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- ItemSomente MetadadadosAcquisition of anoikis resistance promotes alterations in the Ras/ERK and PI3K/Akt signaling pathways and matrix remodeling in endothelial cells(Springer, 2017) de Sousa Mesquita, Ana Paula [UNIFESP]; Lopes, Silvana de Araujo [UNIFESP]; Pernambuco Filho, Paulo Castanho A. [UNIFESP]; Nader, Helena B. [UNIFESP]; Lopes, Carla Cristina [UNIFESP]Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix. Anoikis resistance is a critical mechanism in tumor metastasis. Cancer cells deregulate and adapt their metabolism to survive in the absence of adhesion, spreading metastases to distant organs. These adaptations include abnormal regulation of growth factor receptors activating prosurvival signaling pathways, such as the Ras/ERK and PI3K/Akt pathways, and extracellular matrix remodeling, leading to metastasis by an increase of invasiveness and inhibiting anoikis. This study investigates the possible involvement of ECM components and signaling pathways in the regulation of resistance to anoikis in endothelial cells (EC). Endothelial cells submitted to stressful conditions by blocking adhesion to substrate (anoikis resistance) display an up-regulation of Ras/ERK and PI3k/Akt pathways by high expression of Ras, ERK, PI3K (p110 alpha) and Akt (Thr 308). After ERK and PI3K inhibiting, all EC-derived cell lines studied showed lower growth, a decrease in invasive potential and a higher rate of apoptosis. Furthermore, anoikis-resistant cell lines display a decrease in the expression of fibronectin, collagen IV and hyaluronic acid and an increase in the expression of laminin, perlecan, alpha v, beta 3, alpha 5 and beta 1 integrins subunits, hyaluronidades 1, 2 and 3 and metalloproteinases 2 and 9. These results indicate that the acquisition of anoikis resistance induced remodeling of the extracellular matrix and overexpression of the PI3K/Akt and Ras/ERK pathway components. Acquisition of resistance to anoikis is a potentially crucial step in endothelial cell transformation.
- ItemSomente MetadadadosAging Exacerbates Microvascular Endothelial Damage Induced by Circulating Factors Present in the Serum of Septic Patients(Oxford Univ Press Inc, 2013-06-01) Tucsek, Zsuzsanna; Gautam, Tripti; Sonntag, William E.; Toth, Peter; Saito, Hiroshi; Salomão, Reinaldo [UNIFESP]; Szabo, Csaba; Csiszar, Anna; Ungvari, Zoltan; Univ Oklahoma; Univ Kentucky; Universidade Federal de São Paulo (UNIFESP); Univ Texas Med BranchThe elderly patients show a significantly elevated mortality rate during sepsis than younger patients, due to their higher propensity to microvascular dysfunction and consequential multiorgan failure. We tested whether aging renders vascular endothelial cells more susceptible to damage induced by inflammatory factors present in the circulation during sepsis. Primary microvascular endothelial cells derived from young (3 months) and aged (24 months) Fischer 344 Brown Norway rats were treated with sera obtained from sepsis patients and healthy controls. Oxidative stress (MitoSox fluorescence), death receptor activation (caspase 8 activity), and apoptotic cell death (caspase 3 activity) induced by treatment with septic sera were exacerbated in aged endothelial cells as compared with responses obtained in young cells. Induction of heme oxygenase-1 and thrombomodulin in response to treatment with septic sera was impaired in aged endothelial cells. Treatment with septic sera elicited greater increases in tumor necrosis factor- expression in aged endothelial cells, as compared with young cells, whereas induction of inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule did not differ between the two groups. Collectively, aging increases sensitivity of microvascular endothelial cells (MVECs) to oxidative stress and cellular damage induced by inflammatory factors present in the circulation during septicemia. We hypothesize that these responses may contribute to the increased vulnerability of elderly patients to multiorgan failure associated with sepsis.
