scholarly journals Laser-induced endothelial cell activation supports fibrin formation

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4675-4683 ◽  
Author(s):  
Ben T. Atkinson ◽  
Reema Jasuja ◽  
Vivien M. Chen ◽  
Prathima Nandivada ◽  
Bruce Furie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tissue Factor ◽  
Cell Activation ◽  
Umbilical Vein ◽  
Calcium Mobilization ◽  
Endothelial Cell Activation ◽  
Human Umbilical Vein ◽  
Laser Injury ◽  
Umbilical Vein Endothelial Cells

Laser-induced vessel wall injury leads to rapid thrombus formation in an animal thrombosis model. The target of laser injury is the endothelium. We monitored calcium mobilization to assess activation of the laser-targeted cells. Infusion of Fluo-4 AM, a calcium-sensitive fluorochrome, into the mouse circulation resulted in dye uptake in the endothelium and circulating hematopoietic cells. Laser injury in mice treated with eptifibatide to inhibit platelet accumulation resulted in rapid calcium mobilization within the endothelium. Calcium mobilization correlated with the secretion of lysosomal-associated membrane protein 1, a marker of endothelium activation. In the absence of eptifibatide, endothelium activation preceded platelet accumu-lation. Laser activation of human umbilical vein endothelial cells loaded with Fluo-4 resulted in a rapid increase in calcium mobilization associated cell fluorescence similar to that induced by adenosine diphosphate (10μM) or thrombin (1 U/mL). Laser activation of human umbilical vein endothelial cells in the presence of corn trypsin inhibitor treated human plasma devoid of platelets and cell microparticles led to fibrin for-mation that was inhibited by an inhibitory monoclonal anti–tissue factor antibody. Thus laser injury leads to rapid endothelial cell activation. The laser activated endothelial cells can support formation of tenase and prothrombinase and may be a source of activated tissue factor as well.

scholarly journals Up-regulation of CD81 inhibits cytotrophoblast invasion and mediates maternal endothelial cell dysfunction in preeclampsia

2017 ◽  
Vol 114 (8) ◽  
pp. 1940-1945 ◽  
Author(s):  
Li Shen ◽  
Zhenyu Diao ◽  
Hai-Xiang Sun ◽  
Gui-Jun Yan ◽  
Zhiqun Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Umbilical Vein ◽  
Normal Pregnancy ◽  
Uterine Wall ◽  
High Dose ◽  
Endothelial Cell Activation ◽  
Umbilical Vein Endothelial Cells

Preeclampsia (PE) is initiated by abnormal placentation in the early stages of pregnancy, followed by systemic activation of endothelial cells of the maternal small arterioles in the late second or third trimester (TM) of pregnancy. During normal pregnancy, placental cytotrophoblasts (CTBs) invade the maternal uterine wall and spiral arteries, whereas this process is interrupted in PE. However, it is not known how the malformed placenta triggers maternal endothelial crisis and the associated manifestations. Here, we have focused on the association of CD81 with PE. CD81, a member of the tetraspanin superfamily, plays significant roles in cell growth, adhesion, and motility. The function of CD81 in human placentation and its association with pregnancy complications are currently unknown. In the present study, we have demonstrated that CD81 was preferentially expressed in normal first TM placentas and progressively down-regulated with gestation advance. In patients with early-onset severe PE (sPE), CD81 expression was significantly up-regulated in syncytiotrophoblasts (STBs), CTBs and the cells in the villous core. In addition, high levels of CD81 were observed in the maternal sera of patients with sPE. Overexpressing CD81 in CTBs significantly decreased CTB invasion, and culturing primary human umbilical vein endothelial cells (HUVECs) in the presence of a high dose of exogenous CD81 resulted in interrupted angiogenesis and endothelial cell activation in vitro. Importantly, the phenotype of human PE was mimicked in the CD81-induced rat model.

Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 101-105 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
Fanny E Almus ◽  
Samuel I Rapaport
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Phorbol Ester ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

1998 ◽  
Vol 79 (01) ◽  
pp. 217-221 ◽  
Author(s):  
Koichi Kokame ◽  
Toshiyuki Miyata ◽  
Naoaki Sato ◽  
Hisao Kato
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Mrna Levels ◽  
Down Regulation ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

SummaryThrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 μM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

scholarly journals Association of Tissue Factor Pathway Inhibitor With Human Umbilical Vein Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3568-3578 ◽  
Author(s):  
John-Bjarne Hansen ◽  
Randi Olsen ◽  
Paul Webster
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Coagulation Factors ◽  
Coagulation System ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

