A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells

Blood ◽  
2009 ◽  
Vol 113 (3) ◽  
pp. 714-722 ◽  
Author(s):  
Aya Shibamiya ◽  
Karin Hersemeyer ◽  
Thomas Schmidt Wöll ◽  
Daniel Sedding ◽  
Jan-Marcus Daniel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Hemorrhagic Fever ◽  
Poly I:C ◽  
Toll Like Receptor ◽  
Virus Infections ◽  
Direct Stimulation ◽  
Viral Dsrna ◽  
Stimulation Of

AbstractVarious virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)–mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3−/− mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

Endothelial cells but not platelets are the major source of Toll-like receptor 4 in the arterial thrombosis and tissue factor expression in mice

2014 ◽  
Vol 307 (7) ◽  
pp. R901-R907 ◽  
Author(s):  
Meiping Ren ◽  
Rong Li ◽  
Mao Luo ◽  
Ni Chen ◽  
Xin Deng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Arterial Thrombosis ◽  
Vascular Wall ◽  
Toll Like Receptor ◽  
Occlusion Time ◽  
Significant Difference ◽  
Expression And Activity

It is known that Toll-like receptor (TLR)-4 plays an important role in myocardial infarction and atherothrombosis. The role of TLR-4 in arterial thrombosis is undefined. Both TLR-4-deficient ( TLR-4−/−) and wild-type (WT) mice were subjected to FeCl3carotid artery injury, and the time required to form an occlusive thrombus was measured. The mean time to occlusion in TLR-4−/−mice was significantly greater than that in WT mice after injury (303 ± 32 vs. 165 ± 34 s, P < 0.05). Furthermore, when we used a WT or TLR-4−/−-derived platelet reinfusion in a platelet depletion/reinfusion procedure, there was no significant change in the occlusion time and tissue factor (TF) activity in injured arteries between WT mice and platelet-depleted WT mice. Similarly, no significant difference was observed between TLR-4−/−mice and platelet-depleted TLR-4−/−mice for the WT or TLR-4−/−-derived platelet reinfusion. However, TF expression and activity were significantly reduced in the vascular wall of TLR-4−/−mice compared with WT mice. In vivo, lipopolysaccharide accelerated the occlusion time in WT mice but not TLR-4−/−mice. In vitro, LPS-induced TF activity was reduced in endothelial cells of TLR-4−/−mice relative to WT mice. The data demonstrate that TLR-4 contributes to arterial thrombosis formation in vivo and causes increased TF expression and activity in vitro. The results further suggest that the stimulation is mainly derived by endothelial cells but is not due to platelet-derived TLR-4.

scholarly journals Ethanol raises prostacyclin in vivo and in vitro

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 679-682
Author(s):  
R Landolfi ◽  
M Steiner
Keyword(s):  
Endothelial Cells ◽  
Significant Rise ◽  
Blood Alcohol ◽  
Elevated Plasma ◽  
Direct Stimulation ◽  
Baseline Values ◽  
Induced Changes ◽  
Stimulation Of

Moderate doses of ethanol were shown to induce a significant rise in prostacyclin (PGI2) concentration in cultures of endothelial cells derived from umbilical veins. Administration of 32 g of ethanol to six volunteers elevated plasma levels of PGI2 in parallel with those of blood alcohol. Although not specific for ethanol, this alcohol induced the largest change in PGI2. Withdrawal of the stimulant alcohol caused prompt reduction of the elevated prostacyclin to baseline values. The activity of ethanol appears to be due to a direct stimulation of cyclooxygenase. The release of [14C]arachidonic acid from prelabeled endothelial cells was decreased by ethanol. PGE2 production was also enhanced by exposure of endothelial cells to ethanol. The physiologic significance of these alcohol-induced changes in PGI2 levels remains to be established.

scholarly journals Ethanol raises prostacyclin in vivo and in vitro

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 679-682 ◽  
Author(s):  
R Landolfi ◽  
M Steiner
Keyword(s):  
Endothelial Cells ◽  
Significant Rise ◽  
Blood Alcohol ◽  
Elevated Plasma ◽  
Direct Stimulation ◽  
Baseline Values ◽  
Induced Changes ◽  
Stimulation Of

Abstract Moderate doses of ethanol were shown to induce a significant rise in prostacyclin (PGI2) concentration in cultures of endothelial cells derived from umbilical veins. Administration of 32 g of ethanol to six volunteers elevated plasma levels of PGI2 in parallel with those of blood alcohol. Although not specific for ethanol, this alcohol induced the largest change in PGI2. Withdrawal of the stimulant alcohol caused prompt reduction of the elevated prostacyclin to baseline values. The activity of ethanol appears to be due to a direct stimulation of cyclooxygenase. The release of [14C]arachidonic acid from prelabeled endothelial cells was decreased by ethanol. PGE2 production was also enhanced by exposure of endothelial cells to ethanol. The physiologic significance of these alcohol-induced changes in PGI2 levels remains to be established.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Increased fluid absorption and cell volume in isolated rabbit proximal straight tubules after in vivo DOCA administration

1981 ◽  
Vol 241 (5) ◽  
pp. F502-F508 ◽  
Author(s):  
M. A. Knepper ◽  
M. B. Burg
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Fluid Transport ◽  
Sodium Intake ◽  
Plasma Concentrations ◽  
Dietary Sodium ◽  
Fluid Absorption ◽  
Direct Stimulation ◽  
Stimulation Of