- ItemSomente MetadadadosBauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration(Servicio Publicaciones Universidad Navarra, 2010-12-01) Bilgin, Mehmet; Neuhof, Christiane; Doerr, Oliver; Benscheid, Utz; Andrade, Sheila S. [UNIFESP]; Most, Astrid; Abdallah, Yaser; Parahuleva, Mariana; Guenduez, Dursun; Oliva, Maria L. [UNIFESP]; Erdogan, Ali; Univ Giessen; Universidade Federal de São Paulo (UNIFESP)Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. the Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. the aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). for proliferation experiments, HUVEC were incubated with BbCI (10-100 mu mol/L) for 48 h. the proliferation was detected by cell counting with a Neubauer chamber. the effect of BbCI (10-100 mu M) on the membrane potential was measured with the fluorescence dye DiBAC(4)(3) and the effect on [Ca+2] (i) with the fluorescence probe Fluo-3 AM. the change of the fluorescence intensity was determined with a GENios plate reader (Tecan). the experiments showed that BbCI (10-100 mu mol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 mu mol/L (35.1 +/- 1.8% as compared to control (p a parts per thousand currency signaEuro parts per thousand 0.05; n = 45)). As compared to the control, the addition of BbCI (100 mu mol/L) caused a significant increase of systolic Ca2+ of 28.4 +/- 5.0% after 30 min incubation. HUVEC treatment with BbCI (100 mu mol/L) showed a weak but significant decrease of the membrane potential of 9.5 +/- 0.9% as compared to control (p a parts per thousand currency signaEuro parts per thousand 0.05; n = 80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.
- ItemSomente MetadadadosBrown spider (Loxosceles intermedia) venom triggers endothelial cells death by anoikis(Elsevier B.V., 2012-09-01) Nowatzki, Jenifer; Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Rizzo, Luiz Eduardo; Souza-Fonseca-Guimaraes, Fernando; Veiga, Silvio Sanches; Nader, Helena Bonciani [UNIFESP]; Franco, Celia Regina C.; Trindade, Edvaldo S.; Univ Fed Parana; Universidade Federal de São Paulo (UNIFESP); Estadual Univ ParanaBrown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. the vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. in this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the alpha(5)beta(1) integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. in addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus. L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis. (C) 2012 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosBrown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells(Elsevier B.V., 2010-09-15) Nowatzki, Jenifer; Sene, Reginaldo Vieira de; Paludo, Katia Sabrina [UNIFESP]; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Celia; Nader, Helena Bonciani [UNIFESP]; Trindade, Edvaldo S.; Franco, Celia Regina C.; Univ Fed Parana; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, Including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. the aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom After treating endothelial cells with venom toxins, we observed that the venom Interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells the venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L interned:a venom on endothelial cells is not mediated by venom internalization (C) 2010 Elsevier B.V. All rights reserved
- ItemSomente MetadadadosEfeito da resistência à morte celular por perda de adesão (anoikis) na expressão do sindecam-4 e no ciclo celular de células endoteliais em cultura(Universidade Federal de São Paulo, 2013-04-25) Carneiro, Bruna Ribeiro [UNIFESP]; Azevedo, Carla Cristina Lopes de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix (ECM) by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins and FAK (focal adhesion kinase) in cell adhesion at focal contacts connecting the ECM to the cytoskeleton. Changes in the expression of syndecan-4 have been found in tumor cells, indicating its involvement in cancer. The acquisition of resistance to cell death induced by blockade of adhesion to the substrate (anoikis resistance) is a feature of neoplastic transformation and a critical step during the metastatic process. Aiming to clarify the role of sindecan-4 in the process of anoikis and in the processes of cell transformation, wild endothelial cells (EC) were subjected to transformation induced by blockade of adhesion to the substrate (Adh-EC) and studied in comparison with endothelial cells transfected with the EJ-ras oncogene (EJ-ras EC) in relation to: tumorigenic capacity, adhesive ability, cell cycle, cell proliferation, synthesis and degradation of sulfated glicosaminoglycans (SGAG), expression and localization of syndecam-4 and heparanase expression. After cycles of blocking adhesion, phenotypic changes were observed in the surviving cells. The clones obtained exhibited characteristics similar to tumorigenic cells: multilayer growth, loss of contact inhibition, tumorigenic capacity, uncontrolled cell cycle, differences at the dependence of growth factors present in fetal bovine serum (FBS), decreased adhesive capacity and a major anti-proliferative effect after treatment with specific inhibitors of PI3K/Akt e Ras/Raf/MAPK/ERK pathways. Further, an increase in the synthesis of heparan sulfate (HS) was detected in the clones resistant to anoikis. This increase is observed for both HS chains present in cells and secreted to culture medium. Structural changes in HS chains were also observed. We have seen that there is an increase in the syndecan-4 and heparanase expression in EJ-ras EC and Adh-EC cells. The data suggests that the syndecan-4 is involved in the acquisition of resistance to cell death induced by blockade of adhesion to the substrate (resistance to anoikis), contributing to neoplastic transformation.
