) have been used in Thai traditional medicine for health promotion and for the treatment of digestive disorders and gastric ulcer. This study investigated effect of KP on endothelial function. Studies in human umbilical vein endothelial cells (HUVEC) showed that KP dose-dependently increased nitrite concentrations in culture media after 48 h incubation. eNOS mRNA and protein expression were also enhanced. The induction of eNOS mRNA was detected at 4 h and plateau at 48 h while iNOS expression was not observed. These data demonstrate that KP has a great potential for a supplemental use in vascular endothelial health promotion. PMID:17113256

  8. Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

    International Nuclear Information System (INIS)

    The (35S)glycosaminoglycans ((35S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with 35SO4 for 72 h, the (35S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of (35S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of (35S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author)

  9. Anti-Inflammatory Properties of Sirtuin 6 in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Martha Lappas

    2012-01-01

    Full Text Available A prominent feature of inflammatory diseases is endothelial dysfunction. Factors associated with endothelial dysfunction include proinflammatory cytokines, adhesion molecules, and matrix degrading enzymes. At the transcriptional level, they are regulated by the histone deacetylase sirtuin (SIRT 1 via its actions on the proinflammatory transcription factor nuclear factor-κB (NF-κB. The role of SIRT6, also a histone deacetylase, in regulating inflammation in endothelial cells is not known. The aim of this study was to determine the effect of SIRT6 knockdown on inflammatory markers in human umbilical vein endothelial cells (HUVECs in the presence of lipopolysaccharide (LPS. LPS decreased expression of SIRT6 in HUVECs. Knockdown of SIRT6 increased the expression of proinflammatory cytokines (IL-1β, IL-6, IL-8, COX-prostaglandin system, ECM remodelling enzymes (MMP-2, MMP-9 and PAI-1, the adhesion molecule ICAM-1, and proangiogenic growth factors VEGF and FGF-2; cell migration; cell adhesion to leukocytes. Loss of SIRT6 increased the expression of NF-κB, whereas overexpression of SIRT6 was associated with decreased NF-κB transcriptional activity. Taken together, these results demonstrate that the loss of SIRT6 in endothelial cells is associated with upregulation of genes involved in inflammation, vascular remodelling, and angiogenesis. SIRT6 may be a potential pharmacological target for inflammatory vascular diseases.

  10. Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.

    Science.gov (United States)

    Zhu, Ying-Ting; Chen, Hung-Chi; Chen, Szu-Yu; Tseng, Scheffer C G

    2012-08-01

    Contact inhibition ubiquitously exists in non-transformed cells that are in contact with neighboring cells. This phenomenon explains the poor regenerative capacity of in vivo human corneal endothelial cells during aging, injury and surgery. This study demonstrated that the conventional approach of expanding human corneal endothelial cells by disrupting contact inhibition with EDTA followed by bFGF activated canonical Wnt signaling and lost the normal phenotype to endothelial-mesenchymal transition, especially if TGFβ1 was added. By contrast, siRNA against p120 catenin (CTNND1) also uniquely promoted proliferation of the endothelial cells by activating trafficking of p120 catenin to the nucleus, thus relieving repression by nuclear Kaiso. This nuclear p120-catenin-Kaiso signaling is associated with activation of RhoA-ROCK signaling, destabilization of microtubules and inhibition of Hippo signaling, but not with activation of Wnt-β-catenin signaling. Consequently, proliferating human corneal endothelial cells maintained a hexagonal shape, with junctional expression of N-cadherin, ZO-1 and Na(+)/K(+)-ATPase. Further expansion of human corneal endothelial monolayers with a normal phenotype and a higher density was possible by prolonging treatment with p120 catenin siRNA followed by its withdrawal. This new strategy of perturbing contact inhibition by selective activation of p120-catenin-Kaiso signaling without disrupting adherent junction could be used to engineer surgical grafts containing normal human corneal endothelial cells to meet a global corneal shortage and for endothelial keratoplasties. PMID:22505615

  11. Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

    Directory of Open Access Journals (Sweden)

    Gomes Ronald R

    2010-11-01

    Full Text Available Abstract Background Immobilized recombinant perlecan domain I (PlnDI binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165 enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro. Results In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951, and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2. Conclusions Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.

  12. Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

    Science.gov (United States)

    Jiang, Yi-Zhou; Wang, Kai; Li, Yan; Dai, Cai-Feng; Wang, Ping; Kendziorski, Christina; Chen, Dong-Bao; Zheng, Jing

    2013-05-01

    Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.

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

    Science.gov (United States)

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

    2016-02-01

    The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance: The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field. PMID:26702128

  14. Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

    OpenAIRE

    Stinson, Murray W.; Alder, Susan; Kumar, Sarmishtha

    2003-01-01

    Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic Streptococcus gordonii CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and b...

  15. Generation of iPSC line epiHUVEC from human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Peggy Matz

    2015-11-01

    Full Text Available Human umbilical vein endothelial cells (HUVECs were used to generate the iPSC line epiHUVEC employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHUVEC and the human embryonic stem cell line — H1 have a Pearson correlation of 0.899.

  16. Proangiogenic effects of environmentally relevant levels of bisphenol A in human primary endothelial cells.

    Science.gov (United States)

    Andersson, Helén; Brittebo, Eva

    2012-03-01

    Bisphenol A (BPA) is widely used in the manufacturing of consumer products such as plastic food containers and food cans. Experimental studies suggest a relationship between exposure to BPA and changes in metabolic processes and reproductive organs. Also, epidemiological studies report an association between elevated exposure to BPA and cardiovascular disease and diabetes. Although alterations in the vascular endothelium are implicated in pathological conditions associated with BPA, little is known about the effects of BPA in the human endothelium. This study aimed to investigate the effects of 0.1 nM-1 μM of BPA on selected biomarkers of endothelial dysfunction, inflammation, and angiogenesis in human umbilical vein endothelial cells (HUVEC). The mRNA expression of biomarkers was assayed using qRT-PCR, and the production of nitric oxide and reactive oxygen species was measured using the H(2)DCFDA and the DAF-FM assays. The effect of BPA on phosphorylated eNOS was examined using Western blot and immunofluorescence, and the endothelial tube formation assay was used to investigate in vitro angiogenesis. BPA (≤1 μM) increased the mRNA expression of the proangiogenic genes VEGFR-2, VEGF-A, eNOS, and Cx43 and increased the production of nitric oxide in HUVEC. Furthermore, BPA increased the expression of phosphorylated eNOS and endothelial tube formation in HUVEC. These studies demonstrate that environmentally relevant levels of BPA have direct proangiogenic effects on human primary endothelial cells in vitro suggesting that the human endothelium may be an important target for BPA.

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

    Directory of Open Access Journals (Sweden)

    Naoki Okumura

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

  18. Protection of Salvianolic Acid B for Human Endothelial Cells Against Hydrogen Peroxide-Induced Oxidative Damage

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jungang; ZHAO Guangrong; LIU Jinling; JI Xiangwu

    2009-01-01

    Salvianolic acid B(Sal B) is an active component of traditional Chinese medicine Salvia miltiorrhiza and is used to treat vascular diseases. To better understand its mechanism, the antioxidant capacities of Sal B was evaluated with human endothelial cells under oxidative stress. Human endothelial cells were pretreated with Sal B for 12 h followed by hydrogen peroxide for another 12 h. Production of reactive oxygen species (ROS), activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and concentration of glu-tathione were measured: Protective effect of Sal B on the endothelial cells from hydrogen peroxide-induced damage ' was observed, and ROS production in the cells was found significantly inhibited. Sal B remarkably enhanced the activities of antioxidant enzymes SOD, CAT and GPX. Furthermore, Sal B up-regulated the intracellular glutathione concentration. The results indicate that Sal B protected endothelial cells from oxidative stress by improving the redox status of the cells through enhancing the antioxidant enzyme activities and increasing the reductive glutathione concentration after the oxidative challenge.

  19. MicroRNA expression after ionizing radiation in human endothelial cells

    International Nuclear Information System (INIS)

    Endothelial cells (EC) in tumor and normal tissue constitute critical radiotherapy targets. MicroRNAs have emerged as master switchers of the cellular transcriptome. Here, we seek to investigate the role of miRNAs in primary human dermal microvascular endothelial cells (HDMEC) after ionizing radiation. The microRNA status in HDMEC after 2 Gy radiation treatment was measured using oligo-microarrays covering 361 miRNAs. To functionally analyze the role of radiation-induced differentially regulated miRNAs, cells were transfected with miRNA precursor or inhibitor constructs. Clonogenic survival and proliferation assays were performed. Radiation up-regulated miRNA expression levels included let-7g, miR-16, miR-20a, miR-21 and miR-29c, while miR-18a, miR-125a, miR-127, miR-148b, miR-189 and miR-503 were down-regulated. We found that overexpression or inhibition of let-7g, miR-189, and miR-20a markedly influenced clonogenic survival and cell proliferation per se. Notably, the radiosensitivity of HDMEC was significantly influenced by differential expression of miR-125a, -127, -189, and let-7g. While miR-125a and miR-189 had a radioprotective effect, miR-127 and let-7g enhanced radiosensitivity in human endothelial cells. Our data show that ionizing radiation changes microRNA levels in human endothelial cells and, moreover, exerts biological effects on cell growth and clonogenicity as validated in functional assays. The data also suggest that the miRNAs which are differentially expressed after radiation modulate the intrinsic radiosensitivity of endothelial cells in subsequent irradiations. This indicates that miRNAs are part of the innate response mechanism of the endothelium to radiation

  20. Action of Shiga Toxin Type-2 and Subtilase Cytotoxin on Human Microvascular Endothelial Cells

    Science.gov (United States)

    Amaral, María M.; Sacerdoti, Flavia; Jancic, Carolina; Repetto, Horacio A.; Paton, Adrienne W.; Paton, James C.; Ibarra, Cristina

    2013-01-01

    The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 −a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis. PMID:23936204

  1. In vitro proliferation of adult human beta-cells.

    Directory of Open Access Journals (Sweden)

    Sabine Rutti

    Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

  2. Toxic effects of iron oxide nanoparticles on human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Xinying Wu

    2010-05-01

    Full Text Available Xinying Wu1, Yanbin Tan1, Hui Mao2, Minming Zhang11Department of Radiology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China; 2Department of Radiology, Center for Systems Imaging, Emory University School of Medicine, Atlanta, Georgia, USAAbstract: Iron oxide nanoparticles (IONPs have been employed for hyperthermia treatments, stem cell therapies, cell labeling, and imaging modalities. The biocompatibility and cytotoxic effects of iron oxide nanoparticles when used in biomedical applications, however, are an ongoing concern. Endothelial cells have a critical role in this research dealing with tumors, cardiovascular disease and inflammation. However, there is little information dealing with the biologic effects of IONPs on the endothelial cell. This paper deals with the influence of dextran and citric acid coated IONPs on the behavior and function of human umbilical vein endothelial cells (HUVECs. After exposing endothelial cells to IONPs, dose-dependent effects on HUVECs viability, cytoskeleton and function were determined. Both citric acid and dextran coated particles appeared to be largely internalized by HUVECs through endocytosis and contribute to eventual cell death possibly by apoptosis. Cytoskeletal structures were greatly disrupted, as evidenced by diminished vinculin spots, and disorganized actin fiber and tubulin networks. The capacity of HUVECs to form a vascular network on Matrigel™ diminished after exposure to IONPs. Cell migration/invasion were inhibited significantly even at very low iron concentrations (0.1 mM. The results of this study indicate the great importance of thoroughly understanding nanoparticle-cell interactions, and the potential to exploit this understanding in tumor therapy applications involving IONPs as thermo/chemoembolization agents.Keywords: iron oxide nanoparticles, cytotoxicity, in vitro test, cytoskeleton, human umbilical vein endothelial cell

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2......-ethylhexyl phthalate (DEHP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DnOP), and di-isononyl phthalate (DINP). Gene expression and secretion of inflammatory cytokines were evaluated after exposure to DBP. Decreased cell proliferation was observed for the phthalates DBP, BBP, and DEHP, and cell...... toxicity was observed for DBP and BBP. Upon DBP exposure at nontoxic concentrations, a significant increased gene expression and cytokine cell secretion were observed for interleukin-1ß (IL-1ß) and IL-8, and also an increased IL-6 secretion was observed. In conclusion, the human corneal endothelial cell...

  4. Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

    Science.gov (United States)

    Panorchan, Porntula; Lee, Jerry S H; Kole, Thomas P; Tseng, Yiider; Wirtz, Denis

    2006-11-01

    Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

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

    Directory of Open Access Journals (Sweden)

    Toshio Hayashi

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

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

    Science.gov (United States)

    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.

  7. Advanced oxidation protein products induce endothelial-to-mesenchymal transition in human renal glomerular endothelial cells through induction of endoplasmic reticulum stress.

    Science.gov (United States)

    Liang, Xiujie; Duan, Na; Wang, Yue; Shu, Shuangshuang; Xiang, Xiaohong; Guo, Tingting; Yang, Lei; Zhang, Shaojie; Tang, Xun; Zhang, Jun

    2016-01-01

    Endothelial-to-mesenchymal transition (EndMT) in renal glomerular endothelial cells plays a critical role in the pathogenesis of diabetic nephropathy (DN). Furthermore, advanced oxidation protein products (AOPPs) have been shown to contribute to the progression of DN. However, whether AOPPs induce EndMT in renal glomerular endothelial cells remains unclear. Thus, we investigated the effect of AOPPs on human renal glomerular endothelial cells (HRGECs) and the mechanisms underlying the effects. Our results showed that AOPP treatment lowered the expression of vascular endothelial cadherin, CD31, and claudin 5 and induced the overexpression of α-smooth muscle actin, vimentin, and fibroblast-specific protein 1, which indicated that AOPPs induced EndMT in HRGECs. Furthermore, AOPP stimulation increased the expression of glucose-regulated protein 78 and CCAAT/enhancer-binding protein-homologous protein, which suggested that AOPPs triggered endoplasmic reticulum (ER) stress in HRGECs. Notably, the aforementioned AOPP effects were reversed following the treatment of cells with salubrinal, an inhibitor of ER stress, whereas the effects were reproduced after exposure to thapsigargin, an inducer of ER stress. Collectively, our results indicate that AOPPs trigger EndMT in HRGECs through the induction of ER stress. These findings suggest novel therapeutic strategies for inhibiting renal fibrosis by targeting ER stress. PMID:26861949

  8. Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy

    OpenAIRE

    Cemazar, M; Parkins, C. S.; Holder, A L; Chaplin, D. J.; Tozer, G. M.; Sersa, G

    2001-01-01

    Recent studies have indicated that the antitumour effectiveness of electrochemotherapy, a combination of chemotherapeutic drugs with application of high voltage electric pulses applied to the tumour nodule (electroporation), result in a significant reduction in tumour blood flow and may therefore be mediated by an anti-vascular mechanism. The aim of this study was to evaluate the cytotoxicity of electroporation with bleomycin or cisplatin on cultured human microvascular endothelial cells (HME...

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

    OpenAIRE

    Mohr, Thomas; Desser, Lucia

    2013-01-01

    Background Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein e...

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

    Science.gov (United States)

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

    2007-01-01

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

  11. Endothelial Interleukin-6 defines the tumorigenic potential of primary human cancer stem cells

    OpenAIRE

    Krishnamurthy, Sudha; Warner, Kristy A.; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B.; Helman, Joseph I.; Taichman, Russell S.; Bellile, Emily L.; McCauley, Laurie K.; Polverini, Peter J.; Prince, Mark E.; Wicha, Max S.; Jacques E Nör

    2014-01-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small sub-population of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellul...

  12. Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

    OpenAIRE

    1984-01-01

    Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer c...

  13. Micropatterned 3-Dimensional Hydrogel System to Study Human Endothelial-Mesenchymal Stem Cell Interactions

    OpenAIRE

    Trkov, Sasa; Eng, George; Di Liddo, Rosa; Parnigotto, Pier Paolo; Vunjak-Novakovic, Gordana

    2010-01-01

    The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell type and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (i) bone marrow a...

  14. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    OpenAIRE

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is...

  15. Immunoexpression of vascular endothelial growth factor and Ki-67 in human gingival samples: An observational study

    OpenAIRE

    Kranti, K.; Mani, R; Elizabeth, Anjana

    2015-01-01

    Aim: To evaluate immunohistochemically vascular endothelial growth factor (VEGF) and Ki-67 in human gingival samples and to compare these factors between healthy and diabetic patients. Materials and Methods: A total of 50 subjects were included in the study. They were categorized into three groups: Periodontally healthy group, periodontally diseased gingiva without any systemic disease group and periodontally diseased gingiva with controlled type II diabetes mellitus (DM) group. Gingival biop...

  16. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    Science.gov (United States)

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  17. Radio-sensitivities and angiogenic signaling pathways of irradiated normal endothelial cells derived from diverse human organs

    International Nuclear Information System (INIS)

    The purpose of the present investigation was to study the effects of ionizing radiation on endothelial cells derived from diverse normal tissues. We first compared the effects of radiation on clonogenic survival and tube formation of endothelial cells, and then investigated the molecular signaling pathways involved in endothelial cell survival and angiogenesis. Among the different endothelial cells studied, human hepatic sinusoidal endothelial cells (HHSECs) were the most radio-resistant and human dermal microvascular endothelial cells were the most radio-sensitive. The radio-resistance of HHSECs was related to adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase-mediated expression of MMP-2 and vascular endothelial growth factor receptor-2 (VEGFR-2), whereas the increased radio-sensitivity of HDMECs was related to extracellular signal-regulated kina0se-mediated generation of angiostatin. These observations demonstrate that there are distinct differences in the radiation responses of normal endothelial cells obtained from diverse organs, which may provide important clues for protection of normal tissue from radiation exposure. (author)

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

    Directory of Open Access Journals (Sweden)

    Elvira Pelosi

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

  19. Ultraviolet-A radiation induces adhesion molecule expression on human dermal microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Heckmann, M.; Eberlein-Koenig, B.; Wollenberg, A.; Przybilla, B.; Plewig, G. (Muenchen Univ. (Germany). Dermatologische Klinik und Poliklinik)

    1994-09-01

    In order to investigate putative direct effects of UV radiation on endothelial cells, we studied adhesion molecule expression by immunostaining procedures and FACS analysis, following irradiation of normal human skin and cultured human dermal endothelial cells. Enhanced immunostaining for ICAM-1 and E-selectin was detected in biopsies taken after in vivo UVA and UVB irradiation, compared with non-irradiated control skin. On cultural human dermal endothelial cells, however, ICAM-1 and E-selectin were inducible by UVA but not UVB. The induction was dose-dependent, peaking at 20J/cm[sup 2] for both adhesion molecules, and time-dependent, peaking after 6 and 24h for E-selectin and ICAM-1, respectively. Expression of VCAM-1 and PECAM/EndoCAM/CD31 was unaffected by any UV-radiation modality. The functional integrity of irradiated cells was monitored by an exclusion assay of the fluorescent dye 7-AAD. and by staining for the cytoskeletal proteins actin and vimentin. (author).

  20. Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

    OpenAIRE

    Shono, T; Ono, M; H. Izumi; Jimi, S I; Matsushima, K; Okamoto, T.; Kohno, K.; Kuwano, M.

    1996-01-01

    Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the ...

  1. Vascular Endothelial Growth Factor-A (VEGF-A) Mediates Activin A-Induced Human Trophoblast Endothelial-Like Tube Formation.

    Science.gov (United States)

    Li, Yan; Zhu, Hua; Klausen, Christian; Peng, Bo; Leung, Peter C K

    2015-11-01

    Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation. PMID:26327470

  2. Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Sara; Castiglioni; Clelia; Caspani; Alessandra; Cazzaniga; Jeanette; AM; Maier

    2014-01-01

    AIM: To study the response to silver nanoparticles(Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis. METHODS: We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan(MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2’-7’-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope.RESULTS: Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent

  3. A novel adipocytokine, chemerin exerts anti-inflammatory roles in human vascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp [Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori 034-8628 (Japan); Kameshima, Satoshi; Usui, Tatsuya; Okada, Muneyoshi; Hara, Yukio [Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori 034-8628 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Chemerin is a novel adipocytokine with almost unknown function in vasculature. Black-Right-Pointing-Pointer Chemerin activates Akt/eNOS/NO pathways in endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-{alpha}-induced monocyte adhesion to endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-induced VCAM-1 via suppressing NF-{kappa}B and p38 signal. Black-Right-Pointing-Pointer Chemerin is anti-inflammatory through producing NO in vascular endothelium. -- Abstract: Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-{kappa}B p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-{alpha} (5 ng/ml, 20 min-6 h). Inhibitor of NF-{kappa}B or p38 significantly inhibited the TNF-{alpha}-induced VCAM-1 expression. Chemerin also inhibited TNF-{alpha}-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-{alpha}-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-{alpha}-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-{alpha}-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-{alpha}-induced VCAM-1 expression and monocytes adhesion in vascular

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

    Directory of Open Access Journals (Sweden)

    Saem Mul Park

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

  5. Anti-angiogenic action of plasma hyaluronan binding protein in human umbilical vein endothelial cells.

    Science.gov (United States)

    Jeon, Ji Won; Song, Hyun Seok; Moon, Eun-Joung; Park, Shi-Young; Son, Myung Jin; Jung, Seung Youn; Kim, Ji Tae; Nam, Do-Hyun; Choi-Miura, Nam-Ho; Kim, Kyu-Won; Kim, Yung-Jin

    2006-07-01

    The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo. PMID:16773202

  6. Slit2-Robo4 receptor responses inhibit ANDV directed permeability of human lung microvascular endothelial cells.

    Science.gov (United States)

    Gorbunova, Elena E; Gavrilovskaya, Irina N; Mackow, Erich R

    2013-08-01

    Hantaviruses nonlytically infect human endothelial cells (ECs) and cause edematous and hemorrhagic diseases. Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS), and Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses enhance vascular endothelial growth factor directed EC permeability resulting in the disassembly of inter-endothelial cell adherens junctions (AJs). Recent studies demonstrate that Slit2 binding to Robo1/Robo4 receptors on ECs has opposing effects on AJ disassembly and vascular fluid barrier functions. Here we demonstrate that Slit2 inhibits ANDV and HTNV induced permeability and AJ disassembly of pulmonary microvascular ECs (PMECs) by interactions with Robo4. In contrast, Slit2 had no effect on the permeability of ANDV infected human umbilical vein ECs (HUVECs). Analysis of Robo1/Robo4 expression determined that PMECs express Robo4, but not Robo1, while HUVECs expressed both Robo4 and Robo1 receptors. SiRNA knockdown of Robo4 in PMECs prevented Slit2 inhibition of ANDV induced permeability demonstrating that Robo4 receptors determine PMEC responsiveness to Slit2. Collectively, this data demonstrates a selective role for Slit2/Robo4 responses within PMECs that inhibits ANDV induced permeability and AJ disassembly. These findings suggest Slit2s utility as a potential HPS therapeutic that stabilizes the pulmonary endothelium and antagonizes ANDV induced pulmonary edema.

  7. Proteomic response analysis of endothelial cells of human coronary artery to stimulation with carbachol

    Institute of Scientific and Technical Information of China (English)

    Min YU; Dong-mei CHEN; Gang HU; Hai WANG

    2004-01-01

    AIM: To identify the molecular basis of the endothelial target for acetylcholine (ETA). METHODS: Proteomic methods were used to monitor changes in protein expression in the first 10 h following the stimulation of human coronary endothelial cells with carbachol 100 μmol/L. Thirty proteins showing the largest changes were identified by mass spectrometry. RESULTS: Based on analysis with Image Master 2-D Elite software, about 623 protein spots were detected in control cells and 825 protein spots in carbachol-treated cells, the matching rate was 68.1%.Among all the detected spots, 39 were up-regulated and 29 were down-regulated, showing detectable changes varied from 1.7-3.8 folds. Forty one spots in the peptide mass fingerprints were successfully obtained. The most interesting feature was that all the four newly synthesized proteins belonged to the heat shock protein family.CONCLUSION: These identified proteins played key roles in the molecular mechanism of ETA.

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

    Science.gov (United States)

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

    2016-06-01

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

  9. Ingestion of broccoli sprouts does not improve endothelial function in humans with hypertension

    DEFF Research Database (Denmark)

    Christiansen, Buris; Bellostas Muguerza, Natalia; Petersen, Atheline Major;

    2010-01-01

    UNLABELLED: Ingestion of glucosinolates has previously been reported to improve endothelial function in spontaneously hypertensive rats, possibly because of an increase in NO availability in the endothelium due to an attenuation of oxidative stress; in our study we tried to see if this also would...... be the case in humans suffering from essential hypertension. METHODS: 40 hypertensive individuals without diabetes and with normal levels of cholesterol were examined. The participants were randomized either to ingest 10 g dried broccoli sprouts, a natural donor of glucosinolates with high in vitro...... antioxidative potential, for a 4 week period or to continue their ordinary diet and act as controls. Blood pressure, endothelial function measured by flow mediated dilation (FMD) and blood samples were obtained from the participants every other week and the content of glucosinolates was measured before...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-04

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

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

    Directory of Open Access Journals (Sweden)

    Trine M. Reine

    2016-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Daniel M Reed

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

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

    Directory of Open Access Journals (Sweden)

    Jorge S Burns

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

  14. Differential expression of vascular endothelial growth factor in human fetal skeletal site-specific tissues: Mandible versus femur.

    Science.gov (United States)

    Marini, Mirca; Bertolai, Roberto; Ambrosini, Stefano; Sarchielli, Erica; Vannelli, Gabriella Barbara; Sgambati, Eleonora

    2015-04-01

    Vascular endothelial growth factor (VEGF) is a well-known mediator that signals through pathways in angiogenesis and osteogenesis. Angiogenesis and bone formation are coupled during either skeletal development or bone remodeling and repair occurring in postnatal life. In this study, we examined for the first time the expression of VEGF in human fetal mandibular and femoral bone in comparison with the respective adult tissues. Similarly to other craniofacial bones, but at variance with the axial and appendicular skeleton, during development mandible does not arise from mesoderm but neural crest cells of the neuroectoderm germ layer, and undergoes intramembranous instead of endochondral ossification. By quantitative real-time PCR technique, we could show that VEGF gene expression levels were significantly higher in fetal than in adult samples, especially in femoral tissue. Western blotting analysis confirmed higher protein expression of VEGF in the fetal femur respect to the mandible. Moreover, immunohistochemistry revealed that in both fetal tissues VEGF expression was mainly localized in pre- and osteoblasts. Differential expression of VEGF in femoral and mandibular bone tissues could be related to their different structure, function and development during organogenesis. PMID:25769656

  15. Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

    Science.gov (United States)

    Hu, X Y; Fang, Q; Ma, D; Jiang, L; Yang, Y; Sun, J; Yang, C; Wang, J S

    2016-06-10

    Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

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

    Directory of Open Access Journals (Sweden)

    Elliott H Sohn

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

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

    Science.gov (United States)

    Al Suleimani, Y M; Hiley, C R

    2016-01-01

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

  18. Endothelial to Mesenchymal Transition (EndoMT) in the Pathogenesis of Human Fibrotic Diseases

    Science.gov (United States)

    Piera-Velazquez, Sonsoles; Mendoza, Fabian A.; Jimenez, Sergio A.

    2016-01-01

    Fibrotic diseases encompass a wide spectrum of clinical entities including systemic fibrotic diseases such as systemic sclerosis, sclerodermatous graft versus host disease, nephrogenic systemic fibrosis, and IgG4-associated sclerosing disease, as well as numerous organ-specific disorders including radiation-induced fibrosis, and cardiac, pulmonary, liver, and kidney fibrosis. Although their causative mechanisms are quite diverse, these diseases share the common feature of an uncontrolled and progressive accumulation of fibrous tissue macromolecules in affected organs leading to their dysfunction and ultimate failure. The pathogenesis of fibrotic diseases is complex and despite extensive investigation has remained elusive. Numerous studies have identified myofibroblasts as the cells responsible for the establishment and progression of the fibrotic process. Tissue myofibroblasts in fibrotic diseases originate from several sources including quiescent tissue fibroblasts, circulating CD34+ fibrocytes, and the phenotypic conversion of various cell types including epithelial and endothelial cells into activated myofibroblasts. However, the role of the phenotypic transition of endothelial cells into mesenchymal cells (Endothelial to Mesenchymal Transition or EndoMT) in the pathogenesis of fibrotic disorders has not been fully elucidated. Here, we review the evidence supporting EndoMT’s contribution to human fibrotic disease pathogenesis. PMID:27077889

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

    Directory of Open Access Journals (Sweden)

    Figulla Hans R

    2004-05-01

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

  20. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells.

    Science.gov (United States)

    van den Biggelaar, Maartje; Hernández-Fernaud, Juan Ramon; van den Eshof, Bart L; Neilson, Lisa J; Meijer, Alexander B; Mertens, Koen; Zanivan, Sara

    2014-03-20

    Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function. PMID:24501219

  1. Effect of non-modified and modified nanodiamond particles by Fenton reaction on human endothelial cells

    Directory of Open Access Journals (Sweden)

    K. Solarska

    2010-12-01

    Full Text Available Purpose: The use of carbon nanoparticles in medicine is increasing due to their high biocompatibility. Nanopowders are one of the best materials which can be used in medicine on medical implants and surgical tools. DPP (Diamond Powder Particles obtained by different methods which can be expected to affect their properties, including biocompatibility, were compared. The aim of the present study was to compare the biocompatibility of Diamond Powder Particles (DPP obtained by detonation method and graphite on the basis of their interactions with human endothelial cells.Design/methodology/approach: The effect of nanodiamonds on cell proliferation HUVEC-ST and production of reactive oxygen species (ROS and reactive nitrogen species (RNS was studied. We used FT-IR Spectroscopy attributive chemical function groups.Findings: In this subject the material characterization of nanodiamond powders and influence on endothelial cells.Practical implications: Biological research with endothelial cells and nanodiamond powder are the introduction to application in human’s treatment.Originality/value: Nanodiamond powders with chemical modified surface.

  2. Selected Activities of Citrus Maxima Merr. Fruits on Human Endothelial Cells: Enhancing Cell Migration and Delaying Cellular Aging

    OpenAIRE

    Paiwan Buachan; Linda Chularojmontri; Wattanapitayakul, Suvara K.

    2014-01-01

    Endothelial injury and damage as well as accumulated reactive oxygen species (ROS) in aging play a significant role in the development of cardiovascular disease (CVD). Recent studies show an association of high citrus fruit intake with a lower risk of CVD and stroke but the mechanisms involved are not fully understood. This study investigated the effects of pummelo (Citrus maxima Merr. var. Tubtim Siam, CM) fruit extract on human umbilical vein endothelial cell (HUVECs) migration and aging. T...

  3. Biophysical Properties of Scaffolds Modulate Human Blood Vessel Formation from Circulating Endothelial Colony-Forming Cells

    Science.gov (United States)

    Critser, Paul J.; Yoder, Mervin C.

    A functional vascular system forms early in development and is continually remodeled throughout the life of the organism. Impairment to the regeneration or repair of this system leads to tissue ischemia, dysfunction, and disease. The process of vascular formation and remodeling is complex, relying on local microenvironmental cues, cytokine signaling, and multiple cell types to function properly. Tissue engineering strategies have attempted to exploit these mechanisms to develop functional vascular networks for the generation of artificial tissues and therapeutic strategies to restore tissue homeostasis. The success of these strategies requires the isolation of appropriate progenitor cell sources which are straightforward to obtain, display high proliferative potential, and demonstrate an ability to form functional vessels. Several populations are of interest including endothelial colony-forming cells, a subpopulation of endothelial progenitor cells. Additionally, the development of scaffolds to deliver and support progenitor cell survival and function is crucial for the formation of functional vascular networks. The composition and biophysical properties of these scaffolds have been shown to modulate endothelial cell behavior and vessel formation. However, further investigation is needed to better understand how these mechanical properties and biophysical properties impact vessel formation. Additionally, several other cell populations are involved in neoangiogenesis and formation of tissue parenchyma and an understanding of the potential impact of these cell populations on the biophysical properties of scaffolds will also be needed to advance these strategies. This chapter examines how the biophysical properties of matrix scaffolds can influence vessel formation and remodeling and, in particular, the impact on in vivo human endothelial progenitor cell vessel formation.

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

    Directory of Open Access Journals (Sweden)

    Wei Ruoxin

    2013-02-01

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

  5. TGF-beta is required for vascular barrier function, endothelial survival and homeostasis of the adult microvasculature.

    Directory of Open Access Journals (Sweden)

    Tony E Walshe

    Full Text Available Pericyte-endothelial cell (EC interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-beta signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-beta signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-beta signaling was inhibited by systemic expression of soluble endoglin (sEng and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-beta signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG. Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-beta signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-beta signaling. These results implicate constitutive TGF-beta signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-beta signaling in vasoproliferative and vascular degenerative retinal diseases.

  6. Expression of platelet-endothelial cell adhesion molecule-1 in human umbilical vein endothelial cells by exposure to advanced glycosylation end products and inflammatory mediators

    Institute of Scientific and Technical Information of China (English)

    孟丹; 刘乃丰

    2003-01-01

    Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.Methods Cultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factord-α (TNF-α), interferon (IFN-γ), TNF-α+IFN-γ and AGEs-BSA+TNF-α for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with β-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).Results There were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods (P> 0.05). However, PECAM-1 expression was reduced in the cells treated with TNF-α, IFN-γ, TNF-α+IFN-γ and AGEs-BSA+TNF-α. The level of PECAM-1 treated with AGEs-BSA+TNF-α was lower than that of TNF-α treated alone (P<0.01).Conclusions AGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-α, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.

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

    OpenAIRE

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

    2013-01-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL m....... The results suggest that both EL and LPL participate in the supply of nutrients and steroidogenesis in the testes, and that especially EL may be important for the supply of cholesterol for testosterone production in the Leydig cells. The partial cellular separation of the expression of the two lipases...

  9. A biomimetic physiological model for human adipose tissue by adipocytes and endothelial cell cocultures with spatially controlled distribution.

    Science.gov (United States)

    Yao, Rui; Du, Yanan; Zhang, Renji; Lin, Feng; Luan, Jie

    2013-08-01

    An in vitro model that recapitulates the characteristics of native human adipose tissue would largely benefit pathology studies and therapy development. In this paper, we fabricated a physiological model composed of both human adipocytes and endothelial cells with spatially controlled distribution that biomimics the structure and composition of human adipose tissue. Detailed studies into the cell-cell interactions between the adipocytes and endothelial cells revealed a mutual-enhanced effect which resembles the in vivo routine. Furthermore, comparisons between planar coculture and model coculture demonstrated improved adipocyte function as well as endothelial cell proliferation under the same conditions. This research provided a reliable model for human adipose tissue development studies and potential obesity-related therapy development.

