scholarly journals Oxidative Stress Induces HSP90 Upregulation on the Surface of Primary Human Endothelial Cells: Role of the Antioxidant 7,8-Dihydroxy-4-methylcoumarin in Preventing HSP90 Exposure to the Immune System

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Elisabetta Profumo ◽  
Brigitta Buttari ◽  
Lavinia Tinaburri ◽  
Daniela D’Arcangelo ◽  
Maurizio Sorice ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Protein A ◽  
Heat Shock Protein 90 ◽  
Surface Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Levels ◽  
Umbilical Vein Endothelial Cells

We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.

An In Vitro Toxicity Assay for Human Endothelial Cells

1988 ◽  
Vol 16 (1) ◽  
pp. 38-41
Author(s):  
Rosella Sbarbati ◽  
Maria Luisa Schinetti ◽  
Maria Scarlattini
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Diseases ◽  
In Vitro Toxicity ◽  
Human Endothelial Cells ◽  
Experimental Conditions ◽  
Umbilical Vein Endothelial Cells

Cultured human endothelial cells can replace living animals in studying the toxic role of noxious agents in the pathogenesis of vascular diseases and in the elucidation of the mechanism of action of protective drugs. Preliminary data are presented which examine the effects that oxidative stress produces on human endothelial cells in vitro. Human umbilical vein endothelial cells were subjected to an anoxia-re-oxygenation treatment and tested for the production of Super Oxide Dismutase (SOD)-inhibitable superoxide radicals. The results show that under our experimental conditions endothelial cells produce oxygen-free radicals and that the generation reaches a maximum after an anoxic challenge of 20 minutes. We conclude that the in vitro system presented in this paper could be a suitable tool for further studies on the effects of oxidative stress on the vascular endothelium, which mimics the in vivo conditions of re-perfusion after heart ischemia.

ω-3 Fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation

2002 ◽  
Vol 283 (2) ◽  
pp. H811-H818 ◽  
Author(s):  
Konstantin Mayer ◽  
Martina Merfels ◽  
Marion Muhly-Reinholz ◽  
Stephanie Gokorsch ◽  
Simone Rosseau ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Inflammatory Diseases ◽  
Umbilical Vein ◽  
Platelet Activating Factor ◽  
Surface Expression ◽  
Intercellular Adhesion Molecule ◽  
Umbilical Vein Endothelial Cells

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (ω-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-α) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that ω-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.

Activated protein C induces the release of microparticle-associated endothelial protein C receptor

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1515-1522 ◽  
Author(s):  
Margarita Pérez-Casal ◽  
Colin Downey ◽  
Kenji Fukudome ◽  
Gernot Marx ◽  
Cheng Hock Toh
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor V ◽  
Endothelial Protein C Receptor ◽  
Microparticle Formation ◽  
Umbilical Vein Endothelial Cells

Abstract Activated protein C (APC) treatment is now used for patients with severe sepsis. We investigated its effect in vitro on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release that is not inhibited by metalloproteinase inhibitors. Exposure of human umbilical vein endothelial cells or monocytes to APC (6.25-100 nM) results in the release of EPCR-containing microparticles, as demonstrated by confocal microscopy and characterized through flow cytometry, enzyme-linked immunosorbent assay quantitation of isolated microparticles, and Western blotting. The phenomenon is time- and concentration-dependent and requires the APC active site, EPCR, and protease activated receptor 1 (PAR1) on endothelial cells. Neither protein C nor boiled or d-Phe-Pro-Arg-chloromethylketone–blocked APC can induce microparticle formation and antibody blockade of EPCR or PAR1 cleavage and activation abrogates this APC action. Coincubation with hirudin does not alter the APC effect. The released microparticle bound is full-length EPCR (49 kDa) and APC retains factor V–inactivating activity. Although tumor necrosis factor-α (10 ng/mL) can also induce microparticle-associated EPCR release to a similar extent as APC (100 nM), it is only APC-induced microparticles that contain bound APC. This novel observation could provide new insights into the consequences of APC therapy in the septic patient.

scholarly journals Effect of interleukin-5 exposure during in vitro eosinophilopiesis on MAC-1 adhesion molecule expression and function on cultured human eosinophils

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3575-3582 ◽  
Author(s):  
KJ Hamann ◽  
SP Neeley ◽  
TL Dowling ◽  
JA Grant ◽  
AR Leff
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Surface Expression ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Cd11b Expression ◽  
Interleukin 5 ◽  
Umbilical Vein Endothelial Cells ◽  
Cells Cultured

