EAhy926 Endothelial Cells Research Articles

Suspension state promotes extravasation of breast tumor cells by increasing integrin β1 expression

Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells. However, the effect of suspension state on their extravasation and the mechanisms involved are still unclear. To explore the influence of suspension state on extravasation (including adhesion, spreading and transendothelial migration) of breast tumor cells and its relevant molecular mechanism, MDA-MB-231 cells were cultured on poly (2-hydroxyethyl methacrylate) coated 6-well plates to minic the suspension state. Suspension state promoted adhesion, spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells (ECs) monolayer under both the static condition and 0.5 dyne/cm2 flow shear stress (FSS). The number of cells adhered to ECs monolayer reached 2.15 (static condition, 3 d) and 1.67 (FSS, 3 d) times, and the number of migration reached 10.60 times, respectively, as compared to that in adhesion state. Moreover, MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration, as compared with MDA-MB-231 cells. But it made no difference in cell spreading. Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells, but had no significant influence on their spreading.

Androgen inhibits key atherosclerotic processes by directly activating ADTRP transcription

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. In this study, we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription in a dose- and time-dependent manner, and the effect was abolished by three different androgen inhibitors, including pyrvinium pamoate, bicalutamide, and cyproterone acetate. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE, TGTTCT) located at +324bp from the ADTRP transcription start site. The ARE is required for concentration-dependent transcriptional activation of ADTRP. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP and its downstream genes PIK3R3 and MIA3. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.

Abstract 409: Androgen Inhibits Key Atherosclerotic Processes by Directly Activating ADTRP Transcription

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. Here, we tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE) located at +324bp from the ADTRP transcription start site. The ARE is required for the transcriptional activation of ADTRP . HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was reduced by ADTRP siRNA and enhanced by overexpression of ADTRP . These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.

Does native Trypanosoma cruzi calreticulin mediate growth inhibition of a mammary tumor during infection?

BackgroundFor several decades now an antagonism between Trypanosoma cruzi infection and tumor development has been detected. The molecular basis of this phenomenon remained basically unknown until our proposal that T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum-resident chaperone, translocated-externalized by the parasite, may mediate at least an important part of this effect. Thus, recombinant TcCRT (rTcCRT) has important in vivo antiangiogenic and antitumor activities. However, the relevant question whether the in vivo antitumor effect of T. cruzi infection is indeed mediated by the native chaperone (nTcCRT), remains open. Herein, by using specific modified anti-rTcCRT antibodies (Abs), we have neutralized the antitumor activity of T. cruzi infection and extracts thereof, thus identifying nTcCRT as a valid mediator of this effect.MethodsPolyclonal anti-rTcCRT F(ab’)2 Ab fragments were used to reverse the capacity of rTcCRT to inhibit EAhy926 endothelial cell (EC) proliferation, as detected by BrdU uptake. Using these F(ab’)2 fragments, we also challenged the capacity of nTcCRT, during T. cruzi infection, to inhibit the growth of an aggressive mammary adenocarcinoma cell line (TA3-MTXR) in mice. Moreover, we determined the capacity of anti-rTcCRT Abs to reverse the antitumor effect of an epimastigote extract (EE). Finally, the effects of these treatments on tumor histology were evaluated.ResultsThe rTcCRT capacity to inhibit ECs proliferation was reversed by anti-rTcCRT F(ab’)2 Ab fragments, thus defining them as valid probes to interfere in vivo with this important TcCRT function. Consequently, during infection, these Ab fragments also reversed the in vivo experimental mammary tumor growth. Moreover, anti-rTcCRT Abs also neutralized the antitumor effect of an EE, again identifying the chaperone protein as an important mediator of this anti mammary tumor effect. Finally, as determined by conventional histological parameters, in infected animals and in those treated with EE, less invasive tumors were observed while, as expected, treatment with F(ab’)2 Ab fragments increased malignancy.ConclusionWe have identified translocated/externalized nTcCRT as responsible for at least an important part of the anti mammary tumor effect of the chaperone observed during experimental infections with T. cruzi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2764-5) contains supplementary material, which is available to authorized users.