  10. Lectins as markers of endothelial cells: comparative study between human and animal cells.

    Science.gov (United States)

    Roussel, F; Dalion, J

    1988-04-01

    Vascular endothelial cells were labelled with 10 vegetal lectins and 3 more monoclonal antibodies antiblood group ABO substances, in major organs of 14 common laboratory animals. After fixation in PLPa and paraffin embedding, cells were examined to determine their likeness to human cells. The most interesting reactive used was EEA, whose positivity defines upper mammalians. Blood B substance positivity and CSA negativity defines primates among which man is unique and defined by UEA I positivity and variability in ABO substance. CSA positivity defines non-primate upper mammalians. Rodents and birds were negative with all reactives tested. From the histochemical point of view, the animals closest to humans are monkeys, followed by swine and oxen, then by cat and dog and lastly by sheep. Rodents appear unrelated to humans in this system.

  11. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    Science.gov (United States)

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.

  12. Effects of 60Co γ-ray irradiation on expression of surface antigens in endothelial cells of human umbilical veins

    International Nuclear Information System (INIS)

    Culture of endothelial cells of human umbilical veins and avidin-biotin peroxidase complex (ABC) immunochemical technique were used in the experiment to detect the surface antigens in endothelial cells. Endothelial cells separated from five umbilical cords in original culture were divided into two groups, irradiated and non-irradiated. The cells were irradiated with 15 Gy of 60Co γ-rays at dose rates of 21.78 cGy/min. Then antigens RBC A, HLA-ABC, HLA-DR, CD4 and CD8 were assayed for both groups by the method of ABC. The results showed that the values of integrated optical density (IOD) for the surface antigens in the irradiated cells were lower than those in the non-irradiated cells with the difference in antigen expression in endothelial cells being significant (P<0.05) between the two groups

  13. Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes.

    Directory of Open Access Journals (Sweden)

    Inês G Mollet

    Full Text Available Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10μmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron.Confluent primary human endothelial cells (EC were treated with filter-sterilized iron (II citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated using directional RNA sequencing with no pre-specification of target sequences. Alignments were counted for exons and junctions of the gene strand only, blinded to treatment types.Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10μmol/L iron (II citrate, compared to media-treated cells. Clustering for Gene Ontology (GO performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016, 0.0024 and 0.0032 respectively, and by 6 hours, to cellular response to DNA damage stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR foci, and p53 stabilization.These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium, and induce a DNA damage response.

  14. Rac1 regulates the release of Weibel-Palade Bodies in human aortic endothelial cells

    Institute of Scientific and Technical Information of China (English)

    YANG Shui-xiang 杨水祥; YAN Juan 闫娟; Shailesh S. Deshpande; Kaikobad Irani; Charles J. Lowenstein

    2004-01-01

    Background The release of Weibel-Palade Bodies (WPB) is a form of endothelial cell activation. But the signal transduction pathway leading to WPB release is not yet defined. We hypothesized that small G-protein rac1 and reactive oxygen species (ROS) mediate the ligand induced release of Weibel-Palade Bodies. Methods We tested this hypothesis by using wild-type and mutant adenoviral rac1 expression vectors, and by manipulating the production and destruction of superoxide and hydrogen peroxide in human aortic endothelial cells (HAEC). Results Thrombin (1.0 Unit, 30 min) induced the increase of WPB release by 3.7-fold in HAEC, and that H2O2 (0.1 mmol/L, 30 min) induced by 4.5-fold. These results correlated with thrombin-stimulated activation of rac-GTP binding activity by 3.5-fold, and increase of ROS production by 3.4-fold. The dominant negative adenoviral rac-N17 gene transfer dramatically inhibited the release of WPB by 64.2% (control) and 77.3% (thrombin-stimulation), and decreased ROS production by 65.5% (control) and 83.6% (thrombin-stimulation) compared with non-infected cells, respectively. Anti-oxidants, catalase and N-acetyl-cysteine significantly decreased the release of WPB by 34% and 79% in control cells, and further decreased by 63.6% and 46.7% in rac-N17 transferred cells compared with non-infected cells. We also confirmed that rac1 was located upstream of ROS in the WPB release pathway. Conclusions Small G-protein rac1 medicates ligand-induced release of Weibel-Palade Bodies in human aortic endothelial cells, and the signal pathway of WPB release is a rac1-dependent ROS regulating mechanism.

  15. Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells.

    Science.gov (United States)

    Gentile, Antonietta; Toietta, Gabriele; Pazzano, Vincenzo; Tsiopoulos, Vasileios D; Giglio, Ada Francesca; Crea, Filippo; Pompilio, Giulio; Capogrossi, Maurizio C; Di Rocco, Giuliana

    2011-03-01

    Recent studies have underscored a role for the epicardium as a source of multipotent cells. Here, we investigate the myogenic potential of adult human epicardium-derived cells (EPDCs) and analyze their ability to undergo skeletal myogenesis when cultured with differentiating primary myoblasts. Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative. We demonstrate that EPDCs spontaneously fuse with pre-existing myotubes with an efficiency that is significantly higher than that of other cells. Although at a low frequency, endothelial cells may also contribute to myotube formation. In all cases analyzed, after entering the myotube, nonmuscle nuclei are reprogrammed to express muscle-specific genes. The fusion competence of nonmyogenic cells in vitro parallels their ability to reconstitute dystrophin expression in mdx mice. We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur. Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells. PMID:21209317

  16. Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.

  17. Prunella vulgaris L. Upregulates eNOS expression in human endothelial cells.

    Science.gov (United States)

    Xia, Ning; Bollinger, Larissa; Steinkamp-Fenske, Katja; Förstermann, Ulrich; Li, Huige

    2010-01-01

    The purported effects of "circulation-improving" herbs used in traditional Chinese medicine (TCM) show striking similarities with the vascular actions of nitric oxide (NO) produced by the endothelial NO synthase (eNOS). We have previously reported that Salviae miltiorrhizae radix and Zizyphi spinosae semen upregulate eNOS expression. In the present study, we studied the effect on eNOS gene expression of 15 Chinese herbs with potential effects on the vasculature, and identified Prunella vulgaris L. (PVL) (flowering spike) as a potent eNOS-upregulating agent. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVEC), an aqueous extract of PVL increased eNOS promoter activity, eNOS mRNA and protein expressions, as well as NO production in concentration- and time-dependent manners. We have previously shown that ursolic acid (a constituent of Salviae miltiorrhizae radix), betulinic acid (a compound present in Zizyphi spinosae semen), luteolin and cynaroside (ingredients of artichoke, Cynara scolymus L.) are capable of enhancing eNOS gene expression. These compounds are also present in significant quantities in PVL. Thus, PVL contains active principles that stimulate human eNOS gene expression, and such compounds may have therapeutic potential against cardiovascular diseases. PMID:20503475

  18. Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells

    International Nuclear Information System (INIS)

    Background: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. Methods and results: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24 h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. Conclusion: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses

  19. Hypoxia and the Presence of Human Vascular Endothelial Cells Affect Prostate Cancer Cell Invasion and Metabolism

    Directory of Open Access Journals (Sweden)

    Ellen Ackerstaff

    2007-12-01

    Full Text Available Tumor progression and metastasis are influenced by hypoxia, as well as by interactions between cancer cells and components of the stroma, such as endothelial cells. Here, we have used a magnetic resonance (MRcompatible invasion assay to further understand the effects of hypoxia on human prostate cancer cell invasion and metabolism in the presence and absence of human umbilical vein endothelial cells (HUVECs. Additionally, we compared endogenous activities of selected proteases related to invasion in PC-3 cells and HUVECs, profiled gene expression of PC-3 cells by microarray, evaluated cell proliferation of PC-3 cells and HUVECs by flow cytometry, under hypoxic and oxygenated conditions. The invasion of less-invasive DU-145 cells was not affected by either hypoxia or the presence of HUVECs. However, hypoxia significantly decreased the invasion of PC-3 cells. This hypoxia-induced decrease was attenuated by the presence of HUVECs, whereas under oxygenated conditions, HUVECs did not alter the invasion of PC-3 cells. Cell metabolism changed distinctly with hypoxia and invasion. The endogenous activity of selected extracellular proteases, although altered by hypoxia, did not fully explain the hypoxia-induced changes in invasion. Gene expression profiling indicated that hypoxia affects multiple cellular functions and pathways.

  20. Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Du-Hu Liu; Yu-Bo Chai; Ming Jin; Xue-Yong Zhang; Dai-Ming Fan; Yu-Xin Huang; Jin-Shan Zhang; Wei-Quan Huang; Yuan-Qiang Zhang; Qing-Sheng Huang; Wen-Yu Ma

    2001-01-01

    AIM To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesisof human gastric carcinoma more directly. METHODS The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF165 complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or downregulated. RESULTS VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR,localized in both the cytoplasm and membrane.Introduction of VEGF165 antisense into human gastric cancer cells ( SGC-7901, immunofluorescence intensity,31.6%)) resulted in a significant reduction in VEGFspecific messenger RNA and total and cell surface VEGF protein ( immunofluorescence intensity, 8.9%)(P<0.05). Conversely, stable integration of VEGF165 in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity,75.4%) (P<0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tomor volume: 345.40 ± 136.31 mm3) (P<0.05 vs control SGC7901 group: 1534.40 ± 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tomor volume: 2350.50 ± 637.70mm3) (P<0.05 vs control SGC-7901 group). CONCLUSION This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

  1. Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells

    OpenAIRE

    Pendyala, Srikanth; Gorshkova, Irina A.; Usatyuk, Peter V.; He, Donghong; Pennathur, Arjun; Lambeth, J. David; Thannickal, Victor J.; Natarajan, Viswanathan

    2009-01-01

    In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot a...

  2. Mitotic and antiapoptotic effects of nanoparticles coencapsulating human VEGF and human angiopoietin-1 on vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Khan AA

    2011-05-01

    Full Text Available Afshan Afsar Khan, Arghya Paul, Sana Abbasi, Satya PrakashBiomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering Faculty of Medicine, McGill University Montreal, Québec, CanadaBackground: Research towards the application of nanoparticles as carrier vehicles for the delivery of therapeutic agents is increasingly gaining importance. The angiogenic growth factors, human vascular endothelial growth factor (VEGF and human angiopoietin-1 are known to prevent vascular endothelial cell apoptosis and in fact to stimulate human vascular endothelial cell (HUVEC proliferation. This paper aims to study the combined effect of these bioactive proteins coencapsulated in human serum albumin nanoparticles on HUVECs and to evaluate the potential application of this delivery system towards therapeutic angiogenesis.Methods and results: The angiogenic proteins, human VEGF and human angiopoietin-1, were coencapsulated in albumin nanoparticles for better controlled delivery of the proteins. The application of a nanoparticle system enabled efficient and extended-release kinetics of the proteins. The size of the nanoparticles crosslinked with glutaraldehyde was 101.0 ± 0.9 nm and the zeta potential was found to be -18 ± 2.9 mV. An optimal concentration of glutaraldehyde for the nanoparticle coating process was determined, and this provided stable and less toxic nanoparticles as protein carriers. The results of the study indicate that nanoparticles crosslinked with glutaraldehyde produced nanoparticles with tolerable toxicity which provided efficient and controlled release of the coencapsulated proteins. The nanoparticles were incubated for two weeks to determine the release profiles of the proteins. At the end of the two-week incubation period, it was observed that 49% ± 1.3% of human angiopoietin-1 and 59% ± 2.1% of human VEGF had been released from the nanoparticles. The proliferation and percent apoptosis of the HUVECs in

  3. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    Science.gov (United States)

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-10-01

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

  5. Pro-inflammatory cytokines downregulate Hsp27 and cause apoptosis of human retinal capillary endothelial cells

    Science.gov (United States)

    Nahomi, Rooban B.; Palmer, Allison; Roth, Katelyn E.; Fort, Patrice E.; Nagaraj, Ram H.

    2013-01-01

    The formation of acellular capillaries in the retina, a hallmark feature of diabetic retinopathy, is caused by apoptosis of endothelial cells and pericytes. The biochemical mechanism of such apoptosis remains unclear. Small heat shock proteins play an important role in the regulation of apoptosis. In the diabetic retina, pro-inflammatory cytokines are upregulated. In this study, we investigated the effects of pro-inflammatory cytokines on small heat shock protein 27 (Hsp27) in human retinal endothelial cells (HREC). In HREC cultured in the presence of cytokine mixtures (CM), a significant downregulation of Hsp27 at the protein and mRNA level occurred, with no effect on HSF-1, the transcription factor for Hsp27. The presence of high glucose (25 mM) amplified the effects of cytokines on Hsp27. CM activated indoleamine 2,3-dioxygenase (IDO) and enhanced the production of kynurenine and ROS. An inhibitor of IDO, 1-methyl tryptophan (MT), inhibited the effects of CM on Hsp27. CM also upregulated NOS2 and, consequently, nitric oxide (NO). A NOS inhibitor, L-NAME, and a ROS scavenger blocked the CM-mediated Hsp27 downregulation. While a NO donor in the culture medium did not decrease the Hsp27 content, a peroxynitrite donor and exogenous peroxynitrite did. The cytokines and high glucose-induced apoptosis of HREC were inhibited by MT and L-NAME. Downregulation of Hsp27 by a siRNA treatment promoted apoptosis in HREC. Together, these data suggest that pro-inflammatory cytokines induce the formation of ROS and NO, which, through the formation of peroxynitrite, reduce the Hsp27 content and bring about apoptosis of retinal capillary endothelial cells. PMID:24252613

  6. SODs are involved in the regulation of ICAM-1 expression in human melanoma and endothelial cells.

    Science.gov (United States)

    Morandini, R; Boeynaems, J M; Duhant, X; Jacquemotte, F; Kinnaert, E; Ghanem, G

    1999-11-01

    It is well known that ICAM-1 expression can be stimulated by TNF and by oxidative stress, via the activation of specific transcription factors. Two of these--NFkappaB and AP-1--can also be activated by reactive oxygen species, including the superoxide anion (also produced under TNF challenge). The latter is inactivated by superoxide dismutase of which two forms exist: Cu/Zn-SOD (cytoplasmic) and Mn-SOD (mitochondrial). We investigated whether superoxide anion direct generation or accumulation through specific SOD inhibition, may affect ICAM-1 expression in human melanoma and endothelial cells. Our results show a 20-50% increase in both SOD activities when cells were exposed to TNF or to an oxidative stress produced by Paraquat (a generator of superoxide anion radicals), both in terms of enzymes activity (zymogram) and protein levels (Western blotting and ELISA). Either with TNF or Paraquat, we could measure a significant increase of ICAM-1 expression with maxima ranging from 140 to 200%, depending on the cell line. Specific inhibition of Cu/Zn-SOD activity by DTIC (diethyldithiocarbamic acid), in presence of Paraquat or TNF, was followed by an upregulation of ICAM-1 expression (60 and 20%, respectively). In contrast, the addition of a SOD mimetic (MnTMPyP) completely inhibited Paraquat-stimulated ICAM-1 expression in melanoma cells and significantly decreased it in HUVEC (50%). In presence of TNF however, the same SOD mimetic inhibited TNF-stimulated ICAM-1 expression by 25% in melanoma and 17% in endothelial cells. In conclusion, these data provide evidence that melanoma and endothelial cells exposure to TNF or oxidative stress results in a significant increase of both Mn- and Cu/Zn-SOD activities. This increase seems to be associated with a reduction in the stimulation of ICAM-1 expression by TNF or oxidative stress. PMID:10644010

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

    Science.gov (United States)

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

    2014-04-01

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

  8. Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

    International Nuclear Information System (INIS)

    Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

  9. Coffee bean polyphenols ameliorate postprandial endothelial dysfunction in healthy male adults.

    Science.gov (United States)

    Ochiai, Ryuji; Sugiura, Yoko; Otsuka, Kazuhiro; Katsuragi, Yoshihisa; Hashiguchi, Teruto

    2015-05-01

    To reveal the effect of coffee bean polyphenols (CBPs) on blood vessels, this study aimed to investigate the effect of CBPs on acute postprandial endothelial dysfunction. Thirteen healthy non-diabetic men (mean age, 44.9 ± 1.4 years) consumed a test beverage (active: containing CBPs, placebo: no CBPs) before a 554-kcal test meal containing 14 g of protein, 30 g of fat and 58 g of carbohydrates. Then, a crossover analysis was performed to investigate the time-dependent changes in flow-mediated dilation (FMD) in the brachial artery. In the active group, the postprandial impairment of FMD was significantly improved, the two-hour postprandial nitric oxide metabolite levels were significantly increased and the six-hour postprandial urinary 8-epi-prostaglandin F2α levels were significantly reduced compared to the placebo group. The test meal increased the levels of blood glucose, insulin and triglycerides in both groups with no significant intergroup differences. These findings indicate that CBPs intake ameliorates postprandial endothelial dysfunction in healthy men.

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

    Directory of Open Access Journals (Sweden)

    Shwetank

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

  11. Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in association with neovascularization in human primary astrocytoma

    Institute of Scientific and Technical Information of China (English)

    PAN Jian-wei; ZHAN Ren-ya; TONG Ying; ZHOU Yong-qing; ZHANG Ming

    2005-01-01

    Objective: To investigate the relationship between the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and angiogenesis in primary astrocytoma. Methods: Thirty-seven primary astrocytomas and 4 astrocytic hyperplasia samples were collected and divided into three groups according to histological grade. The expression of eNOS, VEGF and factor Ⅷ related antigen (FVIIIRAg) were assayed by immunohistochemistry. Microvascular density was assessed by FVIIIRAg immunoreactivity. The intensity of immunoreactivity was graded according to the percentage of positive tumor cells. Results: No eNOS and VEGF were expressed in the astrocytes and vascular endothelium in astrocytic hyperplasia.The expression of eNOS or VEGF was light in low-grade astrocytoma and strong in glioblastoma. eNOS expression in astrocytoma was very positively correlated with VEGF. eNOS and VEGF expression in anaplastic astrocytoma was median in contrast to the low grade astrocytoma and glioblastoma. Lower microvascular density was found in low grade astrocytoma than that in higher grade malignant ones. The expressions of eNOS and VEGF were correlated with microvascular density and tumor malignancy.Conclusion: This finding suggests that eNOS and VEGF may have cooperative effect in tumor angiogenesis and play an important role in the pathogenesis of primary astrocytoma.

  12. Staphylococcal SSL5 Binding to Human Leukemia Cells Inhibits Cell Adhesion to Endothelial Cells and Platelets

    Directory of Open Access Journals (Sweden)

    Annemiek M. E. Walenkamp

    2010-01-01

    Full Text Available Bacterial proteins provide promising tools for novel anticancer therapies. Staphylococcal superantigen-like 5 (SSL5 was recently described to bind P-selectin glycoprotein ligand-1 (PSGL-1 on leukocytes and to inhibit neutrophil rolling on a P-selectin surface. As leukocytes and tumor cells share many characteristics in migration and dissemination, we explored the potential of SSL5 as an antagonist of malignant cell behavior. Previously, it was demonstrated that rolling of human HL-60 leukemia cells on activated endothelial cells was mediated by P-selectin. In this study, we show that SSL5 targets HL-60 cells. Binding of SSL5 was rapid and without observed toxicity. Competition of SSL5 with the binding of three anti-PSGL-1 antibodies and P-selectin to HL-60 cells identified PSGL-1 as the ligand on HL-60 cells. Presence of sialyl Lewis x epitopes on PSGL-1 was crucial for its interaction with SSL5. Importantly, SSL5 not only inhibited the interaction of HL-60 cells with activated endothelial cells but also with platelets, which both play an important role in growth and metastasis of cancers. These data support the concept that SSL5 could be a lead in the search for novel strategies against hematological malignancies.

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

    Science.gov (United States)

    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

  14. Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach

    Science.gov (United States)

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

    2016-10-01

    Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells.

  15. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells.

    Science.gov (United States)

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong; Kwon, Hyuk-Sang

    2016-03-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  16. In Vitro Effects of Hollow Gold Nanoshells on Human Aortic Endothelial Cells.

    Science.gov (United States)

    Gu, Chunrong; Wu, Hengfang; Ge, Gaoyuan; Li, Xiongzhi; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Lu, Hua; Chen, Xiangjian; Yang, Di

    2016-12-01

    Gold nanoparticles are emerging as promising biomedical tools due to their unique nanoscale characteristics. Our purpose was to synthesize a hollow-shaped gold nanoparticle and to investigate its effect on human aortic endothelial cells (HAECs) in vitro. Hollow gold nanoshells with average 35-nm diameters and 10-nm shell thickness were obtained by galvanic replacement using quasi-spherical nanosilver as sacrifice-template. Our results showed that hollow gold nanoshells in the culture medium could be internalized into the cytoplasm of HAECs. No cytotoxicity effect of hollow gold nanoshells on HAECs was observed within the test concentrations (0-0.8 μg/mL) and test exposure period (0-72 h) by tetrazolium dye assay. Meanwhile, the release of cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without hollow gold nanoshells). The concentrations of vasodilators, nitric oxide, and prostacyclin I-2 were not changed, but the vasoconstrictor endothelin-1 was decreased by hollow gold nanoshells treatment in HAECs. HAECs exposed to hollow gold nanoshells resulted in suppressing expressions of genes involved in apoptosis and activating expressions of genes of adhesion molecules. Moreover, we demonstrated by in vitro endothelial tube formation that hollow gold nanoshells (0.8 μg/mL) could not inhibit angiogenesis by the HAECs. Altogether, these results indicate that the structure and major function of HAECs would not be disrupted by hollow gold nanoshell treatment. PMID:27624340

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

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    Yumei Zhang

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

  18. Aberrant Phenotype in Human Endothelial Cells of Diabetic Origin: Implications for Saphenous Vein Graft Failure?

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    Anna C. Roberts

    2015-01-01

    Full Text Available Type 2 diabetes (T2DM confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC cultured from T2DM and nondiabetic (ND patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV- EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30% and angiogenesis (~40% compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp., effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.

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

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    Joyce Geelen

    2014-07-01

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

  20. Butein Inhibits Angiogenesis of Human Endothelial Progenitor Cells via the Translation Dependent Signaling Pathway

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    Ching-Hu Chung

    2013-01-01

    Full Text Available Compelling evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs can contribute to postnatal neovascularization and tumor angiogenesis. EPCs have been shown to play a “catalytic” role in metastatic progression by mediating the angiogenic switch. Understanding the pharmacological functions and molecular targets of natural products is critical for drug development. Butein, a natural chalcone derivative, has been reported to exert potent anticancer activity. However, the antiangiogenic activity of butein has not been addressed. In this study, we found that butein inhibited serum- and vascular endothelial growth factor- (VEGF- induced cell proliferation, migration, and tube formation of human EPCs in a concentration dependent manner without cytotoxic effect. Furthermore, butein markedly abrogated VEGF-induced vessels sprouting from aortic rings and suppressed microvessel formation in the Matrigel implant assay in vivo. In addition, butein concentration-dependently repressed the phosphorylation of Akt, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in EPCs. Taken together, our results demonstrate for the first time that butein exhibits the antiangiogenic effect both in vitro and in vivo by targeting the translational machinery. Butein is a promising angiogenesis inhibitor with the potential for treatment of cancer and other angiogenesis-related diseases.

  1. Biocompatibility of pure titanium modified by human endothelial cell-derived extracellular matrix

    International Nuclear Information System (INIS)

    Extracellular matrix (ECM) used to modify biomaterial surface is a promising method for improving cardiovascular material hemocompatibility. In the present work, human umbilical vein endothelial cells (HUVECs) are cultured and native ECM is obtained on pure titanium surface. Fourier infrared spectrum (FTIR) test proves the existence of amide I and amide II band on the modified titanium surface. X-ray photoelectron spectroscopy (XPS) further confirms the chemical composition and binding types of the ECM proteins on the titanium substrate. The results of light microscopy and atomic force microscopy (AFM) exhibit the morphology of HUVEC derived ECM. There are higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) are presented on ECM-covered titanium surface by immunofluorescence staining. The biological behavior of cultured HUVECs and adherent platelets on different samples are investigated by in vitro HUVECs culture and platelet adhesion. Cells exhibit better morphology and their proliferation ability greatly improve on the ECM-covered titanium. At the same time, the platelet adhesion and spreading are inhibited on ECM-covered titanium surface. These investigations demonstrate that ECM produced by HUVECs cannot only improve adhesion and proliferation ability of endothelial cell but also inhibit adhesion and activation of platelets. Thus, the approach described here may provide a basis for preparation of modified surface in cardiovascular implants application.

  2. In Vitro Effects of Hollow Gold Nanoshells on Human Aortic Endothelial Cells

    Science.gov (United States)

    Gu, Chunrong; Wu, Hengfang; Ge, Gaoyuan; Li, Xiongzhi; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Lu, Hua; Chen, Xiangjian; Yang, Di

    2016-09-01

    Gold nanoparticles are emerging as promising biomedical tools due to their unique nanoscale characteristics. Our purpose was to synthesize a hollow-shaped gold nanoparticle and to investigate its effect on human aortic endothelial cells (HAECs) in vitro. Hollow gold nanoshells with average 35-nm diameters and 10-nm shell thickness were obtained by galvanic replacement using quasi-spherical nanosilver as sacrifice-template. Our results showed that hollow gold nanoshells in the culture medium could be internalized into the cytoplasm of HAECs. No cytotoxicity effect of hollow gold nanoshells on HAECs was observed within the test concentrations (0-0.8 μg/mL) and test exposure period (0-72 h) by tetrazolium dye assay. Meanwhile, the release of cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without hollow gold nanoshells). The concentrations of vasodilators, nitric oxide, and prostacyclin I-2 were not changed, but the vasoconstrictor endothelin-1 was decreased by hollow gold nanoshells treatment in HAECs. HAECs exposed to hollow gold nanoshells resulted in suppressing expressions of genes involved in apoptosis and activating expressions of genes of adhesion molecules. Moreover, we demonstrated by in vitro endothelial tube formation that hollow gold nanoshells (0.8 μg/mL) could not inhibit angiogenesis by the HAECs. Altogether, these results indicate that the structure and major function of HAECs would not be disrupted by hollow gold nanoshell treatment.

  3. Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells

    Science.gov (United States)

    Meiring, Muriel; Allers, W.; Le Roux, E.

    2016-01-01

    Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present.

  4. A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

    Institute of Scientific and Technical Information of China (English)

    Clara Bueno; Agustin F Femández; Mario F Fraga; Inmaculada Moreno-Gimeno; Deborah Burks; Maria del Carmen Plaza-Calonge; Juan C Rodríguez-Manzaneque; Pablo Menendez; Rosa Montes; Gustavo J Melen; Verónica Ramos-Mejia; Pedro J Real; Verónica Ayllón; Laura Sanchez; Gertrudis Ligero; Iván Gutierrez-Aranda

    2012-01-01

    The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in inants.Although it is well established that MLL-AF4 arises prenatally during human development,its effects on hematopoieric development in utero remain unexplored.We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs).Functional studies,clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic,functional and gene expression impact.MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs.Functionally,MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate.MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation,as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis.Furthermore,we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells.This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes,known to arise prenatally,regulate human embryonic hematopoietic specification.

  5. Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Zong-Hai Huang; Wen-Yu Yang; Qi Cheng; Jing-Long Yu; Zhou Li; Zong-Yan Tong; Hui-Juan Song; Xiao-Yan Che

    2005-01-01

    AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli(E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDRCDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured.RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.

  6. Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels.

    Science.gov (United States)

    Hewett, Peter W

    2016-01-01

    The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells. PMID

  7. Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels.

    Science.gov (United States)

    Hewett, Peter W

    2016-01-01

    The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells.

  8. Effects of Obstructive Sleep Apneas on Endothelial Function and Autonomic Modulation in Adult Man

    Institute of Scientific and Technical Information of China (English)

    Xu Zhong; Yi Xiao; Rong Huang

    2012-01-01

    Objective To study the effects of obstructive sleep apneas on endothelial function and autonomic modulation.Methods From June 2009 to June 201 1,male patients with obstructive sleep apnea hypopnea syndrome (OSAHS) were consecutively enrolled in this study.Patients with an apnea/hypopnea index (AHI) of greater than 15 and without previous treatment for OSAHS were included as Group OSAHS and obese subjects with an AHI of less than 5 were included as non-OSAHS controls (Group Control).Electrocardiography and beat-to-beat blood pressure were continuously recorded from the radial artery by applanation tonometry which was synchronized with polysomnography recording.Endothelial function was measured by arterial augmentation index (AAI).Spectral analysis of heart rate variability (HRV) and blood pressure variability (BPV) were computed for cardiac parasympathetic modulation (high frequency power,HF); sympathetic modulation (low frequency power,LF),sympathovagal balance (LF/HF power of R-R variability,LF/HF)and BPV sympathetic modulation (BPV LF) in normalized units [total power of the components/(total power-very LF power) × 100].Results Finally,27 moderate-severe OSAHS patients and 22 non-OSAHS obese controls were recruited in the Group OSAHS and Group Control,respectively.In Group OSAHS,the age was 43.3±9.3 year-old,body mass index (BMI) was 36.8±8.7 kg/m2; in Group Control,the age was 42.9±8.6 year-old,BMI was 34.4±7.9 kg/m2; there were no significant differences in age and BMI between the Group OSAHS and Group Control (all P>0.05).The baseline AAI (12.5%±2.2% vs.8.2%±2.1%) and BPV LF (68.3%± 13.5% vs.61.1%±11.7%) of the Group OSAHS were significantly higher than those of the Group Control (all P<0.05).And after overnight sleep,systolic BP (143.7± 14.2 vs.132.8± 13.3 mm Hg),diastolic BP (87.7±7.7 vs.78.6±5.5 mm Hg),HRV LF (69.7%±14.4% vs.64.3%±12.1%),HRV LF/HF (3.7±2.0 vs.2.3± 1.3) and BPV LF (77.8%± 15.6% vs.68.3%±13

  9. Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

    Directory of Open Access Journals (Sweden)

    Fatemeh Kazemi-Lomedasht

    2014-12-01

    Full Text Available Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

  10. Albumin leak across human pulmonary microvascular vs. umbilical vein endothelial cells under septic conditions.

    Science.gov (United States)

    Shelton, Jennifer L; Wang, Lefeng; Cepinskas, Gediminas; Sandig, Martin; Inculet, Richard; McCormack, David G; Mehta, Sanjay

    2006-01-01

    Human pulmonary microvascular endothelial cell (HPMVEC) injury is central to the pathophysiology of human lung injury. However, septic HPMVEC barrier dysfunction and the contribution of neutrophils have not been directly addressed in vitro. Instead, human EC responses are often extrapolated from studies of human umbilical vein EC (HUVEC). We hypothesized that HUVEC was not a good model for investigating HPMVEC barrier function under septic conditions. HPMVEC was isolated from lung tissue resected from lung cancer patients using magnetic bead-bound anti-PECAM-1 antibody. In confluent monolayers in 3-mum cell-culture inserts, we assessed trans-EC Evans-Blue (EB)-conjugated albumin leak under basal, unstimulated conditions and following stimulation with either lipopolysaccharide or a mixture of equal concentrations of TNF-alpha, IL-1beta and IFN-gamma (cytomix). Basal EB-albumin leak was significantly lower across HPMVEC than HUVEC (0.64 +/- 0.06% vs. 1.13 +/- 0.10%, respectively, P neutrophils markedly and dose-dependently enhanced cytomix-induced EB-albumin leak across HPMVEC (P neutrophil presence, and HUVEC is not a suitable model for studying HPMVEC septic barrier responses. The direct study of HPMVEC septic responses will lead to a better understanding of human lung injury. PMID:16376951

  11. Antiangiogenic properties of cafestol, a coffee diterpene, in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shuaiyu [Food Biotechnology, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333 (Korea, Republic of); Korea Food Research Institute, 516 Baekhyun-dong, Bundang-gu, Songnam, Kyungki-do 463-746 (Korea, Republic of); Yoon, Yeo Cho; Sung, Mi-Jeong; Hur, Haeng-Jeon [Korea Food Research Institute, 516 Baekhyun-dong, Bundang-gu, Songnam, Kyungki-do 463-746 (Korea, Republic of); Park, Jae-Ho, E-mail: jaehoparkmail@gmail.com [Korea Food Research Institute, 516 Baekhyun-dong, Bundang-gu, Songnam, Kyungki-do 463-746 (Korea, Republic of)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cafestol inhibits tube formation and migration of VEGF-stimulated HUVEC. Black-Right-Pointing-Pointer Cafestol inhibits phosphorylation of FAK and Akt. Black-Right-Pointing-Pointer Cafestol decreases NO production. -- Abstract: As angiogenesis plays important roles in tumor growth and metastasis, searching for antiangiogenic compounds is a promising tactic for treating cancers. Cafestol, a diterpene found mainly in unfiltered coffee, provides benefit through varied biological activity, including antitumorigenic, antioxidative, and anti-inflammatory effects. This study aimed to investigate the effects of cafestol on angiogenesis and to uncover the associated mechanism. We show that cafestol inhibits angiogenesis of human umbilical vascular endothelial cells. This inhibition affects the following specific steps of the angiogenic process: proliferation, migration, and tube formation. The inhibitory effects of cafestol are accompanied by decreasing phosphorylation of FAK and Akt and by a decrease in nitric oxide production. Overall, cafestol inhibits angiogenesis by affecting the angiogenic signaling pathway.

  12. Have you got any cholesterol? Adults' views of human nutrition

    Science.gov (United States)

    Schibeci, Renato; Wong, Khoon Yoong

    1994-12-01

    The general aim of our human nutrition project is to develop a health education model grounded in ‘everyday’ or ‘situated’ cognition (Hennessey, 1993). In 1993, we began pilot work to document adult understanding of human nutrition. We used a HyperCard stack as the basis for a series of interviews with 50 adults (25 university students, and 25 adults from offcampus). The interviews were transcribed and analysed using the NUDIST computer program. A summary of the views of these 50 adults on selected aspects of human nutrition is presented in this paper.

