Daunorubicin Induces Procoagulant Activity of Cultured Endothelial Cells through Phosphatidylserine Exposure and Microparticles Release

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5185-5185
Author(s):  
Yueyue Fu ◽  
Huibo Li ◽  
Wen Li ◽  
Xiushuai Dong ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Chemotherapeutic Agents ◽  
Significant Inhibition ◽  
Procoagulant Activity ◽  
Clotting Time ◽  
Increased Risk ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact

Abstract Abstract 5185 Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μ M) for 24 h. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μ M of daunorubicin or over. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process. Disclosures: No relevant conflicts of interest to declare.

Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release

2010 ◽  
Vol 104 (12) ◽  
pp. 1235-1241 ◽  
Author(s):  
Huibo Li ◽  
Fenglin Cao ◽  
Yanhua Su ◽  
Shengjin Fan ◽  
Yinghua Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Chemotherapeutic Agents ◽  
Significant Inhibition ◽  
Procoagulant Activity ◽  
Clotting Time ◽  
Increased Risk ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact ◽  
Prothrombotic Effect

SummaryAdministration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μM) for 24 hours. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μM of daunorubicin or more. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process.

scholarly journals Doxorubicin Enhances Procoagulant Activity of Endothelial Cells after Exposure to Tumour Microparticles on Microfluidic Devices

Hemato ◽  
2020 ◽  
Vol 1 (1) ◽  
pp. 23-34
Author(s):  
Abdulrahman Algarni ◽  
John Greenman ◽  
Leigh Madden
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Cell Number ◽  
Procoagulant Activity ◽  
Cytotoxic Agents ◽  
Tumour Spheroid ◽  
Increased Risk ◽  
Huvec Cell ◽  
The Media ◽  
Umbilical Vein Endothelial Cells

The majority of cancer patients undergoing chemotherapy have a significantly increased risk of venous thromboembolism via a mechanism not yet fully elucidated but which most probably involves tumour microparticles (MP) combined with damaged/activated endothelium. Tumour cell lines (ES-2 and U87) were cultured as 3D spheroids and transferred to biochips connected through to a second chip precultured with an endothelial cell layer (human umbilical vein endothelial cells [HUVECs]). Media were introduced with and without doxorubicin (DOX) to the spheroids in parallel chips under constant flow conditions. Media samples collected pre- and post-flow through the biochip were analysed for tissue factor microparticles (TFMP) and procoagulant activity (PCA). HUVECs were also harvested and tested for PCA at a constant cell number. TFMP levels in media decreased after passing over HUVECs in both conditions over time and this was accompanied by a reduction in PCA (indicated by a slower coagulation time) of the media. The relationship between PCA and TFMP was correlated (r = −0.85) and consistent across experiments. Harvested HUVECs displayed increased PCA when exposed to tumour spheroid media containing TFMP, which was increased further after the addition of DOX, suggesting that the TFMP in the media had bound to HUVEC cell surfaces. The enhanced PCA of HUVECs associated with the DOX treatment was attributed to a loss of viability of these cells rather than additional MP binding. The data suggest that tumour MP interact with HUVECs through ligand-receptor binding. The model described is a robust and reproducible method to investigate cytotoxic agents on tumour spheroids and subsequent downstream interaction with endothelial cells.

A novel coagulation assay incorporating adherent endothelial cells in thromboelastometry

2013 ◽  
Vol 109 (05) ◽  
pp. 869-877 ◽  
Author(s):  
Johannes Zipperle ◽  
Wolfgang Holnthoner ◽  
Anna-Maria Husa ◽  
Sylvia Nürnberger ◽  
Heinz Redl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Whole Blood ◽  
Culture Supernatant ◽  
Umbilical Vein ◽  
Blood Clot ◽  
Clotting Time ◽  
Monitoring Tool ◽  
Coagulation Assay ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact

SummaryFollowing vascular injury or activation, endothelial cells (ECs) participate in the modulation of haemostasis and fibrinolysis. Viscoelastic tests (VETs) are a potent bedside monitoring tool that reports haemostatic parameters in real time. However, VETs neglect the influence of the surrounding endothelium. Our aim was therefore to establish an assay that incorporates ECs in a whole blood VET and to assess the impact of ECs on coagulation parameters. Outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs) were seeded onto microbeads to create transferable EC-microcarriers. Microbeads were then added to citrated whole blood in the measurement cup of a thromboelastometry device (ROTEM). After the addition of CaCl2 (star-TEM®) to the blood sample (NATEM assay), standard ROTEM parameters were analysed. Scanning electron microscopy (SEM) was carried out to visualise the interactions of the beads, whole blood components and the ROTEM pin after clotting. SEM showed that the added microbeads were effectively incorporated into the final blood clot. In the presence of activated ECs, the clotting time (CT) of the blood was shortened fourfold compared to that in uncoated control beads. A significant reduction in CT was also observed in the presence of unstimulated ECs. Interestingly, CT was also reduced by the addition of purified EC culture supernatant. CT shortening was prevented by incubating the supernatant with an inhibiting antibody against tissue factor (TF). Our findings demonstrate that ECs can be incorporated into a ROTEM assay via coated microbeads, and whole blood clotting initiation is accelerated by non-activated and activated ECs.

Protease Dependent Activation of Endothelial Cells by Peritoneal Dialysis Effluents

1999 ◽  
Vol 82 (10) ◽  
pp. 1334-1341 ◽  
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.

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.

Neutrophils Clearance by Endothelial Cells Regulates Homeostasis and Coagulation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3782-3782
Author(s):  
Wen Li ◽  
Shuchuan Liu ◽  
Yueyue Fu ◽  
Jinxiao Hou ◽  
Xiushuai Dong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kupffer Cells ◽  
Milk Fat ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Large Fraction ◽  
Background Level ◽  
Procoagulant Activity ◽  
Phagocytic Cells

Abstract Abstract 3782 Neutrophils, also called polymorphonuclear leukocytes (PMN's) have a half-life of only 6 hours in the blood. Inflammation can further shorten the circulating life-time. A large fraction of the bone marrow capacity is committed to ongoing production of these short-lived cells but the manner of their clearance from the circulation is less well understood. We have previously demonstrated that PMN's are cleared by liver macrophages. However, the details of PMN adhesion-induced PMN clearance in the liver are unknown. The aim of this study is to evaluate a pathway of PMNs clearance by endothelial cells, which are not ordinarily considered phagocytes. Lactadherin is a glycoprotein of milk fat globules and is also secreted by stimulated macrophages. Lactadherin binds phosphatidylserine on apoptotic cells via tandem lectin-homology domains with homology to factor VIII and binds αvβ3 and αvβ5 integrins on phagocytic cells via an RGD sequence in an epithelial growth factor domain. Lactadherin aids in engulfment of senescent lymphocytes by splenic macrophages and mediates an anti-inflammatory response. We utilized lactadherin as a probe to detect phosphatidylserine exposure on aging PMN's and evaluated the lactadherin-dependent engulfment of these PMN's by endothelial cells. Cultured human PMNs from healthy donors, with 95% purity, were 40% and 96% PS-exposure positive at 9 and 24 h, respectively. They displayed a parallel increase in procoagulant supporting, activity related to the PS exposure. Coculture of the aging PMNs and human umbilical vein endothelial cells resulted in phagocytosis of the PMN's, observed by confocal microscopy and electron microscopy. Exogenous lactadherin increased phagocytosis by 3–5 fold during 120 minutes of observation. An anti-lactadherin RGD antibody and an anti-lactadherin C2 domain antibody inhibited phagocytosis to approx 1/2 the background level suggesting that lactadherin secreted by PMN's or neutrophils contributes to the base level of phagocytosis. Clearance of the senescent neutrophils by endothelial cells decreased procoagulant activity >70% and blockade of neutrophil PS with lactadherin reduced procoagulant activity by > 90% indicating the potential role of neutrophil uptake in limiting prothrombotic activity. In a rat model of neutrophil homeostasis we injected low dose lipopolysaccharide (LPS) and gadolinium chloride intravenously to increase the number circulating PMN's and block clearance by Kupffer cells. This allowed observation of PMN adhesion and sequestration in the liver. The number of PMNs peaked at 9 h and decreased to the normal range at 24 h after blockade of Kupffer cells. Blocking the endothelial P-selectin significantly delayed PMN's removal in the liver. Injection of lactadherin promoted the PMNs accumlation and removal. The current results suggest that ECs contribute to maintaining the homeostasis of PMNs in the circulation and a possible role of lactadherin in the EC-mediated clearance. Our results also indicate that lactadherin-mediated clearance may limit procoagulant or prothrombotic activity of senescent PMN's. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Impact of Simulated Weightlessness on Endothelium-Dependent Angiogenesis and the Role of Caveolae/Caveolin-1