Intermittent hypoxia induces NF-κB-dependent endothelial activation via adipocyte-derived mediators.

Aberrant release of adipocytokines from adipose tissues dysregulates cardiometabolic functions. The present study hypothesizes that chronic intermittent hypoxia (IH) present in obstructive sleep apnea leads to adipose tissue dysfunction, which in turn contributes to vascular pathogenesis. The effect of IH was evaluated in adipose depots and aortic tissues in lean rats in vivo. Furthermore, the cellular and molecular mechanisms underlying pathophysiological interactions between adipocytes and endothelial cells were investigated in vitro. The in vivo results showed that IH induced upregulation of IL-6 and monocyte chemoattractant protein-1 (MCP-1) in subcutaneous and periaortic adipose tissues and downregulated phosphorylation of endothelial nitric oxide synthase [eNOS (ser1177)] in the aorta with activation of Erk and p38 MAPK. In support, cultured adipocytes demonstrated IH-induced elevations of NADPH oxidase 4, phosphorylation of Erk, NF-κBp65, and inducible NOS (iNOS) and increased expression of IL-6 and MCP-1. Likewise, endothelial EA.hy926 (EA) cells exposed to IH showed eNOS (ser1177) and intracellular cGMP reduction, whereas MCP-1 and iNOS expression were upregulated. Treatment of EA cells with conditioned media derived from IH-exposed cultured adipocytes caused nuclear translocation of NF-κBp65 and elevation of MCP-1, which were prevented by addition of neutralizing IL-6 antibodies to the conditioned media. Recombinant IL-6 in addition to IH induced further MCP-1 release and iNOS protein expression in EA cells, which were prevented by pharmacological inhibition of Erk, p38, and NF-κB. These findings suggest that IH could induce adipose tissue inflammation, which may cross talk with endothelial cells via adipocyte-derived mediators such as IL-6, and promote NF-κB-dependent endothelial dysfunction.

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration: combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies.

Influenza virus aggravates the ox-LDL-induced apoptosis of human endothelial cells via promoting p53 signaling.

Oxidized low-density lipoprotein (ox-LDL) is well recognized to play a key role in the development of atherosclerosis. And influenza virus infection has been also recognized to promote the atherosclerosis onset and progressing. However, little is known about the mechanism into it. In present study, we investigated the infection of A/Porto Rico/8/1934 (H1N1) (PR8) influenza virus in human endothelial Eahy926 cells, and determined the induction of apoptosis by the virus infection in the cell. Then we investigated the apoptosis induced by ox-LDL in Eahy926 cells, determined the influence of influenza virus infection on the ox-LDL-induced apoptosis in Eahy926 cells. Results demonstrated that PR8 virus infected human endothelial Eahy926 cells, forming plaques and replicated efficiently in the cell. And the virus infection promoted apoptosis in the cell, upregulated cytchrome c release, activated caspase 3. And what's more, we found that combined PR8 virus infection and ox-LDL treatment promoted higher level of apoptosis and higher level of the activation of apoptosis-associated molecules. Further examination indicated that the p53 signaling was more significantly promoted by both treatments. Therefore, present study confirmed that influenza virus aggravated the ox-LDL-induced apoptosis of human endothelial Eahy926 cells via promoting p53 signaling.

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

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

Abstract 333: Platelet and Endothelial Aggregation Receptor-1 Tempers Tubulogenesis via the Akt Pathway