  13. Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial Cell proliferation in vitro

    Institute of Scientific and Technical Information of China (English)

    Xuan Wang; Fu-Kun Liu; Xi Li; Jai-Sou Li; Gen-Xin Xu

    2002-01-01

    AIM: To constnuct a stable transfectant of human livercarcinoma cell line SMMC7721 that could secret humanencicstatin and to explore the effect of human encostatinexpressed by the transfectant on enciotheliai cell proliferation.METHODS: Recombinant retroviral plasmid pLncx-Endocontaining the eDNA for human endoslsin gene togetherwith mt albumin signal peptide was engineered andtransferred into SMMC7721 cell by lipofectamine. Afterselection with G418, endcotatin-transfected SMMC7721 ceiiswere chosen and expanded. Immunohistochemical stainingand Western blot were used to detect the expression ofhuman endosatin in transfected SMMC7721 cells and itsmedium. The conditioned medium of endostatin-transfectedand control SMMC7721 cells were collected to cultivate withhuman umbilical vein endothelial cells for 72 hours. Theinhibitory effect of endoststin, expressed by transfectedSMMC7721 cells, on endothelial proliferation in vitro wasobserved by using Mn assay.RESULTS: A 550 bp specific fragment of endostatin gene wasdetected from the PCR product of endostatin-transfeclsdSMMC7721 cells. Immunohistochemistry and Western blotanalysis confirmed the expression and secretion of foreighhuman endostatin protein by endoslstin-transfeclsdSMMC7721 cells. In vitro endothelial proliferation assayshowed that 72 hours after cultivation with human umbilicalvein endothelial cells, the optical density (OD) in groupusing the medium from endostatin-transfected SMMC7721cells was 0.51 ±0.06, lower than that from RPMI 1640 group(0.98 ± 0.09) or that from control plasmid pLncx-transfeotedSMMC7721 cells (0. 88 ± 0. 11). The inhibitory rate formedium from endostatin-transfeclsd SMMC7721 cells was 48%, significantly higher than that from empty plasmid plncx-transfected SMMC7721 cells (10.2 %, P< 0.01).CONCLUSION: Human endoslstin can he stably expressedby SMMC7721 cell tran sferred with human endoslsin geneand its product can significantly inhibit the proliferation ofhuman umbilical vein

  14. Effects of the PPARγ agonist troglitazone on endothelial cells in vivo and in vitro: Differences between human and mouse

    International Nuclear Information System (INIS)

    Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists have been or are being developed for clinical use in the treatment of type 2 diabetes mellitus and hyperlipidemias. A common tumor finding in rodent carcinogenicity studies for these agonists is hemangioma/hemangiosarcoma in mice but not in rats. We hypothesized that increased endothelial cell proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice, and we investigated the effects on endothelial cells utilizing troglitazone, the first clinically used PPARγ agonist, in vivo and in vitro. Troglitazone (400 and 800 mg/kg/day) induced hemangiosarcomas in mice in a 2-year bioassay. We showed that troglitazone increased endothelial cell proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses after 4 weeks of treatment. Troglitazone was cytotoxic both to human dermal microvascular endothelial cells (HMEC1) and mouse mammary fat pad microvascular endothelial cells (MFP MVEC) at high concentrations. However, MFP MVEC were more resistant to the cytotoxic effects of troglitazone based on the much lower LC50 in HMEC1 (17.4 μM) compared to MFP MVEC (92.2 μM). Troglitazone increased the proliferation and survival of MFP MVEC but not HMEC1 in growth factor reduced conditions. Our data demonstrate that troglitazone may induce hemangiosarcomas in mice, at least in part, through enhancement of survival and proliferation of microvascular endothelial cells. Such an effect does not occur with human cells, suggesting that human may react differently to exposure to PPAR agonists compared with mice.

  15. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

    Science.gov (United States)

    Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

    2016-01-01

    Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

  16. Adult Education & Human Resource Development: Overlapping and Disparate Fields

    Science.gov (United States)

    Watkins, Karen E.; Marsick, Victoria J.

    2014-01-01

    Adult education and human resource development as fields of practice and study share some roots in common but have grown in different directions in their histories. Adult education's roots focused initially on citizenship for a democratic society, whereas human resource development's roots are in performance at work. While they have…

  17. Human embryonic and fetal mesenchymal stem cells differentiate toward three different cardiac lineages in contrast to their adult counterparts.

    Science.gov (United States)

    Ramkisoensing, Arti A; Pijnappels, Daniël A; Askar, Saïd F A; Passier, Robert; Swildens, Jim; Goumans, Marie José; Schutte, Cindy I; de Vries, Antoine A F; Scherjon, Sicco; Mummery, Christine L; Schalij, Martin J; Atsma, Douwe E

    2011-01-01

    Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

  18. Immuno-localisation of anti-thyroid antibodies in adult human cerebral cortex.

    Science.gov (United States)

    Moodley, Kogie; Botha, Julia; Raidoo, Deshandra Munsamy; Naidoo, Strinivasen

    2011-03-15

    Expression of thyroid-stimulating hormone receptor (TSH-R) has been demonstrated in adipocytes, lymphocytes, bone, kidney, heart, intestine and rat brain. Immuno-reactive TSH-R has been localised in rat brain and human embryonic cerebral cortex but not in adult human brain. We designed a pilot study to determine whether anti-thyroid auto-antibodies immuno-localise in normal adult human cerebral cortex. Forensic samples from the frontal, motor, sensory, occipital, cingulate and parieto-occipito-temporal association cortices were obtained from five individuals who had died of trauma. Although there were no head injuries, the prior psychiatric history of patients was unknown. The tissues were probed with commercial antibodies against both human TSH-R and human thyroglobulin (TG). Anti-TSH-R IgG immuno-localised to cell bodies and axons of large neurones in all 6 regions of all 5 brains. The intensity and percentage of neurones labelled were similar in all tissue sections. TSH-R immuno-label was also observed in vascular endothelial cells in the cingulate gyrus. Although also found in all 5 brains and all six cortical regions, TG localised exclusively in vascular smooth muscle cells and not on neurones. Although limited by the small sample size and number of brain areas examined, this is the first study describing the presence of antigenic targets for anti-TSH-R IgG on human cortical neurons, and anti-TG IgG in cerebral vasculature. PMID:21196016

  19. Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

    Directory of Open Access Journals (Sweden)

    Nancy A Turner

    Full Text Available The cellular synthesis site and ensuing storage location for human factor VIII (FVIII, the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs and umbilical vein endothelial cells (HUVECs. Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

  20. Inhibitory effects of three diketopiperazines from marine-derived bacteria on endothelial protein C receptor shedding in human endothelial cells and mice.

    Science.gov (United States)

    Lee, Wonhwa; Ku, Sae-Kwang; Choi, Hyukjae; Bae, Jong-Sup

    2016-04-01

    Diketopiperazine is a natural products found from bacteria, fungi, marine sponges, gorgonian and red algae. They are cyclic dipeptides possessing relatively simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity. The endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway and in the activation of protein C. Endothelial cell protein C receptor (EPCR) can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of diketopiperazine on EPCR shedding. We investigated this issue by monitoring the effects of diketopiperazine on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-mediated EPCR shedding in mice and underlying mechanism. Here, three (1-3) of diketopiperazines were isolated from two strains of marine-derived bacteria and 1-3 induced potent inhibition of PMA-, TNF-α-, IL-1β (in HUVECs), and CLP-induced EPCR shedding (in mice) via inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. 1-3 also inhibited the expression and activity of PMA-induced TACE in HUVECs suggesting that p38, ERK1/2, and JNK could be molecular targets of 1-3. These results demonstrate the potential of 1-3 as an anti-EPCR shedding reagent against PMA-mediated and CLP-mediated EPCR shedding. PMID:27012760

  1. Inhibitory effects of three diketopiperazines from marine-derived bacteria on endothelial protein C receptor shedding in human endothelial cells and mice.

    Science.gov (United States)

    Lee, Wonhwa; Ku, Sae-Kwang; Choi, Hyukjae; Bae, Jong-Sup

    2016-04-01

    Diketopiperazine is a natural products found from bacteria, fungi, marine sponges, gorgonian and red algae. They are cyclic dipeptides possessing relatively simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity. The endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway and in the activation of protein C. Endothelial cell protein C receptor (EPCR) can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of diketopiperazine on EPCR shedding. We investigated this issue by monitoring the effects of diketopiperazine on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-mediated EPCR shedding in mice and underlying mechanism. Here, three (1-3) of diketopiperazines were isolated from two strains of marine-derived bacteria and 1-3 induced potent inhibition of PMA-, TNF-α-, IL-1β (in HUVECs), and CLP-induced EPCR shedding (in mice) via inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. 1-3 also inhibited the expression and activity of PMA-induced TACE in HUVECs suggesting that p38, ERK1/2, and JNK could be molecular targets of 1-3. These results demonstrate the potential of 1-3 as an anti-EPCR shedding reagent against PMA-mediated and CLP-mediated EPCR shedding.

  2. Adhesion of subsets of human blood mononuclear cells to porcine endothelial cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cellular immune response is a major barrier to xenotransplantation, and cell adhesion is the first step in intercellular recognition. Flow-cytometric adhesion assay has been used to investigate the differential adhesions of monocyte (Mo), natural killer cell (NK) and T lymphocyte (T) present within human peripheral blood mononuclear cells (PBMC) to porcine aortic endothelial cells (PAEC), and to demonstrate the effect of human interferon-γ(hIFN-γ) or/and tumor necrosis factor-α (hTNF-α) pretreatment of PAEC on their adhesiveness for different PBMC subsets. The preferential sequence for PBMC subset binding to resting PAEC is Mo, NK and T cells, among which T cells show the slightest adherence; hTNF-α can act across the species, and augment Mo, NK and T cell adhesion ratios by 40%, 110% and 3 times, respectively. These results confirm at the cell level that host Mo and NK cells are major participants in the cellular xenograft rejection, thereby, providing a prerequisite for further studying the human Mo/NK-PAEC interactive mechanisms.

  3. Regulation Effect of Vascular Endothelial Growth Factor on Human Fetal Choroid Vascularization

    Institute of Scientific and Technical Information of China (English)

    JinsongZhao; YiWang; 等

    2002-01-01

    Purpose:To investigate the spatial and temporal regulation effect of vascular endothelial growth factor(VEGF) on human fetal choroids vascularization.Methods:The eyeballs of 54 human fetuses from the 9th week to the 40th week due to accidental abortion were studied by immunohistochemically stainin for the expression of VEGF and proliferation cell nuclear antigen (PCNA).Results: (1)The distribution of VEGF expression in the retinal pigment epithelium (RPE) decreased with the incrase of age,the peak of which was between the 9th and 14th week.(2)PCNA immunoreactivity was localized within choriocapillaris endothelium .The expression level decreased alone with fetus age.In this period the choriocapillaris endothelium kept proliferation,differentiation,canalization and remodeled to form the choroids vessels(3)Statistically significant correlations were shown between the expression of VEGF in the PRE and that of PCNA in choriocapillaris endothelium(r=0.933,P<0.01).Couclusin:VEGF expression in PRE was positively involved in modulating human fetal choroids vascularization .Eye Science 2000;16:11-14.

  4. Regulation Effect of Vascular Endothelial Growth Factor on Human Fetal Choroid Vascularization

    Institute of Scientific and Technical Information of China (English)

    Jinsong Zhao; Yue Song; Yi Wang; Xiaoguang Zhang

    2000-01-01

    Purpose: To investigate the spatial and temporal regulation effect of vascular endothelial growth factor(VEGF) on human fetal choroid vascularization. Methods: The eyeballs of 54 human fetuses from the 9th week to the 40th week due to accidental abortion were studied by immunohistochemically staining for the expression of VEGF and proliferation cell nuclear antigen (PCNA). Results: (1) The distribution of VEGF expression in the retinal pigment epithelium (RPE) decreased with the increase of age, the peak of which was between the 9th and 14th week. (2) PCNA immunoreactivity was localized within choriocapillaris endothelium. The expression level decreased alone with fetus age. In this period the choriocapillaris endothelium kept proliferation, differentiation, canalization and remodelled to form the choroid vessels. (3)Statistically significant correlations were shown between the expression of VEGF in the PRE and that of PCNA in choriocapillaris endothelium(r =0. 933, P < 0. 01). Conclusion: VEGF expression in RPE was positively involved in modulating human fetal choroid vascularization. Eye Science 2000; 16:11 ~ 14.

  5. The influence of C-ions and X-rays on human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Alexander eHelm

    2016-01-01

    Full Text Available Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells (HUVEC as a model system to examine the effect of low and high linear energy transfer (LET radiation. Cells were exposed to 250 kV X-rays or C-ions with the energies of either 9.8 MeV/u (LET=170 keV/µm or 91 MeV/u (LET=28 keV/µm. Subculture of cells was performed regularly up to 49 days (~22 population doublings post-irradiation. Immediately after exposure cells were seeded for the colony forming assay. Additionally, at regular intervals mitochondrial membrane potential (JC-1 staining and cellular senescence (senescence associated β-galactosidase staining were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution mFISH technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay as observed in other cell systems. For 9.8 and 91 MeV/u C-ions relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time-points the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the mitochondrial membrane potential was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time-point investigated. Altogether our data indicate that C-ions were LET-dependently more effective in damaging endothelial cells shortly after exposure. Late damage to endothelial cells, however, was not found for the applied conditions and endpoints.

  6. The Influence of C-Ions and X-rays on Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Helm, Alexander; Lee, Ryonfa; Durante, Marco; Ritter, Sylvia

    2016-01-01

    Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells as a model system to examine the effect of low- and high-linear energy transfer (LET) radiation. Cells were exposed to 250 kV X-rays or carbon ions (C-ions) with the energies of either 9.8 MeV/u (LET = 170 keV/μm) or 91 MeV/u (LET = 28 keV/μm). Subculture of cells was performed regularly up to 46 days (~22 population doublings) post-irradiation. Immediately after exposure, cells were seeded for the colony forming assay. Additionally, at regular intervals, mitochondrial membrane potential (MMP) (JC-1 staining) and cellular senescence (senescence-associated β-galactosidase staining) were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay) as observed in other cell systems. For 9.8 and 91 MeV/u C-ions, relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time points, the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the MMP was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time point investigated. Altogether, our data indicate that shortly after exposure C-ions were more effective in damaging endothelial cells than X-rays. However, late damage to endothelial cells was not found for the applied conditions and endpoints.

  7. Desflurane preconditioning induces oscillation of NF-κB in human umbilical vein endothelial cells.

    Directory of Open Access Journals (Sweden)

    Juan Yi

    Full Text Available BACKGROUND: Nuclear factor kappa B (NF-κB has been implicated in anesthetic preconditioning (APC induced protection against anoxia and reoxygenation (A/R injury. The authors hypothesized that desflurane preconditioning would induce NF-κB oscillation and prevent endothelial cells apoptosis. METHODS: A human umbilical vein endothelial cells (HUVECs A/R injury model was used. A 30 minute desflurane treatment was initiated before anoxia. NF-κB inhibitor BAY11-7082 was administered in some experiments before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT assay. The cellular superoxide dismutases (SOD activitiy were tested by water-soluble tetrazolium salt (WST-1 assay. NF-κB p65 subunit nuclear translocation was detected by immunofluorescence staining. Expression of inhibitor of NF-κB-α (IκBα, NF-κB p65 and cellular inhibitor of apoptosis 1 (c-IAP1, B-cell leukemia/lymphoma 2 (Bcl-2, cysteine containing aspartate specific protease 3 (caspases-3 and second mitochondrial-derived activator of caspase (SMAC/DIABLO were determined by western blot. RESULTS: Desflurane preconditioning caused phosphorylation and nuclear translocation of NF-κB before anoxia, on the contrary, induced the synthesis of IκBα and inhibition of NF-κB after reoxygenation. Desflurane preconditioning up-regulated the expression of c-IAP1 and Bcl-2, blocked the cleavage of caspase-3 and reduced SMAC release, and decreased the cell death of HUVECs after A/R. The protective effect was abolished by BAY11-7082 administered before desflurane. CONCLUSIONS: The results demonstrated that desflurane activated NF-κB during the preconditioning period and inhibited excessive activation of NF-κB in reperfusion. And the oscillation of NF-κB induced by desflurane preconditioning finally up-regulated antiapoptotic proteins expression and

  8. Development of Hematopoietic and Endothelial Cells from Human Embryonic Stem Cells: Lessons from the Studies using Mouse as a Model

    OpenAIRE

    Anna Jezierski; Albert Swedani; Lisheng Wang

    2007-01-01

    The current progress using the human embryonic stem cell (hESC) model system has provided much insight into the early origins of the hematopoietic and endothelial lineages, particularly the elusive hemangioblast. Recently, the cellular hierarchy and molecular regulation controlling hematopoietic commitment have been further elucidated. These findings not only provide new insights into early human development, but also advance the knowledge required to develop techniques capable of generating ...

  9. Regulation of Thrombomodulin Expression and Release in Human Aortic Endothelial Cells by Cyclic Strain

    OpenAIRE

    Martin, Fiona A.; Alisha McLoughlin; Rochfort, Keith D.; Colin Davenport; Murphy, Ronan P.; Cummins, Philip M.

    2014-01-01

    Background and Objectives Thrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis. Methods This ...

  10. Effects of antioxidants on homocysteine thiolactone-induced apoptosis in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Weijun GU; Juming LU; Guoqing YANG; Qinghua GUO; Baoan WANG; Yiming MU; Changyu PAN

    2006-01-01

    Background and objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine thiolactone (HcyT), one of the homocysteine metabolites in vivo, is toxic both in vivo and in vitro. The aim of this study was to investigate the effect of HcyT on apoptotic damage in human umbilical vein endothelial cells (HUVECs) and the role of antioxidants in the reduction of HcyT-induced apoptosis. Methods HUVECs were cultured in DMEM supplemented with 20% heat inactivated fetal bovine serum cell cultures were maintained in a humidified 5% CO2 atmosphere at 37 ℃. Cytotoxicity was determined by MTT assay,which consists of hypodiploid cells with propidium iodide labeling and intracellular reactive oxygen species levels using 2',7'-dichlorofluorescein diacetate as the probe by flow cytometry. Results HcyT (250-2000μM) induced HUVECs apoptosis in a time- and concentration-dependent manner. Reactive oxygen species levels rose in response to increasing HcyT concentrations at 24-h incubation.The reduction of cell apoptosis by N-acetylcysteine, vitamin E, or pyrrolidine dithiocarbamate, occurred simultaneously with a significant decrease in intracellular reactive oxygen species levels. Conclusion HcyT exerts its cytotoxic effects on endothelial cells through an apoptotic mechanism involving cellular reactive oxygen species production. The capacity of N-acetylcysteine, vitamin E, and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular reactive oxygen species correlates well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicates HcyT-generated hydrogen peroxide may provoke cell apoptosis via activating nuclear factor-kappa binding protein.

  11. Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

    International Nuclear Information System (INIS)

    Extracellular Ca2+ concentration ([Ca2+]o) regulates the functions of many cell types through a G protein-coupled [Ca2+]o-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca2+]o, neomycin, and gadolinium) failed to increase intracellular Ca2+ concentration ([Ca2+]i), the CaR agonist spermine stimulated an increase in [Ca2+]i that was diminished in buffer without Ca2+ and was abolished after depletion of an intracellular Ca2+ pool with thapsigargin or after blocking IP3- and ryanodine receptor-mediated Ca2+ release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca2+]i and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca2+]i, primarily due to release of IP3- and ryanodine-sensitive intracellular Ca2+ stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC

  12. Ouabain at pathological concentrations might induce damage in human vascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Yan-ping REN; Ruo-wen HUANG; Zhuo-ren L(U)

    2006-01-01

    Aim: To examine the time- and dose-dependent effects of ouabain on human umbilical vein endothelial cells (HUVEC) in vivo, and the changes in aortic endothelium and the different expression levels of Kv4.2 in vitro. Methods: The proliferation of HUVEC and cell death were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the incorporation of [3H]TdR,trypan blue staining, and lactate dehydrogenase (LDH) release. The response of endothelial cells to ouabain was explored with a complementary DNA microarray and a candidate gene was found. "Ouabain-sensitive" hypertensive rats were established by chronic administration of ouabain. Changes in the aortic endothelium were observed by electron microscopy, and the expression level of Ky4.2 in different animals was studied by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: Ouabain stimulated the proliferation of HUVEC at physiological concentrations (0.3-0.9 nmol/L). Ouabain at pathological concentrations (0.9-1.8 nmol/L) inhibited proliferation and induced cell death, mRNA profile analysis indicated that 340 genes were differentially expressed after ouabain treatment: 145 were upregulated, of which 6 were upregulated significantly, including KCND2 (encoding the potassium voltagegated channel shal-related subfamily member 2). The upregulated genes were mainly related to cell metabolism and transcription. In ouabain-sensitive hypertensive rats, the aortic endothelium was damaged and Kv4.2 (coded by KCND2)was over-expressed. Conclusion: The physiological role of ouabain in HUVEC might involve the control of growth and metabolism. Ouabain at pathological concentrations might affect the structure and function of the vascular endothelium by modification of expression of the KCND2 gene, and participate vascular remodeling in hypertension.

  13. Bradykinin preconditioning improves therapeutic potential of human endothelial progenitor cells in infarcted myocardium.

    Directory of Open Access Journals (Sweden)

    Zulong Sheng

    Full Text Available OBJECTIVES: Stem cell preconditioning (PC is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs with bradykinin (BK enhance cell survival, inhibit apoptosis and repair the infarcted myocardium. METHODS: The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging. RESULTS: In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively. CONCLUSIONS: The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used.

  14. Racial differences in the responses to shear stress in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Feairheller DL

    2011-07-01

    Full Text Available Deborah L Feairheller1,4, Joon-Young Park2, Victor Rizzo3, Boa Kim2, Michael D Brown1,31Hypertension, Molecular and Applied Physiology Laboratory, 2Cardiovascular Genomics Laboratory, Department of Kinesiology, 3Cardiovascular Research Center, School of Medicine, Temple University, Philadelphia, PA, USA; 4Exercise and Metabolic Disease Research Laboratory, School of Nursing, University of California Los Angeles, Los Angeles, CA, USABackground: African American ethnicity is an independent risk factor for exaggerated oxidative stress, which is related to inflammation, hypertension, and cardiovascular disease. Recently, we reported that in vitro oxidative stress and inflammation levels differ between African American and Caucasian human umbilical vein endothelial cells (HUVECs, African American HUVECs having higher levels of both. However, it remains to be shown whether the cells would respond differently to external stimuli.Methods: We used a cone and plate viscometer to apply laminar shear stress (LSS as an aerobic exercise mimetic to compare the responses by race. HUVECs were exposed to static conditions (no LSS, low LSS (5 dyne/cm2, and moderate LSS (20 dyne/cm2.Results: It was found that African American HUVECs had higher levels of oxidative stress under static conditions, and when LSS was applied protein expression levels (NADPH oxidase NOX2, NOX4 and p47phox subunits, eNOS, SOD2, and catalase and biomarkers (NO, SOD, and total antioxidant capacity were modulated to similar levels between race.Conclusion: African American HUVECs may be more responsive to LSS stimulus indicating that aerobic exercise prescriptions may be valuable for this population since the potential exists for large in vivo improvements in oxidative stress levels along the endothelial layer in response to increased shear flow.Keywords: shear stress, African American, NADPH oxidase, HUVECs, oxidative stress

  15. Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime

    Directory of Open Access Journals (Sweden)

    M. Mathy-Hartert

    1995-01-01

    Full Text Available We investigated the effects of the antibiotic ceftazidime (CAZ on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−. In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC were capable of taking up active MPO. In presence of H2O2 (10−4 M, this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC. Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2 was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon. So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

  16. Advanced Glycation End Products Induce Endothelial-to-Mesenchymal Transition via Downregulating Sirt 1 and Upregulating TGF-β in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Wei He

    2015-01-01

    Full Text Available In the present study, we examined the advanced glycation end products- (AGEs- induced endothelial-to-mesenchymal transition (EndMT in human umbilical vein endothelial cells (HUVECs. Results demonstrated that AGE-BSAs significantly reduced the cluster of differentiation 31 (CD 31 expression, whereas they promoted the expression of fibroblast-specific protein-1 (FSP-1, α-smooth muscle antibody (α-SMA, and collagen I at both mRNA and protein levels in HUVECs. And the AGE-BSAs also promoted the receptors for AGEs (RAGEs and receptor I for TGF-β (TGFR I markedly with a dose dependence, whereas the Sirt 1 was significantly downregulated by the AGE-BSA at both mRNA and protein levels. Moreover, the Sirt 1 activity manipulation with its activator, resveratrol (RSV, or its inhibitor, EX527, markedly inhibited or ameliorated the AGE-mediated TGF-β upregulation. And the manipulated Sirt 1 activity positively regulated the AGE-induced CD31, whereas it negatively regulated the AGE-induced FSP-1. Thus, Sirt 1 was confirmed to regulate the AGE-induced EndMT via TGF-β. In summary, we found that AGE-BSA induced EndMT in HUVECs via upregulating TGF-β and downregulating Sirt 1, which also negatively regulated TGF-β in the cell. This study implied the EndMT probably as an important mechanism of AGE-induced cardiovascular injury.

  17. Adult Human Neurogenesis: from Microscopy to Magnetic Resonance Imaging

    Directory of Open Access Journals (Sweden)

    Amanda eSierra

    2011-04-01

    Full Text Available Neural stem cells reside in well-defined areas of the adult human brain and are capable of gene-rating new neurons throughout the life span. In rodents, it is well established that the new born neurons are involved in olfaction as well as in certain forms of memory and learning. In humans, the functional relevance of adult human neurogenesis is being investigated, in particular its implication in the etiopathology of a variety of brain disorders. Adult neurogenesis in the human brain was discovered by utilizing methodologies directly imported from the rodent research, such as immunohistological detection of proliferation and cell-type specific biomarkers in postmortem or biopsy tissue. However, in the vast majority of cases, these methods do not support longitudinal studies; thus, the capacity of the putative stem cells to form new neurons under different disease conditions cannot be tested. More recently, new technologies have been specifically developed for the detection and quantification of neural stem cells in the living human brain. These technologies rely on the use of magnetic resonance imaging, available in hospitals worldwide. Although they require further validation in rodents and primates, these new methods hold the potential to test the contribution of adult human neurogenesis to brain function in both health and disease. This review reports on the current knowledge on adult human neurogenesis. We first review the different methods available to assess human neurogenesis, both ex vivo and in vivo and then appraise the changes of adult neurogenesis in human diseases.

  18. Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-hua; WEI Wei; XU Hao; WANG Yan-yan; WU Wen-xi

    2007-01-01

    Background Vascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.Methods Hepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA.The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.Results Serum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.Conclusion This present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

  19. Transplantation of human umbilical cord-derived endothelial progenitor cells promotes re-endothelialization of the injured carotid artery after balloon injury in New Zealand white rabbits

    Institute of Scientific and Technical Information of China (English)

    HU Cheng-heng; KE Xiao; CHEN Kui; YANG Da-ya; DU Zhi-min; WU Gui-fu

    2013-01-01

    Background Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI).The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries.Methods Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women,and EPCs were isolated,cultured,expanded and identified by immunofluorescence.The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon,and the EPCs were injected into the lumen of the injured artery in the transplanted group (n=16),while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n=16).The animals were sacrificed after either 2 or 4 weeks,and the grafted cells were identified by double immunofiuorescence staining with human nuclear antigen (HNA) and CD31 antibodies.Arterial cross sections were analyzed by pathology,immunohistochemisty and morphometry to evaluate the reparative effects of EPCs.Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-β1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results Fluorescence-labeled EPCs were found in the neointima.The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P <0.05).von Willebrand factor (vWF)immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.75±2.92 vs.4.50±1.77,P <0.05).Compared with the control group,the transplanted group had lower expression of PCNA mRNA (0.67±0.11 vs.1.25±0.40,P <0.01) and higher expression of TGF-β1 mRNA (1.10±0.21 vs.0.82±0.07,P <0.05).Conclusions EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels.The transplanted

  20. Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Azizah Ugusman

    2010-01-01

    Full Text Available OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs. METHODS: HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H2O2, treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

  1. Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

    Institute of Scientific and Technical Information of China (English)

    TANG Zehai; LIU Xinyue; ZOU Ping

    2007-01-01

    This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor (VEGF) and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concen- trations (12.5-200 μmol/L) for different time lengths (12-48 h). The proliferation of the U937 leu- kemic cells was determined by MTT assay. Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry (FCM). Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resvera-trol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

  2. Effects of Salvianolic Acid B on Protein Expression in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Tsong-Min Chang

    2011-01-01

    Full Text Available Salvianolic acid B (Sal B, a pure water-soluble compound extracted from Radix Salviae miltiorrhizae, has been reported to possess potential cardioprotective efficacy. To identify proteins or pathways by which Sal B might exert its protective activities on the cardiovascular system, two-dimensional gel electrophoresis-based comparative proteomics was performed, and proteins altered in their expression level after Sal B treatment were identified by MALDI-TOF MS/MS. Human umbilical vein endothelial cells (HUVECs were incubated at Sal B concentrations that can be reached in human plasma by pharmacological intervention. Results indicated that caldesmon, an actin-stabilizing protein, was downregulated in Sal B-exposed HUVECs. Proteins that showed increased expression levels upon Sal B treatment were vimentin, T-complex protein 1, protein disulfide isomerase, tropomyosin alpha, heat shock protein beta-1, UBX domain-containing protein 1, alpha enolase, and peroxiredoxin-2. Additionally, Sal B leads to increased phosphorylation of nucleophosmin in a dose-dependent manner and promotes proliferation of HUVECs. We found that Sal B exhibited a coordinated regulation of enzymes and proteins involved in cytoskeletal reorganization, oxidative stress, and cell growth. Our investigation would provide understanding to the endothelium protection information of Sal B.

  3. Occurring of In Vitro Functional Vasculogenic Pericytes from Human Circulating Early Endothelial Precursor Cell Culture

    Directory of Open Access Journals (Sweden)

    Silvia Cantoni

    2015-01-01

    Full Text Available Pericytes are periendothelial cells of the microcirculation which contribute to tissue homeostasis and hemostasis by regulating microvascular morphogenesis and stability. Because of their multipotential ex vivo differentiation capabilities, pericytes are becoming very interesting in regenerative medicine field. Several studies address this issue by attempting to isolate pericyte/mesenchymal-like cells from peripheral blood; however the origin of these cells and their culture conditions are still debated. Here we showed that early Endothelial Progenitor Cells (EPCs expressing CD45+/CD146+/CD31+ can be a source of cells with pericyte/mesenchymal phenotype and function, identified as human Progenitor Perivascular Cells (hPPCs. We provided evidence that hPPCs have an immunophenotype consistent with Mesenchymal Stem Cells (MSCs from human adipose tissue (hASCs and fetal membranes of term placenta (FM-hMSCs. In addition, hPPCs can be subcultured and exhibit expression of pluripotent genes (OCT-4, KLF-4, and NANOG as well as a remarkable vasculogenic potential. Our findings could be helpful to develop innovative cell-based therapies for future clinical applications with distinct therapeutic purposes.

  4. Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S.; Shachter, Neil S.

    2003-12-01

    We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.

  5. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    Science.gov (United States)

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  6. Proteomics analysis of human endothelial cells after shortterm exposure to mobile phone radiation

    International Nuclear Information System (INIS)

    Mobile phones have been a part of our everyday life in the developed world since the late 1990s. This has raised concerns over the potential health risks of mobile phone use. Biological and health effects potentially caused by mobile phone radiation have been extensively studied and several biological and medical endpoints have been examined. So far, results have not been conclusive on the potential effects of mobile phone radiation. Mobile phones generate a modulated radio frequency electromagnetic field (RF-EMF), which is a form of non-ionizing radiation. This means that mobile phone radiation does not have enough energy to ionize atoms and it cannot break chemical bonds directly (e.g., in DNA strands). There could, however, be other mechanisms by which mobile phone radiation may affect cellular and physiological functions. Whether these mechanisms exist is unknown. In this thesis, large-scale screening techniques, such as proteomics, were applied to examine changes on the proteome level after exposure to mobile phone radiation. Proteomics techniques allow the screening of several hundreds, and even thousands, of proteins simultaneously, and are thus more efficient than single endpoint techniques. Four different types of human endothelial cells (two cell lines, two types of primary cells) were exposed to two types of mobile phone radiation (900 and 1800 MHz GSM). The proteome of these cells was examined immediately after short-term exposure using two-dimensional gel electrophoresis (2DE). Two protein detection/analysis techniques were used: silver staining for the cell line samples and difference gel electrophoresis (DIGE) for the primary cells. 2DE-DIGE technology is currently a state-of-the-art technique in 2DE studies. Several changes were found in the proteome of the human endothelial cell line EA.hy926 after exposure to 900 MHz GSM mobile phone radiation. In addition, the proteome of a variant of the same cell line, the EA.hy926v1, was affected after 900 MHz

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

    Science.gov (United States)

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

    2010-12-01

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

  8. Gustofacial and olfactofacial responses in human adults.

    Science.gov (United States)

    Weiland, Romy; Ellgring, Heiner; Macht, Michael

    2010-11-01

    Adults' facial reactions in response to tastes and odors were investigated in order to determine whether differential facial displays observed in newborns remain stable in adults who exhibit a greater voluntary facial control. Twenty-eight healthy nonsmokers (14 females) tasted solutions of PROP (bitter), NaCl (salty), citric acid (sour), sucrose (sweet), and glutamate (umami) differing in concentration (low, medium, and high) and smelled different odors (banana, cinnamon, clove, coffee, fish, and garlic). Their facial reactions were video recorded and analyzed using the Facial Action Coding System. Adults' facial reactions discriminated between stimuli with opponent valences. Unpleasant tastes and odors elicited negative displays (brow lower, upper lip raise, and lip corner depress). The pleasant sweet taste elicited positive displays (lip suck), whereas the pleasant odors did not. Unlike newborns, adults smiled with higher concentrations of some unpleasant tastes that can be regarded as serving communicative functions. Moreover, adults expressed negative displays with higher sweetness. Except for the "social" smile in response to unpleasant tastes, adults' facial reactions elicited by tastes and odors mostly correspond to those found in newborns. In conclusion, adults' facial reactions to tastes and odors appear to remain stable in their basic displays; however, some additional reactions might reflect socialization influences.

  9. Adult Literacy Education and Human Rights: A View from Afghanistan

    Science.gov (United States)

    Andersen, Susan M.; Kooij, Christina S.