2016 ◽  
Vol 38 (2) ◽  
pp. 502-513 ◽  
Author(s):  
Fei Shi ◽  
Tian-Zhi Zhao ◽  
Yong-Chun Wang ◽  
Xin-Sheng Cao ◽  
Chang-Bin Yang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Microgravity Environment ◽  
Caveolin 1 ◽  
Endothelial Functions ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact

Background/Aims: The potential role of caveolin-1 in modulating angiogenesis in microgravity environment is unexplored. Methods: Using simulated microgravity by clinostat, we measured the expressions and interactions of caveolin-1 and eNOS in human umbilical vein endothelial cells. Results: We found that decreased caveolin-1 expression is associated with increased expression and phosphorylation levels of eNOS in endothelial cells stimulated by microgravity, which causes a dissociation of eNOS from caveolin-1 complexes. As a result, microgravity induces cell migration and tube formation in endothelial cell in vitro that depends on the regulations of caveolin-1. Conclusion: Our study provides insight for the important endothelial functions in altered gravitational environments.

Melatonin inhibits high glucose-induced ox-LDL/LDL expression and apoptosis in human umbilical endothelial cells

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Yee Lian Tiong ◽  
Khuen Yen Ng ◽  
Rhun Yian Koh ◽  
Gnanajothy Ponnudurai ◽  
Soi Moi Chye
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Annexin V ◽  
Diabetic Patients ◽  
Therapeutic Effects ◽  
Pathway Activation ◽  
Increased Risk ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells

AbstractBackgroundCardiovascular disease (CVD) is one of the major cause of mortality in diabetic patients. Evidence suggests that hyperglycemia in diabetic patients contributes to increased risk of CVD. This study is to investigate the therapeutic effects of melatonin on glucose-treated human umbilical vein endothelial cells (HUVEC) and provide insights on the underlying mechanisms.Materials and methodsCell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and membrane potential was detected using 2′,7′-dichlorofluorescein diacetate and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) dye staining, respectively. While, cell apoptosis was determined by Annexin-V staining and protein expression was measured using Western blot.ResultsOur results suggested that melatonin inhibited glucose-induced ROS elevation, mitochondria dysfunction and apoptosis on HUVEC. Melatonin inhibited glucose-induced HUVEC apoptosis via PI3K/Akt signaling pathway. Activation of Akt further activated BcL-2 pathway through upregulation of Mcl-1 expression and downregulation Bax expression in order to inhibit glucose-induced HUVEC apoptosis. Besides that, melatonin promoted downregulation of oxLDL/LOX-1 in order to inhibit glucose-induced HUVEC apoptosis.ConclusionsIn conclusion, our results suggested that melatonin exerted vasculoprotective effects against glucose-induced apoptosis in HUVEC through PI3K/Akt, Bcl-2 and oxLDL/LOX-1 signaling pathways.

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