Platelet endothelial aggregation receptor-1 (PEAR1) is a transmembrane receptor expressed on platelets and endothelial cells (ECs). We aim to unravel its function in ECs. qPCR analyses for PEAR1 in cultured human endothelial progenitors (blood outgrowth endothelial cells (BOECs)), in ECs from human umbilical cord (HUVECs and HUAECs) and in ECs freshly isolated from multiple vascular beds showed the lowest expression in BOECs and the highest in macro- and microvascular ECs from heart and liver. HUVECs and HUAECs showed intermediate PEAR1-expression. Staining of fast growing skin hemangiomas versus normal skin ECs revealed low expression of PEAR1 in rapidly proliferating tumor ECs. Short hairpin based miRNA lentiviral PEAR1-knockdown (shPEAR1) doubled the hybrid EA.hy926 and HUVEC EC proliferation rate compared to control lentiviral-transduced ECs. This was associated with downregulation of HES1-expression (a downstream regulator of the NOTCH-pathway), with increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and with decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the nuclear proliferation suppressor p21CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR and Western blot), whereas the band intensity for p34/CDC2 significantly rose on Western blots (increased mitosis). PEAR1-knockdown also enhanced EC migration via reduced expression of β1- and β3-integrins both in HUVECs and EAhy926 ECs. Both enhanced proliferation and migration contributed to increased tube formation in shPEAR1 HUAECs (in vitro matrigel tube formation assays). During HUAEC tube formation, PEAR1-expression rose 5-fold, associated with a 3-fold increase of PTEN. However, PTEN-levels dropped 1,5-fold during tube formation of shPEAR1 HUAECs, compatible with increased phosphorylation levels of Akt in shPEAR1 ECs. Our study provides the first evidence that PEAR1-expression is variable in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, migration and tube formation of ECs through regulation of the PI3K/Akt/PTEN-pathway. Our data suggest a regulatory role for PEAR1 in the NOTCH-pathway via HES1. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.

Etomidate deteriorates the toxicity of advanced glycation end products to human endothelial Eahy926 cells

Patients with diabetes mellitus, particularly those with cardiovascular complications, have increased risk of mortality when subject to anesthetics and surgery, compared with non-diabetic patients. Anesthetics may exert pressure on the cardiovascular system of diabetic patients, directly or by aggravating pre-existing cardiovascular complications. Advanced glycation end products (AGEs) are extremely accumulated in diabetes mellitus, and are confirmed to play an important role in the pathogenesis of diabetic microvascular and macrovascular complications. The purpose of the present study was to investigate the regulatory role of etomidate, which is widely used as intravenous general anesthetics, on the viability and apoptosis of human endothelial Eahy926 cells, in the presence of AGEs. The results demonstrated that etomidate and Glu-BSA (one type of AGE) synergistically reduced the human endothelial Eahy926 cell viability and induced cell apoptosis. In addition, western blot assay of apoptosis-associated molecules indicated that both agents synergistically upregulated the cytochrome c release, activated the apoptosis executor, caspase 3, and promoted the poly-ADP-ribose polymerase (PARP) lysis. Further results confirmed that the two agents synergistically promoted oxidative stress by decreasing mitochondrial respiratory chain complex IV and mitochondrial membrane potential (MMP), while upregulating reactive oxygen species (ROS) and mitochondrial superoxide. In conclusion, the results presented in this study offer a novel insight into the mechanisms of endothelial cell apoptosis in response to etomidate in the presence of AGEs. These results suggest that oxidative stress has important role in the synergistic promotion of apoptosis by etomidate and AGEs in endothelial Eahy926 cells.

Human cytomegalovirus-encoded miR-US25-1 aggravates the oxidised low density lipoprotein-induced apoptosis of endothelial cells.

Human cytomegalovirus (HCMV) infection is linked to the development and severity of the cardiovascular disease atherosclerosis; however, there is little known about the promotion of atherosclerosis. miR-US25-1 is one of HCMV-encoded miRNAs and targets cellular genes that are essential for virus growth to control the life cycle of the virus and host cells. The prominent regulation on cell cycle genes of the miR-US25-1 attracts us to explore its role in the atherosclerosis promotion. It was indicated that miR-US25-1 level was upregulated in subjects or in endothelial cells with HCMV infection; and the miR-US25-1 downregulated the expression of BRCC 3 by targeting the 5′ UTR of BRCC 3. And a miR-US25-1 mimics transfection could reduce the EAhy926 cell viability but did not induce apoptosis in EAhy926 cells. And what is more, miR-US25-1 mimicis transfection deteriorated the ox-LDL-induced apoptosis and aggravated the upregulation of apoptosis-associated molecules by oxidised low density lipoprotein (ox-LDL) in EAhy926 cells. And we have also confirmed the deregulation of BRCC 3 expression by miR-US25-1 by targeting the 5′ UTR of it. Given the vital role of BRCC 3 in DNA damage repairing, we speculated that the targeting inhibition of BRCC 3 by miR-US25-1 may contribute to the aggravation of ox-LDL-promoted apoptosis of endothelial EAhy926 cells.

Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

To investigate the effects of serum deprivation (SD) on microvesicles (MVs) secreted from human myeloma cells and the implications for disease progression. RPMI 8226, U266, and KM3 human myeloma cells were incubated in medium containing 10% (non-SD) or 1% fetal bovine serum (SD) and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using 2D SDS-PAGE, MALDI-TOF-MS, and Western blotting. NF-κB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926 endothelial cells. Exposure of RPMI 8226 cells to SD for 24 h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI 8226, U266, and KM3 cells to 2.5-, 4.3-, and 3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM, and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduction and metabolism, respectively. Co-culture of SD MVs and RPMI 8226 cells increased NF-κB activation in the target RPMI 8226 cells. Furthermore, SD MVs from RPMI 8226 cells significantly increased the microtubule formation capacity of Eahy926 endothelial cells compared with non-SD MVs. SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in human myeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD.

TNFα induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149

MicroRNAs have been proposed as novel regulators of vascular inflammation and dysfunction. This study aimed to evaluate the role of miR-149 in regulating the expression of key molecules associated with TNFα-induced endothelial activation. miR-149 was selected by in silico analysis and microRNA target prediction. Endothelial dysfunction was induced by TNFα treatment in Eahy926 endothelial cells and HUVEC. miR-149 level was evaluated by quantitative real time-polymerase chain reaction (RT-qPCR). Metalloproteinase-9 (MMP-9) was measured by zymography, Inducible Nitric Oxide Synthase (iNOS) by immunoblotting, Interleukin-6 (IL-6) and Interleukin-8 (IL-8) by ELISA. miR-149 regulatory effect was evaluated by gain-of-function technique upon miR-149 mimics transfection.TNFα down-modulated miR-149 level in Eahy926 and HUVEC. This effect was significantly abolished in Eahy926 by treatment with p38MAPK inhibitor. miR-149 mimic transfection counteracted the TNFα-induced expression of MMP-9, iNOS and IL-6. No effect was detected on IL-8 expression. Our results suggest that miR-149 represents an important new regulator of endothelial function through negative regulation of molecules associated with TNFα-induced endothelial dysfunction.

Hsa-Let-7g miRNA Targets Caspase-3 and Inhibits the Apoptosis Induced by ox-LDL in Endothelial Cells

It has been well confirmed ox-LDL plays key roles in the development of atherosclerosis via binding to LOX-1 and inducing apoptosis in vascular endothelial cells. Recent studies have shown ox-LDL can suppress microRNA has-let-7g, which in turn inhibits the ox-LDL induced apoptosis. However, details need to be uncovered. To determine the anti-atherosclerosis effect of microRNA has-let-7g, and to evaluate the possibility of CASP3 as an anti-atherosclerotic drug target by has-let-7g, the present study determined the role of hsa-let-7g miRNA in ox-LDL induced apoptosis in the vascular endothelial cells. We found that miRNA has-let-7g was suppressed during the ox-LDL-induced apoptosis in EAhy926 endothelial cells. In addition, overexpression of has-let-7g negatively regulated apoptosis in the endothelial cells by targeting caspase-3 expression. Therefore, miRNA let-7g may play important role in endothelial apoptosis and atherosclerosis.

