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Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.

Correlations between exposure to particle matter (PM) with an aerodynamic diameter

Introduction: Mesenchymal stem cells have the potential to differentiate into several types of cells including osteoblasts. These stem cells have cell surface markers found on cells of endothelial and subendothelial origin of the umbilical cord vein. Taking this into consideration we have postulated that human umbilical vein endothelial cells (HUVEC) could present osteogenic differentiation as well. Gene activation that could drive osteogenic differentiation is regulated by exogenous and endogenous factors., Objective: The induction osteogenesis in HUVEC., Material and Methods: We used: a) an osteogenic medium containing 0.1 microM dexamethasone, 10 microM beta-glycerophosphate, 50 microM L-ascorbic-acid 2-phosphate, 20% MCGS serum; and b) a treatment with DNA demethylating agents hydralazine and 5'-aza-2'-deoxycytidine (0.39-200 microM). Phenotypic characteristics of HUVEC were their spindle and stellate shapes with fine homogenous cytoplasm, typically associated with fibroblast-like cells., Results: The control cells (without osteogenic treatment) exhibited little extracellular matrix, whereas the osteogenically treated cells appeared shortened and flattened, and they were surrounded by extracellular matrix that subsequently became mineralized in vitro. After 28 days in culture, morphologic and histochemical studies confirmed that osteogenic medium had a strong stimulatory effect on the alkaline phosphatase activity of endothelial cells, a very early marker of cell differentiation into the osteogenic lineage. Hydralazine and 5'-aza-2'-deoxycytidine, two drugs utilized in chromatin remodeling leading to gene re-expression of inactivated DNA hypermethylated islands, did not favor osteoblast differentiation., Conclusion: Our study shows that HUVEC can differentiate along an osteogenic lineage and thus provide an alternative source for cell-based therapies and tissue engineering strategies.

Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis, most likely mediating an anti-inflammatory action. In order to understand the mechanisms involved in this protection, we evaluated the effects of DHEA on several molecules involved in the inflammatory response. Reactive oxygen species (ROS), expression of adhesion molecules, activation of the NF-kappaB/IkappaB-alpha pathway and of the AP-1 transcription factor were evaluated in human umbilical vein endothelial cells (HUVECs) treated with oxidized low density lipoproteins (oxLDL) and DHEA. We also determined if DHEA affected LDL oxidation in vitro. 100 microM DHEA-treatment inhibited the oxLDL-induced expression of ICAM-1, VCAM-1, PECAM-1, ROS production, and U937 cells adhesion to HUVECs. DHEA also delayed the kinetics of LDL oxidation in vitro. While DHEA did not affect the translocation of NF-kappaB neither the degradation IkappaB-alpha, it led to an increased translocation of AP-1. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process in endothelial cells activated with oxLDL, therefore its potential anti-inflammatory properties should be evaluated for the treatment of chronic inflammatory diseases such as atherosclerosis.

Background: Recent findings indicate that atherosclerosis, a chronic inflammatory process, might start during childhood. Nevertheless, the expression of inflammation-related molecules of endothelial cell isolated from healthy neonates with a strong family history of myocardial infarction (SFHMI) has been rarely analyzed., Methods: Human umbilical vein endothelial cells (HUVECs) from children with SFHMI were assessed for the expression of CD40 and CD40L, in the presence of TNF-alpha and oxLDL. The intracellular content of CD80, CXCL8 and tissue factor by HUVECs stimulated with a CD40 agonist monoclonal antibody as well as monocytes/lymphocyte adhesion to TNF-alpha-stimulated HUVECs was also evaluated., Results: The basal expression of CD40 and CD40L was higher in SFHMI-positive HUVECs in comparison to controls. TNF-alpha and oxLDL upregulated the expression of CD40 and CD40L in SFHMI versus control HUVECs (p<0.001). The intracellular expression of CXCL8, tissue factor and CD80 was also higher than in controls, and the adhesion of lymphocyte- and monocyte-like cells augmented upon TNF-alpha stimulation., Conclusions: It is possible that the modifications observed in the SFHMI-positive HUVECs, all of them relevant to the atherosclerosis process, may lead to early inflammatory reactions, thus contributing to the premature initiation of atherosclerotic lesions in these children.