    2007-01-01

    In this article, we argue that adult literacy as part of international development is an issue of both human rights and women's rights. We explore this by presenting a case study of the effects of one innovative adult literacy program in Afghanistan that places men and women, as well as various ethnicities, together in the same classroom as…

  10. Dopaminerge Differenzierung adulter humaner hippocampaler Stammzellen

    OpenAIRE

    Türk, Matthias

    2013-01-01

    Hintergrund und Ziele: Nachdem seit der ersten Hälfte des letzten Jahrhunderts durch mehrere Experimente adulte Neurogenese schließlich nachgewiesen und somit Cajals Dogma widerlegt werden konnte, erlebten die Neurowissenschaften durch die Möglichkeit zur Isolation adulter neuraler Stammzellen ein exponentielles Wachstum. Gleichzeitig mit der basiswissenschaftlichen Aufarbeitung der adulten Neurogenese sowohl im Tier, als auch im Menschen, kam die Idee der therapeutischen Verwendung dieser, v...

  11. A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells

    Directory of Open Access Journals (Sweden)

    Fanny Ehret

    2015-11-01

    Full Text Available The niche concept of stem cell biology proposes a functional unit between the precursor cells and their local microenvironment, to which several cell types might contribute by cell–cell contacts, extracellular matrix, and humoral factors. We here established three co-culture models (with cell types separated by membrane for both adherent monolayers and neurospheres to address the potential influence of different niche cell types in the neurogenic zone of the adult hippocampus of mice. Astrocytes and endothelial cells enhanced precursor cell proliferation and neurosphere formation. Endothelial factors also led to a prolonged increase in proliferation after growth factor withdrawal, which otherwise induces differentiation. All niche cell types enhanced cell survival in monolayer cultures, endothelial cells also stimulated neuronal differentiation. A parallel trend elicited by astrocytes did not reach conventional statistical significance. Pericytes had variable effects here. We did not observe changes in differentiation in neurosphere co-cultures. In summary, our data indicate that in precursor cell culture protocols survival could be improved by adding as yet unknown factors physiologically contributed by astrocytes and endothelial cells. Our findings also underscore the complexity of the niche and the differential impact of factors from the different sources on distinct aspects of neuronal development. With the help of the models presented here, identification of these factors and their specific biological activity can now be initiated.

  12. Endothelial progenitor cells (EPCs as gene carrier system for rat model of human glioma.

    Directory of Open Access Journals (Sweden)

    Nadimpalli Ravi S Varma

    Full Text Available BACKGROUND: Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1 intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2 whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities. METHODS AND RESULTS: Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS. Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors. CONCLUSION: EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for

  13. Berberine Protects Human Umbilical Vein Endothelial Cells against LPS-Induced Apoptosis by Blocking JNK-Mediated Signaling

    Science.gov (United States)

    Guo, Junping; Wang, Lijun; Wang, Linyao; Qian, Senmi; Fang, Jie

    2016-01-01

    Endothelial dysfunction is a critical factor during the initiation of atherosclerosis. Berberine has a beneficial effect on endothelial function; however, the underlying mechanisms remain unclear. In this study, we investigated the effects of berberine on lipopolysaccharide- (LPS-) induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the molecular mechanisms mediating the effect. The effects of berberine on LPS-induced cell apoptosis and viability were measured with 5-ethynyl-2′-deoxyuridine staining, flow cytometry, and Cell Counting Kit-8 assays. The expression and/or activation of proapoptotic and antiapoptotic proteins or signaling pathways, including caspase-3, poly(ADP-ribose) polymerase, myeloid cell leukemia-1 (MCL-1), p38 mitogen-activated protein kinase, C-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase, were determined with western blotting. The malondialdehyde levels, superoxide dismutase (SOD) activity, and production of proinflammatory cytokines were measured with enzyme-linked immunosorbent assays. The results demonstrated that berberine pretreatment protected HUVECs from LPS-induced apoptosis, attenuated LPS-induced injury, inhibited LPS-induced JNK phosphorylation, increased MCL-1 expression and SOD activity, and decreased proinflammatory cytokine production. The effects of berberine on LPS-treated HUVECs were prevented by SP600125, a JNK-specific inhibitor. Thus, berberine might be a potential candidate in the treatment of endothelial cell injury-related vascular diseases. PMID:27478481

  14. Berberine Protects Human Umbilical Vein Endothelial Cells against LPS-Induced Apoptosis by Blocking JNK-Mediated Signaling.

    Science.gov (United States)

    Guo, Junping; Wang, Lijun; Wang, Linyao; Qian, Senmi; Zhang, Dayong; Fang, Jie; Pan, Jianping

    2016-01-01

    Endothelial dysfunction is a critical factor during the initiation of atherosclerosis. Berberine has a beneficial effect on endothelial function; however, the underlying mechanisms remain unclear. In this study, we investigated the effects of berberine on lipopolysaccharide- (LPS-) induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the molecular mechanisms mediating the effect. The effects of berberine on LPS-induced cell apoptosis and viability were measured with 5-ethynyl-2'-deoxyuridine staining, flow cytometry, and Cell Counting Kit-8 assays. The expression and/or activation of proapoptotic and antiapoptotic proteins or signaling pathways, including caspase-3, poly(ADP-ribose) polymerase, myeloid cell leukemia-1 (MCL-1), p38 mitogen-activated protein kinase, C-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase, were determined with western blotting. The malondialdehyde levels, superoxide dismutase (SOD) activity, and production of proinflammatory cytokines were measured with enzyme-linked immunosorbent assays. The results demonstrated that berberine pretreatment protected HUVECs from LPS-induced apoptosis, attenuated LPS-induced injury, inhibited LPS-induced JNK phosphorylation, increased MCL-1 expression and SOD activity, and decreased proinflammatory cytokine production. The effects of berberine on LPS-treated HUVECs were prevented by SP600125, a JNK-specific inhibitor. Thus, berberine might be a potential candidate in the treatment of endothelial cell injury-related vascular diseases. PMID:27478481

  15. Bacteriology of moderate (chronic) periodontitis in mature adult humans.

    OpenAIRE

    Moore, W E; Holdeman, L V; Cato, E P; Smibert, R M; Burmeister, J A; Ranney, R R

    1983-01-01

    A total of 171 taxa was represented among 1,900 bacterial isolates from 60 samples of sites affected with moderate periodontitis in 22 mature adult humans. The composition of the subgingival sulcus flora was statistically significantly different from that of the adjacent supragingival flora and the subgingival flora of 14 people with healthy gingiva, but was not significantly different from that of sulci affected with severe periodontitis in 21 young human adults. The sulcus floras of moderat...

  16. Early reversal cells in adult human bone remodeling

    DEFF Research Database (Denmark)

    Abdelgawad, Mohamed Essameldin; Delaisse, Jean-Marie; Hinge, Maja;

    2016-01-01

    . Earlier preclinical studies indicate that reversal cells degrade the organic matrix left behind by the osteoclasts and that this degradation is crucial for the initiation of the subsequent bone formation. To our knowledge, this study is the first addressing these catabolic activities in adult human bone...... demonstrates that reversal cells colonizing bone surfaces right after resorption are osteoblast-lineage cells, and extends to adult human bone remodeling their role in rendering eroded surfaces osteogenic....

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

    Science.gov (United States)

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

    2016-09-01

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

  18. Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it. Methods  By inserting hVEGF165 cDNA coding 165 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 was expressed under the induction of 1% methanol. Results  SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by Heparin-Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC. Conclusion  hVEGF165 was successfully expressed. The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.

  19. Morroniside protects cultured human umbilical vein endothelial cells from damage by high ambient glucose

    Institute of Scientific and Technical Information of China (English)

    Hui-qin XU; Hai-ping HAO; Xu ZHANG; Yang PAN

    2004-01-01

    AIM: To determine whether morroniside, a compound in Comus officinalis Sieb et Zucc can prevent cultured human umbilical vein endothelial cells (HUVEC) from damage by high ambient glucose. METHODS: HUVEC was incubated in glucose, 5 or 30 mmol/L, either alone or in the presence of morroniside (final concentration 100, 10,and 1 μmol/L, respectively) for 48 h. The proliferation of HUVEC was quantified by MTT method; its cycle was analyzed by flow cytometry; morphological change was observed with fluorescence microscopy. RESULTS:Survival of HUVEC cultured in high ambient glucose was significantly decreased when compared to that in normal concentration of glucose (P<0.01). High ambient glucose also lowered the rate of cells entering into S-phase, along with severe morphological damage. With the intervention of morroniside (final concentration 100 and 10 μmol/L),the cell survival was significantly recovered (P<0.01, P<0.05, respectively), accompanied with increased S-phase rate and less extent of morphological damage. CONCLUSION: Morroniside protected HUVEC against high ambient glucose induced injury, which suggested that morroniside could exert a beneficial effect on preventing diabetic angiopathies.

  20. Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Revtyak, G.E.; Hughes, M.J.; Johnson, A.R.; Campbell, W.B.

    1988-08-01

    Endothelial cells (EC) cultured from human umbilical artery (UA) and vein (UV) metabolized (/sup 14/C)arachidonic acid to prostaglandins (PGs), monohydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Major radioactive products were identified as 6-keto-PGF1 alpha, PGE2, PGF2 alpha, 12-hydroxy heptadecatrienoic acid, 15-HETE, and 11-HETE. In addition, extracts from UV ECs contained 12-HETE, 5-HETE, 14,15-EET, and 5,6-EET as minor products, whereas extracts from UA ECs contained only 12-HETE as a minor product. UA ECs also produced metabolites comigrating with 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. Histamine increased the release of (/sup 14/C)PGs and (/sup 14/C)HETEs from (/sup 14/C)arachidonic acid-labeled ECs. Indomethacin, aspirin, and nordihydroguauretic acid completely inhibited synthesis of both (/sup 14/C)PGs and (/sup 14/C)HETEs from exogenous (/sup 14/C)arachidonic acid in these cells. Microsomes metabolized (/sup 14/C)arachidonic acid to the same (/sup 14/C)PGs and (/sup 14/C)HETEs as intact cells. Pretreatment of microsomes with indomethacin completely inhibited formation of these products. These data indicate that UA ECs and UV ECs metabolize endogenous and exogenous arachidonic acid to both PGs and HETEs. Also 15-HETE and 11-HETE appear to be synthesized by a microsomal enzyme with the properties of cyclooxygenase.

  1. Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Jing; Wang; Ting-Jun; Fan; Xiu-Xia; Yang; Shi-Min; Chang

    2014-01-01

    AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P <0.01) and the length of F-actin,reduced the mean optical density(P <0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

  2. Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells

    International Nuclear Information System (INIS)

    Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium. (author)

  3. Cloning and Identification of A Novel Variant of Human Vascular Endothelial Growth Factor

    Institute of Scientific and Technical Information of China (English)

    GUO; Jianli; QU; Shen

    2001-01-01

    A novel variant of human vascular endothelial growth factor (h'VEGF165) cDNA was amplified by nested PCR method from the HL601 cells and was cloned into a eukaryotic expressing vector pcDNA3 to construct a recombinant plasmid pCD-h'VEGF165. The amplified h'VEGF165cDNA fragment was identified by enzyme digestion and DNA sequencing methods. Also, wild-type hVEGF165 cDNA was obtained, identified and cloned into a eukaryotic expressing vector pcDNA3by using the same methods. The results of DNA sequencing showed that h'VEGF165 cDNA cloned from HL601 was 600 bp in size with 8 % of the base sequence in h'VEGF165 cDNA being changed as compared with the base sequence in the wild-type hVEGF165 cDNA. The results of sequencing of hVEGF165 which was cloned from HL60 by us were consistent with the reports completely.

  4. Evidence for regulated secretion of proteins by human umbilical vein endothelial cells

    International Nuclear Information System (INIS)

    There are two methods of protein secretion, regulated and constitutive. The purpose of this study was to determine if there is a regulated pathway of protein secretion in human umbilical vein endothelial cells (EC). EC were cultured as previously described and then incubated with 3H-leucine. Media and cell were analyzed with time (30 sec-16 hrs) for secreted TCA precipitable labelled protein which was then separated by SDS-PAGE, α-thrombin (Th) (5 x 10-12M). DFP-Th (5 x 10-12M), calcium ionophore A23187 (10-7M) but not Th stimulated rapid (125I-fibrin well assay of 1 mm gel slices of conditioned medium following SDS-PAGE. Th because of EC receptor binding, and possibly because of Ca+2 influx, stimulates a rapid secretion of EC proteins, some of which have been identified as u-PA and t-PA. This may represent a major regulatory function of ECs in maintaining normal hemostasis

  5. Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration.

    Science.gov (United States)

    Detaille, D; Vial, G; Borel, A-L; Cottet-Rouselle, C; Hallakou-Bozec, S; Bolze, S; Fouqueray, P; Fontaine, E

    2016-01-01

    Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of β-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy. PMID:27551496

  6. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Khang, Gilson; Soker, Shay

    2013-02-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.

  7. Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes.

    Science.gov (United States)

    Turpaev, K; Litvinov, D; Dubovaya, V; Panasyuk, A; Ivanov, D; Prassolov, V

    2001-06-01

    We investigated the role of nitric oxide (NO) in the control of vascular endothelial growth factor A (VEGF) gene expression in cultured human articular chondrocytes. Cell treatment with the NO-generating compound nitrosoglutathione (GSNO) caused a significant accumulation of 4.4 kb VEGF mRNA, a major VEGF mRNA isoform expressing in chondrocytes. This is the first demonstration that NO can induce VEGF mRNA expression in chondrocytes. VEGF mRNA level was not affected in cells exposed to dibutyryl cGMP, a non-hydrolyzable analog of cGMP, suggesting that the cGMP system is not involved in NO-dependent transcriptional activation of VEGF gene. The GSNO-stimulated induction of VEGF mRNA was slightly attenuated by MAP protein kinase inhibitors PD98058 and SB203580, but was completely blocked in cells incubated with GSNO in the presence of catalase and superoxide dismutase, enzymes scavenging reactive oxygen species (ROS), or in the presence of thiol-containing antioxidants, N-acetyl cysteine and reduced glutathione. These results suggest that in articular chondrocytes the GSNO-induced VEGF gene transcriptional activation is dependent on endogenous ROS production and oxidative thiol modifications.

  8. Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Bing; Qi; Qing-Shan; Ji; Guang-Hui; Hou; Liu; Li; Xian-Fen; Cao; Jing; Wu

    2014-01-01

    AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conventional freezing(CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide(DMSO), 60% fetal bovine serum(FBS) and 30%Dulbecco’s modified Eagle’s medium(DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program:precool in 4℃ for 30 min,-20℃ for 1h, and then immersion in-80℃ refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in-80℃ refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups.RESULTS:There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CFgroup in adherent rates, morphological changes and proliferative ability.CONCLUSION:In the conventional cryopreserved method, cells are slow equilibrium cooling by steps(4℃,-20℃ and finally-80℃), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.

  9. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    Science.gov (United States)

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

  10. Proteomics analysis of human umbilical vein endothelial cells (HUVEC) after treatment with low molecular weight heparin

    Institute of Scientific and Technical Information of China (English)

    YanPAN; Jun-huaWANG; He-mingYU; Xue-junLI

    2004-01-01

    AIM: The endothelium is involved in the generation and the regulation of multiple physiological and pathological processes of blood vessels. Previously we confirmed low molecular weight heparin (LMWH) could inhibit tumor metastasis by protecting human umbilical vein endothelial cells (HUVEC). To understand the effects of LMWH on the protein expression of HUVEC, we performed a comprehensive proteomics to survey global changes in proteins after LMWH treatment in HUVEC cells. METHODS:

  11. Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells.

    OpenAIRE

    Cheung, A L; Krishnan, M.; Jaffe, E A; Fischetti, V A

    1991-01-01

    The propensity of Staphylococcus aureus to cause acute endovascular infections during transient bacteremia is poorly understood. To examine the events leading to the attachment of staphylococci to endothelium, adherence assays were developed to study the role of blood factors in the mediation of staphylococcal adherence to cultured human umbilical vein endothelium in vitro. Results indicate that the preferential attachment of S. aureus to endothelial cells is mediated by fibrinogen adsorbed f...

  12. Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

    OpenAIRE

    Fay, Jakob; Varoga, Deike; Wruck, Christoph J.; Kurz, Bodo; Goldring, Mary B.; Pufe, Thomas

    2006-01-01

    Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartil...

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

    Directory of Open Access Journals (Sweden)

    Eduard Urich

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

  14. Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.

  15. Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

    Institute of Scientific and Technical Information of China (English)

    冯志民; 张晓峰; 王宏芳; 丰美福

    2000-01-01

    Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the different

  16. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Ok-Nam [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Eun-Sun [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Jeong, Tae Cheon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Chun, Young-Jin [College of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Ai-Young, E-mail: leeay@duih.org [Department of Dermatology, Dongguk University Ilsan Hospital, Goyang 410-773 (Korea, Republic of); Noh, Minsoo, E-mail: minsoo@alum.mit.edu [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  17. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    International Nuclear Information System (INIS)

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  18. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke. PMID:27038754

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

    Directory of Open Access Journals (Sweden)

    Dennis J Grab

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

  20. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.

  1. Pre-weaning growth hormone treatment reverses hypertension and endothelial dysfunction in adult male offspring of mothers undernourished during pregnancy.

    Directory of Open Access Journals (Sweden)

    Clint Gray

    Full Text Available Maternal undernutrition results in elevated blood pressure (BP and endothelial dysfunction in adult offspring. However, few studies have investigated interventions during early life to ameliorate the programming of hypertension and vascular disorders. We have utilised a model of maternal undernutrition to examine the effects of pre-weaning growth hormone (GH treatment on BP and vascular function in adulthood. Female Sprague-Dawley rats were fed either a standard control diet (CON or 50% of CON intake throughout pregnancy (UN. From neonatal day 3 until weaning (day 21, CON and UN pups received either saline (CON-S, UN-S or GH (2.5 ug/g/day(CON-GH, UN-GH. All dams were fed ad libitum throughout lactation. Male offspring were fed a standard diet until the end of the study. Systolic blood pressure (SBP was measured at day 150 by tail cuff plethysmography. At day 160, intact mesenteric vessels mounted on a pressure myograph. Responses to pressure, agonist-induced constriction and endothelium-dependent vasodilators were investigated to determine vascular function. SBP was increased in UN-S groups and normalised in UN-GH groups (CON-S 121±2 mmHg, CON-GH 115±3, UN-S 146±3, UN-GH 127±2. Pressure mediated dilation was reduced in UN-S offspring and normalised in UN-GH groups. Vessels from UN-S offspring demonstrated a reduced constrictor response to phenylephrine and reduced vasodilator response to acetylcholine (ACh. Furthermore, UN-S offspring vessels displayed a reduced vasodilator response in the presence of L-NG-Nitroarginine Methyl Ester (L-NAME, carbenoxolone (CBX, L-NAME and CBX, Tram-34 and Apamin. UN-GH vessels showed little difference in responses when compared to CON and significantly increased vasodilator responses when compared to UN-S offspring. Pre-weaning GH treatment reverses the negative effects of maternal UN on SBP and vasomotor function in adult offspring. These data suggest that developmental cardiovascular programming is

  2. Association between anthropometry, cardiometabolic risk factors, & early life factors & adult measures of endothelial function: Results from the New Delhi Birth Cohort

    Directory of Open Access Journals (Sweden)

    Mark D Huffman

    2015-01-01

    Full Text Available Background & objectives: Abnormal endothelial function represents a preclinical marker of atherosclerosis. This study was conducted to evaluate associations between anthropometry, cardiometabolic risk factors, and early life factors and adult measures of endothelial function in a young urban Indian cohort free of clinical cardiovascular disease. Methods: Absolute changes in brachial artery diameter following cuff inflation and sublingual nitroglycerin (400 µg were recorded to evaluate endothelium-dependent and -independent measures of endothelial function in 600 participants (362 men; 238 women from the New Delhi Birth Cohort (2006-2009. Data on anthropometry, cardiometabolic risk factors, medical history, socio-economic position, and lifestyle habits were collected. Height and weight were recorded at birth, two and 11 yr of age. Age- and sex-adjusted linear regression models were developed to evaluate these associations. Results: The mean age of participants was 36±1 yr. Twenty two per cent men and 29 per cent women were obese (BMI th > 30 kg/m [2] . Mean systolic blood pressure (SBP was 131±14 and 119±13 mmHg, and diabetes prevalence was 12 and 8 per cent for men and women, respectively. Brachial artery diameter was higher for men compared with women both before (3.48±0.37 and 2.95±0.35 cm and after hyperaemia (3.87±0.37 vs. 3.37±0.35 cm. A similar difference was seen before and after nitroglycerin. Markers of increased adiposity, smoking, SBP, and metabolic syndrome, but not early life anthropometry, were inversely associated with endothelial function after adjustment for age and sex. Interpretation & conclusions: The analysis of the current prospective data from a young urban Indian cohort showed that cardiometabolic risk factors, but not early life anthropometry, were associated with worse endothelial function.

  3. Serelaxin (recombinant human relaxin-2) prevents high glucose-induced endothelial dysfunction by ameliorating prostacyclin production in the mouse aorta.

    Science.gov (United States)

    Ng, Hooi Hooi; Leo, Chen Huei; Parry, Laura J

    2016-05-01

    Diabetes-induced endothelial dysfunction is a critical initiating factor in the development of cardiovascular complications. Treatment with relaxin improves tumour necrosis factor α-induced endothelial dysfunction by enhancing endothelial nitric oxide synthase (eNOS) activity and restoring superoxide dismutase 1 protein in rat aortic rings ex vivo. It is, therefore, possible that relaxin treatment could alleviate endothelial dysfunction in diabetes. This study aimed to test the hypothesis that serelaxin (recombinant human relaxin-2) prevents high glucose-induced vascular dysfunction in the mouse aorta. Abdominal aortae were isolated from C57BL/6 male mice and incubated in M199 media for 3days with either normal glucose (5.5mM) or high glucose (30mM), and co-incubated with placebo (20mM sodium acetate) or 10nM serelaxin at 37°C in 5% CO2. Vascular function was analysed using wire-myography. High glucose significantly reduced the sensitivity to the endothelium-dependent agonist, acetylcholine (ACh) (pEC50; normal glucose=7.66±0.10 vs high glucose=7.29±0.10, n=11-12, Paortae, but had no effect in serelaxin treated aortae. This suggests that high glucose incubation alters the superoxide and COX-sensitive pathway, which was normalized by co-incubation with serelaxin. Neither high glucose incubation nor serelaxin treatment had an effect on cyclooxygenase 1 and 2 (Ptgs1, Ptgs2), prostacyclin synthase (PTGIS) and receptor (Ptgir) as well as thromboxane A2 receptor (Tbxa2r) mRNA expression. Importantly, production of prostacyclin was significantly (Paortae, which was prevented by serelaxin treatment. Our data show that serelaxin treatment for 3 days restores high glucose-induced endothelial dysfunction by ameliorating vasodilator prostacyclin production and possibly through the reduction of superoxide in the mouse aorta. PMID:26993102

  4. Temporal and pharmacological characterization of angiostatin release and generation by human platelets: implications for endothelial cell migration.

    Directory of Open Access Journals (Sweden)

    Aneta Radziwon-Balicka

    Full Text Available Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF. Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo

  5. Enhanced cellular responses and distinct gene profiles in human fetoplacental artery endothelial cells under chronic low oxygen.

    Science.gov (United States)

    Jiang, Yi-Zhou; Wang, Kai; Li, Yan; Dai, Cai-Feng; Wang, Ping; Kendziorski, Christina; Chen, Dong-Bao; Zheng, Jing

    2013-12-01

    Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O2) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O2) for ∼20-25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O2 decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O2.

  6. Genome-wide analysis of the effect of esophageal squamous cell carcinoma on human umbilical vein endothelial cells.

    Science.gov (United States)

    Jin, Guoguo; Yang, Yi; Liu, Hangfan; Liu, Kangdong; Zhao, Jimin; Chen, Xinhuan; Zhang, Xiaoyan; Zhang, Yanyan; Lu, Jing; Dong, Ziming

    2016-07-01

    A large volume of data indicates that controlling tumor-associated angiogenesis is a promising therapy against cancer. However, angiogenesis is a complex process, little is known about the differential gene expression in the process of normal endothelial cell differentiation toward tumor vascular endothelial cells induced by tumor microenvironment. The aim of this study is to investigate the effect of tumor microenvironment simulated by the supernatant of esophageal squamous cancer cells (KYSE70) on normal endothelial cells (HUVECs) at the whole genome level. The gene expression profile was studied through gene ontology and signal pathway analysis. Compared with the normal HUVECs, a total of 3769 differentially expressed genes in induced HUVECs were detected, including 1609 upregulated genes and 2160 downregulated genes. Moreover, the microarray data analysis showed that 11 significant biological processes and 10 significant signaling pathways changed most, which are associated with angiogenesis and cell differentiation. According to the different expression levels in the microarrays and their functions, four differentially expressed genes involved in tumor angiogenesis and cell differentiation (IL6, VEGFA, S1PR1, TYMP) were selected and analyzed by qRT-PCR. The qRT-PCR results were consistent with the microarray data. Furthermore, we simulated the tumor microenvironment by human esophageal carcinoma tissue homogenate to investigate its effect on HUVECs, the qRT-PCR results indicated that the above genes were highly expressed in HUVECs after induction by esophageal carcinoma tissue homogenate. In conclusion, tumor microenvironment impact on normal endothelial cells differentiated toward tumor vascular endothelial cells, and the selected genes, which are associated with tumor angiogenesis, would be anti-angiogenesis targets against esophageal carcinoma.

  7. Interaction of human endothelial cells and nickel-titanium materials modified with silicon ions

    Energy Technology Data Exchange (ETDEWEB)

    Lotkov, Aleksandr I., E-mail: lotkov@ispms.tsc.ru; Kashin, Oleg A., E-mail: okashin@ispms.tsc.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); Kudryavtseva, Yuliya A., E-mail: yulia-k1970@mail.ru; Antonova, Larisa V., E-mail: antonova.la@mail.ru; Matveeva, Vera G., E-mail: matveeva-vg@mail.ru; Sergeeva, Evgeniya A., E-mail: sergeewa.ew@yandex.ru [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation); Kudryashov, Andrey N., E-mail: kudryashov@angioline.ru [Angioline Interventional Device Ltd, Novosibirsk, 630090 (Russian Federation)

    2015-10-27

    The paper studies the influence of chemical and phase compositions of NiTi surface layers modified with Si ions by plasma immersion implantation on their interaction with endothelial cells. It is shown that certain technological modes of Si ion implantation enhance the adhesion, proliferation, and viability of endothelial cells. It is found that the Si-modified NiTi surface is capable of stimulating the formation of capillary-like structures in the cell culture.

  8. Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

    OpenAIRE

    Cicmil, Milenko; Stevens, Jo; Leduc, Mireille; Bon, Cassian; Gibbins, Jonathan M.

    2002-01-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the r...

  9. Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

    KAUST Repository

    Malara, N.M.

    2015-03-01

    Aim: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. Methods and results: Isolated primary fragment of different vessel types was expanded in Ham\\'s F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. Conclusion: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications. © 2015 The Authors. Published by Elsevier Ireland Ltd.

  10. Comparison of the effects of recombinant human endostatin and docetaxel on human umbilical vein endothelial cells in different growth states

    Institute of Scientific and Technical Information of China (English)

    XU Wen-jing; HUANG Chun; WANG Jing; JIANG Ri-cheng; WANG Liu-chun; LIN Li; LIU Zhu-jun; SUN Bao-cun; LI Kai

    2011-01-01

    Background Recombinant human endostatin (rh-endostatin, Endostar) has been proved to be an inhibitor of angiogenesis. Docetaxel has been also considered as a common chemotherapeutic agent with inhibition of angiogenesis of malignancies. However, their function has been seldom compared and a best synergism protocol is not determined.This study aimed to compare the effects of two drugs, investigate their combined impact on human umbilical vein endothelial cells (HUVECs), a molecular basis and find ideal protocols to inhibit endothelial cell proliferation.Methods HUVECs on confluent growth or activated by vascular endothelial growth factor (VEGF) were treated by rh-endostatin or/and docetaxel at respective gradient concentration in following operations as cell proliferation determined by MTT assay, cell cycle distribution, apoptosis and markers of CD146, CD62E and CD105 detected by flow cytometery, the structure of the channel formed by HUVECs measured by tube formation count.Results Rh-endostatin exhibited time dependent inhibition of proliferation while docetaxel showed both time and dose dependent inhibition. HUVECs accumulated in Go-G1 with decreased numbers of cells in G2 after a single treatment of rh-endostatin or that followed by docetaxel treatment. Cells accumulated in G2 after both a single docetaxel and simultaneous administration. Both the number of cells in G0-G1 and apoptotic cells were increased by docetaxel followed by rh-endostatin treatment. The number of non-apoptotic cells at G0-G1 was increased by first administering rh-endostatin then docetaxel. Sequential treatment of docetaxel followed by rh-endostatin resulted in the greatest increase in apoptosis (34.7%) and the second highest apoptosis was seen with simultaneous administration (18.2%). Expression of CD146 and CD105 on confluent HUVECs was reduced at certain doses of rh-endostatin and/or docetaxel. However,rh-endostatin reduced CD105 without any apparent impact on either CD146 or CD62E

  11. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells.

    Science.gov (United States)

    Finoli, Anthony; Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  12. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Directory of Open Access Journals (Sweden)

    Anthony Finoli

    2016-01-01

    Full Text Available Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  13. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  14. Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

    Directory of Open Access Journals (Sweden)

    Ryohei Numata

    Full Text Available The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

  15. Anionic Sites, Fucose Residues and Class I Human Leukocyte Antigen Fate During Interaction of Toxoplasma gondii with Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Stumbo Ana Carolina

    2002-01-01

    Full Text Available Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocytte antigen (HLA before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containig vacuoles.

  16. Inhibitive Effects of Mulberry Leaf-Related Extracts on Cell Adhesion and Inflammatory Response in Human Aortic Endothelial Cells

    Directory of Open Access Journals (Sweden)

    P.-Y. Chao

    2013-01-01

    Full Text Available Effects of mulberry leaf-related extracts (MLREs on hydrogen peroxide-induced DNA damage in human lymphocytes and on inflammatory signaling pathways in human aortic endothelial cells (HAECs were studied. The tested MLREs were rich in flavonols, especially bombyx faces tea (BT in quercetin and kaempferol. Polyphenols, flavonoids, and anthocyanidin also abounded in BT. The best trolox equivalent antioxidant capacity (TEAC was generated from the acidic methanolic extracts of BT. Acidic methanolic and water extracts of mulberry leaf tea (MT, mulberry leaf (M, and BT significantly inhibited DNA oxidative damage to lymphocytes based on the comet assay as compared to the H2O2-treated group. TNF-α-induced monocyte-endothelial cell adhesion was significantly suppressed by MLREs. Additionally, nuclear factor kappa B (NF-κB expression was significantly reduced by BT and MT. Significant reductions were also observed in both NF-κB and activator protein (AP-1 DNA binding by MLREs. Significant increases in peroxisome proliferator-activated receptor (PPAR α and γ DNA binding by MLREs were also detected in M and MT extracts, but no evidence for PPAR α DNA binding in 50 μg/mL MT extract was found. Apparently, MLREs can provide distinct cytoprotective mechanisms that may contribute to its putative beneficial effects on suppressing endothelial responses to cytokines during inflammation.

  17. Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

    Institute of Scientific and Technical Information of China (English)

    BIAN Chang; ZHAO Kui; TONG Guo-xin; ZHU Yong-liang; CHEN Peng

    2005-01-01

    Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods:Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor,hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

  18. Capture of circulatory endothelial progenitor cells and accelerated re-endothelialization of a bio-engineered stent in human ex vivo shunt and rabbit denudation model

    NARCIS (Netherlands)

    K. Larsen (Katarína); K.L. Cheng (Caroline); D. Tempel (Dennie); S. Parker (Sherry); S. Yazdani (Saami); W.K. den Dekker (Wijnand); H.J. Houtgraaf (Jaco); R. de Jong (Renate); S. Swager-ten Hoor (Stijn); E. Ligtenberg (Erik); S.R. Hanson (Stephen); R. Rowland (Steve); F. Kolodgie (Frank); P.W.J.C. Serruys (Patrick); R. Virmani (Renu); H.J. Duckers (Henricus)

    2012-01-01

    textabstractThe Genous™ Bio-engineered R™ stent (GS) aims to promote vascular healing by capture of circulatory endothelial progenitor cells (EPCs) to the surface of the stent struts, resulting in accelerated re-endothelialization. Here, we assessed the function of the GS in comparison to bare-metal

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  20. Exercise but not prostanoids enhance levels of vascular endothelial growth factor and other proliferative agents in human skeletal muscle interstitium

    DEFF Research Database (Denmark)

    Höffner, Lotte; Nielsen, Jens Jung; Langberg, Henning;

    2003-01-01

    In the present study we examined whether exercise and prostanoids have an effect on the muscle interstitial concentration of vascular endothelial growth factor (VEGF) and on the proliferative effect of muscle interstitial fluid. Dialysate from resting and exercising human skeletal muscle, obtained...... either during control conditions or during cyclooxygenase inhibition, was examined for its content of VEGF and for its effect on endothelial cell proliferation. Microdialysis probes with high (960 kDa) and low (5 kDa) molecular-mass cut-off membranes were placed in the vastus lateralis muscle of healthy......Da dialysate from resting muscle than with perfusate and was 5.8-fold higher (P muscle. VEGF was not enhanced with exercise in the 5 kDa dialysate, yet the exercise dialysate induced a 1.9-fold higher (P

  1. Cadmium chloride alters mRNA levels of angiogenesis related genes in primary human endometrial endothelial cells grown in vitro.

    Science.gov (United States)

    Helmestam, Malin; Stavreus-Evers, Anneli; Olovsson, Matts

    2010-11-01

    Cadmium, is known to cause adverse reproductive effects, and classified as an endocrine disrupting chemical (EDC). Human endometrial endothelial cells (HEEC) have a key role in the regulation of endometrial angiogenesis. These cells are known to express estrogen receptors, a feature that makes them potential targets for EDCs such as cadmium. We have designed a co-culture system, in which HEEC were grown in the same cell culture medium as endometrial stromal cells but in separate, communicating chambers. With quantitative PCR, we investigated changes in mRNA expression of genes associated with angiogenesis, sex steroids and endothelial cell specific functions. We found that cadmium altered the mRNA expression of the two important angiogenic molecules VEGF-A and PLGF. Cadmium might thus affect endometrial angiogenesis and as a consequence cause endometrial dysfunction with an increased risk for fertility problems. PMID:20580663

  2. Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.