- ItemSomente MetadadadosEffect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration(Verduci Publisher, 2014-01-01) Bilgin, M.; Burgazli, K. M.; Rafiq, A.; Mericliler, M.; Neuhof, C.; Oliva, M. L. [UNIFESP]; Parahuleva, M.; Soydan, N.; Doerr, O.; Abdallah, Y.; Erdogan, A.; Univ Giessen; Bezmialem Vakif Univ; Wuppertal Res & Med Ctr; Universidade Federal de São Paulo (UNIFESP)OBJECTIVES: Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium.MATERIALS AND METHODS: HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 mu M 12% and 13%. Furthermore, BbKI (100 mu M) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control.RESULTS: The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential.CONCLUSIONS: BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.
- ItemSomente MetadadadosEndothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis(Elsevier B.V., 2014-09-15) Moraes, Miriam S.; Costa, Paulo E. [UNIFESP]; Batista, Wagner L. [UNIFESP]; Paschoalin, Taysa [UNIFESP]; Curcio, Marli F. [UNIFESP]; Borges, Roberta E. [UNIFESP]; Taha, Murched O. [UNIFESP]; Fonseca, Fabio V.; Stern, Arnold; Monteiro, Hugo P. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); NYU; Univ Spiritu Santo Ecuador; Case Western UnivNitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel (R)-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway. (C) 2014 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosEstudos in vitro da participação da leptina em células endoteliais pulmonares de ratos nutridos e desnutridos intrauterinamente(Universidade Federal de São Paulo, 2014-03-02) Santos, Leila Aparecida dos [UNIFESP]; Landgraf, Richardt Gama [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Studies suggest that intrauterine malnutrition can "program" the fetal tissues making them more prone to disorders associated with food, such as type 2 diabetes, metabolic syndrome and other chronic diseases in adulthood , and induce changes in the immune response and inflammatory. Leptin, the main hormone secreted by adipose tissue, has a pleiotropic action, working in various systems such as the regulation of energy balance, reproduction, hematopoiesis, lymphopoiesis and has a modulating action of the immune system. Pulmonary endothelial cells were obtained from Wistar rats, male or intrauterine undernourished fed rats at 12 weeks of age. These cells were stimulated with leptin or LPS or leptin plus LPS, furthermore, cells with no stimulus were used as controls. Two hours after stimulation, production of inflammatory mediators PGE2, LTB4, IL1?, TNF? and the expression of ERK ½, and leptin receptors were analyzed. We observed that expression of the leptin receptor is 63 % lower in primary cultures of endothelial cells derived from intrauterine undernourished rats. Leptin alone did not induce any change in levels of inflammatory mediators evaluated, whereas LPS increased levels of PGE2 (250 %) and LTB4 (29%). Only endothelial cells of rats fed LPS administration + leptin production increased lipid mediators compared to LPS group (PGE 2 - and LTB4 28 % - 18%). The assay for IL1? showed that only cells from the control animals showed expression of IL1?. The expression of TNF and activation of ERK ½ showed a contradictory result because only the endothelial cells from the malnourished animals showed higher expression and activation, respectively. This result may suggest an expression of TNFa stimulation via LPS via another receptor with activation of ERK. These results suggest that the lower expression of the leptin receptor in lung endothelial cells in intrauterine undernourished rats could negatively modulate lipid mediator production in these cells.
- ItemSomente MetadadadosGlycosaminoglycan backbone is not required for the modulation of hemostasis: Effect of different heparin derivatives and non-glycosaminoglycan analogs(Elsevier B.V., 2012-06-01) Boucas, Rodrigo I.; Jarrouge-Boucas, Thais R.; Lima, Marcelo A.; Trindade, Edvaldo S.; Moraes, Fabio A.; Cavalheiro, Renan P.; Tersariol, Ivarne L. S.; Hoppenstead, Debra; Fareed, Jawed; Nader, Helena B. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Parana; Univ Mogi das Cruzes; Loyola Med SchHeparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. in the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide. C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. in addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. the data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. the results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis. (C) 2012 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Heparin affects the interaction of kininogen on endothelial cells(Elsevier B.V., 2011-10-01) Gozzo, Andrezza Justino [UNIFESP]; Motta, Guacyara da [UNIFESP]; Cruz-Silva, Ilana [UNIFESP]; Nunes, Viviane Abreu [UNIFESP]; Barros, Nilana Meza Tenório de [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Shimamoto, Kazuaki; Nader, Helena Bonciani [UNIFESP]; Araujo, Mariana da Silva [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Sapporo Med UnivIn the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PM) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. in the presence of Zn(+2), HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PM binding to cell- or ECM-bound-HK and PM activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn(2+). Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. in conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment. (C) 2011 Elsevier Masson SAS. All rights reserved.