To investigate whether mineralocorticoids affect the intrinsic capacity of the proximal tubule to absorb sodium and fluid, rabbits were chronically treated a number of ways to systematically vary plasma concentrations of mineralocorticoid hormones. The rate of fluid absorption and tubule dimensions were measured in superficial S2 segments from these rabbits. Chronic administration of deoxycorticosterone acetate (DOCA) was associated with a 67% increase in fluid absorption and a 29% increase in cell volume per unit tubule length. However, neither adrenalectomy nor low sodium diet significantly affected either fluid absorption or cell volume. Furthermore, marked dietary sodium restriction prevented the response to DOCA. We conclude that the DOCA-induced increases in fluid absorption and cell volume do not result from a direct stimulation of the proximal tubular cells by the steroid but more likely are responses to systemic effects of DOCA administration that are dependent on the level of sodium intake. Thus, we find no evidence for a direct mineralocorticoid stimulation of sodium and fluid transport by the S2 portion of the proximal tubule.

scholarly journals Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

2002 ◽  
Vol 160 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Carsten Brock ◽  
Michael Schaefer ◽  
H. Peter Reusch ◽  
Cornelia Czupalla ◽  
Manuela Michalke ◽  
...  
Keyword(s):  
G Protein ◽  
Living Cells ◽  
Membrane Lipid ◽  
Cellular Functions ◽  
Direct Stimulation ◽  
Membrane Recruitment ◽  
Ph Domains ◽  
Phosphoinositide 3 Kinase ◽  
Stimulation Of

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ.

Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities

2003 ◽  
Vol 284 (1) ◽  
pp. L100-L107 ◽  
Author(s):  
Judy R. Creighton ◽  
Nanako Masada ◽  
Dermot M. F. Cooper ◽  
Troy Stevens
Keyword(s):  
Endothelial Cells ◽  
Adenylyl Cyclase ◽  
Enzyme Activation ◽  
Microvascular Endothelial Cells ◽  
Phosphodiesterase Activity ◽  
Camp Concentration ◽  
Direct Stimulation ◽  
Coordinate Regulation ◽  
Turn Over ◽  
Stimulation Of

Activation of store-operated Ca2+ entry inhibits type 6 adenylyl cyclase (EC 4.6.1.1 ; AC6; Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712–6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC6 and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca2+ entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca2+ inhibition of AC6 is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca2+ inhibition of AC6. Enzyme activity was assessed using PMVEC membranes, where Ca2+ and cAMP concentrations were independently controlled. Endogenous AC6 activity exhibited high- and low-affinity Ca2+ inhibition, similar to that observed in C6-2B cells, which predominantly express AC6. Ca2+ inhibition of AC6 in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca2+ inhibition of AC6 is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC6 to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.

scholarly journals Direct Stimulation of Adult Neural Stem Cells In Vitro and Neurogenesis In Vivo by Vascular Endothelial Growth Factor

2006 ◽  
Vol 14 (3) ◽  
pp. 237-248 ◽  
Author(s):  
Anne Schänzer ◽  
Frank-Peter Wachs ◽  
Daniel Wilhelm ◽  
Till Acker ◽  
Christiana Cooper-Kuhn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Neural Stem Cells ◽  
Vascular Endothelial ◽  
Direct Stimulation ◽  
Endothelial Growth Factor ◽  
Stimulation Of

scholarly journals Induction of Innate Immune Response Genes by Sin Nombre Hantavirus Does Not Require Viral Replication

2005 ◽  
Vol 79 (24) ◽  
pp. 15007-15015 ◽  
Author(s):  
Joseph Prescott ◽  
Chunyan Ye ◽  
Ganes Sen ◽  
Brian Hjelle
Keyword(s):  
Endothelial Cells ◽  
Uv Irradiation ◽  
Early Time ◽  
Hemorrhagic Fever ◽  
Etiologic Agent ◽  
Hantavirus Infection ◽  
Live Virus ◽  
Rna Integrity ◽  
Reverse Transcription Pcr

ABSTRACT Maladaptive immune responses are considered to be important factors in the pathogenesis of the two diseases caused by hantaviruses, hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome (HCPS). While the intensity of adaptive antiviral T-cell responses seems to correlate with the severity of HCPS, there is increasing evidence that innate antiviral responses by endothelial cells, the native targets for hantavirus infection in vivo, are induced within hours of exposure to infectious hantaviruses. To investigate early events in the innate response to Sin Nombre virus (SNV), the principal etiologic agent of HCPS in North America, we treated human endothelial cells with live virus, or virus subjected to inactivation by UV irradiation at minimal doses required to inhibit replication, and assayed host expression of interferon-stimulated genes (ISG) by microarray and reverse transcription-PCR. We show herein that a variety of ISG are induced between 4 and 24 h after exposure to both live and killed virus. The levels of such induction at early time points (before 24 h) were generally higher in cells treated with SNV particles that had been killed by exposure to UV irradiation. Additionally, SNV exposed to increasing doses of UV irradiation induced ISG better than live virus despite increased disruption of viral RNA integrity. However, SNV replication was required for continued ISG overexpression by 3 days posttreatment. These results suggest that hantavirus particles may themselves be capable of early induction of ISG and that ongoing production of viral particles during infection could contribute to the pathogenic process.

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