    Science.gov (United States)

    Donahower, Brian C; McCullough, Sandra S; Hennings, Leah; Simpson, Pippa M; Stowe, Cindy D; Saad, Ali G; Kurten, Richard C; Hinson, Jack A; James, Laura P

    2010-07-01

    We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

  3. Effects of pentoxifylline on proliferation of human umbilical vein endothelial cells (HUVEC

    Directory of Open Access Journals (Sweden)

    Eyup Çağatay Zengin

    2016-09-01

    Full Text Available SUMMARY Objective: The aim of this study was to investigate the effect of pentoxifylline (PNX with a pharmacological dose range on proliferation of human umblical venous endothelial cells (HUVEC. Method: The cells were maintained in M199 supplemented with 20% fetal bovine serum, penicillin, and streptomycin. The cultures were cultivated in an incubator at 37°C and with 5% CO2, until cell monolayers attained confluence which occurred after 7 days. The assays were performed in the exponential growth phase of the cells. The cell viability was assessed using the cleavage of tetrazolium salts added to the culture medium. Pentoxifylline with concentrations of 10-4M, 10-5M, 10-6M and 10-7M were used for the proliferation assay in which cells were incubated for 24, 48, and 72-hours with these drugs. The experiments were conducted in six replicates. Results: Only the 10-4M dose of PNX at 72 h significantly reduced the viability of HUVEC (p0.05. Conclusions: Overall, PTX with a pharmacological dose range has no cytotoxic effect on HUVEC. We think that this is also in accordance with the findings of several studies performed in animal models and clinical settings, indicating positive effects of PTX on tissues in normal and ischemic conditions. Keywords: Pentoxifylline, proliferation, human umbilical venous endothelial cells ÖZET Amaç: Bu çalışmanın amacı farmokolojik doz aralığında uygulanan pentoksifilinin insan umblikal ven endotel hücrelerinin (HUVEC proliferasyonu üzerine etkisini araştırmaktır. Yöntem: Hücreler içerisine %20 fetal bovine serum, penisilin ve streptomisin eklenen M199 besiyeri içinde, %5 CO2 içeren 37 ̊C ‘lik inkübatörde kültüre edildi. Deneyler hücreler gelişim fazında iken gerçekleştirildi. Hücrelerin canlılığı kültür ortamına tetrazolium tuzları eklenerek değerlendirildi. Pentoksifilinin 10-4M, 10-5M, 10-6M and 10-7M konsantrasyonları, 24, 48 ve 72. saatlerde değerlendirildi. Deneyler alt

  4. Artesunate reduces chicken chorioallantoic membrane neovascularisation and exhibits antiangiogenic and apoptotic activity on human microvascular dermal endothelial cell.

    Science.gov (United States)

    Huan-huan, Chen; Li-Li, You; Shang-Bin, Li

    2004-08-10

    Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. ART has now been analyzed for its anti-angiogenic activity in vivo and in vitro. The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model. ART started to significantly inhibit CAM angiogenesis at a low concentration of 10 nm/100 microl/egg, and completely inhibited the angiogenesis at 80 nm/100 microl/egg. The inhibitory effect of in vitro angiogenesis was tested on the models of proliferation and differentiation of human microvascular dermal endothelial cell line, an important representive of endothelial cells, as well as immunocytochemistry assay for two major VEGF receptors (Flt-1 and KDR/flk-1) expressions. The results showed that ART could remarkably inhibit proliferation and differentiation of endothelial cells in a dose-dependent form in a range of 12.5-100 microM. ART also could reduce Flt-1 and KDR/flk-1 expressions in a range of 0.1-0.5 microM. Furthermore, we examined the apoptosis of human microvascular dermal endothelial cell line induced by ART. The apoptosis was detected by morphological assay of ethidium bromide (EB)/acridine orange (AO) dual staining as well as DNA fragmentation assay of TUNEL labeling and quantified by flowcytometric PI assay. Our results suggest that the antiangiogenic effect induced by ART might occur by the induction of cellular apoptosis. These findings and the known low toxicity indicated ART might be a promising candidate for angiogenesis inhibitors. PMID:15219940

  5. Effects of asiaticoside on human umbilical vein endothelial cell apoptosis induced by Aβ1-42.

    Science.gov (United States)

    Zhang, Zhuo; Cai, Pengfei; Zhou, Jie; Liu, Minghua; Jiang, Xian

    2015-01-01

    This study is to investigate the potential role of asiaticoside (AS) in Aβ1-42-induced apoptosis on the human umbilical vein endothelial cell (HUVEC). HUVEC cells were divided into Aβ1-42 group (treated with 50 μM Aβ1-42), AS groups (treated with 50 μM Aβ1-42 and 10 mM, 1 mM, 0.1 mM or 0.01 mM AS), and negative control group (without treatments). Cell proliferation was detected by CCK-8 assay. Apoptosis was analyzed by Hochest33342 staining and flow cytometry. Western Blot was carried out to detect the expression of Bcl-2 and Bax protein. Aβ1-42 treatment inhibited cell proliferation and increased cell apoptosis of HUVEC cells. Interestingly, AS at concentrations of 10 mM, 1 mM, 0.1 mM and 0.01 mM reversed the effects of Aβ1-42 by increasing cell survival rate and reducing apoptosis of HUVEC cells. Furthermore, the expression of Bcl-2 protein was increased whereas the expression of Bax protein was decreased in AS groups. Compared with Aβ1-42 group, the ratio of Bcl-2/Bax was significantly increased in AS groups (P < 0.05). These results suggested that AS may be effective in protecting cells from damage caused by aggregated Aβ1-42. And this effect may be attributed to the increase of Bcl-2 and decrease of Bax under AS treatment.

  6. Aspirin inhibits tumor necrosis factor-α-stimulated fractalkine expression in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    JIANG De-qian; LIU Hong; ZHANG She-bing; ZHANG Xiao-lian

    2009-01-01

    Background Fractalkine is an important chemokine mediating local monocyte accumulation and inflammatory reactions in the vascular wall. Aspirin inhibits inflammatory cytokine expression closely related to atherosclerosis through the way independent of platelet and cyclooxygenase (COX). There has been no report about the effect of aspirin on fractalkine expression. We aimed to determine the fractalkine expression in human umbilical vein endothelial cell (HUVEC) stimulated by tumor necrosis factor (TNF)-α and the effect of aspirin intervention.Methods Six of 8 HUVEC groups received either different concentrations of aspirin (0.02, 0.2, 1.0, 5.0 mmol/L) or 40 μmol/L pyrrolidinecarbodithioc acid (PDTC) or 0.5 μmol/L NS-398. The other two groups were negative control and positive control (TNF-α-stimulated). After being incubated for 24 hours, cells of the 8 groups except the negative control one were stimulated with TNF-a (4 ng/ml) for another 24 hours. After that, the cells were collected for RNA isolation and protein extraction.Results Both mRNA and protein expressions of fractalkine in HUVEC were upregulated by 4 ng/ml TNF-α stimulation,Aspirin inhibited fractalkine expression in a dose-dependent manner at mRNA and protein levels. Nuclear factor-kappa B inhibitor, PDTC, effectively decreased the fractalkine expression. Fractalkine expression was not influenced by COX-2 selective inhibitor NS-398. COX-1 protein expression was not changed by either TNF-α stimulation or aspirin, PDTC,NS-398 intervention. Both mRNA and protein expression of COX-2 in HUVEC were upregulated by 4 ng/ml TNF-α stimulation. Aspirin decreased COX-2 expression in a dose-dependent manner at mRNA and protein levels.Conclusions TNF-α-stimulated fractalkine expression is suppressed by aspirin in a dose-dependent manner through the nuclear factor-kappa B p65 pathway.

  7. Expression of vascular endothelial growth factor in cultured human dental follicle cells and its biological roles

    Institute of Scientific and Technical Information of China (English)

    Xue-peng CHEN; Hong QIAN; Jun-jie WU; Xian-wei MA; Ze-xu GU; Hai-yan SUN; Yin-zhong DUAN; Zuo-lin JIN

    2007-01-01

    Aim: To investigate the expression of vascular endothelial growth factor (VEGF) in cultured human dental follicle cells (HDFC), and to examine the roles of VEGF in the proliferation, differentiation, and apoptosis of HDFC in vitro. Methods: Immunocytochemistry, ELISA, and RT-PCR were used to detect the expression and transcription of VEGF in cultured HDFC. The dose-dependent and the time-course effect of VEGF on cell proliferation and alkaline phosphatase (ALP) activ-ity in cultured HDFC were determined by MTT assay and colorimetric ALP assay, respectively. The effect of specific mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0126) on the VEGF-mediated HDFC proliferation was also determined by MTT assay. The effect of VEGF on HDFC apoptosis was measured by flow cytometry. Results: VEGF was transcribed and expressed in cultured HDFC. VEGF at 10-300 μg/L significantly increased HDFC proliferation and ALP activity compared to the control. Following 1, 3, 5, or 7 d of stimulation, VEGF induced a significant increase in HDFC proliferation compared with the corresponding control, while VEGF was effective at increasing ALP activity at the incubation time point of 3, 5, or 7 d. PD98059 and U0126 could attenuate the VEGF-mediated HDFC proliferation. Fewer apoptotic cells were observed in the VEGF-treated groups compared to the controls, although the difference was not statistically significant. Conclusion: VEGF is expressed in cultured HDFC, and at a proper concentration range can stimulate HDFC proliferation, induce HDFC to differentiate in a "cementoblast/osteoblast" pathway and protect HDFC from apoptosis. The MAPK signaling pathway might be involved in the VEGF-medi-ated HDFC proliferation.

  8. Red Raspberry Phenols Inhibit Angiogenesis: A Morphological and Subcellular Analysis Upon Human Endothelial Cells.

    Science.gov (United States)

    Sousa, M; Machado, V; Costa, R; Figueira, M E; Sepodes, B; Barata, P; Ribeiro, L; Soares, R

    2016-07-01

    Polyphenols are a class of natural compounds whose potential as antioxidant, anti-inflammatory, and anti-angiogenesis has been reported in many pathological conditions. Red raspberry extract, rich in polyphenols, has been reported to exert anti-inflammatory effects and prevent cell proliferation in distinct animal models. However, the signaling pathways involved remain unknown. Herein, we used human microvascular endothelial cells (HMVECs) to determine the influence of red raspberry phenolic compound extract concentrations, ranging from 10 to 250 µg gallic acid equivalents (GAE)/mL, on endothelium viability (MTS assay), proliferation (BrdU incorporation), migration (injury assay), and capillary-like structures formation (Matrigel assay). Protein expression in cell lysates was determined by Western blot analysis. We showed that red raspberry extracts reduced cell viability (GI50  = 87,64 ± 6,59 μg GAE/mL) and proliferation in a dose-dependent manner. A significant abrogation of cells ability to migrate to injured areas, even at low concentrations, was observed by injury assay. Cell assembly into capillary-like structures on Matrigel also decreased in a dose dependent-manner for higher extract concentrations, as well as the number of branching points per unit of area. Protein expression analysis showed a dose-dependent decrease in Phospho-VEGFR2 expression, implying abrogation of VEGF signaling activity. We also showed for the first time that red raspberry phenolic compounds induce the rearrangement of filamentous actin cytoskeleton, with an isotropy increase found for higher testing concentrations. Taken together, our findings corroborate the anti-angiogenic potential of red raspberry phenolic compounds and provide new insights into their mode of action upon endothelium. J. Cell. Biochem. 117: 1604-1612, 2016. © 2015 Wiley Periodicals, Inc. PMID:26590362

  9. Influence of gestational diabetes mellitus on human umbilical vein endothelial cell miRNA.

    Science.gov (United States)

    Tryggestad, Jeanie B; Vishwanath, Anu; Jiang, Shaoning; Mallappa, Ashwini; Teague, April M; Takahashi, Yusuke; Thompson, David M; Chernausek, Steven D

    2016-11-01

    We aimed to identify miRNAs whose expression levels in fetal tissues are altered by exposure to a diabetic milieu and elucidate the impact on target protein expression. Gestational diabetes mellitus (GDM) affects both immediate and future disease risk in the offspring. We hypothesized that GDM alters miRNA expression in human umbilical vein endothelial cells (HUVECs) that may influence metabolic processes. A cross-sectional design compared differences in miRNA expression in HUVECs and target protein abundance in placentae between infants of women with GDM (IGDM) and infants born to normoglycaemic controls. miRNAs were identified using microarray profiling and literature review and validated by quantitative PCR (qPCR). In vitro transfection studies explored the impact of the miRNA on target protein expression. Expression of seven miRNA species, miR-30c-5p, miR-452-5p, miR-126-3p, miR-130b-3p, miR-148a-3p, miR-let-7a-5p and miR-let-7g-5p, was higher in the HUVECs of IGDM. Abundance of the catalytic subunit of AMP-activated protein kinase α1 (AMPKα1) was decreased in the HUVECs and BeWo cells (transformed trophoblast cell line) transfected with miR-130b and miR-148a mimics. AMPKα1 expression was also decreased in placental tissues of IGDM. The expression of several miRNAs were altered by in utero exposure to DM in infants of women whose dysglycaemia was very well controlled by current standards. Decreased expression of AMPKα1 as a result of increased levels of miR-130b and miR-148a may potentially explain the decrease in fat oxidation we reported in infants at 1 month of age and, if persistent, may predispose offspring to future metabolic disease. PMID:27562513

  10. Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo.

    Science.gov (United States)

    Ferreira, Lino S; Gerecht, Sharon; Shieh, Hester F; Watson, Nicki; Rupnick, Maria A; Dallabrida, Susan M; Vunjak-Novakovic, Gordana; Langer, Robert

    2007-08-01

    We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34+ cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34+ cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (alpha-SM actin, SM myosin heavy chain, calponin, caldesmon, SM alpha-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering.

  11. Expression of the cystic fibrosis gene in adult human lung.

    OpenAIRE

    Engelhardt, J F; Zepeda, M; Cohn, J.A.; Yankaskas, J R; Wilson, J. M.

    1994-01-01

    Critical to an understanding of the pulmonary disease in cystic fibrosis (CF) and the development of effective gene therapies is a definition of the distribution and regulation of CF gene expression in adult human lung. Previous studies have detected the product of the CF gene, the CF transmembrane conductance regulator (CFTR), in submucosal glands of human bronchi. In this report, we have characterized the distribution of CFTR RNA and protein in the distal airway and alveoli of human lungs. ...

  12. Short Communication: Initiation of an Abacavir-Containing Regimen in HIV-Infected Adults Is Associated with a Smaller Decrease in Inflammation and Endothelial Activation Markers Compared to Non-Abacavir-Containing Regimens

    OpenAIRE

    HILEMAN, Corrilynn O; Wohl, David A.; Tisch, Daniel J.; Debanne, Sara M.; McComsey, Grace A

    2012-01-01

    Abacavir has been associated with myocardial infarction in several studies. This may be related to inflammation and endothelial cell activation. We compared changes in inflammation and endothelial activation markers between antiretroviral-naive adults initiating zidovudine, lamivudine, abacavir, and nonnucleoside reverse transcriptase inhibitor (NNRTI) or this regimen without abacavir. Changes in soluble tumor necrosis factor receptors-I, -II (sTNFR-I, -II), high sensitivity C-reactive protei...

  13. Anti inflammatory and anti angiogenic effect of black raspberry extract on human esophageal and intestinal microvascular endothelial cells.

    Science.gov (United States)

    Medda, Rituparna; Lyros, Orestis; Schmidt, Jamie L; Jovanovic, Nebojsa; Nie, Linghui; Link, Benjamin J; Otterson, Mary F; Stoner, Gary D; Shaker, Reza; Rafiee, Parvaneh

    2015-01-01

    Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat's digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial cell-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer.

  14. Three-dimensional model of angiogenesis: coculture of human retinal cells with bovine aortic endothelial cells in the NASA bioreactor.

    Science.gov (United States)

    Dutt, Kamla; Sanford, Gary; Harris-Hooker, Sandra; Brako, Lawrence; Kumar, Ravindra; Sroufe, Angela; Melhado, Caroline

    2003-10-01

    Ocular angiogenesis is the leading cause of blindness and is associated with diabetic retinopathy and age-related macular degeneration. We describe, in this report, our preliminary studies using a horizontally rotating bioreactor (HRB), developed by the National Aeronautics and Space Administration (NASA), to explore growth and differentiation-associated events in the early phase of ocular angiogenesis. Human retinal (HRet) cells and bovine endothelial cells (ECs) were cocultured on laminin-coated Cytodex-3 microcarrier beads in an HRB for 1-36 days. Endothelial cells grown alone in the HRB remained cuboidal and were well differentiated. However, when HRet cells were cocultured with ECs, cordlike structures formed as early as 18-36 h and were positive for von Willebrand factor. In addition to the formation of cords and capillary-like structures, ECs showed the beginning of sprouts. The HRB seems not only to promote accelerated capillary formation, but also to enhance differentiation of retinal precursor cells. This leads to the formation of rosette-like structures (which may be aggregates of photoreceptors that were positive for rhodopsin). Upregulation of vascular endothelial growth factor and basic fibroblast growth factor was seen in retinal cells grown in the HRB as compared with monolayers and could be one of the factors responsible for accelerated capillary formation. Hence, the HRB promotes three-dimensional assembly and differentiation, possibly through promoting cell-to-cell interaction and/or secretion of growth and differentiation factors. PMID:14633374

  15. Trichostatin A enhances vascular repair by injected human endothelial progenitors through increasing the expression of TAL1-dependent genes.

    Science.gov (United States)

    Palii, Carmen G; Vulesevic, Branka; Fraineau, Sylvain; Pranckeviciene, Erinija; Griffith, Alexander J; Chu, Alphonse; Faralli, Hervé; Li, Yuhua; McNeill, Brian; Sun, Jie; Perkins, Theodore J; Dilworth, F Jeffrey; Perez-Iratxeta, Carol; Suuronen, Erik J; Allan, David S; Brand, Marjorie

    2014-05-01

    A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential. PMID:24792117

  16. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  17. Proliferation, behavior, and cytokine gene expression of human umbilical vascular endothelial cells in response to different titanium surfaces.

    Science.gov (United States)

    An, Na; Schedle, Andreas; Wieland, Marco; Andrukhov, Oleh; Matejka, Michael; Rausch-Fan, Xiaohui

    2010-04-01

    Success of dental implantation is initially affected by wound healing of both, hard and soft tissues. Endothelial cells (ECs) are involved as crucial cells in the angiogenesis and inflammation process of wound healing. In the present study, proliferation, mobility, cluster formation, and gene expression of angiogenesis-related molecules of human umbilical vascular endothelial cells (HUVECs) were investigated on titanium surfaces with different roughnesses: acid-etched (A), coarse-grit-blasted and acid-etched (SLA) surfaces, as well as on hydrophilic modified modA and modSLA surfaces. Cell behaviors were analyzed by proliferation assay and time-lapse microscopy, gene expression was analyzed by real time PCR. Results showed that cell proliferation, mobility, and cluster formation were highest on modA surfaces compared with all other surfaces. HUVECs moved slowly and exhibited seldom cell aggregation on SLA and modSLA surfaces during the whole observing period of 120 h. The gene expressions of the angiogenesis-related factors von Willebrand factor, thrombomodulin, endothelial cell protein C receptor, and adhesion molecules intercellular adhesion molecule-1 and E-selectin were most enhanced on modSLA surfaces. These results suggest that modA surface is optimal for proliferation and angiogenic behavior of ECs. However, modSLA surface seems to promote ECs to express angiogenesis-related factor genes, which play essential roles in controlling inflammation and revascularization of wound healing. PMID:19569217

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

    Directory of Open Access Journals (Sweden)

    Hongxiu Wen

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

  19. Selected Activities of Citrus Maxima Merr. Fruits on Human Endothelial Cells: Enhancing Cell Migration and Delaying Cellular Aging

    Directory of Open Access Journals (Sweden)

    Paiwan Buachan

    2014-04-01

    Full Text Available Endothelial injury and damage as well as accumulated reactive oxygen species (ROS in aging play a significant role in the development of cardiovascular disease (CVD. Recent studies show an association of high citrus fruit intake with a lower risk of CVD and stroke but the mechanisms involved are not fully understood. This study investigated the effects of pummelo (Citrus maxima Merr. var. Tubtim Siam, CM fruit extract on human umbilical vein endothelial cell (HUVECs migration and aging. The freeze-dried powder of fruit extract was characterized for antioxidant capacity (FRAP assay and certain natural antioxidants, including ascorbic acid, gallic acid, hesperidin, and naringin (HPLC. Short-term (48 h co-cultivation of HUVECs with CM enhanced cell migration as evaluated by a scratch wound assay and Boyden chamber assay. A long-term treatment with CM for 35 days significantly increased HUVEC proliferation capability as indicated by population doubling level (PDL. CM also delayed the onset of aging phenotype shown by senescence-associated β-galactosidase (SA-β-gal staining. Furthermore, CM was able to attenuate increased ROS levels in aged cells when determined by 2′,7′-dichlorodihydrofluorescein diacetate (DCDHF while eNOS mRNA expression was increased but the eNOS protein level was not changed. Thus, further in vivo and clinical studies are warranted to support the use of pummelo as a functional fruit for endothelial health and CVD risk reduction.

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

    Directory of Open Access Journals (Sweden)

    Yongjie Li

    2015-01-01

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

  1. Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

    Directory of Open Access Journals (Sweden)

    Gary S. L. Peh

    2012-01-01

    Full Text Available The culture of human corneal endothelial cells (CECs is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

  2. DHA and EPA Down-regulate COX-2 Expression through Suppression of NF-κB Activity in LPS-treated Human Umbilical Vein Endothelial Cells

    OpenAIRE

    Lee, Soon Ae; Kim, Hye Jung; Chang, Ki Churl; Baek, Jong Chul; Park, Ji Kwon; Shin, Jeong Kyu; Choi, Won Jun; Lee, Jong Hak; Paik, Won Young

    2009-01-01

    Inflammatory processes of vascular endothelial cells play a key role in the development ofatherosclerosis. We determined the anti-inflammatory effects and mechanisms of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on LPS-treated human umbilical vein endothelial cells (HUVECs) to evaluate their cardioprotective potential. Cells were pretreated with DHA, EPA, or troglitazone prior to activation with LPS. Expression of COX-2, prostaglandin E2 (PGE2) and IL-6 production, and NF-κB a...

  3. Atorvastatin induces thrombomodulin expression in the aorta of cholesterol-fed rabbits and in TNFa-treated human aortic endothelial cells

    OpenAIRE

    Lin, Shing-Jong; Hsieh, Fang-Yu; Chen, Yung-Hsiang; Lin, Chia-Chi; Kuan, I-I; Wang, Shu-Huei; Wu, Chau-Chung; Chien, Hsiung-Fei; Lin, Fen-Yen; Chen, Yuh-Lien

    2009-01-01

    Expression of functionally active thrombomodulin (TM) on endothelial cells is critical for vascular thromboresistance. 3-Hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) can protect the vasculature from inflammation and atherosclerosis caused by cholesterol-dependent and cholesterolindependent mechanisms. In the present study, the effects of atorvastatin on TM expression in the aorta of cholesterol-fed rabbits and in TNFa-treated human aortic endothelial ...

  4. Interleukin-8 increases vascular endothelial growth factor and neuropilin expression and stimulates ERK activation in human pancreatic cancer.

    Science.gov (United States)

    Li, Min; Zhang, Yuqing; Feurino, Louis W; Wang, Hao; Fisher, William E; Brunicardi, F Charles; Chen, Changyi; Yao, Qizhi

    2008-04-01

    Interleukin-8 (IL-8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up-regulation of IL-8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL-8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL-8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL-8 mRNA than normal human pancreatic ductal epithelium cells. IL-8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic-adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL-8 up-regulated the expression of vascular endothelial growth factor(165), and neuropilin (NRP)-2 in BxPC-3 cells, one of human pancreatic cancer cell lines. IL-8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL-8 activated extracellular signal-regulated kinase (ERK)1/2 signaling pathway in BxPC-3 cells. Our studies suggest that IL-8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP-2 expression and ERK activation. Targeting IL-8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy. PMID:18307536

  5. Interleukin-8 increases vascular endothelial growth factor and neuropilin expression and stimulates ERK activation in human pancreatic cancer.

    Science.gov (United States)

    Li, Min; Zhang, Yuqing; Feurino, Louis W; Wang, Hao; Fisher, William E; Brunicardi, F Charles; Chen, Changyi; Yao, Qizhi

    2008-04-01

    Interleukin-8 (IL-8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up-regulation of IL-8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL-8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL-8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL-8 mRNA than normal human pancreatic ductal epithelium cells. IL-8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic-adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL-8 up-regulated the expression of vascular endothelial growth factor(165), and neuropilin (NRP)-2 in BxPC-3 cells, one of human pancreatic cancer cell lines. IL-8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL-8 activated extracellular signal-regulated kinase (ERK)1/2 signaling pathway in BxPC-3 cells. Our studies suggest that IL-8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP-2 expression and ERK activation. Targeting IL-8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy.

  6. Adult human metapneumonovirus (hMPV) pneumonia mimicking Legionnaire's disease.

    Science.gov (United States)

    Cunha, Burke A; Irshad, Nadia; Connolly, James J

    2016-01-01

    In adults hospitalized with viral pneumonias the main differential diagnostic consideration is influenza pneumonia. The respiratory viruses causing viral influenza like illnesses (ILIs), e.g., RSV may closely resemble influenza. Rarely, extrapulmonary findings of some ILIs may resemble Legionnaire's disease (LD), e.g., adenovirus, human parainfluenza virus (HPIV-3). We present a most unusual case of human metapneumonovirus pneumonia (hMPV) with some characteristic extrapulmonary findings characteristic of LD, e.g., relative bradycardia, as well as mildly elevated serum transaminases and hyphosphatemia. We believe this is the first reported case of hMPV pneumonia in a hospitalized adult that had some features of LD. PMID:26988110

  7. The weight of nations: an estimation of adult human biomass

    Directory of Open Access Journals (Sweden)

    Walpole Sarah

    2012-06-01

    Full Text Available Abstract Background The energy requirement of species at each trophic level in an ecological pyramid is a function of the number of organisms and their average mass. Regarding human populations, although considerable attention is given to estimating the number of people, much less is given to estimating average mass, despite evidence that average body mass is increasing. We estimate global human biomass, its distribution by region and the proportion of biomass due to overweight and obesity. Methods For each country we used data on body mass index (BMI and height distribution to estimate average adult body mass. We calculated total biomass as the product of population size and average body mass. We estimated the percentage of the population that is overweight (BMI > 25 and obese (BMI > 30 and the biomass due to overweight and obesity. Results In 2005, global adult human biomass was approximately 287 million tonnes, of which 15 million tonnes were due to overweight (BMI > 25, a mass equivalent to that of 242 million people of average body mass (5% of global human biomass. Biomass due to obesity was 3.5 million tonnes, the mass equivalent of 56 million people of average body mass (1.2% of human biomass. North America has 6% of the world population but 34% of biomass due to obesity. Asia has 61% of the world population but 13% of biomass due to obesity. One tonne of human biomass corresponds to approximately 12 adults in North America and 17 adults in Asia. If all countries had the BMI distribution of the USA, the increase in human biomass of 58 million tonnes would be equivalent in mass to an extra 935 million people of average body mass, and have energy requirements equivalent to that of 473 million adults. Conclusions Increasing population fatness could have the same implications for world food energy demands as an extra half a billion people living on the earth.

  8. Discovery of molecular markers to discriminate corneal endothelial cells in the human body

    NARCIS (Netherlands)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Clevers, J.C.; van de Wetering, M.L.

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant nu

  9. The interaction of human endothelial cells with chemical gradient surfaces during exposure to flow

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; Van der Meer, J; Van der Mei, HC; Busscher, HJ; Olij, WJV; Anderson, HR

    1998-01-01

    In this study, the position bound shape, spreading, detachment and migration of adhering HUVEC endothelial cells on dichlorodimethylsilane (DDS) chemical gradient surfaces was investigated during exposure to flow in a parallel plate flow chamber in the presence of` serum proteins. Gradient surfaces

  10. Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

    NARCIS (Netherlands)

    Meijer, L.A.T.; Zhou, H.; Chan, O.Y.A.; Altelaar, A.F.M.; Hennrich, M.L.; Mohammed, S.; Bos, J.L.; Heck, A.J.R.

    2013-01-01

    The small GTPase Rap1 is required for proper cell–cell junction formation and also plays a key role in mediating cAMP-induced tightening of adherens junctions and subsequent increased barrier function of endothelial cells. To further study how Rap1 controls barrier function, we performed quantitativ

  11. IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation

    DEFF Research Database (Denmark)

    Hammer, Troels; Tritsaris, Katerina; Hübschmann, Martin V;

    2009-01-01

    IL-20 is an arteriogenic cytokine that remodels collateral networks in vivo, and plays a role in cellular organization. Here, we investigate its role in lymphangiogenesis using a lymphatic endothelial cell line, hTERT-HDLEC, which expresses the lymphatic markers LYVE-1 and podoplanin. Upon stimul...

  12. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can rec...

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

    LENUS (Irish Health Repository)

    Corcoran, T B

    2012-02-03

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

  14. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring.

    Science.gov (United States)

    Gorlin, J B; Yamin, R; Egan, S; Stewart, M; Stossel, T P; Kwiatkowski, D J; Hartwig, J H

    1990-09-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin

  15. Toxicity of antiglaucoma drugs with and without benzalkonium chloride to cultured human corneal endothelial cells

    Directory of Open Access Journals (Sweden)

    Masahiko Ayaki

    2010-10-01

    Full Text Available Masahiko Ayaki1, Atsuo Iwasawa2, Yoichi Inoue31Department of Ophthalmology, Saitama National Hospital, Wako, Japan; 2Life Particle Interaction Engineering Creation, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan; 3Department of Ophthalmology, Olympia Eye Hospital, Tokyo, JapanPurpose: The toxicity of antiglaucoma medications to ocular surface cells has been evaluated extensively; however, the toxicity to corneal endothelial cells (CECs remains elusive. Our aim is to evaluate the toxicity of antiglaucoma medications to CECs using an in vitro toxicity assay.Methods: Primary cultures of human (H CECs derived from eye bank specimens were established. Following exposure of HCECs to test solutions for 10, 30, or 60 minutes, or 48 hours, we measured cell viability using a WST-1 assay. Test solutions were diluted in culture media and included 0.5% Timoptol®, preservative-free 0.5% timolol maleate, 1% Trusopt®, preservative-free 1% dorzolamide, Travatan®, Travatan Z®, Xalatan®, and benzalkonium chloride (BAK. To assess cell viability, the value of the test culture well after treatment was expressed as a percentage of that of the control well. Toxicity of each solution was compared using the cell viability score (CVS.Results: After exposure to 10-fold dilutions of test solutions for 48 hours, HCEC viabilities were 48.5% for 0.5% Timoptol, 80.9% for preservative-free 0.5% timolol maleate, 47.0% for 1% Trusopt, 71.7% for preservative-free 1% dorzolamide, 55.5% for Travatan, 88.5% for Travatan Z, and 52.5% for Xalatan. Exposure to test solutions diluted 100-fold or more resulted in HCEC viabilities > 80%, with the exception of preservative-free 1% dorzolamide, which resulted in a viability of 72.0% at a dilution of 100-fold. Based on CVS, the order of cell viability was Travatan Z ≥ preservative-free timolol maleate = preservative-free dorzolamide > 0.5% Timoptol = 1% Trusopt > Travatan ≥ Xalatan. Assessment of the

  16. Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

    OpenAIRE

    Encabo, A.; Romanin, C; Birke, F. W.; Kukovetz, W. R.; Groschner, K

    1996-01-01

    1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations...

  17. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    Directory of Open Access Journals (Sweden)

    Tiago M Fortunato

    Full Text Available Endothelial colony-forming cells (ECFCs are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin. The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2 in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml but not basic Fibroblast Growth Factor (FGF (25-100 ng/ml rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

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

    Directory of Open Access Journals (Sweden)

    Yuen Kit M

    2009-10-01

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

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

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

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

  20. Expression of Interleukin-15 and Its Receptor on the Surface of Stimulated Human Umbilical Vein Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Xiuping LIU; Yumei ZUO; Weina ZHANG; Deguang YANG; Changyun XIONG; Xiaozhou ZHANG

    2009-01-01

    Human interleukin-15 (hlL-15) is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ,(IFN-γ), and tumor necrosis factor-ct (TNF-α) to induce the production of human interleukin-15 (hlL-15)and IL-15 receptor (IL-15Rα) by human umbilical vein endothelial cells (HUVECs). The data are summarized as follows: 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α. 2. lntracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expres-sion of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting (FACS),and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-γ and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-γ and TNF-α play an important role in regulating the expres-sion of IL-15 and IL-15Rα on the surface of HUVECs.

  1. Pigment epithelium derived factor upregulates expression of vascular endothelial growth factor by human mesenchymal stem cells: Possible role in PEDF regulated matrix mineralization.

    Science.gov (United States)

    Li, Feng; Armstrong, Gillian B; Tombran-Tink, Joyce; Niyibizi, Christopher

    2016-09-23

    Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization. PMID:27530920

  2. Uptake and cytotoxicity of citrate-coated gold nanospheres: Comparative studies on human endothelial and epithelial cells

    Directory of Open Access Journals (Sweden)

    Freese Christian

    2012-07-01

    Full Text Available Abstract Background The use of gold nanoparticles (AuNPs for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm, to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier. Results Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm, the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the

  3. An anatomically comprehensive atlas of the adult human brain transcriptome

    NARCIS (Netherlands)

    Hawrylycz, M.J.; Beckmann, C.F.; et al., et al.

