Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture

1997 ◽  
Vol 110 (22) ◽  
pp. 2807-2818 ◽  
Author(s):  
M. Sandig ◽  
E. Negrou ◽  
K.A. Rogers
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Adhesion Molecules ◽  
Laser Scanning ◽  
Transendothelial Migration ◽  
Cell Contact ◽  
Beta Catenin ◽  
Immunofluorescence Labeling ◽  
Cell Cell

To determine changes in the spatial and temporal distribution of cell-cell adhesion molecules during transendothelial migration of monocytes, we examined an in vitro model system of diapedesis using high resolution laser scanning confocal microscopy. Human arterial endothelial cells were cultured to confluence on coverslips coated with Matrigel and activated with IL-1beta before the addition of monocytic THP-1 cells. Seventy per cent of monocytes transmigrated through the endothelium within one hour. Diapedesis, but not adhesion and spreading, was inhibited 8-fold in co-cultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor. Double immunofluorescence labeling with antibodies to LFA-1, alpha- and beta-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regions. This passage was formed by an LFA-1-containing pseudopodium that penetrated between endothelial cells. Apical to the transmigration passage, monocytes remained round in shape, while underneath the endothelium, they spread along the Matrigel. The margins of the transmigration passage contained high levels of LFA-1 and F-actin, suggesting a major role of these molecules during the transmigration process itself. Endothelial adherens junctions, as judged by the presence of VE-cadherin and alpha-catenin adjacent to the passage, remained intact during diapedesis. The presence of catenins at heterotypic contact regions between monocytes and endothelial cells during diapedesis suggested cadherin-mediated interactions between the two cell types. These results reveal dynamic changes in the distribution of adhesion molecules and the actin cytoskeleton during monocyte transendothelial migration in culture.

The roles of adhesion molecules and proteinases in lymphocyte transendothelial migration

1996 ◽  
Vol 74 (6) ◽  
pp. 749-757 ◽  
Author(s):  
Joseph A. Madri ◽  
Donnasue Graesser ◽  
Tara Haas
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
T Cell ◽  
Matrix Metalloproteinase ◽  
Adhesion Molecules ◽  
Cell Monolayer ◽  
Transendothelial Migration ◽  
Matrix Metalloproteinase 2

T cell extravasation into perivascular tissue during inflammation involves transmigration through the endothelial cell (EC) layer and basement membrane. We have demonstrated that matrix metalloproteinase-2 (MMP-2) is induced in T cells upon adhesion to endothelial cells and that the induction of MMP-2 is mediated by binding of T cell VLA-4 to VCAM-1. Cloned murine Th1 cells antigenic to myelin basic protein, either expressing VLA-4 on their cell surface and causing experimental autoimmune encephalomyelitis (EAE) or not expressing VLA-4 and not causing EAE, were used. VLA-4 positive (+) T cells that adhered to VCAM-1 positive (+) endothelial cells exhibited an induction in MMP-2 mRNA, protein, and activity, whereas MMP-2 was not induced in the T cells that adhered to the VCAM-1 negative (−) endothelial cells or VLA-4 negative (−) T cells that adhered to VCAM-1+ endothelial cells. Incubating T cells with rVCAM-1-coated dishes showed that VLA-4+ T cells adhered to the molecule and that adhesion to rVCAM-1 was sufficient to induce MMP-2. VLA-4+ T cells that had transmigrated through a VCAM-1+ endothelial cell monolayer exhibited MMP-2 activity. TTMP-2 was shown to reduce T cell transmigration in vitro. Transmigrated T cells exhibited downregulation of VLA-4 and LFA-1 integrin surface expression and decreased binding to rVCAM-1 and rICAM-1 and increased binding to collagens I and IV, fibronectin, and laminin. Brain sections of mice demonstrated that as T cells migrated farther into the tissue, VLA-4 expression was lost, although CD4 expression remained unchanged. These results demonstrate that binding to VCAM-1 on endothelial cells induces MMP-2 in T cells, which, in turn, may facilitate T cell migration into perivascular tissue. The significance of these findings in the modulation of the inflammatory response is discussed.Key words: T lymphocyte, endothelial cell, matrix metalloproteinase, inflammation, transendothelial migration, integrins, cell adhesion molecules.

scholarly journals PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jesús Gómez-Escudero ◽  
Cristina Clemente ◽  
Diego García-Weber ◽  
Rebeca Acín-Pérez ◽  
Jaime Millán ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Inflammatory Disease ◽  
Chronic Inflammatory Disease ◽  
Cell Junctions ◽  
Cell Junction ◽  
Collective Migration ◽  
Sprouting Angiogenesis ◽  
Cell Cell

Abstract Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.

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.

Endothelial adhesion molecules and their role in inflammation

1993 ◽  
Vol 71 (1) ◽  
pp. 76-87 ◽  
Author(s):  
C. Wayne Smith
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Adhesion Molecules ◽  
Surrounding Tissue ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Endothelial Cell Surface

The emigration of leukocytes such as neutrophils into inflammatory sites requires adhesion to the endothelium of small venules. The initial adhesive event is margination characterized by rolling of neutrophils along the luminal surface of the endothelium. Each member of the selectin family of adhesion molecules has been shown to support neutrophil rolling under conditions of flow. E-selectin is synthesized by endothelial cells following cytokine stimulation, P-selectin is rapidly mobilized from Weibel–Palade bodies to the endothelial cell surface following stimulation with agents such as histamine, and L-selectin is constitutively expressed on the surface of leukocytes. Each selectin functions primarily as a lectin, recognizing carbohydrate structures on the leukocyte or endothelial cell surface. Once the marginated neutrophil forms a stationary adhesion with endothelial cells, it is stimulated by chemotactic factors to downregulate the selectin-based adhesion and upregulate adherence dependent on β2-integrins, principally CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1). These adhesion molecules interact with intercellular adhesion molecule 1 (ICAM-1) and possibly other structures on the endothelial cell, and the leukocyte rapidly emigrates into surrounding tissue. Transendothelial migration in vitro is markedly inhibited by monoclonal antibodies against CD18 integrins or ICAM-1. Monoclonal antibodies against the selectins, CD18, CD11a, CD11b, and ICAM-1 have all been shown to significantly reduce the influx of neutrophils into sites of inflammation in various animal models.Key words: adhesion, integrins, selectins, leukocytes, endothelial cells.

scholarly journals Monocytes enhance the bidirectional release of type I plasminogen activator inhibitor by endothelial cells

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2272-2278
Author(s):  
BC Hakkert ◽  
JM Rentenaar ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Umbilical Vein ◽  
Plasminogen Activator Inhibitor ◽  
Cell Contact ◽  
Type I ◽  
Activator Inhibitor ◽  
Pai 1 ◽  
Cell Cell

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the effects of the interaction between human monocytes and endothelial cells on the production of type 1 plasminogen activator inhibitor (PAI-1) by endothelial cells. The effects of adherence and transendothelial migration of monocytes on endothelial PAI-1 release were compared with those of other leukocytes, conditioned media from monocytes, and interleukin-1 beta (IL-1 beta). Because the cell culture system used allows simultaneous analysis of the lumenal and the subendothelial compartment of endothelial cell monolayers, we also studied into which direction PAI-1 is released by endothelial cells. Under quiescent conditions, the net amount of PAI-1 accumulated at the lumenal side was twofold higher than that accumulated at the subendothelial side (about 2.0 micrograms PAI-1/10(6) cells and 1.1 microgram PAI-1/10(6) cells, respectively, in 24 hours), as analyzed by a quantitative immunoradiometric assay (IRMA). Direct cell-cell contact between highly purified monocytes and endothelial cells strongly enhanced the PAI-1 release by endothelial cells in a dose-dependent way, whereas lymphocytes and neutrophils did not affect endothelial PAI- 1 production. The monocyte-mediated increase was first detected after 12 hours of incubation and lasted for at least 48 hours. In the presence of two monocytes per endothelial cell, the increases of PAI-1 at the lumenal side and at the subendothelial side were 87% and 32% in 24 hours, respectively. The effect of IL-1 beta on PAI-1 release by endothelial cells closely resembled that observed for monocytes. Monocyte-conditioned medium contained heat-labile product(s) which also, although to a much lesser extent than intact monocytes, enhanced endothelial PAI-1 release. Similarly, monocytes cultured on top endothelial cell separated by a microporous filter enhanced the release of PAI-1 to a lesser extent. Thus, these findings indicate that monocytes enhance endothelial PAI-1 release by mechanisms that are, at least in part, dependent on cell-cell contact.