AbstractTissue factor pathway inhibitor (TFPI) is a serine protease inhibitor of the extrinsic coagulation system, synthesized in endothelial cells, which has recently been shown to play an important role in the regulation of activated coagulation factors at the endothelial cell surface. In the present study we investigated the subcellular localization and metabolism of TFPI in human umbilical vein endothelial cells (HUVEC). Immunocytochemical labeling of HUVEC with anti-TFPI showed specific labeling associated with the cell surface and with many intracellular organelles including the Golgi complex. Further characterization of these organelles was performed by colocalizing the anti-TFPI with 3-(2,4-dinitroanilino)′-amino-N-methyldipropylamine (DAMP; to demonstrate low pH), mannose phosphate receptor (endosomes), and LAMP 1 (late endocytic compartments). TFPI also colocalized with antibodies to the human transferrin receptor, a marker for early endocytic, recycling compartment. Endogenous TFPI colocalized with biotin in intracellular vesicles during endocytosis after biotinylation of the cell surface, which indicated that TFPI was being co-internalized with the surface biotin. The binding of exogenously added 125I-TFPI increased linearly to HUVEC over the concentration range of 0 to 32 nmol/L without saturation, the binding was not affected by up to a thousand-fold molar excess of unlabeled TFPI, and heparin inhibited the binding dose dependently. An intact C-terminal domain was important for the interaction between TFPI and the cell surface of HUVEC, because less than 10% of a C-terminal truncated form of TFPI (TFPI1-161 ) was bound after addition of equimolar concentrations of full-length TFPI. Exogenously added 125I-TFPI was not degraded in HUVEC during 4 hours at 37°C. The presence of TFPI in endocytic and recycling compartments support the hypothesis that endogenous, membrane-anchored TFPI could be internalized for subsequent recycling back to the cell surface.

MODULATION of PROSTACYCLIN PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS WITH ECGF/HEPARIN MEDIUM : ROLE OF CELLULAR DENSITY AT CONFLUENCE

1987 ◽  
Author(s):  
O BOUTHERIN-FALSON ◽  
N BLAES
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Lower Amount ◽  
Endothelial Cell Proliferation ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in vascular endothelial cells. In addition to the role of exogenous agents, its production could be modulated by culture conditions : proliferative state, medium renewal, subcultivation... The use of endothelial cell growth factor (ECGF) associated with heparin has been shown to improve human endothelial cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) grown in that medium produce less prostacyclin than without growth factor.HUVEC were cultured in RPMI-199 1:1 + 20% fetal calf serum, added or not with ECGF (Bovine hypothalamus extract BTI Cambridge, 24 ug/ml) and heparin (from porcine intestinal mucosa, Signa, 90 ug/ml). After 4 days in culture, medium was removed and replaced by Tyrode Hepes buffer and basal production was measured after 20 min. Cells were then submitted to 5 min thrombin to assess PGI2 production in stimulated conditions. PGI2 production was estimated by specific radioimmunoassay for 6 keto PGFjalpha. For each point, cell number in the culture was counted after Trypsin EDTA treatment. In the present study, cells grown in ECGF-heparin medium produce lower amount of PGI2, compared to heparin or control medium. This result was observed in both basal and stimulated conditions. For each medium (ECGF-heparin, heparin, control), correlations between PGI2 production per cell and log cell density were shown to be significantly negative.These observations suggest that ECGF effect on PGI2 production could be a consequence of its growth factor activity, notably by the fact that it leads to an endothelial monolayer made of more numerous cells. Since it is now suggested by a number of clinical observations that PGI2 is rather produced in pathological conditions, culture models showing a weak production of PGI2 appear in that connection doser to the physiological conditions.

CD40 Engagement on Endothelial Cells Promotes Tissue Factor-dependent Procoagulant Activity

1998 ◽  
Vol 79 (05) ◽  
pp. 1025-1028 ◽  
Author(s):  
Ling Zhou ◽  
Patrick Stordeur ◽  
Aurore de Lavareille ◽  
Kris Thielemans ◽  
Paul Capel ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tissue Factor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
The Impact

SummaryThe CD40 molecule expressed on endothelial cells has been shown to transduce activation signals resulting in upregulation of adhesion molecules. Herein, we studied the impact of CD40 engagement on the induction of tissue factor (TF)-dependent procoagulant activity (PCA) at the surface of human umbilical vein endothelial cells (HUVECs). First, we found that co-incubation of HUVECs with 3T6 fibroblasts transfected with the CD40L gene (3T6-CD40L) resulted in a clear induction of PCA which was not observed with control untransfected fibroblasts. The specificity of this finding was established by inhibition experiments using monoclonal antibodies (mAbs) blocking CD40 or CD40L. PCA induced by CD40 ligation was TF-related as it was not observed in factor VII-deficient plasma and was associated with the accumulation of TF mRNA. To investigate the role of CD40/CD40L interactions in the induction of endothelial cell PCA by lymphocytes, interferon (IFN)-γ-stimulated EC were incubated with T cells in the absence or presence of anti-CD40 or anti-CD40L mAb. The 60-70% inhibition of PCA induced by these mAbs but not their isotype-matched control indicated that the CD40 pathway is involved in the induction of PCA resulting from interactions between activated HUVECs and T cells. We conclude that activation signals elicited by CD40 engagement on endothelial cells result in the induction of TF-dependent PCA. The CD40/CD40L pathway might therefore be involved in the development of prothrombic states during diseases associated with endothelial cell and T cell activation.

Dimerization of P-selectin in platelets and endothelial cells

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3070-3077 ◽  
Author(s):  
Fern J. Barkalow ◽  
Kurt L. Barkalow ◽  
Tanya N. Mayadas
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Platelet Activation ◽  
Cell Activation ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Monomeric Form ◽  
Intact Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.

Dimerization of P-selectin in platelets and endothelial cells

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3070-3077 ◽  
Author(s):  
Fern J. Barkalow ◽  
Kurt L. Barkalow ◽  
Tanya N. Mayadas
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Platelet Activation ◽  
Cell Activation ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Monomeric Form ◽  
Intact Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Abstract P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.

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