    2012-01-01

    Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising

  4. Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells

    International Nuclear Information System (INIS)

    Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation

  5. VEGF gene expression in adult human thymus fat: a correlative study with hypoxic induced factor and cyclooxygenase-2.

    Directory of Open Access Journals (Sweden)

    Francisco Tinahones

    Full Text Available UNLABELLED: It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. DESIGN: Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. RESULTS: We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1alpha, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARgamma1/gamma2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. CONCLUSION: Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new

  6. C-reactive protein decreases interleukin-8 production in human endothelial progenitor cells by inhibition of p38 MAPK pathway

    Institute of Scientific and Technical Information of China (English)

    NAN Jing-long; LI Jian-jun; HE Jian-guo

    2009-01-01

    Background C-reactive protein (CRP) has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation,and it is also speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs).Interleukin-8 (IL-8) is an important mediator of the paracrine mitogenic effect of EPCs,which has direct angiogenic effects on mature endothelial cells.We,herein,investigated the direct effect of CRP on IL-8 production and gene expression in cultured human EPCs.Methods EPCs were isolated from the peripheral venous blood of healthy male volunteers.Cells were cultured in EndoCultTM liquid medium in the absence and presence of CRP at clinically relevant concentrations (5 to 25 μg/ml) for different durations (3 to 48 hours).IL-8 protein and mRNA of cultured EPCs were evaluated using ELISA and real-time PCR.Results The results showed that CRP at a concentration of 10 pg/ml significantly reduced IL-8 secretion of cultured EPCs with a peak at 25 μg/ml,and also decreased mRNA expression in EPCs with a peak at 12 hours.In addition,preincubation of EPCs with SB203580,an inhibitor of p38 mitogen-activated protein kinase (MAPK) decreased CRP inhibition of IL-8 mRNA expression at 12 hours in EPCs.Conclusions Our study,for the first time,demonstrates that CRP directly inhibits EPCs IL-8 secretion,a key cytokine player of angiogenesis induced by EPCs.Inhibition occurred in part via an effect of CRP to active the p38 MAPK signal transduction pathway in EPC.The ability of CRP to inhibit EPCs IL-8 secretion may represent an important mechanism that further links inflammation to cardiovascular disease.

  7. Human umbilical cord-derived endothelial progenitor cells promote growth cytokines-mediated neorevascularization in rat myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    HU Cheng-heng; LI Zhi-ming; DU Zhi-min; ZHANG Ai-xia; YANG Da-ya; WU Gui-fu

    2009-01-01

    Background Cell-based vascular therapies of endothelial progenitor cells (EPCs) mediated neovascularization is still a novel but promising approach for the treatment of ischemic disease. The present study was designed to investigate the therapeutic potentials of human umbilical cord blood-derived EPCs (hUCB-EPCs) in rat with acute myocardial infarction.Methods Human umbilical cord blood (hUCB) mononuclear cells were isolated using density gradient centrifugation from the fresh human umbilical cord in healthy delivery woman, and cultured in M199 medium for 7 days. The EPCs were identified by double-positive staining with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholorate-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and fluorescein isothiocyanate-conjugated Ulex europaeus lectin (FITC-UEA-I). The rat acute myocardial infarction model was established by the ligation of the left anterior descending artery. The hUCB-EPCs were intramyocardially injected into the peri-infarct area. Four weeks later, left ventricular function was assessed by a pressure-volume catheter. The average capillary density (CAD) was evaluated by anti-VⅢ immunohistochemistry staining to reflect the development of neovascularization at the peri-infarct area. The graft cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibody,representing human origin of EPCs and vascular endothelium, respectively. Expressions of cytokines, proliferating cell nuclear angigen (PCNA), platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor (VEGF) were detected to investigate the underlying mechanisms of cell differentiation and revascularization.Results The donor EPCs were detectable and integrated into the host myocardium as confirmed by double-positive immunofluorescence staining with HNA and CD31. And the anti-VⅢ staining demonstrated a higher degree of microvessel formation in EPCs transplanted

  8. Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells.

    Science.gov (United States)

    Tseng, Shuei-Kuen; Chang, Mei-Chi; Su, Cheng-Yao; Chi, Lin-Yang; Chang, Jenny Zwei-Ching; Tseng, Wan-Yu; Yeung, Sin-Yuet; Hsu, Ming-Lun; Jeng, Jiiang-Huei

    2012-08-01

    Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases. PMID:21847594

  9. Regulatory T Cells Protect Fine Particulate Matter-Induced Inflammatory Responses in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Wen-cai Zhang

    2014-01-01

    Full Text Available Objective. To investigate the role of CD4+CD25+ T cells (Tregs in protecting fine particulate matter (PM- induced inflammatory responses, and its potential mechanisms. Methods. Human umbilical vein endothelial cells (HUVECs were treated with graded concentrations (2, 5, 10, 20, and 40 µg/cm2 of suspension of fine particles for 24h. For coculture experiment, HUVECs were incubated alone, with CD4+CD25− T cells (Teff, or with Tregs in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without suspension of fine particles for 24 hours. The expression of adhesion molecules and inflammatory cytokines was examined. Results. Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1 and intercellular adhesion molecule-1 (ICAM-1, and inflammatory cytokines, such as interleukin (IL- 6 and IL-8, were increased in a concentration-dependent manner. Moreover, the adhesion of human acute monocytic leukemia cells (THP-1 to endothelial cells was increased and NF-κB activity was upregulated in HUVECs after treatment with fine particles. However, after Tregs treatment, fine particles-induced inflammatory responses and NF-κB activation were significantly alleviated. Transwell experiments showed that Treg-mediated suppression of HUVECs inflammatory responses impaired by fine particles required cell contact and soluble factors. Conclusions. Tregs could attenuate fine particles-induced inflammatory responses and NF-κB activation in HUVECs.

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

    Directory of Open Access Journals (Sweden)

    Catherine A. Brissette

    2013-08-01

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

  11. Microcystins Induces Vascular Inflammation in Human Umbilical Vein Endothelial Cells via Activation of NF-κB

    Directory of Open Access Journals (Sweden)

    Jun Shi

    2015-01-01

    Full Text Available Microcystins (MCs produced by toxic cyanobacteria cause serious water pollution and public health hazard to humans and animals. However, direct molecular mechanisms of MC-LR in vascular endothelial cells (ECs have not been understood yet. In this study, we investigated whether MC-LR induces vascular inflammatory process in cultured human umbilical vein endothelial cells (HUVECs. Our data demonstrated that MC-LR decreased HUVECs proliferation and tube formation and enhanced apoptosis. MC-LR also induced intracellular reactive oxygen species formation (ROS in HUVECs. The MC-LR directly stimulated phosphorylation of NF-κB. Furthermore, MC-LR also increased cell adhesion molecules (ICAM-1 and VCAM-1 expression in HUVECs. Taken together, the present data suggested that MC-LR induced vascular inflammatory process, which may be closely related to the oxidative stress, NF-κB activation, and cell adhesion molecules expression in HUVECs. Our findings may highlight that MC-LR causes potential damage to blood vessels.

  12. Alphastatin downregulates vascular endothelial cells sphingosine kinase activity and suppresses tumor growth in nude mice bearing human gastric cancer xenografts

    Institute of Scientific and Technical Information of China (English)

    Lin Chen; Tao Li; Rong Li; Bo Wei; Zheng Peng

    2006-01-01

    AIM: To investigate whether alphastatin could inhibit human gastric cancer growth and furthermore whether sphingosine kinase (SPK) activity is involved in this process.METHODS: Using migration assay, MTT assay and Matrigel assay, the effect of alphastatin on vascular endothelial cells (ECs) was evaluated in vitro. SPK and endothelial differentiation gene (EDG)-1, -3, -5 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). SPK activity assay was used to evaluate the effect of alphastatin on ECs. Matrigel plug assay in nude mice was used to investigate the effect of alphastatin on angiogenesis in vivo. Female nude mice were subcutaneously implanted with human gastric cancer cells (BGC823) for the tumor xenografts studies.Micro vessel density was analyzed in Factor Ⅷ-stained tumor sections by the immunohistochemical SP method.RESULTS: In vitro, alphastatin inhibited the migration and tube formation of ECs, but had no effect on proliferation of ECs. RT-PCR analysis demonstrated that ECs expressed SPK and EDG-1, -3, -5 mRNAs. In vivo,alphastatin sufficiently suppressed neovascularization of the tumor in the nude mice. Daily administration of alphastatin produced significant tumor growth suppression. Immunohistochemical studies of tumor tissues revealed decreased micro vessel density in alphastatin-treated animals as compared with controls.CONCLUSION: Downregulating ECs SPK activity may be one of the mechanisms that alphastatin inhibits gastric cancer angiogenesis. Alphastatin might be a useful and relatively nontoxic adjuvant therapy in the treatment of gastric cancer.

  13. Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

    Directory of Open Access Journals (Sweden)

    Shi Xianglin

    2009-01-01

    Full Text Available Abstract Background Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human. Results The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery. Conclusion Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.

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

    Directory of Open Access Journals (Sweden)

    Mathieu Barbier

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

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

    Directory of Open Access Journals (Sweden)

    Yazi Huang

    2014-01-01

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

  16. A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

    Science.gov (United States)

    Geng, Yijie; Feng, Bradley

    2016-07-01

    The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

  17. Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

    Institute of Scientific and Technical Information of China (English)

    Guan-Bin Song; Jian Qin; Qing Luo; Xiao-Dong Shen; Run-Bin Yan; Shao-Xi Cai

    2005-01-01

    AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-7721cells and its contribution to this adhesive course.METHODS: Adhesive force of SMMC-7721 cells to endothelialcells was measured using micropipette aspiration technique.Synchronous G1 and S phase SMMC-7721 cells wereachieved by thymine-2-deoxyriboside and colchicinessequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronousrates of SMMC-7721 cells and expression of integrinβ1 inSMMC-7721 cells were detected by flow cytometer.RESULTS: The percentage of cell cycle phases of generalSMMC-7721 cells was 11.01% in G2/M phases, 53.51% inG0/G1 phase, and 35.48% in S phase. The synchronous ratesof G1 and S phase SMMC-7721 cells amounted to 74.09%and 98.29%, respectively. The adhesive force of SMMC-7721cells to endothelial cells changed with the variations ofadhesive time and presented behavior characteristics ofadhesion and de-adhesion. S phase SMMC-7721 cells had higheradhesive forces than G1 phase cells [(307.65±92.10)× 10-10Nvs (195.42±60.72)×10-10N, P<0.01]. The expressivefluorescent intensity of integrinβ1 in G1 phase SMMC-7721cells was depressed more significantly than the values ofS phase and general SMMC-7721cells. The contribution ofadhesive integrinβ1 was about 53% in this adhesive course.CONCLUSION: SMMC-7721 cells can be synchronizedpreferably in G1 and S phases with thymine-2-deoxyribosideand colchicines. The adhesive molecule integrinβ1 expressesa high level in SMMC-7721 cells and shows differences invarious cell cycles, suggesting integrin β1 plays an importantrole in adhesion to endothelial cells. The change of adhesiveforces in different cell cycle SMMC-7721 cells indicatesthat S phase cells play predominant roles possibly whilethey interact with endothelial cells.

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

    Directory of Open Access Journals (Sweden)

    Pittet Camille L

    2011-11-01

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

  19. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  20. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  1. Towards hemerythrin-based blood substitutes: Comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells

    Indian Academy of Sciences (India)

    Eva Fischer-Fodor; Augustin Mot; Florina Deac; Mariann Arkosi; Radu Silaghi-Dumitrescu

    2011-06-01

    Hemerythrin is a dioxygen-carrying protein whose oxidative/nitrosative stress-related reactivity is lower than that of hemoglobin, which may warrant investigation of hemerythrin as raw material for artificial oxygen carriers (‘blood substitutes’). We report here the first biological tests for hemerythrin and its chemical derivatives, comparing their performance with that of a representative competitor, glutaraldehyde-polymerized bovine hemoglobin. Hemerythrin (native or derivatized) exhibits a proliferative effect on human umbilical vein endothelial cell (HUVEC) cultures, as opposed to a slight inhibitory effect of hemoglobin. A similar positive effect is displayed on human lymphocytes by glutaraldehyde-polymerized hemerythrin, but not by native or polyethylene glycol-derivatized hemerythrin.

  2. Knockdown of GSK3β increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells

    Science.gov (United States)

    Weikel, Karen A.; Cacicedo, José M.; Ruderman, Neil B.; Ido, Yasuo

    2016-01-01

    High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308, C249–C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis. PMID:27534430

  3. Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

    Science.gov (United States)

    Pillon, Nicolas J; Azizi, Paymon M; Li, Yujin E; Liu, Jun; Wang, Changsen; Chan, Kenny L; Hopperton, Kathryn E; Bazinet, Richard P; Heit, Bryan; Bilan, Philip J; Lee, Warren L; Klip, Amira

    2015-07-01

    Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue

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

    International Nuclear Information System (INIS)

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

  5. 成体干细胞向血管内皮细胞定向分化的研究现状%Oriented differentiation of adult stem cells into vascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    宋秀军; 江其生

    2009-01-01

    Adult stem cells identified in tissue organs possess obviously plasticity. They can differentiate into the cells in other systems and germ layers, which are known to support the continuous repair and regeneration of tissues. In the vascular tissue engineering, the research was more and more widespread and deep on adult stem cells as seed cells~ Research indicated that there are many kinds of adult stem cells which can differentiate into vascular endothelial cells, such as bone marrow mesenchymal stem cells, hematopoietic stem cells, human adipose derived stem cells, and amnion derived stem calls. Thus, this study reviewed the current situation of adult stem cells as seed cells in vascular tissue engineering.%成体干细胞存在于已分化的组织器官中,具有明显的可塑性,能跨系统、跨胚层分化,在组织工程和损伤修复领域具有重要的应用价值.在血管组织工程研究领域中,成体干细胞作为种子细胞得到越来越广泛而深入的研究.目前研究表明,具有向血管内皮细胞分化的成体干细胞主要有骨髓间充质干细胞、造血干细胞、脂肪来源的干细胞以及羊膜来源的干细胞等基于此,就成体干细胞作为种子细胞在血管组织工程领域研究现状进行综述.

  6. Olive Oil Supplements Ameliorate Endothelial Dysfunction Caused by Concentrated Ambient Particulate Matter Exposure in Healthy Human Volunteers

    Science.gov (United States)

    Context: Exposure to ambient particulate matter (PM) induces endothelial dysfunction, a risk factor for clinical cardiovascular events and progression of atherosclerosis. Dietary supplements such as olive oil and fish oil have beneficial effects on endothelial function, and ther...

  7. Culture supernatants of breast cancer cell line MDA-MB-231 treated with parthenolide inhibit the proliferation, migration, and lumen formation capacity of human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    LI Cai-juan; GUO Su-fen; SHI Tie-mei

    2012-01-01

    Background Parthenolide has been tested for anti-tumor activities,such as anti-proliferation and pro-apoptosis in recent studies.However,little is known about its role in the process of tumor angiogenesis.This study aims to investigate the effects and potential mechanisms of parthenolide on the proliferation,migration and lumen formation capacity of human umbilical vein endothelial cells.Methods Different concentrations of parthenolide were applied to the human breast cancer cell line MDA-MB-231 cells.After 24-hour incubation,the culture supematants were harvested and used to treat human umbilical vein endothelial cells for 24 hours.Then an inverted fluorescence phase contrast microscope was used to evaluate the human umbilical vein endothelial cells.The secretion of vascular endothelial growth factor (VEGF),interleukin (IL)-8 and matrix metalloproteinases (MMP)-9 in the culture supernatant of the MDA-MB-231 cells was then measured with enzyme-linked immunosorbent assay (ELISA) assays.Results Suppression of proliferation,migration,and the lumen formation capacity of human umbilical vein endothelial cells was observed in the presence of the culture supernatants from the breast cancer cell line treated with different concentrations of parthenolide.Parthenolide decreased the levels of the angiogenic factors MMP-9,VEGF,and IL-8secreted by the MDA-MB-231 cells.Conclusions Parthenolide may suppress angiogenesis through decreasing angiogenic factors secreted by breast cancer cells to interfere with the proliferation,migration and lumen-like structure formation of endothelial cells,thereby inhibiting tumor growth.It is a promising potential anti-angiogenic drug.

  8. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets

    OpenAIRE

    Möhle, Robert; Green, David; Moore, Malcolm A. S.; Nachman, Ralph L.; Rafii, Shahin

    1997-01-01

    We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growt...

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

    Directory of Open Access Journals (Sweden)

    Pospischil Andreas

    2006-06-01

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

  10. Editorial: Technology for higher education, adult learning and human performance

    Directory of Open Access Journals (Sweden)

    Minhong Wang

    2013-09-01

    Full Text Available This special issue is dedicated to technology-enabled approaches for improving higher education, adult learning, and human performance. Improvement of learning and human development for sustainable development has been recognized as a key strategy for individuals, institutions, and organizations to strengthen their competitive advantages. It becomes crucial to help adult learners and knowledge workers to improve their self-directed and life-long learning capabilities. Meanwhile, advances in technology have been increasingly enabling and facilitating learning and knowledge-related initiatives.. They have largely extended learning opportunities through the provision of resource-rich and learner-centered environment, computer-based learning support, and expanded social interactions and networks. Papers in this special issue are representative of ongoing research on integration of technology with learning for innovation and sustainable development in higher education institutions and organizational and community environments.

  11. Matrix-degrading and pro-inflammatory changes in human vascular endothelial cells exposed to cigarette smoke condensate.

    Science.gov (United States)

    Nordskog, Brian K; Blixt, Allison D; Morgan, Walter T; Fields, Wanda R; Hellmann, Gary M

    2003-01-01

    Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation of this finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 microg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation. PMID:14501029

  12. The angiogenic effect of dracorhodin perchlorate on human umbilical vein endothelial cells and its potential mechanism of action.

    Science.gov (United States)

    Li, Feng; Jiang, Tao; Liu, Wei; Hu, Quan; Yin, Huinan

    2016-08-01

    Hyperglycemia is the key clinical feature of diabetes, and may induce refractory wound lesions and impaired angiogenesis. Dracorhodin perchlorate (Dra) is the major ingredient of dragon's blood and it has been used as a medicine to treat chronic wounds, such as diabetic foot, since ancient times in many cultures. The current study aimed to investigate the effect of Dra on human umbilical vein endothelial cells (HUVECs) under high‑glucose (HG) stimulation and its potential mechanism. Dra was observed to increase the multiplication capacity of HUVECs both under low glucose (LG) and HG concentrations. Additionally, migration and tube formation in HUVECs was facilitated by Dra. The expression levels of Ras, mitogen‑activated protein kinase (MAPK) and vascular endothelial growth factor, which are key components of the Ras/MAPK pathway, were upregulated following Dra treatment. The present study is the first report, to the best of our knowledge, of the effects of Dra on wound healing, and the association with the Ras/MAPK signaling pathway. PMID:27357516

  13. Scutellarein inhibits hypoxia- and moderately-high glucose-induced proliferation and VEGF expression in human retinal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Rong GAO; Bang-hao ZHU; Shi-bo TANG; Jiang-feng WANG; Jun REN

    2008-01-01

    Aim: This study was designed to examine the effect of scutellarein on high glu-cose- and hypoxia-stimulated proliferation of human retinal endothelial cells (HREC). Methods: HREC were cultured under normal glucose (NG), moderate, and high glucose (NG supplemented with 10 or 25 mmol/L D-glucose) and/or hypoxic (cobalt chloride treated) conditions. Cell proliferation was evaluated by a cell counting kit. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. Results: The proliferation of HREC was significantly elevated in response to moderately-high glucose and hypoxic conditions. The combination of high glucose and hypoxia did not have any additive effects on cell proliferation. Consistent with the proliferation data, the expression of VEGF was also upregulated under both moderately-high glucose and hypoxic conditions. The treatment with scutellarein (1 × 10-11-1 × 10-5 mol/L) significantly inhibited high glucose- or hypoxia-induced cell proliferation and VEGF expression. Conclusion: Both hypoxia and moderately-high glucose were potent stimuli for cell proliferation and VEGF expression in HREC without any significant additive effects. Scutellarein is capable of inhibiting the proliferation of HREC, which is possibly related to its ability to suppress the VEGF expression.

  14. High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Braun, Alexander; Caesar, Nicole M; Dang, Kyvan; Myers, Kenneth A

    2016-01-01

    The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration. PMID:27584860

  15. Matrix-degrading and pro-inflammatory changes in human vascular endothelial cells exposed to cigarette smoke condensate.

    Science.gov (United States)

    Nordskog, Brian K; Blixt, Allison D; Morgan, Walter T; Fields, Wanda R; Hellmann, Gary M

    2003-01-01

    Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation of this finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 microg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation.

  16. Comprehensive and computational analysis of genes in human umbilical vein endothelial cells responsive to X-irradiation

    Directory of Open Access Journals (Sweden)

    Yukihiro Furusawa

    2016-06-01

    Full Text Available Radiation exposure such as A-bomb or radiation therapy is considered a major health-risk factor for cardiovascular disease. In order to understand the molecular mechanisms underlying the inflammatory reaction frequently encountered in the vascular system after exposure to ionizing radiation, we carried out a global scale microarray and computational gene expression analyses on human umbilical endothelial cells (HUVECs exposed to X-ray (2.5 Gy. The gene ontology analysis revealed that the down-regulated genes were associated with cell cycle regulation, whereas the up-regulated genes were associated with inflammatory responses, in particular, the type 1 interferon response. The computational analysis using ingenuity pathway analysis also identified a gene network containing the interferon response factor 7 (IRF7 and its transcriptional targets such as interferon-induced transcripts (IFITs and Mx1, which have been known to be associated with inflammation in endothelial cells. The up-regulated genes and the gene network identified here may explain the inflammatory response induced by X-irradiation. These findings uncover part of the molecular basis of the mechanism(s of the inflammatory disorder in response to X-irradiation in HUVECs. The dataset is publicly available at the Gene Expression Omnibus (GEO repository (http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE76484.

  17. Amorphous silica nanoparticles trigger nitric oxide/peroxynitrite imbalance in human endothelial cells: inflammatory and cytotoxic effects

    Directory of Open Access Journals (Sweden)

    Corbalan JJ

    2011-11-01

    Full Text Available J Jose Corbalan1,2, Carlos Medina1, Adam Jacoby2, Tadeusz Malinski2, Marek W Radomski11School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences, Panoz Institute, Trinity College Dublin, Dublin, Ireland; 2Department of Chemistry and Biochemistry, Ohio University, Athens, OH, USABackground: The purpose of this study was to investigate the mechanism of noxious effects of amorphous silica nanoparticles on human endothelial cells.Methods: Nanoparticle uptake was examined by transmission electron microscopy. Electrochemical nanosensors were used to measure the nitric oxide (NO and peroxynitrite (ONOO- released by a single cell upon nanoparticle stimulation. The downstream inflammatory effects were measured by an enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and flow cytometry, and cytotoxicity was measured by lactate dehydrogenase assay.Results: We found that the silica nanoparticles penetrated the plasma membrane and rapidly stimulated release of cytoprotective NO and, to a greater extent, production of cytotoxic ONOO-. The low [NO]/[ONOO-] ratio indicated increased nitroxidative/oxidative stress and correlated closely with endothelial inflammation and necrosis. This imbalance was associated with nuclear factor κB activation, upregulation of key inflammatory factors, and cell death. These effects were observed in a nanoparticle size-dependent and concentration-dependent manner.Conclusion: The [NO]/[ONOO-] imbalance induced by amorphous silica nanoparticles indicates a potentially deleterious effect of silica nanoparticles on vascular endothelium.Keywords: amorphous silica nanoparticles, nanotoxicology, nitric oxide, peroxynitrite, inflammation, risk factors

  18. Antithrombogenic investigation and biological behavior of cultured human umbilical vein endothelial cells on Ti-O film

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Our previous research results have shown that the Ti-O films with appropriate characteristics possess great potentials for biomaterials application. In this paper, using plasma immersion ion implantation and deposition (PIII-D), titanium oxide thin films are fabricated onto silicon wafer. The antithrombogenesis of films is evaluated in vitro through the platelet adhesion investigation. The biological behavior of human umbilical vein endothelial cells (HUVEC) on the film surface is investigated in vitro by endothelial cell (EC) culture. Our results reveal that the crystalline Ti-O films exhibit attractive blood compatibility. The in vitro HUVEC-cultured investigation of Ti-O film surface has justified that the biological behavior of HUVECs on different structure surfaces is significantly different. The adherence, growth and proliferation of HUVECs to the crystalline Ti-O film surface are in order, by forming a perfect single layer, preserving the natural original shape and displaying the cobblestone road metal rank, and obviously superior to that on the amorphous Ti-O film surface. According to our study, the crystalline Ti-O film, with proper microstructure, is helpful for seeding Ecs and can be used as a functional surface for the adherence and growth of ECS.

  19. Amorphous silica nanoparticles trigger nitric oxide/peroxynitrite imbalance in human endothelial cells: inflammatory and cytotoxic effects

    Science.gov (United States)

    Corbalan, J Jose; Medina, Carlos; Jacoby, Adam; Malinski, Tadeusz; Radomski, Marek W

    2011-01-01

    Background The purpose of this study was to investigate the mechanism of noxious effects of amorphous silica nanoparticles on human endothelial cells. Methods Nanoparticle uptake was examined by transmission electron microscopy. Electrochemical nanosensors were used to measure the nitric oxide (NO) and peroxynitrite (ONOO−) released by a single cell upon nanoparticle stimulation. The downstream inflammatory effects were measured by an enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and flow cytometry, and cytotoxicity was measured by lactate dehydrogenase assay. Results We found that the silica nanoparticles penetrated the plasma membrane and rapidly stimulated release of cytoprotective NO and, to a greater extent, production of cytotoxic ONOO−. The low [NO]/[ONOO−] ratio indicated increased nitroxidative/oxidative stress and correlated closely with endothelial inflammation and necrosis. This imbalance was associated with nuclear factor κB activation, upregulation of key inflammatory factors, and cell death. These effects were observed in a nanoparticle size-dependent and concentration-dependent manner. Conclusion The [NO]/[ONOO−] imbalance induced by amorphous silica nanoparticles indicates a potentially deleterious effect of silica nanoparticles on vascular endothelium. PMID:22131828

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Brazilin Ameliorates High Glucose-Induced Vascular Inflammation via Inhibiting ROS and CAMs Production in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Thanasekaran Jayakumar

    2014-01-01

    Full Text Available Vascular inflammatory process has been suggested to play a key role in the initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Recent studies have shown that brazilin exhibits antihepatotoxic, antiplatelet, cancer preventive, or anti-inflammatory properties. Thus, we investigated whether brazilin suppresses vascular inflammatory process induced by high glucose (HG in cultured human umbilical vein endothelial cells (HUVEC. HG induced nitrite production, lipid peroxidation, and intracellular reactive oxygen species formation in HUVEC cells, which was reversed by brazilin. Western blot analysis revealed that brazilin markedly inhibited HG-induced phosphorylation of endothelial nitric oxide synthase. Besides, we investigated the effects of brazilin on the MAPK signal transduction pathway because MAPK families are associated with vascular inflammation under stress. Brazilin blocked HG-induced phosphorylation of extracellular signal-regulated kinase and transcription factor NF-κB. Furthermore, brazilin concentration-dependently attenuated cell adhesion molecules (ICAM-1 and VCAM-1 expression induced by various concentrations of HG in HUVEC. Taken together, the present data suggested that brazilin could suppress high glucose-induced vascular inflammatory process, which may be closely related with the inhibition of oxidative stress, CAMs expression, and NF-κB activation in HUVEC. Our findings may highlight a new therapeutic intervention for the prevention of vascular diseases.

  2. Binding of human coronary artery endothelial cells to plasma-treated titanium dioxide nanotubes of different diameters.

    Science.gov (United States)

    Flašker, Ajda; Kulkarni, Mukta; Mrak-Poljšak, Katjuša; Junkar, Ita; Čučnik, Saša; Žigon, Polona; Mazare, Anca; Schmuki, Patrik; Iglič, Aleš; Sodin-Semrl, Snezna

    2016-05-01

    Nanoscale topography in improving vascular response in vitro was established previously on various titanium surfaces. In the present study different surface nanotopographies that is different diameters of titanium dioxide (TiO2 ) nanotubes (NTs) were fabricated by electrochemical anodization and conditioned with highly reactive gaseous oxygen plasma. The morphology of different diameter NTs was studied by scanning electron microscopy and atomic force microscopy, while changes in chemical composition on the surface before and after plasma treatment were determined by X-ray photoelectron spectroscopy. Performance of human coronary artery endothelial cells (HCAEC) on those conditioned surfaces was studied in regard to cell proliferation, released IL-6 protein and immunofluorescence microscopy (IFM). We show that HCAEC function is dependent on the diameter of the TiO2 NTs, functioning far less optimally when bound to 100 nm TiO2 NTs as compared to Ti foil, 15 nm NTs or 50 nm NTs. There were improved, morphological cell shape changes, observed with IFM, between HCAEC growing on oxygen-rich plasma-treated versus nontreated 100 nm NTs. These endothelialized conditioned Ti nanosurfaces could elucidate optimization conditions necessary for vascular implants in coronary arteries. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1113-1120, 2016. PMID:26748552

  3. Binding of human coronary artery endothelial cells to plasma-treated titanium dioxide nanotubes of different diameters.

    Science.gov (United States)

    Flašker, Ajda; Kulkarni, Mukta; Mrak-Poljšak, Katjuša; Junkar, Ita; Čučnik, Saša; Žigon, Polona; Mazare, Anca; Schmuki, Patrik; Iglič, Aleš; Sodin-Semrl, Snezna

    2016-05-01

    Nanoscale topography in improving vascular response in vitro was established previously on various titanium surfaces. In the present study different surface nanotopographies that is different diameters of titanium dioxide (TiO2 ) nanotubes (NTs) were fabricated by electrochemical anodization and conditioned with highly reactive gaseous oxygen plasma. The morphology of different diameter NTs was studied by scanning electron microscopy and atomic force microscopy, while changes in chemical composition on the surface before and after plasma treatment were determined by X-ray photoelectron spectroscopy. Performance of human coronary artery endothelial cells (HCAEC) on those conditioned surfaces was studied in regard to cell proliferation, released IL-6 protein and immunofluorescence microscopy (IFM). We show that HCAEC function is dependent on the diameter of the TiO2 NTs, functioning far less optimally when bound to 100 nm TiO2 NTs as compared to Ti foil, 15 nm NTs or 50 nm NTs. There were improved, morphological cell shape changes, observed with IFM, between HCAEC growing on oxygen-rich plasma-treated versus nontreated 100 nm NTs. These endothelialized conditioned Ti nanosurfaces could elucidate optimization conditions necessary for vascular implants in coronary arteries.

  4. Naringenin exerts anti-angiogenic effects in human endothelial cells: Involvement of ERRα/VEGF/KDR signaling pathway.

    Science.gov (United States)

    Li, Qunyi; Wang, Yi; Zhang, Liudi; Chen, Lu; Du, Yongli; Ye, Ting; Shi, Xiaojin

    2016-06-01

    Naringenin (Nar), most abundant in oranges and tomatoes, are known for the hypocholesterolemic, anti-estrogenic, hypolipidemic, anti-hypertensive, and anti-inflammatory activities. Here, the present study was designed to investigate the in vitro and in vivo anti-angiogenesis of Nar. Inhibition of angiogenesis was determined in vitro by using proliferation, apoptosis, migration, and tube-formation assays in Nar-treated human endothelial cell. Finally, CAM assays were used to assess inhibitory effect of Nar on physiological angiogenesis in vivo. The data suggest that Nar should be a direct ERRα inhibitor capable of inhibiting angiogenesis in vitro and in vivo, including endothelial cell proliferation, survival, migration and capillary-like structures formation of HUVECs, as well as reduced neovascularization of the CAM. Furthermore, the effects exerted by Nar are cell cycle related and mediated by VEGF/KDR signaling pathway along with downregulation of certain proangiogenic inflammatory cytokines. Our data thus provide potential molecular mechanisms through which Nar manifests it as a promising anti-angiogenic and anti-cancer agent. PMID:27105956

  5. Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

    Science.gov (United States)

    Encabo, A; Romanin, C; Birke, F W; Kukovetz, W R; Groschner, K

    1996-10-01

    1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells. PMID:8904644

  6. Neuropeptide Y in the Adult and Fetal Human Pineal Gland

    OpenAIRE

    Morten Møller; Pansiri Phansuwan-Pujito; Corin Badiu

    2014-01-01

    Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passag...

  7. Screening of human vascular endothelial growth factor (VEGF)receptor Flt-1 domain and study on its biological activity

    Institute of Scientific and Technical Information of China (English)

    马骊; 张智清; 王小宁; 孙大军; 周小明; 陈爱君; 姚立红

    2001-01-01

    Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2,1-2,2-3 and 1-3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system.The result showed that,not only loop 1-3,but also the smaller fragment loop 2-3 could bind bo hVEGF165.Recombinant expression plasmids pPIC9K/Flt-1(1-3)and pPIC9K/Flt-1(2-3)were constructed and transformed to Pichia.pastoris host strain GS115,cultured in flasks,and expressed under the induction of 1% methanol.The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d.After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography,its purity reached above 90%.Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1(2-3)to hVEGF165 and its nhibiting effect on the proliferation of human umbilical reins endothelial cells(HUVEC)stimulated with hVEGF165 were close to those of s Flt-1(1-3).Animal test showed that sFlt-1(2-3)could nhibit the formation of regenerate blood vessels stimulated with hVEGF165 significantly.

  8. Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli.

    Science.gov (United States)

    Henriksen, Peter A; Hitt, Mary; Xing, Zhou; Wang, Jun; Haslett, Chris; Riemersma, Rudolph A; Webb, David J; Kotelevtsev, Yuri V; Sallenave, Jean-Michel

    2004-04-01

    Atherosclerosis is a chronic inflammatory disease affecting arterial vessels. Strategies to reduce the inflammatory responses of endothelial cells and macrophages may slow lesion development and prevent complications such as plaque rupture. The human protease human neutrophil elastase (HNE), oxidized low density lipoprotein, LPS, and TNF-alpha were chosen as model stimuli of arterial wall inflammation and led to production of the chemokine IL-8 in endothelial cells. To counteract the activity of HNE, we have examined the effects of adenoviral gene delivery of the anti-elastases elafin, previously demonstrated within human atheroma, and murine secretory leukocyte protease inhibitor (SLPI), a related molecule, on the inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. We developed a technique of precomplexing adenovirus with cationic lipid to augment adenoviral infection efficiency in endothelial cells and to facilitate infection in macrophages. Elafin overexpression protected endothelial cells from HNE-induced IL-8 production and cytotoxicity. Elafin and murine SLPI also reduced endothelial IL-8 release in response to oxidized low density lipoprotein, LPS, and TNF-alpha and macrophage TNF-alpha production in response to LPS. This effect was associated with reduced activation of the inflammatory transcription factor NF-kappaB, through up-regulation of IkappaBalpha, in both cell types. Our work suggests a novel and extended anti-inflammatory role for these HNE inhibitors working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. Our findings indicate that elafin and SLPI may be gene therapy targets for the treatment of atheroma. PMID:15034071

  9. Attenuation of Hind-Limb Ischemia in Mice with Endothelial-Like Cells Derived from Different Sources of Human Stem Cells

    OpenAIRE

    Wing-Hon Lai; Ho, Jenny C. Y.; Yau-Chi Chan; Ng, Joyce H. L.; Ka-Wing Au; Lai-Yung Wong; Chung-Wah Siu; Hung-Fat Tse

    2013-01-01

    Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo ca...

