Evidence for New Endothelial Cell Binding Sites on Fibrinogen

2000 ◽  
Vol 84 (11) ◽  
pp. 819-825 ◽  
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.

scholarly journals Role of glycocalyx in leukocyte-endothelial cell adhesion

2002 ◽  
Vol 283 (4) ◽  
pp. H1282-H1291 ◽  
Author(s):  
A. W. Mulivor ◽  
H. H. Lipowsky
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Inflammatory Response ◽  
Topical Application ◽  
Binding Sites ◽  
Subsequent Application ◽  
Endothelial Cell Adhesion

The binding of fluorescently labeled microspheres (FLMs, 0.1-μm diameter) coated with antibody (1a29) to ICAM-1 was studied in postcapillary venules during topical application of the chemoattractant N-formylmethionyl-leucyl-phenylalanine (fMLP). FLM adhesion to endothelial cells (ECs) increased dramatically from 50 to 150 spheres per 100-μm length of venule after superfusion of the mesentery with fMLP and equaled or exceeded levels of leukocyte (WBC) adhesion. Removal of the EC glycocalyx by micropipette infusion of the venule with heparinase increased FLM-EC adhesion to levels attained with fMLP. Subsequent application of fMLP did not increase FLM adhesion further, suggesting that the FLMs saturated all ICAM-1 binding sites. Perfusion with heparinase after suffusion with fMLP significantly increased FLM-EC adhesion above levels attained with fMLP. However, WBC adhesion fell because of possible removal of selectins necessary to maintain WBC rolling at the wall. It is concluded that the glycocalyx serves as a barrier to adhesion and that its shedding during natural activation of ECs may be an essential part of the inflammatory response.

Neuropeptides promote neutrophil adherence to endothelial cell monolayers

1992 ◽  
Vol 263 (5) ◽  
pp. G678-G682 ◽  
Author(s):  
B. J. Zimmerman ◽  
D. C. Anderson ◽  
D. N. Granger
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Substance P ◽  
Adhesion Molecules ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Human Neutrophils ◽  
Neutrophil Adherence ◽  
Endothelial Cell Adhesion

The objective of this study was to determine whether substance P and calcitonin gene-related peptide (CGRP), at physiologically relevant concentrations, affect leukocyte-endothelial cell adhesion. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated (40 min) with freshly isolated human neutrophils in the presence or absence of substance P or CGRP (10(-11) M). Both substance P and CGRP caused a significant increase (2-fold) in neutrophil adherence to HUVEC. Monoclonal antibodies (MAb) directed against the leukocyte adhesion glycoproteins CD11/CD18 (MAb IB4) and L-selectin (MAb DREG56) did not attenuate substance P-induced adhesion. Antibodies directed against the endothelial cell adhesion molecules E-selectin (MAb CL2) and ICAM-1 (MAb R6.5) were also without effect on substance P-induced neutrophil adhesion. Similar results were obtained when either MAb IB4, DREG56, CL2, or R6.5 was coincubated with CGRP-stimulated neutrophils and endothelial cells. Phorbol 12-myristate 13-acetate-stimulated neutrophil adherence was significantly attenuated by MAb IB4, indicating that CD11/CD18 participates in this adhesion process. The results of this study indicate that 1) the neuropeptides substance P and CGRP promote neutrophil adherence to venular endothelium and 2) the neuropeptide-induced adhesion is not mediated by the adhesion molecules CD11/CD18, L-selectin, E-selectin, or ICAM-1.

scholarly journals Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.

1997 ◽  
Vol 8 (7) ◽  
pp. 1329-1341 ◽  
Author(s):  
N Sheibani ◽  
P J Newman ◽  
W A Frazier
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cell Contact ◽  
Platelet Endothelial Cell Adhesion ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion ◽  
Cell Cell

Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.

scholarly journals Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

1998 ◽  
Vol 9 (4) ◽  
pp. 701-713 ◽  
Author(s):  
Nader Sheibani ◽  
William A. Frazier
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Down Regulation ◽  
Platelet Endothelial Cell Adhesion ◽  
Thrombospondin 1 ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

scholarly journals Peroxisome Proliferator-Activated Receptor γ and Retinoid X Receptor Agonists Have Minimal Effects on the Interaction of Endothelial Cells with Plasmodium falciparum- Infected Erythrocytes

2005 ◽  
Vol 73 (2) ◽  
pp. 1209-1213 ◽  
Author(s):  
Lena Serghides ◽  
Kevin C. Kain
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Umbilical Vein ◽  
Retinoid X Receptor ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cell Adhesion Molecule 1

ABSTRACT Peroxisome proliferator-activated receptor γ-retinoid X receptor (PPARγ-RXR) agonists had minimal effects on the surface levels of CD36, intercellular cell adhesion molecule-1, or platelet-endothelial cell adhesion molecule-1 and had no effect on the cytoadherence of infected erythrocytes to either human umbilical vein endothelial cells or human microvascular endothelial cells or on malaria-induced interleukin-6 secretion from these cells. PPARγ-RXR agonists do not significantly modify malaria-infected erythrocyte-endothelial cell interactions in vitro.

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Sharon Harel ◽  
Veronica Sanchez ◽  
Alaa Moamer ◽  
Javier E. Sanchez-Galan ◽  
Mohammad N. Abid Hussein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Endothelial Cell ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Intracellular Localization ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Angiogenic Response ◽  
Angiopoietin 1

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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