Toward computer simulation of high-LET in vitro survival curves

We developed a Monte Carlo based computer program called MCSC (Monte Carlo Survival Curve) able to predict the survival fraction of cells irradiated in vitro with a broad beam of high linear energy transfer particles. Three types of cell responses are studied: the usual high dose response, the bystander effect and the low-dose hypersensitivity (HRS). The program models the broad beam irradiation and double strand break distribution following Poisson statistics. The progression of cells through the cell cycle is taken into account while the repair takes place. Input parameters are experimentally determined for A549 lung carcinoma cells irradiated with 10 and 20 keV µm−1 protons, 115 keV µm−1 alpha particles and for EAhy926 endothelial cells exposed to 115 keV µm−1 alpha particles. Results of simulations are presented and compared with experimental survival curves obtained for A549 and EAhy296 cells. Results are in good agreement with experimental data for both cell lines and all irradiation protocols. The benefits of MCSC are several: the gain of time that would have been spent performing time-consuming clonogenic assays, the capacity to estimate survival fraction of cell lines not forming colonies and possibly the evaluation of radiosensitivity parameters of given individuals.

Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

Background/purposeBetel quid (BQ) chewing is a popular habit in South-Asian and Southeast Asian countries, and Taiwan. BQ chewing can cause oral cancer and oral submucous fibrosis, and increases the risk of cardiovascular diseases. However, how BQ chewing affects endothelial cells and is involved in cardiovascular diseases and vascular changes is not fully understood. The effects of arecoline, a component of BQ, on the growth and migration of endothelial cells (EAhy 926), and their adherence by U937 cells were investigated. Materials and methodsEAhy926 endothelial cells were cultured and exposed to various concentrations of arecoline for 24 hours. Morphological changes were observed using phase-contrast microscopy. Cytotoxic effects were analyzed using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. A wound closure assay was used to evaluate the cellular migration of EAhy926 cells. The attachment of 2′,7′-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein-labeled U937 cells to EAhy926 endothelial cells that were pretreated with various concentrations of arecoline was further studied. ResultsThe addition of arecoline at concentrations >0.4 mM significantly decreased cellular viability. EAhy endothelial cells showed marked morphological changes, and cellular migration decreased after 24 hours and 48 hours of exposure to arecoline. The number of U937 cells attached to EAhy 926 cells increased when endothelial cells were pretreated by arecoline. ConclusionArecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

Aerobic Training Workload Affects Human Endothelial Cells Redox Homeostasis

Moderate aerobic exercise reduces oxidative stress, whereas intense physical activity may produce the opposite result. At present, the effects of different exercise loads on oxidative stress markers and the response of human cells to different exercise volumes have not been fully elucidated. Human (Eahy-926) endothelial cells (EC), exposed or not exposed to oxidative stress, were conditioned with sera from two groups of triathletes practicing at different workloads. Although no differences in functional and hemodynamic variables were observed between the two groups of triathletes, significant changes in some markers for oxidative stress were found in their sera. Thiobarbituric acid reactive substances and superoxide dismutase activity were similar, but triathletes practicing the sport at lower volume (T1) had higher serum nitric oxide and lower catalase activity than triathletes performing the training at greater load (T2). The EC conditioned with serum from T1 (T1-EC) showed higher survival and proliferation rates and lower senescence levels than the EC supplemented with T2 (T2-EC) serum both before and after oxidative stress induction. These effects depended on catalase as demonstrated via enzyme activity inhibition using 3-amino-1,2,4-triazole. After oxidative stress induction, Sirt1 activity, a regulator of the oxidative stress response, was significantly increased in the T1-EC but not in the T2-EC. Moreover, the T1-EC required less catalase activity than the T2-EC to counteract an equal amount of oxidative stress after H2O2 administration. This study demonstrates that the beneficial effects of aerobic exercise are eliminated when the training is performed at a greater workload. Moreover, we suggest an oxidative stress marker, serum catalase activity, as a valid tool to use in the supervision of changes to exercise volume.

High phosphate directly affects endothelial function by downregulating annexin II

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.

Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

Cytotoxicity testing of nanoparticles (NPs) by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs) are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA) and automated microscopy (Cell-IQ) compared to formazan bioreduction (MTS assay). For validation of the label-free systems different concentrations of ethanol and of amine (AMI) polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (<8 nm) CNTs as more cytotoxic than thick (>20 nm) CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs.

S-Nitrosation of β-Catenin and p120 Catenin

Rationale: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. Objective: We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results: We measured PAF-stimulated SNO of β-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-α increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of β-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin. Conclusions: Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.