  10. Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: a mechanism for uric acid-induced cardiovascular disease development.

    Science.gov (United States)

    Park, Jung-Hyun; Jin, Yoon Mi; Hwang, Soojin; Cho, Du-Hyong; Kang, Duk-Hee; Jo, Inho

    2013-08-01

    The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.

  11. ICAM-1 expression and organization in human endothelial cells is sensitive to gravity

    Science.gov (United States)

    Zhang, Yu; Sang, Chen; Paulsen, Katrin; Arenz, Andrea; Zhao, Ziyan; Jia, Xiaoling; Ullrich, Oliver; Zhuang, Fengyuan

    2010-11-01

    Transendothelial migration (TEM) of immune cells is a crucial process during a multitude of physiological and pathological conditions such as development, defense against infections and wound healing. Migration within the body tissues and through endothelial barriers is strongly dependent and regulated both by cytoskeletal processes and by expression of surface adhesion molecules such as ICAM-1 and VCAM-1. Space flight experiments have confirmed that TEM will be inhibited and may cause astronauts' immune function decreased and make them easy for infection. We used NASA RCCS to provide a simulated microgravity environment; endothelial cells were cultured on microcarrier beads and activated by TNF-α. Results demonstrate after clinorotation ICAM-1 expression increased, consistent with the notion in parabolic flights. However, VCAM-1 showed no significant change between activated or inactivated cells. Depolymerization of F-actin and clustering of ICAM-1 on cell membrane were also observed in short-term simulated microgravity, and after 24 h clinorotation, actin fiber rearrangement was initiated and clustering of ICAM-1 became stable. ICAM-1 mRNA and VCAM-1 mRNA were up-regulated after 30 min clinorotation, and returned to the same level with controls after 24 h clinorotation.

  12. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMARTTM PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder,cDNA xProfiler, Digital GENE Expression Displayer (DGED),and Digital Differential Display (DDD) were used.RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocardnoma.

  14. Vascular endothelial growth factor receptor-1 in human cancer: concise review and rationale for development of IMC-18F1 (Human antibody targeting vascular endothelial growth factor receptor-1).

    Science.gov (United States)

    Schwartz, Jonathan D; Rowinsky, Eric K; Youssoufian, Hagop; Pytowski, Bronislaw; Wu, Yan

    2010-02-15

    The human vascular endothelial growth factor receptor-1 (VEGFR-1, or Flt-1) is widely expressed in normal and pathologic tissue and contributes to the pathogenesis of both neoplastic and inflammatory diseases. In human cancer, VEGFR-1 mediated signaling is responsible for both direct tumor activation and angiogenesis. VEGFR-1 mediated activation of nonmalignant supporting cells, particularly stromal, dendritic, hematopoietic cells, and macrophages, is also likely important for cancer pathogenesis. VEGFR-1 is also hypothesized to enable the development of cancer metastases by means of activation and premetastatic localization in distant organs of bone marrow-derived hematopoietic progenitor cells, which express VEGFR-1. IMC-18F1 is a fully human IgG(1) antibody that binds to VEGFR-1 and has been associated with the inhibition of cancer growth in multiple in vitro and human tumor xenograft models. The preliminary results of phase 1 investigations have also indicated a favorable safety profile for IMC-18F1 at doses that confer antibody concentrations that are associated with relevant antitumor activity in preclinical models.

  15. Effects of fosinopril and valsartan on expressions of ICAM-1 and NO in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    管思明; 王斌

    2003-01-01

    ObjectiveTo investigate the effects of fosinopril and valsartan on the expression of intercellular adhesion molecule-1 (ICAM-1) and nitric oxide (NO) induced by oxidizlls. MethodsThe levels of NO, ICAM-1, and nitric oxide synthase (NOS) were determined using the nitrate reductase method, ELISA, immunohistochemical and image analyses.ResultsThe ox-LDL can significantly increase the expression of ICAM-1 and inhibit theexpression of NO and NOS in a dose-dependent manner. Fosinopril and valsartancan significantly inhibit these roles of ox-LDL. The roles of fosinopril and valsartan were not significantly different. ConclusionFosinopril and valsartan inhibit oxidized LDL-induced expression of ICAM-1and increase the expression of NO in human umbilical vein endothelial cells, which is one of the mechanisms of antiatherosclerosis.

  16. Recombinant human erythropoietin suppresses endothelial cell apoptosis and reduces the ratio of Bax to Bcl-2 proteins in the aortas of apolipoprotein E-deficient mice

    OpenAIRE

    Warren, Jeffrey S.; Zhao, Ying; Yung, Raymond; Desai, Anjali

    2011-01-01

    Recent clinical trials have raised concern that therapy with recombinant human erythropoietin (EPO) may increase cardiovascular disease risk, event rate, and mortality. Endothelial cell (EC) apoptosis has been implicated in both atherogenesis as well as in the destabilization and rupture of atheromatous plaques.

  17. IL-17A potentiates TNFα-induced secretion from human endothelial cells and alters barrier functions controlling neutrophils rights of passage

    DEFF Research Database (Denmark)

    Bosteen, Markus H; Tritsaris, Katerina; Hansen, Anker J;

    2014-01-01

    Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine that regulates leukocyte mobilization and recruitment. To better understand how IL-17A controls leukocyte trafficking across capillaries in the peripheral blood circulation, we used primary human dermal microvascular endothelial...

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

    Directory of Open Access Journals (Sweden)

    Kanmogne GD

    2012-05-01

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

  19. An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Grazia Marano

    2012-05-01

    Full Text Available Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell’s microenvironment resulting in an increased malignancy. Schmidt’s imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethylfuran as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis.The most active compound, (4-{[(β-D-galactopyranosyloxy]methyl}furan-3-ylmethyl hydrogen sulfate (GSF, inhibited the activation of matrix-metalloproteinase-2 (MMP-2 as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM proteins, fibrinogen and fibronectin.In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyloxy]methyl}furan (BGF nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethylfuran, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin αvβ3 was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site.These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis

  20. An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells

    Science.gov (United States)

    Marano, Grazia; Gronewold, Claas; Frank, Martin; Merling, Anette; Kliem, Christian; Sauer, Sandra; Wiessler, Manfred; Frei, Eva

    2012-01-01

    Summary Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell’s microenvironment resulting in an increased malignancy. Schmidt’s imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis. The most active compound, (4-{[(β-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin. In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin αvβ3 was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site. These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis

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

    Science.gov (United States)

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

    2015-01-01

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

  2. Effect of anti-sense oligodeoxynucleotides homeobox B2 on the proliferation and expression of primary human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    刘旭盛; 张晓

    2002-01-01

    Objective: To explore the effect of homeobox B2 (HOXB2) antisense oligodeoxynucleotides (asodn) on the proliferation and expression of primary human umbilical vein endothelial cells (HUVECs).   Methods: Various concentrations of HOXB2 asodn modified by thiophosphate transfected the induction of liposome into HUVECs. MTT and RT-PCR methods were employed to determine the effect of different concentrations of asodn on the endothelial proliferation and the expression level of HOXB2 mRNA.   Results: After the transfection of HOXB2 asodn, the endothelial proliferation was inhibited in a dose-dependent fashion. Simultaneously, the expression of HOXB2 mRNA decreased significantly.   Conclusions: HOXB2 plays an important role in the proliferation of endothelia.

  3. Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

    International Nuclear Information System (INIS)

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

  4. Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy, E-mail: shivanna@bcm.edu

    2015-07-15

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

  5. The effect of chronic exposure to high palmitic acid concentrations on the aerobic metabolism of human endothelial EA.hy926 cells.

    Science.gov (United States)

    Broniarek, Izabela; Koziel, Agnieszka; Jarmuszkiewicz, Wieslawa

    2016-09-01

    A chronic elevation of circulating free fatty acids (FFAs) is associated with diseases like obesity or diabetes and can lead to lipotoxicity. The goals of this study were to assess the influence of chronic exposure to high palmitic acid (PAL) levels on mitochondrial respiratory functions in endothelial cells and isolated mitochondria. Human umbilical vein endothelial cells (EA.hy926 line) were grown for 6 days in a medium containing either 100 or 150 μM PAL. Growth at high PAL concentrations induced a considerable increase in fatty acid-supplied respiration and a reduction of mitochondrial respiration during carbohydrate and glutamine oxidation. High PAL levels elevated intracellular and mitochondrial superoxide generation; increased inflammation marker, acyl-coenzyme A (CoA) dehydrogenase, uncoupling protein 2 (UCP2), and superoxide dismutase 2 expression; and decreased hexokinase I and pyruvate dehydrogenase expression. No change in aerobic respiration capacity was observed, while fermentation was decreased. In mitochondria isolated from high PAL-treated cells, an increase in the oxidation of palmitoylcarnitine, a decrease in the oxidation of pyruvate, and an increase in UCP2 activity were observed. Our results demonstrate that exposure to high PAL levels induces a shift in endothelial aerobic metabolism toward the oxidation of fatty acids. Increased levels of PAL caused impairment and uncoupling of the mitochondrial oxidative phosphorylation system. Our data indicate that FFAs significantly affect endothelial oxidative metabolism, reactive oxygen species (ROS) formation, and cell viability and, thus, might contribute to endothelial and vascular dysfunction. PMID:27417103

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

    Science.gov (United States)

    Zheng, Wei-Wan; Li, Xin-Yuan; Liu, Hui-Bin; Wang, Zi-Rui; Hu, Qing-Qing; Li, Yu-Xia; Song, Bin-Lin; Lou, Jie; Wang, Qiu-Shi; Ma, He-Ping; Zhang, Zhi-Ren

    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 (P O ). 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 P O . 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. PMID:27635187

  7. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    Directory of Open Access Journals (Sweden)

    Sarah L Appleby

    Full Text Available Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+ population of non-adherent endothelial forming cells (naEFCs which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38 together with mature endothelial cell markers (VEGFR2, CD144 and CD31. These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8 or myeloid markers (CD11b and CD14 which distinguishes them from 'early' endothelial progenitor cells (EPCs. Functional studies demonstrated that these naEFCs (i bound Ulex europaeus lectin, (ii demonstrated acetylated-low density lipoprotein uptake, (iii increased vascular cell adhesion molecule (VCAM-1 surface expression in response to tumor necrosis factor and (iv in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs. Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  8. Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Manuel Castro de Moura

    2011-11-01

    Full Text Available 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.

  9. Effect of endotoxin on the celluar activity and secretion of endothelin-1 in cultured human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM and METHODS: To investigate the effect of endotoxin on the celluar activity and secretion of endothelin-1 by radioimmunoassay and MTT methods in cultured human umbilical vein endothelial cells stimulated by E coli endotoxin (E coli O55:B5, Sigma) of various concentrations (1 g/L, 100 mg/L, 10 mg/L,1 mg/L,100 μg/L,10 μg/L, 1 μg/L) and at the same time interval (HUVEC stimulated by endotoxin for 6 hours) in vitro.RESULTS:Endotoxin showed a slightly inhibitory effect on the viability of endothelial cells at low doses (1 μg/L, 10 μg/L, 100 μg/L, 1 mg/L). The viabilities were 92.00%±1.45%, 91.81%±2.03%, 89.52%±1.49%, 88.35%±1.88%, respectively, versus control group, P<0.01. The cells were impaired significantly at the higher dose of LPS (100 mg/L), the viability was 80.49%±8.76%, versus control group, P<0.01. The cells were killed evidently at the concentration of LPS (1 g/L), the viability was 73%±8%, versus control group, P<0.01. The secretion of ET-1 increased gradually with the concentration of endotoxin manifolding. The concentration of ET-1 reached its peak at the dose of 100 μg/L, and it was (324.384±17.023) ng/L, versus control group (251.636±17.023) ng/L, P<0.01. Endotoxin was effective in stimulating the endothelial cells to secret ET-1 in a dose dependent manner. CONCLUSION: These findings suggested ET-1 may be one of the important factors in endotoxic shock, and the increase in plasma ET-1 level in endotoxemia may be associated with increase in ET-1 secretion.

  10. Caveolin-1 is important for nitric oxide-mediated angiogenesis in fibrin gels with human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Yi-ming PAN; Yong-zhong YAO; Zhang-hua ZHU; Xi-tai SUN; Yu-dong QIU; Yi-tao DING

    2006-01-01

    Aim: The role of caveolin-l (Cav-1) in angiogenesis remains poorly understood. The endothelial nitric oxide (NO) synthase (eNOS), a caveolin-interacting protein, was demonstrated to play a predominant role in vascular endothelial growth factor (VEGF) -induced angiogenesis. The purpose of our study was to examine the role of Cav-1 and the eNOS complex in NO-mediated angiogenesis. Methods: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in 3-D fibrin gels to form capillary-like tubules by VEGF stimulation. The expression of Cav-1 and eNOS was detected by semiquantitative RT-PCR. The HUVEC were treated with antisense oligonucleotides to downregulate Cav-1 expression. Both transduced and non-infected HUVEC were cultured in fibrin gels in the presence or absence of VEGF (20 ng/mL) and NG-nitro-L-arginine methyl ester (L-NAME; 5 mmol/L). NO was measured using a NO assay kit and capillary-like tubules were quantified by tubule formation index using the Image J program. Results: RT-PCR analysis revealed that Cav-1 levels steadily increased in a time-dependent manner and reached their maximum after 5 d of incubation, but there were no obvious changes in eNOS mRNA expression in response to VEGF in the fibrin gel model. VEGF (20 ng/mL) can promote NO production and the formation of capillary-like tubules, and this promoting effect of VEGF was blocked by the addition of L-NAME (5 mmol/L). When transduced HUVEC with the antisense Cav-1 oligonucleotides were plated in the fibrin gels, the capillary-like tubules were significantly fewer than those of the non-infected cells. The capillary-like tubules formation and NO production of transduced HUVEC with the antisense Cav-1 oligonucleotides cultured in fibrin gels showed no responses to the addition of VEGF (20 ng/mL) and L-NAME (5.0 mmol/L). Conclusion: NO was a critical angiogenic mediator in this model. Cav-1 was essential for NO-mediated angiogenesis and may be an important target of anti

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

    Energy Technology Data Exchange (ETDEWEB)

    Pecorelli, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Child Neuropsychiatry Unit, University Hospital, AOUS, Siena (Italy); Bocci, Velio [Department of Physiology, University of Siena (Italy); Acquaviva, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Belmonte, Giuseppe [Department of Biomedical Sciences, University of Siena (Italy); Gardi, Concetta [Department of Molecular and Developmental Medicine, University of Siena (Italy); Virgili, Fabio [INRAN, Rome (Italy); Ciccoli, Lucia [Department of Molecular and Developmental Medicine, University of Siena (Italy); Valacchi, Giuseppe, E-mail: giuseppe.valacchi@unife.it [Department of Life Sciences and Biotechnology, University of Ferrara (Italy); Department of Food and Nutrition, Kyung Hee University, Seoul (Korea, Republic of)

    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.

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

    Science.gov (United States)

    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

  13. Methicillin resistant Staphylococcus aureus adhesion to human umbilical vein endothelial cells demonstrates wall shear stress dependent behaviour

    Directory of Open Access Journals (Sweden)

    Martinuzzi Robert M

    2011-03-01

    Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA is an increasingly prevalent pathogen capable of causing severe vascular infections. The goal of this work was to investigate the role of shear stress in early adhesion events. Methods Human umbilical vein endothelial cells (HUVEC were exposed to MRSA for 15-60 minutes and shear stresses of 0-1.2 Pa in a parallel plate flow chamber system. Confocal microscopy stacks were captured and analyzed to assess the number of MRSA. Flow chamber parameters were validated using micro-particle image velocimetry (PIV and computational fluid dynamics modelling (CFD. Results Under static conditions, MRSA adhered to, and were internalized by, more than 80% of HUVEC at 15 minutes, and almost 100% of the cells at 1 hour. At 30 minutes, there was no change in the percent HUVEC infected between static and low flow (0.24 Pa, but a 15% decrease was seen at 1.2 Pa. The average number of MRSA per HUVEC decreased 22% between static and 0.24 Pa, and 37% between 0.24 Pa and 1.2 Pa. However, when corrected for changes in bacterial concentration near the surface due to flow, bacteria per area was shown to increase at 0.24 Pa compared to static, with a subsequent decline at 1.2 Pa. Conclusions This study demonstrates that MRSA adhesion to endothelial cells is strongly influenced by flow conditions and time, and that MSRA adhere in greater numbers to regions of low shear stress. These areas are common in arterial bifurcations, locations also susceptible to generation of atherosclerosis.

  14. Protein Profiling of Isolated Leukocytes, Myofibroblasts, Epithelial, Basal, and Endothelial Cells from Normal, Hyperplastic, Cancerous, and Inflammatory Human Prostate Tissues

    Directory of Open Access Journals (Sweden)

    Zahraa I. Khamis, Kenneth A. Iczkowski, Ziad J. Sahab, Qing-Xiang Amy Sang

    2010-01-01

    Full Text Available In situ neoplastic prostate cells are not lethal unless they become invasive and metastatic. For cells to become invasive, the prostate gland must undergo degradation of the basement membrane and disruption of the basal cell layer underneath the luminal epithelia. Although the roles of proteinases in breaking down the basement membrane have been well-studied, little is known about the factors that induce basal cell layer disruption, degeneration, and its eventual disappearance in invasive cancer. It is hypothesized that microenvironmental factors may affect the degradation of the basal cell layer, which if protected may prevent tumor progression and invasion. In this study, we have revealed differential protein expression patterns between epithelial and stromal cells isolated from different prostate pathologies and identified several important epithelial and stromal proteins that may contribute to inflammation and malignant transformation of human benign prostate tissues to cancerous tissues using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and proteomics methods. Cellular retinoic acid-binding protein 2 was downregulated in basal cells of benign prsotate. Caspase-1 and interleukin-18 receptor 1 were highly expressed in leukocytes of prostate cancer. Proto-oncogene Wnt-3 was downregulated in endothelial cells of prostatitis tissue and tyrosine phosphatase non receptor type 1 was only found in normal and benign endothelial cells. Poly ADP-ribose polymerase 14 was downregulated in myofibroblasts of prostatitis tissue. Interestingly, integrin alpha-6 was upregulated in epithelial cells but not detected in myofibroblasts of prostate cancer. Further validation of these proteins may generate new strategies for the prevention of basal cell layer disruption and subsequent cancer invasion.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-02

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

  16. Inhibition of miR-7 promotes angiogenesis in human umbilical vein endothelial cells by upregulating VEGF via KLF4.

    Science.gov (United States)

    Li, Yi-Ze; Wen, Lei; Wei, Xu; Wang, Qian-Rong; Xu, Long-Wen; Zhang, Hong-Mei; Liu, Wen-Chao

    2016-09-01

    Recent lentiviral-based microRNA (miRNA) library screening has identified miRNA-7 (miR-7) as an anti‑angiogenic miRNA in human umbilical vein endothelial cells (HUVECs). However, the underlying mechanism of miR-7 in the suppression of angiogenesis remains largely unknown. In the present study, we report that miR-7 inhibition promoted angiogenesis by upregulating vascular endothelial growth factor (VEGF) and directly targeting Krüppel-like factor 4 (KLF4). Downregulation of miR-7 promoted tube formation of HUVECs, accompanied by upregulation of mRNA and protein levels of both VEGF and KLF4. miR-7 directly targeted KLF4 as demonstrated by luciferase reporter assay and miR-7 mimics decreased KLF4. Furthermore, bioinformatic analysis revealed the presence of multiple DNA-binding elements of KLF4 in the VEGF promoter. Chromatin immunoprecipitation (ChIP) demonstrated that the KLF4 antibody specifically pulled down the VEGF promoter in the HUVECs. Furthermore, ectopic overexpression of KLF4 induced VEGF mRNA and protein levels. In addition, KLF4 silencing inhibited the angiogenesis induced by the miR-7 inhibitor in the HUVECs. Our results demonstrated that KLF4 is a direct target of miR-7 and a transcription activator of VEGF. These findings indicate that the miR-7-KLF4-VEGF signaling axis plays an important role in the regulation of angiogenesis in HUVECs, suggesting that miR-7 is a potential agent for the development of anti-angiogenic therapeutics in vascular diseases and solid tumors. PMID:27431648

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 m......RNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors...... GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress...

  18. Vascular endothelial growth factor in skeletal muscle following glycogen-depleting exercise in humans

    DEFF Research Database (Denmark)

    Jensen, Line

    2015-01-01

    Vascular endothelial growth factor (VEGF) is traditionally considered important for skeletal muscle angiogenesis. VEGF is released from vascular endothelium as well as the muscle cells in response to exercise. The mechanism and the physiological role of VEGF secreted from the muscle cells remain...... unclear. However, as VEGF is also considered very important for the regulation of vascular permeability, it is possible that metabolic stress may trigger muscle VEGF release. PURPOSE: To study the role of metabolic stress induced by glycogen-depleting exercise on muscle VEGF expression. METHODS: Fifteen...... males (age 27.0±0.8; VO2max 66.0±1.2 ml•kg-1•min-1) carried out 4h of cycling exercise supplied with H2O only followed by 4h of recovery with either carbohydrate (CHO) (n=8) or H2O (n=7) supplementation. Hereafter both groups received CHO. Muscle biopsies were collected pre and post as well as 4 and 24...

  19. Effects of human vascular endothelial growth factor on reparative dentin formation.

    Science.gov (United States)

    Zhang, Juan; Liu, Xia; Yu, Weixian; Zhang, Yingli; Shi, Ce; Ni, Shilei; Liu, Qilin; Li, Xiangwei; Sun, Yingjian; Zheng, Changyu; Sun, Hongchen

    2016-01-01

    It is a challenge for dentists to save dental pulp in patients with pulp disease without resorting to root canal therapy. Formation of tertiary dentin to maintain pulp vitality is a key odontoblast response to dental pulp injury. Vascular endothelial growth factor (VEGF) is the most potent angiogenic and vasculogenic factor involved in tertiary dentin formation. It was hypothesized that VEGF may be used to treat pulp diseases such as pulpitis. To explore this hypothesis, the first step was to assess whether VEGF affects dental pulp cells to promote reparative dentin formation. In the current study, an AdCMV‑hVEGF vector was constructed to deliver hVEGF into dental pulp cells of exfoliated deciduous teeth (hDPCs) in vitro and dental pulp cells in a rat model in vivo. The collected data clearly demonstrated that hVEGF increased alkaline phosphatase and mineralization by enzymatic activity. RT‑qPCR data demonstrated that hVEGF significantly increased the expression levels of genes commonly involved in osteogenesis/odontogenesis. Data from the in vivo assays indicated that hVEGF enhanced pulp cell proliferation and neovascularization, and markedly increased formation of reparative dentin in dental pulp. The in vitro and in vivo data suggest that hVEGF may have potential clinical applications, thus may aid in the development of novel treatment strategies for dental pulpitis. PMID:26647730

  20. Fibronectin Binding Is Required for Acquisition of Mesenchymal/Endothelial Differentiation Potential in Human Circulating Monocytes

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    Noriyuki Seta

    2012-01-01

    Full Text Available We previously reported monocyte-derived multipotential cells (MOMCs, which include progenitors capable of differentiating into a variety of mesenchymal cells and endothelial cells. In vitro generation of MOMCs from circulating CD14+ monocytes requires their binding to extracellular matrix (ECM protein and exposure to soluble factor(s derived from circulating CD14- cells. Here, we investigated the molecular factors involved in MOMC generation by examining the binding of monocytes to ECM proteins. We found that MOMCs were obtained on the fibronectin, but not on type I collagen, laminin, or poly-L-lysine. MOMC generation was followed by changes in the expression profiles of transcription factors and was completely inhibited by either anti-α5 integrin antibody or a synthetic peptide that competed with the RGD domain for the β1-integrin binding site. These results indicate that acquisition of the multidifferentiation potential by circulating monocytes depends on their binding to the RGD domain of fibronectin via cell-surface α5β1 integrin.

  1. Ontogeny of morningness-eveningness across the adult human lifespan

    Science.gov (United States)

    Randler, Christoph

    2016-02-01

    Sleep timing of humans can be classified alongside a continuum from early to late sleepers, with some people (larks) having an early activity, early bed, and rise times and others (owls) with a more nocturnally orientated activity. Only a few studies reported that morningness-eveningness changes significantly during the adult lifespan based on community samples. Here, I applied a different methodological approach to seek for evidence for the age-related changes in morningness-eveningness preferences by using a meta-data from all available studies. The new aspect of this cross-sectional approach is that only a few studies themselves address the age-related changes of the adult lifespan development, but that many studies are available that provide exactly the data needed. The studies came from 27 countries and included 36,939 participants. Age was highly significantly correlated with scores on the Composite Scale of Morningness ( r = 0.70). This relationship seems linear, because a linear regression explained nearly the same amount of variance compared to other models such as logarithmic, quadratic, or cubic models. The standard deviation of age correlated with the standard deviation of CSM scores ( r = 0.55), suggesting when there is much variance in age in a study; in turn, there is much variance in morningness. This meta-analytical approach shows that morningness-eveningness changes across the adult lifespan and that older age is related to higher morningness.

  2. Association of inflammation and endothelial dysfunction with metabolic syndrome, prediabetes and diabetes in adults from Inner Mongolia, China

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    Kelly Tanika N

    2011-10-01

    Full Text Available Abstract Background We examined the association of biomarkers of inflammation and endothelial dysfunction with diabetes and metabolic syndrome (MetS in persons from Inner Mongolia. Methods A cross-sectional study was conducted among 2,536 people aged 20 years and older from Inner Mongolia, China. Overnight fasting blood samples were obtained to measure plasma concentrations of high sensitivity C-reactive protein (hsCRP, soluble inter-cellular adhesion molecule-1 (sICAM-1, sE-selectin, angiotensin II, high density lipoprotein cholesterol, triglycerides, and blood glucose. Waist circumference and blood pressure were measured by trained staff. MetS was defined according to the modified ATP III definition for Asians. Elevated level of the biomarker was defined as values in the upper tertile of the distribution. Participants were categorized into one of four groups based on the presence or absence of metabolic and glycemic abnormalities: 1 free of prediabetes, diabetes and MetS (reference group, 2 prediabetes or diabetes only, 3 MetS without prediabetes or diabetes, and 4 MetS plus prediabetes or diabetes. The multivariable models are adjusted for age, gender, smoking, drinking, family history of hypertension, and body mass index. Results Among study participants, 18.5% had prediabetes, 3.6% had diabetes, and 27.4% of the entire study population had 3 or more components of the MetS. Elevated hsCRP was associated with an increased odds of prediabetes or diabetes only, MetS without prediabetes or diabetes, and MetS plus prediabetes or diabetes with multivariable adjusted odds ratios (95% confidence intervals of 2.3 (1.7-3.1, 3.0 (2.4-3.8, and 5.8 (4.5-7.5, respectively. Elevated sICAM-1 was associated with increased odds (95% CI of prediabetes or diabetes only (2.1, 1.6-2.9 and MetS plus prediabetes or diabetes (4.2, 3.2-5.3 but was not associated with MetS alone. Elevated sE-selectin was associated with a modestly increased risk of MetS (OR 1.7, 95

  3. Uncaria tomentosa alkaloidal fraction reduces paracellular permeability, IL-8 and NS1 production on human microvascular endothelial cells infected with dengue virus.

    Science.gov (United States)

    Lima-Junior, Raimundo Sousa; Mello, Cintia da Silva; Siani, Antonio Carlos; Valente, Ligia M Marino; Kubelka, Claire Fernandes

    2013-11-01

    Dengue is the major Arbovirus in the world, annually causing morbidity and death. Severe dengue is associated with changes in the endothelial barrier function due to the production of inflammatory mediators by immune cells and by the endothelium. Dengue virus (DENV) replicates efficiently in human endothelial cells in vitro and elicits immune responses resulting in endothelial permeability. Uncaria tomentosa (Willd.) DC.(Rubiaceae), known as cat's claw, has been used in folk medicine for the treatment of a wide-array of symptoms, and several scientific studies reported its antiviral, immunomodulatory, anti-inflammatory and antioxidant properties. Here we infected a human lineage of dermal microvascular endothelial cells (HMEC-1) with DENV-2 and treated it with an alkaloidal fraction from U. tomentosa bark (AFUT). We showed antiviral and immunomodulatory activities of U. tomentosa by determining the NS1 antigen and IL-8 in supernatant of DENV-2 infected HMEC-1. Furthermore, by measurement of transendothelial electrical resistance (TEER) we demonstrated, for the first time, that a plant derivative contributed to the reduction of paracellular permeability in DENV-2 infected HMEC-1. We also showed that IL-8 contributed significantly to the induction of permeability. Although further investigations should be conducted before a new drug can be suggested, our in vitro data support evidence that AFUT could be potentially useful in developing a treatment for severe dengue.

  4. Caveolae-mediated albumin transcytosis is enhanced in dengue-infected human endothelial cells: A model of vascular leakage in dengue hemorrhagic fever.

    Science.gov (United States)

    Chanthick, Chanettee; Kanlaya, Rattiyaporn; Kiatbumrung, Rattanaporn; Pattanakitsakul, Sa-Nga; Thongboonkerd, Visith

    2016-01-01

    Vascular leakage is a life-threatening complication of dengue virus (DENV) infection. Previously, association between "paracellular" endothelial hyperpermeability and plasma leakage had been extensively investigated. However, whether "transcellular" endothelial leakage is involved in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) remained unknown. We thus investigated effects of DENV (serotype 2) infection on transcellular transport of albumin, the main oncotic plasma protein, through human endothelial cell monolayer by Western blotting, immunofluorescence staining, fluorescence imaging, and fluorometry. The data showed that Alexa488-conjugated bovine serum albumin (Alexa488-BSA) was detectable inside DENV2-infected cells and its level was progressively increased during 48-h post-infection. While paracellular transport could be excluded using FITC-conjugated dextran, Alexa488-BSA was progressively increased and decreased in lower and upper chambers of Transwell, respectively. Pretreatment with nystatin, an inhibitor of caveolae-dependent endocytic pathway, significantly decreased albumin internalization into the DENV2-infected cells, whereas inhibitors of other endocytic pathways showed no significant effects. Co-localization of the internalized Alexa488-BSA and caveolin-1 was also observed. Our findings indicate that DENV infection enhances caveolae-mediated albumin transcytosis through human endothelial cells that may ultimately induce plasma leakage from intravascular compartment. Further elucidation of this model in vivo may lead to effective prevention and better therapeutic outcome of DHF/DSS. PMID:27546060

  5. Isoflavone genistein protects high glucose-induced human aortic endothelial cell apoptosis through estrogen receptor-mediated pathway

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Guang Yang; Hui Tian

    2008-01-01

    Objective The aim of this study was to determine if isoflavone genistien has protective effects against high glucose-induced cell apoptosis in human aortic endlthelial cells,and investigate the possible mechanism for this protection.Methods Human aortic endothelial cells subjected to normal (5mmol/L) or high glucose (25mmol/L) were treated with genistein at 0,50,100nmol/L.Parallel experiments were performed with 100nM 17b-estradiol,and also in the presence and absence of the pure anti-estrogen ICI-182,780 (100nmol/L).The effects on cell apoptotic DNA fragmentation were determined using cell death ELISA,and the effects on cellular proliferation were determined using tritiated thymidine incorporation assay.Estrogen receptor expression was detected by Taqman quantitative PCR.Results Genistein at 100nmol/L significantly reduced high glucose-induced DNA fragmentation,and reversed cell DNA synthesis inhibition (P<0.001) after 24 hours' incubation.The effect of genistein was completely blocked by ICI-182,780administration.Estrogen receptor beta,but not alpha was found to be expressed in these cells.Conclusion Isoflavone genistein shows protection against high glucose-induced cell damage through estrogen receptor beta,reducing apoptotic DNA damage and protecting from the inhibition of cell proliferation.

  6. TNFα signals via p66(Shc to induce E-Selectin, promote leukocyte transmigration and enhance permeability in human endothelial cells.

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    Luigi Laviola

    Full Text Available Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC. Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc on Ser(36 and the stress kinase c-Jun NH2-terminal protein kinase (JNK-1/2, and higher intracellular reactive oxygen species (ROS levels. Overexpression of p66(Shc in HUVEC resulted in enhanced p66(Shc phosphorylation on Ser(36, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc protein, in which Ser(36 was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc on Ser(36.

  7. Development of a xeno-free autologous culture system for endothelial progenitor cells derived from human umbilical cord blood.

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    Sung-Hwan Moon

    Full Text Available Despite promising preclinical outcomes in animal models, a number of challenges remain for human clinical use. In particular, expanding a large number of endothelial progenitor cells (EPCs in vitro in the absence of animal-derived products is the most critical hurdle remaining to be overcome to ensure the safety and efficiency of human therapy. To develop in vitro culture conditions for EPCs derived from human cord blood (hCB-EPCs, we isolated extracts (UCE and collagen (UC-collagen from umbilical cord tissue to replace their animal-derived counterparts. UC-collagen and UCE efficiently supported the attachment and proliferation of hCB-EPCs in a manner comparable to that of animal-derived collagen in the conventional culture system. Our developed autologous culture system maintained the typical characteristics of hCB-EPCs, as represented by the expression of EPC-associated surface markers. In addition, the therapeutic potential of hCB-EPCs was confirmed when the transplantation of hCB-EPCs cultured in this autologous culture system promoted limb salvage in a mouse model of hindlimb ischemia and was shown to contribute to attenuating muscle degeneration and fibrosis. We suggest that the umbilical cord represents a source for autologous biomaterials for the in vitro culture of hCB-EPCs. The main characteristics and therapeutic potential of hCB-EPCs were not compromised in developed autologous culture system. The absence of animal-derived products in our newly developed in vitro culture removes concerns associated with secondary contamination. Thus, we hope that this culture system accelerates the realization of therapeutic applications of autologous hCB-EPCs for human vascular diseases.

