Ebola Virus GP Activates Endothelial Cells via Host Cytoskeletal Signaling Factors

Ebola virus disease (EVD) is a lethal disease caused by the highly pathogenic Ebola virus (EBOV), and its major symptoms in severe cases include vascular leakage and hemorrhage. These symptoms are caused by abnormal activation and disruption of endothelial cells (ECs) whose mediators include EBOV glycoprotein (GP) without the need for viral replication. However, the detailed molecular mechanisms underlying virus–host interactions remain largely unknown. Here, we show that EBOV-like particles (VLPs) formed by GP, VP40, and NP activate ECs in a GP-dependent manner, as demonstrated by the upregulation of intercellular adhesion molecules-1 (ICAM-1) expression. VLPs-mediated ECs activation showed a different kinetic pattern from that of TNF-α-mediated activation and was associated with apoptotic ECs disruption. In contrast to TNF-α, VLPs induced ICAM-1 overexpression at late time points. Furthermore, screening of host cytoskeletal signaling inhibitors revealed that focal adhesion kinase inhibitors were found to be potent inhibitors of ICAM-1 expression mediated by both TNF-α and VLPs. Our results suggest that EBOV GP stimulates ECs to induce endothelial activation and dysfunction with the involvement of host cytoskeletal signaling factors, which represent potential therapeutic targets for EVD.

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Microvascular Endothelial Cells Express Phosphatidylserine Receptor: A Functionally Active Receptor for Phosphatidylserine-Positive Erythrocytes.

Blood ◽

10.1182/blood.v110.11.1720.1720 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1720-1720

Author(s):

Yamaja B.N. Setty ◽

Suhita Gayen Betal

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Constitutive Expression ◽

Fold Increase ◽

Endothelial Activation ◽

Surface Expression ◽

Dependent Manner ◽

Adhesive Interaction ◽

Adhesion Receptor ◽

Tnf Α

Abstract Phosphatidylserine (PS)-positive erythrocytes adhere to both endothelial cells and the sub-endothelial matrix components. While thrombospondin mediates these interactions, it is not known whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control erythrocytes, we demonstrate that PS-positive erythrocytes adhered directly to human lung micro-endothelial cells in the absence of plasma ligands, that this adhesion was further enhanced following endothelial activation with IL-1α, TNF-α, LPS, and hypoxia (2.5- to 8-fold increase), and that this adhesive interaction was selective to erythrocyte-PS. We next explored whether micro-endothelial cells express an adhesion receptor that recognizes cell surface expressed PS (PSR) similar to that expressed on activated macrophages. Using RT-PCR and Western blotting, we demonstrate constitutive expression of both PSR mRNA and protein which were up-regulated in a time-dependent manner following endothelial activation (with maximal increases of 3-fold in mRNA and 2-fold in protein noted at 4 and 6 hour, respectively). While minimal PSR expression was noted on un-stimulated cell surface (8% positivity), endothelial activation up-regulated surface expression of this receptor (35–40% positivity in IL-1α and TNF-α activated cultures). In antibody blocking studies, using both artificially generated PS-positive erythrocytes and also using PS-positive erythrocytes from patient with sickle cell disease, we demonstrate that PSR was functionally active supporting PS-mediated erythrocyte adhesion to activated endothelial cells. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the micro-endothelium which is up-regulated by such pathologically relevant agonists as hypoxia and cytokines.

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Abstract 3380: Acetylation-dependent Regulation Of Notch1 Signaling In Endothelial Cells

Circulation ◽

10.1161/circ.118.suppl_18.s_415-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Virginia Guarani ◽

Franck Dequiedt ◽

Andreas M Zeiher ◽

Stefanie Dimmeler ◽

Michael Potente

Keyword(s):

Endothelial Cells ◽

Notch Signaling ◽

Cell Fate ◽

Molecular Mechanisms ◽

Trichostatin A ◽

Notch Pathway ◽

Dependent Manner ◽

Class Iii ◽

Cell Fate Decisions ◽

Knock Down

The Notch signaling pathway is a versatile regulator of cell fate decisions and plays an essential role for embryonic and postnatal vascular development. As only modest differences in Notch pathway activity suffice to determine dramatic differences in blood vessel development, this pathway is tightly regulated by a variety of molecular mechanisms. Reversible acetylation has emerged as an important post-translational modification of several non-histone proteins, which are targeted by histone deacetylases (HDACs). Here, we report that specifically the Notch1 intracellular domain (NICD) is itself an acetylated protein and that its acetylation level is tightly regulated by the SIRT1 deacetylase, which we have previously identified as a key regulator of endothelial angiogenic functions during vascular growth. Coexpression of NICD with histone acetyltransferases such as p300 or PCAF induced a dose- and time-dependent acetylation of NICD. Blocking HDAC activity using the class III HDAC inhibitor nicotinamid (NAM), but not the class I/II HDAC inhibior trichostatin A, resulted in a significant increase of NICD acetylation suggesting that NICD is targetd by class III HDACs for deacetylation. Among the class III HDACs with deacetylase activity (SIRT1, 2, 3, 5), knock down of specifically SIRT1 resulted in enhanced acetylation of NICD. Moreover, wild type SIRT1, but not a catalytically inactive mutant catalyzed the deacetylation of NICD in a nicotinamid-dependent manner. SIRT1, but SIRT2, SIRT3 or SIRT5, associated with NICD through its catalytic domain demonstrating that SIRT1 is a direct NICD deacetylase. Enhancing NICD acetylation by either overexpression of p300 or inhibition of SIRT1 activity using NAM or RNAi-mediated knock down resulted in enhanced NICD protein stability by blocking its ubiquitin-mediated degradation. Consistent with these results, loss of SIRT1 amplified Notch target gene expression in endothelial cells in response to NICD overexpression or treatment with the Notch ligand Dll4. In summary, our results identify reversible acetylation of NICD as a novel molecular mechanism to control Notch signaling and suggest that deacetylation of NICD by SIRT1 plays a key role in the dynamic regulation of Notch signaling in endothelial cells.

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Abstract 660: Local Hemodynamic Forces and Aqueous Cigarette Smoke Extract Affect Development of Endothelial Dysfunction

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.660 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Sindy Giebe ◽

Coy Brunssen ◽

Melanie Brux ◽

Natalia Cockcroft ◽

Katherine Hewitt ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Laminar Flow ◽

Tobacco Smoking ◽

Molecular Mechanisms ◽

Low Flow ◽

Dependent Manner ◽

Flow Conditions ◽

Hemodynamic Forces ◽

Plaque Phenotype

Endothelial dysfunction is one of the first steps in the development of atherosclerosis. This proinflammatory phenotype is associated with decreased bioavailability of nitric oxide and a corresponding expression profile in the endothelial cells. Tobacco smoking promotes development of atherosclerotic plaques and local hemodynamic forces are key stimuli in this process. Low laminar flow is involved in the development of an unstable plaque phenotype, while high laminar flow has atheroprotective role. The molecular mechanisms controlling plaque stability in response to tobacco smoking remain largely unknown so far. Therefore, we exposed human endothelial cells to cigarette smoke extract (CSEaq) under disturbed flow conditions. Primary human endothelial cells were stimulated with increasing dosages of CSEaq for 24h. Cell viability was reduced by CSEaq in a dose-dependent manner. The impact of specific flow conditions and different doses of CSEaq on the expression of atherosclerosis-related genes was investigated using a cone-and-plate viscometer. High laminar flow induced elongation of endothelial cells in the direction of flow, increased eNOS expression and NO release in a time-dependent manner. This increase was inhibited by CSEaq. Low laminar flow showed no effect on eNOS expression and NO release. The NRF2 antioxidative defense system was also induced by high laminar flow. NRF2 and NRF2 target genes HMOX1 and NQO1 were strongly activated by CSEaq. Furthermore, we monitored the expression of proinflammatory genes. CSEaq strongly induced adhesion molecule ICAM-1. Interestingly, VCAM-1 was unaffected by CSEaq. Induction of endothelial NADPH oxidase isoform 4 by CSEaq was prevented by high laminar flow. Catalase expression was not affected by flow and CSEaq, whereas CSEaq transiently increased SOD1 expression. Endothelial wound healing was improved by atheroprotective high laminar flow. Low flow did not affect wound healing. Furthermore, high laminar flow decreased adhesion of monocytes to endothelial cells, compared to low flow. We suggest novel molecular mechanisms how tobacco smoking promotes the development of endothelial dysfunction. This can contribute to the formation of an unstable atherosclerotic plaque phenotype.

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MALAT1 modulates miR-146’s protection of microvascular endothelial cells against LPS-induced NF-κB activation and inflammatory injury

Innate Immunity ◽

10.1177/1753425919861427 ◽

2019 ◽

Vol 25 (7) ◽

pp. 433-443

Author(s):

Lin-Lin Feng ◽

Wei-Na Xin ◽

Xiu-Li Tian

Keyword(s):

Endothelial Cells ◽

Cell Viability ◽

Combined Treatment ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Microvascular Endothelial Cells ◽

Dependent Manner ◽

Inflammatory Injury ◽

Tnf Α ◽

Microvascular Endothelial

To investigate the role of miR-146 and its possible relationship with MALAT1 in LPS-induced inflammation in human microvascular endothelial cells (HMECs), HMEC-1 cells were treated with LPS to construct an inflammatory injury cell model, and the cell viability, TNF-α and IL-6 secretion and the expression levels of VCAM-1, SELE and ICAM-1 were analysed as markers of inflammatory injury. The regulation mechanisms of miR-146 interacted with MALAT1 and the downstream NF-κB signalling were also verified by dual-luciferase assay and knockdown technology. LPS significantly decreased the cell viability, increased levels of VCAM-1, SELE and ICAM-1 and also up-regulated miR-146a/b, TNF-α and IL-6 in a dose-dependent manner. Over-expression of miR-146a resulted in down-regulation of TNF-α and IL-6, as well as VCAM-1, SELE and ICAM-1, while inhibition of miR-146a led to opposite results. The dual-luciferase reporter assay showed both miR-146a and miR-146b directly targeted and negatively regulated the expression of MALAT1. Silencing of MALAT1 suppressed LPS-induced NF-κB activation and TNF-α and IL-6 secretion, reducing the cell inflammatory injury, but these changes were reversed after combined treatment with miR-146a inhibitor. Taken together, we demonstrate that miR-146 protects HMECs against inflammatory injury by inhibiting NF-κB activation. This process is modulated by MALAT1.

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Vascular Protective Effects of Xanthotoxin and Its Action Mechanism in Rat Aorta and Human Vascular Endothelial Cells

Journal of Vascular Research ◽

10.1159/000509112 ◽

2020 ◽

Vol 57 (6) ◽

pp. 313-324

Author(s):

Li-Hua Cao ◽

Ho Sub Lee ◽

Zhe-Shan Quan ◽

Yun Jung Lee ◽

Yu Jin

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Neuroprotective Effect ◽

Intercellular Adhesion Molecule ◽

Dependent Manner ◽

Protective Effects ◽

Concentration Dependent Manner

Objective: Xanthotoxin (XAT) is a linear furanocoumarin mainly extracted from the plants Ammi majus L. XAT has been reported the apoptosis of tumor cells, anti-convulsant, neuroprotective effect, antioxidative activity, and vasorelaxant effects. This study aimed to investigate the vascular protective effects and underlying molecular mechanisms of XAT. Methods: XAT’s activity was studied in rat thoracic aortas, isolated with aortic rings, and human umbilical vein endothelial cells (HUVECs). Results: XAT induced endothelium-dependent vasodilation in a concentration-dependent manner in the isolated rat thoracic aortas. Removal of endothelium or pretreatment of aortic rings with L-NAME, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, and wortmannin significantly inhibited XAT-induced relaxation. In addition, treatment with thapsigargin, 2-aminoethyl diphenylborinate, Gd3+, and 4-aminopyridine markedly attenuated the XAT-induced vasorelaxation. XAT increased nitric oxide production and Akt- endothelial NOS (eNOS) phosphorylation in HUVECs. Moreover, XAT attenuated the expression of TNF-α-induced cell adhesion molecules such as intercellular adhesion molecule, vascular cell adhesion molecule-1, and E-selectin. However, this effect was attenuated by the eNOS inhibitors L-NAME and asymmetric dimethylarginine. Conclusions: This study suggests that XAT induces vasorelaxation through the Akt-eNOS-cGMP pathway by activating the KV channel and inhibiting the L-type Ca2+ channel. Furthermore, XAT exerts an inhibitory effect on vascular inflammation, which is correlated with the observed vascular protective effects.