Exposure to PM10 is associated with cardiovascular effects. We evaluated the effects of PM10 on E-Selectin expression and monocytic cell adhesion in human umbilical vein endothelial cells (HUVECs). HUVEC were exposed to PM10 (5-40 microg/cm2) for 6 h, following which surface E-Selectin expression was detected by fluorescence microscopy and flow cytometry. The effects of total particles, particles treated with polymixin-B to block the effects of endotoxin, and both soluble and insoluble fractions of particles, were assessed. Incubation with PM10 lead to a concentration-related increase of E-Selectin expression (>seven-fold increase at 40 microg/cm2). Particles pre-treated with polymixin-B inhibited E-Selectin expression to a level slightly higher than untreated particles. An increase in fluorescence was also observed with the insoluble fraction, while the soluble fraction had no significant effect. HUVEC exposed to PM10 were also evaluated for adhesivity of monocytic cells (U937). PM10 strongly increased the adhesion of U937 cells to HUVEC. In conclusion, PM10 induces endothelial cell activation, evidenced by enhanced E-Selectin expression. This activation is manifested functionally as an increase in monocytic cell adhesion. Insoluble components as well as endotoxins appear to be responsible for this activity.

Exposure to airborne particles has been associated with an increase in cardiopulmonary events. Endothelial cells could be playing an important role in the response to airborne particles due their involvement in proinflammatory events, and there is some evidence of particle translocation from lung into circulation. One of the initiating events of inflammation is endothelial activation. We determined the concentration-response effect of a particulate matter with different aerodynamic sizes (PM2.5 [particulate matter with aerodynamic diameter of 2.5 microm and less] and PM10 [particulate matter with aerodynamic diameter of 10 microm and less]) obtained from Mexico City on human umbilical vein endothelial cells (HUVEC). The adhesion of monocytic U937 cells to HUVEC and the expression of early (E- and P-selectins) and late (ICAM-1, PECAM-1, VCAM-1) adhesion molecules were tested. Adhesion of U937 cells to HUVEC was evaluated by coculture experiments using [3H]thymidine-labeled U937 cells and the expression of adhesion molecules was evaluated by flow cytometry. Tumor necrosis factor (TNF)-alpha was used as a positive control of endothelial activation. Our results showed that both PM2.5 and PM10 induced the adhesion of U937 cells to HUVEC, and their maximal effect was observed at 20 microg/cm2. This adhesion was associated with an increase in the expression of all adhesion molecules evaluated for PM10, and E-selectin, P-selectin, and ICAM-1 for PM2.5. In general, maximum expression of adhesion molecules induced by PM2.5 and PM10 was obtained with 20 microg/cm2; however, PM10-induced expression was observed from 5 microg/cm2. E-selectin and ICAM-1 had the strongest expression in response to particles. In conclusion, PM2.5 and PM10 induce the activation of HUVEC, leading to monocytic adhesion via the expression of adhesion molecules, suggesting that these particles may participate in the development of inflammatory diseases. The role of these events in the development of diseases such as atherosclerosis is likely to be evaluated.

Curcumin has protective effects against toxic agents and shows preventive properties for various diseases. Particulate material with an aerodynamic diameter of ≤10 μm (PM10) and titanium dioxide nanoparticles (TiO2-NPs) induce endothelial dysfunction and activation. We explored whether curcumin is able to attenuate different events related to endothelial activation. This includes adhesion, expression of adhesion molecules and oxidative stress induced by PM10 and TiO2-NPs. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 10 and 100 μM curcumin for 1 h and then exposed to PM10 at 3 μg/cm2 or TiO2-NPs at 10 μg/cm2. Cell adhesion was evaluated by co-culture with U937 human myelomonocytic cells. Adhesion molecules expression was measured by flow cytometry after 3 or 24 h of exposure. Oxidative stress was determined by 2,7-dichlorodihydrofluorescein (H2DCF) oxidation. PM10 and TiO2-NPs induced the adhesion of U937 cells and the expression of E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1). The expression of E- and P-selectins matched the adhesion of monocytes to HUVEC after 3 h. In HUVEC treated with 1 or 10 μM curcumin, the expression of adhesion molecules and monocytes adhesion was significantly diminished. Curcumin also partially reduced the H2DCF oxidation induced by PM10 and TiO2-NPs. Our results suggest an anti-inflammatory and antioxidant role by curcumin attenuating the activation caused on endothelial cells by exposure to particles. Therefore, curcumin could be useful in the treatment of diseases where an inflammatory process and endothelial activation are involved. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor (TNF)-α induces a transient increase in N-octanoylsphingosine (C8-ceramide) which has been postulated as an intracellular mediator in TNF-α signalling. We tested the ability of C8-ceramide to reproduce the TNF-α-mediated interference with endothelial cell proliferation and DNA synthesis. TNF-α (10 ng·mL-1) and C8-ceramide (20 µm) inhibited the incorporation of [3H]thymidine into DNA and led to an accumulation of cells in the G1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF-α and C8-ceramide induced increased expression of p53. Treatment with TNF-α or C8-ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF-α prevented RB phosphorylation whereas C8-ceramide did not interfere with this process, suggesting that it follows a ceramide-independent pathway. [ABSTRACT FROM AUTHOR]