  8. Thrombocytes Correlate with Lymphangiogenesis in Human Esophageal Cancer and Mediate Growth of Lymphatic Endothelial Cells In Vitro.

    Science.gov (United States)

    Schoppmann, Sebastian F; Alidzanovic, Lejla; Schultheis, Andrea; Perkmann, Thomas; Brostjan, Christine; Birner, Peter

    2013-01-01

    Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC) were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC), and 136 squamous cell carcinomas (SCC). Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs) were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC) were evident in 82 samples (25.6%), and vascular thrombocytic clusters (VTC) in 56 (17.5%). STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001), PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test), and VTC with shorter DFS in in SCC (p = 0.025, Breslow test). In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.

  9. Thrombocytes Correlate with Lymphangiogenesis in Human Esophageal Cancer and Mediate Growth of Lymphatic Endothelial Cells In Vitro.

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    Sebastian F Schoppmann

    Full Text Available Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC, and 136 squamous cell carcinomas (SCC. Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC were evident in 82 samples (25.6%, and vascular thrombocytic clusters (VTC in 56 (17.5%. STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001, PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test, and VTC with shorter DFS in in SCC (p = 0.025, Breslow test. In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.

  10. Hyperglycemia Induces Toll-Like Receptor-2 and -4 Expression and Activity in Human Microvascular Retinal Endothelial Cells: Implications for Diabetic Retinopathy

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    Uthra Rajamani

    2014-01-01

    Full Text Available Diabetic retinopathy (DR causes visual impairment in working age adults and hyperglycemia-mediated inflammation is central in DR. Toll-like receptors (TLRs play a key role in innate immune responses and inflammation. However, scanty data is available on their role in DR. Hence, in this study, we examined TLR2 and TLR4 mRNA and protein expression and activity in hyperglycemic human retinal endothelial cells (HMVRECs. HMVRECs were treated with hyperglycemia (HG or euglycemia and mRNA and protein levels of TLR-2, TLR-4, MyD88, IRF3, and TRIF as well as NF-κB p65 activation were measured. IL-8, IL-1β, TNF-α and MCP-1, ICAM-1, and VCAM-1 as well as monocyte adhesion to HMVRECs were also assayed. HG (25 mM significantly induced TLR2 and TLR4 mRNA and protein in HMVRECs. It also increased both MyD88 and non-MyD88 pathways, nuclear factor-κB (NF-κB, biomediators, and monocyte adhesion. This inflammation was attenuated by TLR-4 or TLR-2 inhibition, and dual inhibition by a TLR inhibitory peptide as well as TLR2 and 4 siRNA. Additionally, antioxidant treatment reduced TLR-2 and TLR4 expression and downstream inflammatory markers. Collectively, our novel data suggest that hyperglycemia induces TLR-2 and TLR-4 activation and downstream signaling mediating increased inflammation possibly via reactive oxygen species (ROS and could contribute to DR.

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

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    Ken Kono

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

  12. KR-31543 reduces the production of proinflammatory molecules in human endothelial cells and monocytes and attenuates atherosclerosis in mouse model.

    Science.gov (United States)

    Choi, Jae-Hoon; Yoo, Ji-Young; Kim, Sun-Ok; Yoo, Sung-Eun; Oh, Goo Taeg

    2012-12-31

    KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)- N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro- 2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemiareperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H₂O₂-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr ⁻/⁻) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.

  13. Effects of high-intensity interval training and moderate-intensity continuous training on endothelial function and cardiometabolic risk markers in obese adults.

    Science.gov (United States)

    Sawyer, Brandon J; Tucker, Wesley J; Bhammar, Dharini M; Ryder, Justin R; Sweazea, Karen L; Gaesser, Glenn A

    2016-07-01

    We hypothesized that high-intensity interval training (HIIT) would be more effective than moderate-intensity continuous training (MICT) at improving endothelial function and maximum oxygen uptake (V̇o2 max) in obese adults. Eighteen participants [35.1 ± 8.1 (SD) yr; body mass index = 36.0 ± 5.0 kg/m(2)] were randomized to 8 wk (3 sessions/wk) of either HIIT [10 × 1 min, 90-95% maximum heart rate (HRmax), 1-min active recovery] or MICT (30 min, 70-75% HRmax). Brachial artery flow-mediated dilation (FMD) increased after HIIT (5.13 ± 2.80% vs. 8.98 ± 2.86%, P = 0.02) but not after MICT (5.23 ± 2.82% vs. 3.05 ± 2.76%, P = 0.16). Resting artery diameter increased after MICT (3.68 ± 0.58 mm vs. 3.86 ± 0.58 mm, P = 0.02) but not after HIIT (4.04 ± 0.70 mm vs. 4.09 ± 0.70 mm; P = 0.63). There was a significant (P = 0.02) group × time interaction in low flow-mediated constriction (L-FMC) between MICT (0.63 ± 2.00% vs. -2.79 ± 3.20%; P = 0.03) and HIIT (-1.04 ± 4.09% vs. 1.74 ± 3.46%; P = 0.29). V̇o2 max increased (P obese adults, with HIIT improving FMD and MICT increasing resting artery diameter and enhancing L-FMC. HIIT required 27.5% less total exercise time and ∼25% less energy expenditure than MICT. PMID:27255523

  14. Evidence-based antioxidant activity of the essential oil from Fructus A. zerumbet on cultured human umbilical vein endothelial cells’ injury induced by ox-LDL

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    Shen Xiang-chun

    2012-10-01

    Full Text Available Abstract Background The essential oil from Fructus Alpiniae zerumbet (FAZ is its principal bioactive ingredient, and is widely used in Miao folk herbs in Guizhou province for the treatment of gastrointestinal and cardiovascular diseases. Several studies have confirmed that FAZ ameliorates hyperlipidemia and atherosclerosis. Because endothelial dysfunction often accompanies cardiovascular diseases, especially hyperlipidemia and atherosclerosis, the present study concentrated on evaluating the endothelial protective effects of the essential oil from FAZ (EOFAZ on oxidized low-density lipoprotein (ox-LDL-induced injury of cultured human umbilical vein endothelial cells (HUVECs and on the regulation of oxidative stress. Methods Cell viability was analyzed with the MTT assay and trypan blue exclusion staining (TBES. Cell injury was assessed by lactate dehydrogenase (LDH release. Biochemical enzymatic methods were used to evaluate the oxidative stress, including the lipid peroxidation product, malondialdehyde (MDA, reduced glutathione (GSH, superoxide dismutase (SOD, catalase (CAT and glutathione peroxidase (GSH-Px. Results The redox status of HUVECs was significantly exacerbated after exposure to ox-LDL. EOFAZ protected HUVECs against ox-LDL injury as assessed by the MTT assay, TBES and LDH release. Furthermore, EOFAZ ameliorated the oxidative stress by elevating the activities of SOD, CAT and GSH-Px, and increasing the GSH levels, in addition to attenuating the MDA contents. Conclusions The present data provide the first experimental evidence that EOFAZ protects endothelial cells against ox-LDL-induced injury, and indicate that this protection involves ameliorating the redox status.

  15. Neuropeptide Y in the Adult and Fetal Human Pineal Gland

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    Morten Møller

    2014-01-01

    Full Text Available Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally.

  16. Enhanced in vitro angiogenic behaviour of human umbilical vein endothelial cells on thermally oxidized TiO2 nanofibrous surfaces

    Science.gov (United States)

    Tan, Ai Wen; Liau, Ling Ling; Chua, Kien Hui; Ahmad, Roslina; Akbar, Sheikh Ali; Pingguan-Murphy, Belinda

    2016-02-01

    One of the major challenges in bone grafting is the lack of sufficient bone vascularization. A rapid and stable bone vascularization at an early stage of implantation is essential for optimal functioning of the bone graft. To address this, the ability of in situ TiO2 nanofibrous surfaces fabricated via thermal oxidation method to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) was investigated. The cellular responses of HUVECs on TiO2 nanofibrous surfaces were studied through cell adhesion, cell proliferation, capillary-like tube formation, growth factors secretion (VEGF and BFGF), and angiogenic-endogenic-associated gene (VEGF, VEGFR2, BFGF, PGF, HGF, Ang-1, VWF, PECAM-1 and ENOS) expression analysis after 2 weeks of cell seeding. Our results show that TiO2 nanofibrous surfaces significantly enhanced adhesion, proliferation, formation of capillary-like tube networks and growth factors secretion of HUVECs, as well as leading to higher expression level of all angiogenic-endogenic-associated genes, in comparison to unmodified control surfaces. These beneficial effects suggest the potential use of such surface nanostructures to be utilized as an advantageous interface for bone grafts as they can promote angiogenesis, which improves bone vascularization.

  17. Inhibition of PAI-1 release from human endothelial cells by Angelica keiskei Koidzumi (Ashitaba chalcones is structure-dependent

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    Naoki Ohkura

    2015-12-01

    Full Text Available Angelica keiskei Koidzumi (Ashitaba is a traditional herbal medicine and it is also regarded in Japan as a health food that might have antithrombotic properties. Ashitaba exudate suppresses lipopolysaccharide (LPS-induced plasma plasminogen activator inhibitor 1 (PAI-1, a risk factor for thrombotic diseases in mice. Xanthoangelol (XA and 4-hydroxyderricin (4-HD comprise > 95% of total chalcones from Ashitaba exudates that also contain trace amounts of other chalcone subtypes. The present study aimed to determine the effects of Ashitaba chalcones including xanthoangelols B (XB, D (XD, E (XE, F (XF and XA as well as 4-HD on PAI-1 levels in the medium of stimulated human EA.hy926 endothelial cells. Xanthoangelol (10 and #61549;M inhibited PAI-1 production at a rate of 77.1%, whereas the inhibition rates of XB, XD, XE and 4-HD were not significant. Xanthoangelol F was highly cytotoxic and thus its ability to inhibit PAI-1 production could not be evaluated. The side hydrocarbon chain of XA played an important role in the excretion of inhibitory activity. Small modifications of the hydrocarbon chain or small functional groups on the A ring measurably influenced the inhibitory activity of xanthoangelols. These findings warrant future research towards an understanding of the mechanism of antithrombotic action of Ashitaba as herbal medicine or antithrombotic health food. [J Intercult Ethnopharmacol 2015; 4(4.000: 355-357

  18. Investigation of the effect of tanshinone IIA on nitric oxide production in human vascular endothelial cells by fluorescence imaging

    Science.gov (United States)

    Huang, Ke-Jing; Wang, Hong; Xie, Wan-Zhen; Zhang, Hua-Shan

    2007-12-01

    Nitric oxide (NO) has been proved to be a potent vasodilator that played an important role in regulating vascular tones. Tanshinone, one of the active components of Radix Salvia miltiorrhiza, was used widely in clinics in China for treating cardiovascular diseases. The objective of this study was to sensitively and specifically investigate the effects of tanshinone IIA, one important pharmacological constituent of tanshinone, on the release of NO from human vascular endothelial cells (HVECs) by fluorescence imaging with an excellent fluorescent probe 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza- s-indacence (TMDCDABODIPY). After cells were incubated with tanshinone IIA, TMDCDABODIPY was employed to label NO. Following the tagging, real-time imaging of NO release from the cells was performed with inverted fluorescence microscope. The results of the experiments showed that tanshinone IIA could induce NO production significantly enhanced in HVECs. The activation of NO by tanshinone IIA may be employed therapeutically in modulating NO production in HVECs.

  19. Effects of netrin-1 and netrin-1 knockdown on human umbilical vein endothelial cells and angiogenesis of rat placenta.

    Science.gov (United States)

    Xie, H; Zou, L; Zhu, J; Yang, Y

    2011-08-01

    Angiogenesis is an important process essential for the development of placenta. Netrin-1 was first discovered in nervous system and was later found to play roles in angiogenesis. In order to better understand the functional relevance of netrin-1 in placental angiogenesis, we investigated the effect of netrin-1 on human umbilical vein endothelial cells (HUVECs) and rat placenta by employing up-regulation and down-regulation strategies. HUVECs and rat placenta were treated with recombinant netrin-1, and netrin-1 expression in the cells and placenta was reduced by short hairpin RNA (shRNA) in vitro and in vivo. The inhibition efficiency was determined by real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting. The expression of netrin-1 was immunohistochemically located. The results demonstrated that netrin-1 promoted viability, proliferation, migration and tube formation of HUVECs. A strong reduction in cell capability was observed in vitro after netrin-1 expression was inhibited with shRNA. Netrin-1 accelerated neovascularization of placenta in pregnant rats. Suppression of netrin-1 expression in placenta resulted in reduced vascular sprouting in vivo. These findings suggest that netrin-1 is essential for the proper functioning of HUVECs and angiogenesis of rat placenta, and it is involved in the development of placenta and fetus. The proangiogenic effect of netrin-1 might offer an alternative therapeutic approach for the treatment of vascular disease of placenta.

  20. Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor.

    Science.gov (United States)

    Sivarapatna, Amogh; Ghaedi, Mahboobe; Le, Andrew V; Mendez, Julio J; Qyang, Yibing; Niklason, Laura E

    2015-01-01

    Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications.

  1. GENISTEIN INHIBITS EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN HER-2/NEU TRANSFECTED HUMAN BREAST CANCER MCF-7 CELLS

    Institute of Scientific and Technical Information of China (English)

    ZHU Jun-dong; YU Xiao-ping; MI Man-tian

    2006-01-01

    Objective: our previous studies have demonstrated that HER-2/neu gene expression in human breast cancer MCF-7 cells promotes angiogenesis in MCF-7 cells xenograft tumors, and genistein inhibits angiogenesis in MCF-7 cells with HER-2/neu expression xenograft tumors. Here, the effects of genistein on the expression of vascular endothelial growth factor (VEGF) inMCR-7 cells with HER-2/neu expression were further studied for exploring the molecular mechanism of anti-angiogenesis in HER-2/neu-overexpressing breast cancer by genistein. Methods: HER-2/neu-overexpressing MCF-7 cells (MCF-7/HER-2)were established by transfecting HER-2/neu gene into HER-2/neu negative expression breast cancer MCF-7 cells.Immunocytochemical staining, western blot and reverse transcription-polymerase chain reaction (RT-PCR) were adopted to measure the expression of VEGF in MCF-7/HER-2 cells treated by genistein for 24, 48 and 72h. Results: HER-2/neu expression up-regulated VEGF mRNA and protein in MCF-7 cells, genistein decreased VEGF mRNA and protein level in MCF-7/HER-2 cells in a time-dependent manner. Conclusion: These results suggest that VEGF plays an important role in HER-2/neu gene expression promoted antiogenesis in breast cancer and genistein induced down-regulation of the expression of VEGF may be one of the molecular mechanisms of its anti-angiogenesis in HER-2/neu-overexpressing breast cancer.

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

    Directory of Open Access Journals (Sweden)

    Wenying Luo

    2015-01-01

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

  3. Poly(ADP-ribose) polymerase 1 inhibition protects human aortic endothelial cells against LPS-induced inflammation response

    Institute of Scientific and Technical Information of China (English)

    Xiaonu Peng; Wenjun Li; Wei Zhang

    2012-01-01

    Atherosclerosis is a chronic inflammatory disease.Tolllike receptor 4 (TLR4) is an important signaling receptor and plays a critical role in the inflammatory response.Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that can regulate the expression of various inflammatory genes.In this study,we investigated the role and the underlying mechanisms of PARP1 on lipopolysaccharide (LPS)-induced inflammation in human aortic endothelial cells.Compared with the control,LPS stimulation increased the protein expression of TLR4 and PARP1.TLR4 inhibition reduced LPS-induced upregulation of inducible nitric oxide synthase (iNOS) and ICAM-1 as well as PARP1. Nuclear factor κB (NF-κB) inhibition decreased ICAM-1 and iNOS expression.Inhibition of PARP1 decreased protein expression of inflammatory cytokines induced by LPS stimulation,probably through preventing NF-KB nuclear translocation. Our study demonstrated that LPS increased ICAM-1 and iNOS expression via TLR4/PARP1/NF-KB pathway.PARP1 might be an indispensable factor in TLR4-mediated inflammation after LPS stimulation.PARP1 inhibition might shed light on the treatment of LPS-induced inflammatory cytokines expression during atherosclerosis.

  4. Cinnamon extract inhibits angiogenesis in zebrafish and human endothelial cells by suppressing VEGFR1, VEGFR2, and PKC-mediated MAP kinase

    OpenAIRE

    Bansode, R. R.; Leung, T; Randolph, P.; L. L. Williams; Ahmedna, M.

    2013-01-01

    Angiogenesis is a process of new blood vessel generation and under pathological conditions, lead to tumor development, progression, and metastasis. Many bioactive components have been studied for its antiangiogenic properties as a preventive strategy against tumor development. This study is focused on the effects of cinnamon extract in modulating the pathway involved in angiogenesis. Human umbilical vein endothelial cells (HUVEC) were treated with cinnamon extract at a concentration of 25 μg/...

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

    OpenAIRE

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

    2016-01-01

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

  6. Up-regulation of Heme Oxygenase-1 by Korean Red Ginseng Water Extract as a Cytoprotective Effect in Human Endothelial Cells

    OpenAIRE

    Yang, Hana; Lee, Seung Eun; Jeong, Seong Il; Park, Cheung-Seog; Jin, Young-Ho; Park, Yong Seek

    2011-01-01

    Korean red ginseng (KRG) is used worldwide as a popular traditional herbal medicine. KRG has shown beneficial effects on cardiovascular diseases, such as atherosclerosis, diabetes, and hypertension. Up-regulation of a cytoprotective protein, heme oxygenase (HO)-1, is considered to augment the cellular defense against various agents that may induce cytotoxic injury. In the present study, we demonstrate that KRG water extract induces HO-1 expression in human umbilical vein endothelial cells (HU...

  7. Protection of human umbilical vein endothelial cells by glycine and structurally similar amino acids against calcium and hydrogen peroxide-induced lethal cell injury.

    OpenAIRE

    Weinberg, J M; Varani, J.; Johnson, K.J.; Roeser, N F; Dame, M K; Davis, J. A.; Venkatachalam, M. A.

    1992-01-01

    Cultured human umbilical vein endothelial cells treated with either the calcium ionophore, ionomycin, or ionomycin plus cyanide-m-chlorophenylhydrazone had immediate severe depletion of adenosine triphosphate, (ATP) and increases of cytosolic free calcium (Caf) and then sustained lethal cell injury as manifested by release of lactate dehydrogenase and failure to exclude vital dyes within 15 minutes. Inclusion of glycine in the experimental medium prevented the enzyme leakage for at least 60 m...

  8. Replication of macrophage-tropic and T-cell-tropic strains of human immunodeficiency virus type 1 is augmented by macrophage-endothelial cell contact.

    OpenAIRE

    Gilles, P N; Lathey, J L; Spector, S. A.

    1995-01-01

    Macrophages perform a central role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and have been implicated as the cell type most prominent in the development of central nervous system impairment. In this study, we evaluated the effect of interaction between macrophages and endothelial cells on HIV-1 replication. Upregulation of HIV-1 replication was consistently observed in monocyte-derived macrophages (hereafter called macrophages) cocultured with either umbilic...

  9. Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled.positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells

    DEFF Research Database (Denmark)

    Flebus, Luca; Lombart, Francois; Sevrin, Chantal;

    2015-01-01

    ) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein.The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and h...... to a minor cytotoxicity compared to the higher ones.As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes....

  10. Neuropeptide Y in the adult and fetal human pineal gland

    DEFF Research Database (Denmark)

    Møller, Morten; Phansuwan-Pujito, Pansiri; Badiu, Corin

    2014-01-01

    Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we...... show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma...... inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland...

  11. Anti-angiogenic effect of Nelumbo nucifera leaf extracts in human umbilical vein endothelial cells with antioxidant potential.

    Directory of Open Access Journals (Sweden)

    Jong Suk Lee

    Full Text Available Nelumbo nucifera Gaertn (Nymphaeaceae has long been used as a traditional herb in Chinese, Japanese, Indian, and Korean medicinal practices since prehistoric times and flourishes today as the primary form of medicine. This study reports for the first time the potent ability of N. nucifera leaf extracts to inhibit vascular endothelial growth factor (VEGF-induced angiogenesis in vitro and in vivo, as well as their antioxidant efficacy in various scavenging models and an analysis of their chemical composition. In vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM model using fertilized chicken eggs, in human umbilical vein endothelial cells (HUVECs by using cell viability, cell proliferation and tube formation assays, and by determining intracellular reactive oxygen species (ROS in vitro. The antioxidant efficacy of N. nucifera leaf extracts was determined in various scavenging models, including total phenolic and flavonoid content. The chemical composition of N. nucifera leaf extracts was determined by GC-MS analysis, which revealed the presence of different phytochemicals. The IC50 values for the DPPH radical scavenging activities of water and methanol extracts were found to be 1699.47 and 514.36 μg ml(-1, and their total phenolic and flavonoid contents were 85.01 ± 2.32 and 147.63 ± 2.23 mg GAE g dry mass(-1 and 35.38 ± 1.32 and 41.86 ± 1.07 mg QA g dry mass(-1, respectively. N. nucifera leaf extracts (10-100 μg ml(-1 exhibited significant dose-dependent inhibition of VEGF-induced angiogenesis, as well as VEGF-induced proliferation and tube formation in HUVECs. In this study, N. nucifera leaf extracts displayed potent antioxidant and inhibitory effects on VEGF-induced angiogenesis. N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis in vivo. Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROS production in HUVECs

  12. Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wen-Yu Yang; Zong-Hai Huang; Li-Jun Lin; Zhou Li; Jing-Long Yu; Hui-Juan Song; Yong Qian; Xiao-Yan Che

    2006-01-01

    AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector.METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified.HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured.RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVCDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P<0.001).CONCLJSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

  13. Influence of Carbon Monoxide on Growth and Apoptosis of Human Umbilical Artery Smooth Muscle Cells and Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Yajuan Li, Hai Wang, Bin Yang, Jichen Yang, Xiuyan Ruan, Yadong Yang, Edward K. Wakeland, Quanzhen Li, Xiangdong Fang

    2012-01-01

    Full Text Available Carbon monoxide (CO is a vasoactive molecule that is generated by vascular cells as a byproduct of heme catabolism and it plays an important physiological role in circulation system. In order to investigate whether exogenous CO can mediate the growth and proliferation of vascular cells, in this study, we used 250 parts per million (ppm of CO to treat human umbilical artery smooth muscle cell (hUASMC and human umbilical vein endothelial cell (HuVEC and further evaluated the growth and apoptosis status of SMC and HuVEC. After SMC and HuVEC were exposed to CO for 7-day, the growth of SMC and HuVEC was significantly inhibited by CO in vitro on day 5 of CO exposure. And CO blocked cell cycle progress of SMC and HuVEC, more SMC and HuVEC stagnated at G0/G1 phase by flow cytometric analysis. Moreover, CO treatment inhibited SMC and HuVEC apoptosis caused by hydrogen peroxide through decreasing caspase 3 and 9 activities. To confirm the molecular mechanism of CO effect on SMC and HuVEC growth, we compared the gene expression profile in SMC and CO-treated SMC, HuVEC and CO-treated HuVEC. By microarray analysis, we found the expression level of some genes which are related to cell cycle regulation, cell growth and proliferation, and apoptosis were changed during CO exposure. We further identified that the down-regulated CDK2 contributed to arresting cell growth and the down-regulated Caspase 3 (CASP3 and Caspase 9 (CASP9 were associated with the inhibition of cell apoptosis. Therefore, CO exerts a certain growth arrest on SMC and HuVEC by inhibiting cell cycle transition from G0/G1 phase to S phase and has regulatory effect on cell apoptosis by regulating the expression of apoptosis-associated genes.

  14. Neuropeptide Y reduces the expression of PLCB2, PLCD1 and selected PLC genes in cultured human endothelial cells.

    Science.gov (United States)

    Lo Vasco, V R; Leopizzi, M; Puggioni, C; Della Rocca, C; Businaro, R

    2014-09-01

    Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes' production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    , there was a positive association between the expression of EL mRNA and CD68 mRNA in plaques from 26 patients. The impact of differentiation from monocytes into macrophages, and subsequently foam cells (by incubation with acetylated LDL) on expression was studied using THP-1 monocytes and primary human monocytes. EL m...... on sections of carotid endarterectomy specimens from patients with symptomatic cerebrovascular disease. In each of eight patients, EL mRNA and/or protein were seen in areas between the necrotic core and the fibrotic cap where they colocalized with LPL and macrophage-specific CD68. Moreover......RNA expression increased markedly when either type of monocytes was differentiated into macrophages. Upon further differentiation into foam cells EL mRNA decreased whereas protein levels remained high compared to monocytes. In conclusion, macrophages in advanced human atherosclerotic lesions display high levels...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    RNA expression increased markedly when either type of monocytes was differentiated into macrophages. Upon further differentiation into foam cells EL mRNA decreased whereas protein levels remained high compared to monocytes. In conclusion, macrophages in advanced human atherosclerotic lesions display high levels......, there was a positive association between the expression of EL mRNA and CD68 mRNA in plaques from 26 patients. The impact of differentiation from monocytes into macrophages, and subsequently foam cells (by incubation with acetylated LDL) on expression was studied using THP-1 monocytes and primary human monocytes. EL m...... of EL expression, and the level of EL expression varies greatly during transformation of blood monocytes into foam cells....

  17. Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Hui-Xiong Xu; Ming-De Lu; Qing Tang

    2008-01-01

    AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript 11 KDR-TK plasmid by enzymatic digestion with XhoI and Sa/I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble,pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP)expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTr) assay.RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively.CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs.Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

  18. Human herpesvirus 7 is a constitutive inhabitant of adult human saliva.

    OpenAIRE

    Wyatt, L S; Frenkel, N

    1992-01-01

    We report the frequent isolation of human herpesvirus 7 from the saliva of healthy adults. Virus isolates recovered from different individuals exhibited minimal restriction enzyme polymorphism, which was mostly confined to heterogeneous (het) sequences in the genome. DNAs of isolates recovered from the same individual over a period of several months showed the same characteristic het fragments, indicating the stability of the het sequences upon virus replication and shedding in vivo. In contr...

  19. Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt inactivation and down-regulation of VEGF and VEGF-R2.

    Science.gov (United States)

    Xiao, Dong; Li, Mengfeng; Herman-Antosiewicz, Anna; Antosiewicz, Jedrzej; Xiao, Hui; Lew, Karen L; Zeng, Yan; Marynowski, Stanley W; Singh, Shivendra V

    2006-01-01

    We have shown recently that diallyl trisulfide (DATS), a cancer-chemopreventive constituent of garlic, inactivates Akt to trigger mitochondrial translocation of proapoptotic protein BAD in human prostate cancer cells. Because Akt activation is implicated in the promotion of endothelial cell survival and angiogenesis, we hypothesized that DATS may inhibit angiogenesis. In the present study, we tested this hypothesis using human umbilical vein endothelial cells (HUVECs) as a model. Survival of HUVECs was reduced significantly in the presence of DATS in a concentration-dependent manner, with an IC50 of approximately 4 microM. The DATS-mediated suppression of HUVEC survival was associated with apoptosis induction characterized by accumulation of subdiploid cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and poly-(ADP-ribose)-polymerase. The DATS-induced DNA fragmentation was significantly attenuated in the presence of pan-caspase inhibitor zVAD-fmk and specific inhibitors of caspase-9 (zLEHD-fmk) and caspase-8 (zIETD-fmk). DATS treatment inhibited the formation of capillary-like tube structure and migration by HUVECs in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level and inactivation of Akt kinase. DATS treatment also caused activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38MAPK).DATS-mediatedapoptosis induction and inhibition of HUVEC tube formation was partially but statistically significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or p38MAPK. The present study demonstrates, for the first time, that DATS has the ability to inhibit angiogenic features of human endothelial cells. PMID:16965246

  20. A biokinetic model for systemic technetium in adult humans

    International Nuclear Information System (INIS)

    The International Commission on Radiological Protection (ICRP) currently is updating its biokinetic and dosimetric models for internally deposited radionuclides. Technetium (Tc), the lightest element that exists only in radioactive form, has two important isotopes from the standpoint of potential risk to humans: the long-lived isotope99Tm(T1/2=2.1x105 y) is present in high concentration in nuclear waste, and the short-lived isotope 99mTc (T1/2=6.02 h) is the most commonly used radionuclide in diagnostic nuclear medicine. This paper reviews data on the biological behavior of technetium and proposes a biokinetic model for systemic technetium in the adult human body for use in radiation protection. Compared with the ICRP s current occupational model for systemic technetium, the proposed model provides a more realistic description of the paths of movement of technetium in the body; provides greater consistency with experimental and medical data; and, for most radiosensitive organs, yields substantially different estimates of cumulative activity (total radioactive decays within the organ) following uptake of 99Tm or 99mTc to blood

  1. Comprehensive cellular-resolution atlas of the adult human brain.

    Science.gov (United States)

    Ding, Song-Lin; Royall, Joshua J; Sunkin, Susan M; Ng, Lydia; Facer, Benjamin A C; Lesnar, Phil; Guillozet-Bongaarts, Angie; McMurray, Bergen; Szafer, Aaron; Dolbeare, Tim A; Stevens, Allison; Tirrell, Lee; Benner, Thomas; Caldejon, Shiella; Dalley, Rachel A; Dee, Nick; Lau, Christopher; Nyhus, Julie; Reding, Melissa; Riley, Zackery L; Sandman, David; Shen, Elaine; van der Kouwe, Andre; Varjabedian, Ani; Write, Michelle; Zollei, Lilla; Dang, Chinh; Knowles, James A; Koch, Christof; Phillips, John W; Sestan, Nenad; Wohnoutka, Paul; Zielke, H Ronald; Hohmann, John G; Jones, Allan R; Bernard, Amy; Hawrylycz, Michael J; Hof, Patrick R; Fischl, Bruce; Lein, Ed S

    2016-11-01

    Detailed anatomical understanding of the human brain is essential for unraveling its functional architecture, yet current reference atlases have major limitations such as lack of whole-brain coverage, relatively low image resolution, and sparse structural annotation. We present the first digital human brain atlas to incorporate neuroimaging, high-resolution histology, and chemoarchitecture across a complete adult female brain, consisting of magnetic resonance imaging (MRI), diffusion-weighted imaging (DWI), and 1,356 large-format cellular resolution (1 µm/pixel) Nissl and immunohistochemistry anatomical plates. The atlas is comprehensively annotated for 862 structures, including 117 white matter tracts and several novel cyto- and chemoarchitecturally defined structures, and these annotations were transferred onto the matching MRI dataset. Neocortical delineations were done for sulci, gyri, and modified Brodmann areas to link macroscopic anatomical and microscopic cytoarchitectural parcellations. Correlated neuroimaging and histological structural delineation allowed fine feature identification in MRI data and subsequent structural identification in MRI data from other brains. This interactive online digital atlas is integrated with existing Allen Institute for Brain Science gene expression atlases and is publicly accessible as a resource for the neuroscience community. J. Comp. Neurol. 524:3127-3481, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  2. Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity

    Science.gov (United States)

    Régnier, Philippe; Bastias, Jorge; Rodriguez-Ruiz, Violeta; Caballero-Casero, Noelia; Caballo, Carmen; Sicilia, Dolores; Fuentes, Axelle; Maire, Murielle; Crepin, Michel; Letourneur, Didier; Gueguen, Virginie; Rubio, Soledad; Pavon-Djavid, Graciela

    2015-01-01

    Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI)+)/ion trap-MS) characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC) assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM). No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases. PMID:25962124

  3. Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity

    Directory of Open Access Journals (Sweden)

    Philippe Régnier

    2015-05-01

    Full Text Available Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI+/ion trap-MS characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM. No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.

  4. Gene expression programs of mouse endothelial cells in kidney development and disease.

    Science.gov (United States)

    Brunskill, Eric W; Potter, S Steven

    2010-01-01

    Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease. PMID:20706631

  5. Gene expression programs of mouse endothelial cells in kidney development and disease.

    Directory of Open Access Journals (Sweden)

    Eric W Brunskill

    Full Text Available Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.

  6. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-30

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

  7. The presence and absence of lymphatic vessels in the adult human intervertebral disc: relation to disc pathology

    Energy Technology Data Exchange (ETDEWEB)

    Kliskey, Karolina; Williams, Kelly; Yu, J.; Urban, Jill; Athanasou, Nick [University of Oxford, Nuffield Department of Orthopaedic, Rheumatology and Musculoskeletal Science, Oxford (United Kingdom); Jackson, David [Weatherall Institute of Molecular Medicine, Human Immunology Unit, Oxford (United Kingdom)

    2009-12-15

    Although the normal adult human intervertebral disc is considered to be avascular, vascularised cellular fibrous tissue can be found in pathological conditions involving the disc such as disc herniation. Whether lymphatics vessels form a component of this reparative tissue is not known as the presence or absence of lymphatics in herniated and normal disc tissue is not known. We examined spinal tissues and discectomy specimens for the presence of lymphatics. The examination used immunohistochemistry to identify the specific lymphatic endothelial cell markers, podoplanin and LYVE1. Lymphatic vessels were not found in the nucleus pulposus or annulus fibrosus of intact, non-herniated lumbar and thoracic discs but were present in the surrounding ligaments. Ingrowth of fibrous tissue was seen in 73% of herniated disc specimens of which 36% contained LYVE1+/podoplanin + lymphatic vessels. Lymphatic vessels were not seen in the sacrum and coccyx or biopsies of four sacrococcygeal chordomas, but they were noted in surrounding extra-osseous fat and fibrous tissue at the edge of the infiltrating tumour. Our findings indicate that lymphatic vessels are not present in the normal adult intervertebral disc but that, when there is extrusion of disc material into surrounding soft tissue, there is ingrowth of reparative fibrous tissue containing lymphatic vessels. Our findings also indicate that chordoma, a tumour of notochordal origin, spreads to regional lymph nodes via lymphatics in para-spinal soft tissues. (orig.)

  8. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    International Nuclear Information System (INIS)

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior. (paper)

  9. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    Science.gov (United States)

    Laranjeira, Marta S.; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-04-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol-gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.

  10. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    Directory of Open Access Journals (Sweden)

    Marta S Laranjeira

    2014-03-01

    Full Text Available Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirc