ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

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Rickettsia rickettsii infection of cultured human endothelial cells induces tissue factor expression

Blood ◽

10.1182/blood.v83.6.1527.bloodjournal8361527 ◽

1994 ◽

Vol 83 (6) ◽

pp. 1527-1534 ◽

Cited By ~ 1

Author(s):

LA Sporn ◽

PJ Haidaris ◽

RJ Shi ◽

Y Nemerson ◽

DJ Silverman ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Tissue Factor ◽

Thrombus Formation ◽

Umbilical Vein ◽

Spotted Fever ◽

Clinical Manifestations ◽

Mrna Levels ◽

Rocky Mountain Spotted Fever ◽

Rickettsia Rickettsii

Microvascular thrombi underlie many of the clinical manifestations of Rocky Mountain spotted fever (RMSF), a disease characterized by Rickettsia rickettsii infection of vascular endothelial cells. Studies were designed to determine whether R rickettsii-infection of cultured human umbilical vein endothelial cells results in tissue factor (TF) induction, a process that could directly activate coagulation in infected vessels. Whereas uninfected endothelial cell cultures showed essentially undetectable TF mRNA and activity, both TF mRNA and activity were present after R rickettsii infection. TF mRNA levels were transient, peaking at 4 hours after the initiation of infection, whereas the peak of TF activity occurred at 8 hours. Induction of the TF response requires the intracellular presence of R rickettsii organisms, because uninfected rickettsia were ineffective and the response was blocked by inhibiting rickettsial entry using cytochalasin B. TF induction was not mediated by endothelial cell release of soluble factor, because no response was induced using culture medium conditioned by R rickettsii-infected cells. Furthermore, preadsorption of suspensions of R rickettsii with polymyxin B to remove contaminating lipopolysaccharide did not eliminate the TF response. Induction of TF in vital endothelial cells during R rickettsii infection could be the trigger for vascular thrombus formation of RMSF.

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Rickettsia rickettsii infection of cultured human endothelial cells induces tissue factor expression

Blood ◽

10.1182/blood.v83.6.1527.1527 ◽

1994 ◽

Vol 83 (6) ◽

pp. 1527-1534 ◽

Cited By ~ 32

Author(s):

LA Sporn ◽

PJ Haidaris ◽

RJ Shi ◽

Y Nemerson ◽

DJ Silverman ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Tissue Factor ◽

Thrombus Formation ◽

Umbilical Vein ◽

Spotted Fever ◽

Clinical Manifestations ◽

Mrna Levels ◽

Rocky Mountain Spotted Fever ◽

Rickettsia Rickettsii

Abstract Microvascular thrombi underlie many of the clinical manifestations of Rocky Mountain spotted fever (RMSF), a disease characterized by Rickettsia rickettsii infection of vascular endothelial cells. Studies were designed to determine whether R rickettsii-infection of cultured human umbilical vein endothelial cells results in tissue factor (TF) induction, a process that could directly activate coagulation in infected vessels. Whereas uninfected endothelial cell cultures showed essentially undetectable TF mRNA and activity, both TF mRNA and activity were present after R rickettsii infection. TF mRNA levels were transient, peaking at 4 hours after the initiation of infection, whereas the peak of TF activity occurred at 8 hours. Induction of the TF response requires the intracellular presence of R rickettsii organisms, because uninfected rickettsia were ineffective and the response was blocked by inhibiting rickettsial entry using cytochalasin B. TF induction was not mediated by endothelial cell release of soluble factor, because no response was induced using culture medium conditioned by R rickettsii-infected cells. Furthermore, preadsorption of suspensions of R rickettsii with polymyxin B to remove contaminating lipopolysaccharide did not eliminate the TF response. Induction of TF in vital endothelial cells during R rickettsii infection could be the trigger for vascular thrombus formation of RMSF.