We examined the selective effects of interleukin (IL-5) in regulating the maturational expression of surface adhesion molecules on human eosinophils and adhesion to endothelial cells during eosinophiiopolesis in vitro. Expression of the beta 2 integrins (CD11/CD18) and the beta 1 integrin, VLA-4 (CD49d/ CD29), was assessed during development in culture with IL-3, IL-5, and granulocyte-macrophage colony stimulating factor in cultures of human umbilical cord blood-derived eosinophil (CDE) precursor cells. Expression of both CD11b and CD18 subunits of Mac-1 was lower on CDE which were continuously (= chronically) exposed to IL-5 than on CDE which were cultured without IL-5 for the final week of culture. CD11b expression on cells grown without IL-5 was 71.3 +/- 5.92 (mean specific fluorescence value [MSF] as measured by flow cytometry) versus 52.5 +/- 4.48 MSF for Mac-1 alpha (CD11b) on CDE grown in the continued presence of 2 x 10 – 11 mol/L IL-5 (P < .01). Although expression of VLA-4 decreased as CDE matured, expression of CD29 and CD49d were similar regardless of cytokine exposure for the final week of culture. For eosinophils cultured without IL-5, acute stimulation with 10 – 8 mol/L IL-5 increased CD11b surface expression and increased the number of cells adhering to unstimulated human umbilical vein endothelial cells (HUVEC) from 4,570 +/- 780 cells (9.14 +/- 1.56% adhesion) to 8,385 +/- 515 cells (16.8 +/- 1.03% adhesion) (P < .01). Basal adhesion to unstimulated HUVEC of CDE cultured continuously with IL-5 was comparable (8.62 +/- 1.12% adhesion; P = NS), but neither CD11b expression (50.3 +/- 11.8 MSF; P = NS v control) nor adhesion to HUVEC (6.77 +/- 1.35%; P = NS) was enhanced in these eosinophils after acute stimulation with IL-5. Blockade of adhesion to IL-1-stimulated HUVEC caused by the anti-CD49d monoclonal antibody (MoAb), HP2/1, was comparable for cells cultured with IL-5 and without IL-5. However, the anti-CD18 MoAb, R15.7, caused 47.6 +/- 5.08% inhibition of adhesion of eosinophils cultured without IL-5 and only 25.8 +/- 5.20% for cells cultured continuously with IL-5 (P < .01), and failed to block significantly the adhesion of only the latter cells to IL-4-stimulated HUVEC. Our data show that continuous, chronic exposure to low concentrations of IL-5 causes decreased expression of Mac-1 and refractoriness to acute stimulation with IL-5 of adhesion to HUVEC. These data further demonstrate that CDE maturing in the continued presence of IL-5 adhere to HUVEC predominantly through VLA-4 ligation.

scholarly journals Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens

2021 ◽  
Author(s):  
Susan Gallogly ◽  
Takeshi Fujisawa ◽  
John D. Hung ◽  
Mairi Brittan ◽  
Elizabeth M. Skinner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
In Vitro Model ◽  
Umbilical Vein ◽  
Coronary Syndrome ◽  
Coronary Endothelial Cells ◽  
Vitro Model ◽  
Umbilical Vein Endothelial Cells

Abstract Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

scholarly journals Effect of TNFα stimulation on expression of kidney risk inflammatory proteins in human umbilical vein endothelial cells cultured in hyperglycemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zaipul I. Md Dom ◽  
Caterina Pipino ◽  
Bozena Krolewski ◽  
Kristina O’Neil ◽  
Eiichiro Satake ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Systemic Exposure ◽  
In Vitro Study ◽  
Human Umbilical Vein ◽  
Intracellular Fraction ◽  
Inflammatory Proteins ◽  
Umbilical Vein Endothelial Cells ◽  
Extracellular Fraction

AbstractWe recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

scholarly journals Cytoprotective Role of Edible Seahorse (Hippocampus abdominalis)-Derived Peptides in H2O2-Induced Oxidative Stress in Human Umbilical Vein Endothelial Cells

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 86
Author(s):  
Yunok Oh ◽  
Chang-Bum Ahn ◽  
Jae-Young Je
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Cardiovascular Diseases ◽  
Combination Treatment ◽  
Umbilical Vein ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Nuclear Staining ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Oxidative stress-induced endothelial dysfunction is strongly linked to the pathogenesis of cardiovascular diseases. A previous study revealed that seahorse hydrolysates ameliorated oxidative stress-mediated human umbilical vein endothelial cells (HUVECs) injury. However, the responsible compounds have not yet been identified. This study aimed to identify cytoprotective peptides and to investigate the molecular mechanism underlying the cytoprotective role in H2O2-induced HUVECs injury. After purification by gel filtration and HPLC, two peptides were sequenced by liquid chromatography-tandem mass spectrometry as HGSH (436.43 Da) and KGPSW (573.65 Da). The synthesized peptides and their combination (1:1 ratio) showed significant HUVECs protection effect at 100 μg/mL against H2O2-induced oxidative damage via significantly reducing intracellular reactive oxygen species (ROS). Two peptides and their combination treatment resulted in the increased heme oxygenase-1 (HO-1), a phase II detoxifying enzyme, through the activation of nuclear transcription factor-erythroid 2-related factor (Nrf2). Additionally, cell cycle and nuclear staining analysis revealed that two peptides and their combination significantly protected H2O2-induced cell death through antiapoptotic action. Two peptides and their combination treatment led to inhibit the expression of proapoptotic Bax, the release of cytochrome C into the cytosol, the activation of caspase 3 by H2O2 treatment in HUVECs, whereas antiapoptotic Bcl-2 expression was increased with concomitant downregulation of Bax/Bcl-2 ratio. Taken together, these results suggest that seahorse-derived peptides may be a promising agent for oxidative stress-related cardiovascular diseases.

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