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

Abstract: The effects of particulate matter (PM) on endothelial cells have been evaluated in vitro by exposing isolated endothelial cells to different types of PM. Although some of the findings from these experiments have been corroborated by in vivo studies, an in vitro model that assesses the interaction among different cell types is necessary to achieve more realistic assays. We developed an in vitro model that mimics the alveolar–capillary interface, and we challenged the model using TiO2 nanoparticles (TiO2-NPs). Human umbilical endothelial cells (HUVECs) were cultured on the basolateral side of a membrane and pneumocytes (A549) on the apical side. Confluent co-cultures were exposed on the apical side to 10μg/cm2 of TiO2-NPs or 10ng/mL of TNFα for 24h. Unexposed cultures were used as negative controls. We evaluated monocyte adhesion to HUVECs, adhesion molecule expression, nitric oxide concentration and proinflammatory cytokine release. The TiO2-NPs added to the pneumocytes induced a 3- to 4-fold increase in monocyte adhesion to the HUVECs and significant increases in the expression of adhesion molecules (4-fold for P-selectin at 8h, and about 8- and 10-fold for E-selectin, ICAM-1, VCAM-1 and PECAM-1 at 24h). Nitric oxide production also increased significantly (2-fold). These results indicate that exposing pneumocytes to TiO2-NPs causes endothelial cell activation. [Copyright &y& Elsevier]

Abstract: Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. Adhesion on U937 cells, expression of adhesion molecules, production of ROS and NO, expression of eNOS, and translocation of NF-κB were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, or both. High concentrations of glucose (>20mM) induced an increase in adhesion, an increment in mainly E-selectin and PECAM-1 expression, as well as in ROS and NO production, eNOS expression, translocation of NF-κB, and degradation of its inhibitor IκB-α. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative stress and decreased translocation of NF-κB and the degradation of IκB-α induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes. [Copyright &y& Elsevier]

Abstract: Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis. We determined the effect of pharmacological doses of DHEA upon the adhesion of monocytic U937 cells to human umbilical vein endothelial cells (HUVEC), as well as the expression of adhesion and chemoattractant molecules, the translocation of NF-κB, the degradation of IκB-α and the production of reactive oxygen species (ROS) in HUVEC. Adhesion of U937 cells to DHEA-treated HUVEC was evaluated by co-culture experiments using [3H]-thymidine-labeled U937 cells. The expression of adhesion and chemoattractant molecules was evaluated by flow cytometry and RT-PCR, respectively; NF-κB translocation was determined by Electrophoretic Mobility Shift Assay (EMSA) and IκB-α degradation by Western blot. ROS production was determined by the reduction of fluorescent DCFDA. TNF-α was used to induce inflammatory responses in HUVEC. One hundred micromolar of DHEA-treatment inhibited the TNF-α-induced expression of ICAM-1, E-selectin, ROS production and U937 cells adhesion to HUVEC, and interfered with NF-κB translocation and IκB-α degradation. DHEA at the above mention concentration also inhibited the mRNA expression of MCP-1 and IL-8 in basal conditions but not in TNF-α-stimulated conditions. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process, therefore it could be used as an alternative in the treatment of chronic inflammatory diseases such as atherosclerosis. [Copyright &y& Elsevier]