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Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid

Blood ◽

10.1182/blood.v88.11.4173.4173 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4173-4182 ◽

Cited By ~ 3

Author(s):

LV Rao ◽

AD Hoang ◽

SI Rapaport

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Surface ◽

Binding Sites ◽

Lupus Anticoagulant ◽

Umbilical Vein ◽

Protein A ◽

Thrombotic Risk ◽

Cell Monolayers ◽

Umbilical Vein Endothelial Cells

Abstract We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

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Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid

Blood ◽

10.1182/blood.v88.11.4173.bloodjournal88114173 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4173-4182 ◽

Cited By ~ 70

Author(s):

LV Rao ◽

AD Hoang ◽

SI Rapaport

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Surface ◽

Binding Sites ◽

Lupus Anticoagulant ◽

Umbilical Vein ◽

Protein A ◽

Thrombotic Risk ◽

Cell Monolayers ◽

Umbilical Vein Endothelial Cells

We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

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Evidence for New Endothelial Cell Binding Sites on Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614123 ◽

2000 ◽

Vol 84 (11) ◽

pp. 819-825 ◽

Cited By ~ 8

Author(s):

Michael Rooney ◽

Susan Lord ◽

M. W. Mosesson ◽

T. Gartner ◽

Richard Smith

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Endothelial Cell ◽

Binding Site ◽

Binding Sites ◽

Time Course ◽

Umbilical Vein ◽

In Vitro Assays ◽

Cell Binding ◽

Endothelial Cell Adhesion

SummaryIn vitro assays were used to characterize adhesion of human aortic, microvascular and umbilical vein endothelial cells to various forms of immobilized fibrinogen. All three types of endothelial cells adhered to fibrinogen in a manner that was independent of the Aα-chain 572-574 RGD cell binding site. In fact, all three adhered to a fragment of the molecule which is composed of only one D domain (D1) of fibrinogen. A time course study revealed that extensive adhesion of endothelial cells on the ligand coated surface occurred between one and two hours incubation. The anti-fibrinogen γA-chain monoclonal antibody 4A5 as well as 4A5 Fabs, blocked adhesion of endothelial cells to fibrinogen, not vitronectin. The inhibitory effects of 4A5 seemed to be indirect because the endothelial cells adhered to the recombinant fibrinogen γ407 (which lacks the γ-chain AGDV sequence of the carboxyl terminal 4A5 binding site) as well as they did to normal recombinant fibrinogen. A recombinant fibrinogen lacking the γ-chain AGDV sequence, containing RGE in place of RGD at the γ-chain 572-574 and 95-97 positions, also supported endothelial cell adhesion. The anti-αvβ3 antibody, LM609, blocked adhesion of endothelial cells to fibrinogen. The peptide GRGDSP inhibited endothelial cell adhesion on fibrinogen and vitronectin. These results demonstrate that αvβ3 mediated adhesion (attachment and spreading) of HUVECs to fibrinogen may use a site in the D domain of fibrinogen and is not dependent on the Aα-chain RGD (95-97 and 572-574) sequences, as has been shown in shorter term (where cells were rounded) experiments, or the γA-chain 408-411 cell binding sites. Thus, the data reveal the existence of another unidentified site(s) on fibrinogen which can support the irreversible adhesion (attachment and spreading) of endothelial cells.

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Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646886 ◽

1989 ◽

Vol 62 (02) ◽

pp. 699-703 ◽

Cited By ~ 8

Author(s):

Rob J Aerts ◽

Karin Gillis ◽

Hans Pannekoek

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Binding Sites ◽

Umbilical Vein ◽

Tissue Type ◽

Single Chain ◽

Human Endothelial Cells ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

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Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650304 ◽

1996 ◽

Vol 75 (03) ◽

pp. 497-502 ◽

Cited By ~ 10

Author(s):

Hadewijch L M Pekelharing ◽

Henne A Kleinveld ◽

Pieter F C.C.M Duif ◽

Bonno N Bouma ◽

Herman J M van Rijn

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Plasminogen Activator ◽

Binding Sites ◽

Umbilical Vein ◽

Single Step ◽

Plasminogen Activation ◽

Structural Homology ◽

Tissue Plasminogen ◽

Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

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Abstract 315: Efficacy and Safety of Rivaroxaban in in vitro Experiments With the Endothelial Cells

Circulation Research ◽

10.1161/res.119.suppl_1.315 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Kaname Seki ◽

Yosuke Mizuno ◽

Toku Sakashita ◽

Jun Tanno ◽

Shintaro Nakano ◽

...