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Exchange Protein Directly Activated by cAMP Modulates Ebola Virus Uptake into Vascular Endothelial Cells

Viruses ◽

10.3390/v10100563 ◽

2018 ◽

Vol 10 (10) ◽

pp. 563 ◽

Cited By ~ 5

Author(s):

Aleksandra Drelich ◽

Barbara Judy ◽

Xi He ◽

Qing Chang ◽

Shangyi Yu ◽

...

Keyword(s):

Endothelial Cells ◽

Ebola Virus ◽

Vascular Endothelial Cells ◽

Ex Vivo ◽

Marburg Virus ◽

Dependent Manner ◽

Vascular Endothelial ◽

Ultrastructural Studies ◽

Ebov Infection ◽

Exchange Protein

Members of the family Filoviridae, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates. Given their high lethality, a comprehensive understanding of filoviral pathogenesis is urgently needed. In the present studies, we revealed that the exchange protein directly activated by cAMP 1 (EPAC1) gene deletion protects vasculature in ex vivo explants from EBOV infection. Importantly, pharmacological inhibition of EPAC1 using EPAC-specific inhibitors (ESIs) mimicked the EPAC1 knockout phenotype in the ex vivo model. ESI treatment dramatically decreased EBOV infectivity in both ex vivo vasculature and in vitro vascular endothelial cells (ECs). Furthermore, postexposure protection of ECs against EBOV infection was conferred using ESIs. Protective efficacy of ESIs in ECs was observed also in MARV infection. Additional studies using a vesicular stomatitis virus pseudotype that expresses EBOV glycoprotein (EGP-VSV) confirmed that ESIs reduced infection in ECs. Ultrastructural studies suggested that ESIs blocked EGP-VSV internalization via inhibition of macropinocytosis. The inactivation of EPAC1 affects the early stage of viral entry after viral binding to the cell surface, but before early endosome formation, in a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent manner. Our study delineated a new critical role of EPAC1 during EBOV uptake into ECs.

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Protease Dependent Activation of Endothelial Cells by Peritoneal Dialysis Effluents

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614386 ◽

1999 ◽

Vol 82 (10) ◽

pp. 1334-1341 ◽

Cited By ~ 7

Author(s):

Michael Krebs ◽

Christoph Kaun ◽

Matthias Lorenz ◽

Marianne Haag-Weber ◽

Bernd Binder ◽

...

Keyword(s):

Endothelial Cells ◽

Peritoneal Dialysis ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Partial Purification ◽

Complement Factor ◽

Dependent Manner ◽

Facs Analysis ◽

Tnf Α ◽

Umbilical Vein Endothelial Cells

SummaryIncubation of cultured human umbilical vein endothelial cells (HUVECs) with dilutions of peritoneal dialysis effluents (PDEs) from 11 individual patients undergoing continuous ambulatory peritoneal dialysis (CAPD) induced cellular procoagulant activity in a dose and time dependent manner. This procoagulant activity could be attributed to tissue factor (TF) expression since it was blocked by rabbit anti-TF IgG. These data was confirmed by FACS analysis yielding surface TF expression; In addition PDEs induced the expression of E-selectin in HUVECs. This TF and selectin inducing activity was heat labile and could be inhibited by protease inhibitors. Partial purification could be achieved using a benzamidine-Sepharose column. The TF inducing activity could not be attributed to LPS, IL-1, TNF-α, mast cell tryptase, active thrombin, or complement factor D. We therefore conclude that the peritoneal cavity contains a protease activity that induces a procoagulatory and proinflammatory phenotype in HUVECs.