Keyword(s):

Endothelial Cells ◽

Time Course ◽

Umbilical Vein ◽

Endothelial Damage ◽

Coagulation Cascade ◽

Elisa Assay ◽

Mrna Levels ◽

Factor X ◽

Clotting Factors ◽

Inflammatory Phase

Aim: Activated factor X (FXa) plays important roles in the thrombin generation and in inflammation, which is evoked during the endothelial damage. Although rivaroxaban is a selective FXa antagonist, it is one of the key therapies in ischemic heart disease, and yet its function in the state of inactivated coagulation cascade is uncertain. Rivaroxaban blocks FXa in the blood but not the tissue, while factor X is converted to FXa only when glutamic acid is changed to γ-carboxyglutamic acid by vitamin K following the intrinsic clotting factors and/or cellular injury activation. To uncover this aspect, we performed the following experiments. Methods and results: Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Co., Ltd. The cells were grown to 80% confluence and were treated with rivaroxaban (100nM, 500nM, 1000nM, 2000nM respectively) without FXa stimulation for 4 h, 10 h or 24 h. Cells and medium were collected and then their RNA was extracted from the cells. The qPCR of MCP-1, PAR1-4 and the DNA micro arrays (The GeneChip Human Gene 2.0 ST Array, Affymetrix) were performed. There was neither increased nor decreased gene expression significantly in either experimental time course of the qPCRs or the the DNA micro arrays. The ELISA assay of MCP-1 with medium showed non-activated MCP-1. As a next step, cells were treated with 100nM FXa and with/without rivaroxaban in same time course, and cells and medium were collected for further experiments. FXa evoked induction of mRNA levels for several pro-inflammatory cytokines including MCP-1 maximally at 4h, whereas MCP-1 was maximally evoked at 24 h in ELISA assay. Interestingly rivaroxaban inhibited both in all time course, at 4 hour inflammatory phase and at 24 hour inflammatory phase. Conclusion: Collectively, these results suggest that rivaroxaban may be safe in the inactivated coagulation state, and has the efficacy to attenuate the endothelial damage evoked by FXa and by pro-inflammatory cytokine genes.

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Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System

Circulation Research ◽

10.1161/res.115.suppl_1.116 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Ha-Rim Seo ◽

Hyo Eun Jeong ◽

Hyung Joon Joo ◽

Seung-Cheol Choi ◽

Jong-Ho Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Assay System ◽

Vascular Density ◽

Angiogenic Potential ◽

Angiogenesis Assay

Background: Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared in vitro angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms. Method: Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis. Results: In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat. Conclusion: HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression

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Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide

Journal of Applied Physiology ◽

10.1152/japplphysiol.00829.2001 ◽

2002 ◽

Vol 92 (3) ◽

pp. 1152-1158 ◽

Cited By ~ 15

Author(s):

Scott Earley ◽

Leif D. Nelin ◽

Louis G. Chicoine ◽

Benjimen R. Walker

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Promoter Activity ◽

Umbilical Vein ◽

Hypoxia Inducible Factor ◽

Hypoxic Exposure ◽

Mrna Levels ◽

Protective Effects ◽

No Donor ◽

Umbilical Vein Endothelial Cells

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O2) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso- N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O2) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O2) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O2) exposure. ET-1 promoter activity after S-nitroso- N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.

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