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Resveratrol attenuates TNF-α-induced activation of coronary arterial endothelial cells: role of NF-κB inhibition

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00340.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. H1694-H1699 ◽

Cited By ~ 163

Author(s):

Anna Csiszar ◽

Kira Smith ◽

Nazar Labinskyy ◽

Zsuzsanna Orosz ◽

Aracelie Rivera ◽

...

Keyword(s):

Endothelial Cells ◽

Reporter Gene ◽

Endothelial Activation ◽

Bone Morphogenetic Protein 2 ◽

Luciferase Expression ◽

Pyrrolidine Dithiocarbamate ◽

Tnf Α ◽

Gene Assay ◽

Cardioprotective Effects ◽

Arterial Endothelial Cells

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its cardioprotective effects are not completely understood. Because TNF-α-induced endothelial activation and vascular inflammation play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits TNF-α-induced signal transduction in human coronary arterial endothelial cells (HCAECs). We found that TNF-α significantly increased adhesiveness of the monocytic THP-1 cells to HCAECs, an effect that could be inhibited by pretreatment with resveratrol and the NF-κB inhibitor pyrrolidine dithiocarbamate. Previously, we found that TNF-α activates NAD(P)H oxidases, and our recent data showed that TNF-α-induced endothelial activation was prevented by the NAD(P)H oxidase inhibitor apocynin or catalase plus SOD. Resveratrol also inhibited H2O2-induced monocyte adhesiveness. Using a reporter gene assay, we found that, in HCAECs, TNF-α significantly increased NF-κB activity, which could be inhibited by resveratrol (>50% inhibition at 10−6 mol/l) and pyrrolidine dithiocarbamate. Resveratrol also inhibited TNF-α-induced, NF-κB-driven luciferase expression in rat aortas electroporated with the reporter gene construct. In TNF-α-treated HCAECs, resveratrol (in the submicromolar range) significantly attenuated expression of NF-κB-dependent inflammatory markers inducible nitric oxide synthase, IL-6, bone morphogenetic protein-2, ICAM-1, and VCAM. Thus resveratrol at nutritionally relevant concentrations inhibits TNF-α-induced NF-κB activation and inflammatory gene expression and attenuates monocyte adhesiveness to HCAECs. We propose that these anti-inflammatory actions of resveratrol are responsible, at least in part, for its cardioprotective effects.

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Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets

Blood ◽

10.1182/blood-2004-12-4866 ◽

2005 ◽

Vol 106 (2) ◽

pp. 550-557 ◽

Cited By ~ 50

Author(s):

Swaminathan Murugappan ◽

Haripriya Shankar ◽

Surya Bhamidipati ◽

Robert T. Dorsam ◽

Jianguo Jin ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Thromboxane A2 ◽

Human Platelets ◽

Time Dependent ◽

Dependent Manner ◽

Src Tyrosine Kinase ◽

Functional Implications

Abstract Thrombin has been known to cause tyrosine phosphorylation of protein kinase C δ (PKCδ) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCδ and the role of this event in platelet function. PKCδ was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCδ did not occur in Gαq-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5′-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ′, N ′-tetraacetic acid), suggesting a role for Gαq pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCδ. Inhibition of tyrosine phosphorylation or the kinase activity of PKCδ dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCδ in a calcium- and Src-family kinase–dependent manner in platelets, with functional implications in thromboxane A2 generation.

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Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function

Journal of Virology ◽

10.1128/jvi.79.16.10442-10450.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10442-10450 ◽

Cited By ~ 129

Author(s):

Victoria M. Wahl-Jensen ◽

Tatiana A. Afanasieva ◽

Jochen Seebach ◽

Ute Ströher ◽

Heinz Feldmann ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Barrier Function ◽

Cell Activation ◽

Matrix Protein ◽

Ebola Virus ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Endothelial Cell Activation ◽

Tnf Α

ABSTRACT Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP1,2 has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP1,2 were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and Δ-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-α), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not Δ-peptide, induced a recovery of endothelial barrier function following treatment with TNF-α. Our results demonstrate that Ebola virus GP1,2 in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.

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