scholarly journals Septin Roles and Mechanisms in Organization of Endothelial Cell Junctions

2020 ◽  
Author(s):  
Joanna Kim ◽  
John A. Cooper
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Adherens Junctions ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cell Contact ◽  
Junction Molecules ◽  
Tnf Α ◽  
Multiple Cell ◽  
Cell Cell

AbstractSeptins play an important role in regulating the barrier function of the endothelial monolayer of the microvasculature. Depletion of septin 2 protein alters the organization of vascular endothelial (VE)-cadherin at cell-cell adherens junctions as well as the dynamics of membrane protrusions at endothelial cell-cell contact sites. Here, we report the discovery that localization of septin 2 at endothelial cell junctions is important for the distribution of a number of other junctional molecules. We also found that treatment of microvascular endothelial cells with the inflammatory mediator TNF-α led to sequestration of septin 2 away from cell junctions and into the cytoplasm, without an effect on the overall level of septin 2 protein. Interestingly, TNF-α treatment of endothelial monolayers produced effects similar to those of depletion of septin 2 on various molecular components of adherens junctions (AJs) and tight junctions (TJs). Immunofluorescence staining revealed disruption of the integrity of AJs and TJs at cell-cell junctions without significant changes in protein expression except for VE-cadherin and nectin-2. To investigate the mechanism of junctional localization of septin 2, we mutated the polybasic motif of septin 2, which is proposed to interact with PIP2 in the plasma membrane. Overexpression of PIP2-binding mutant (PIP2BM) septin 2 led to loss of septin 2 from cell junctions with accumulation in the cytoplasm. This redistribution of septin 2 away from the membrane led to effects on cell junction molecules similar to those observed for depletion of septin 2. We conclude that septin localization to the membrane is essential for function and that septins support the localization of multiple cell junction molecules in endothelial cells.

PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin

1999 ◽  
Vol 112 (18) ◽  
pp. 3005-3014 ◽  
Author(s):  
N. Ilan ◽  
S. Mahooti ◽  
D.L. Rimm ◽  
J.A. Madri
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Nuclear Translocation ◽  
Adherens Junction ◽  
Cell Contact ◽  
Beta Catenin ◽  
Dependent Manner

Catenins function as regulators of cellular signaling events in addition to their previously documented roles in adherens junction formation and function. Evidence to date suggests that beta and gamma catenins can act as signaling molecules, bind transcriptional factors and translocate to the nucleus. Beta- and gamma-catenin are also major substrates for protein tyrosine kinases, and tyrosine phosphorylation of junctional proteins is correlated with decreased adhesiveness. One way in which catenin functions are modulated is by dynamic incorporation into junctional complexes which controls, in part, the cytoplasmic levels of catenins. Here we show that: (1) vascular endothelial growth factor (VEGF) induces beta-catenin tyrosine phosphorylation in a time-, and dose-dependent manner and that VEGF receptors co-localize to areas of endothelial cell-cell contact in vitro and in vivo. (2) Platelet-endothelial cell adhesion molecule (PECAM)-1 can function as a reservoir for, and modulator of, tyrosine phosphorylated beta-catenin. (3) PECAM-1 can prevent beta-catenin nuclear translocation in transfected SW480 colon carcinoma cells. We suggest that PECAM-1 may play a role in modulating beta-catenin tyrosine phosphorylation levels, localization and signaling and by doing so, functions as an important modulator of the endothelium.

scholarly journals Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3371-3381 ◽  
Author(s):  
Malika Oubaha ◽  
Michelle I. Lin ◽  
Yoran Margaron ◽  
Dominic Filion ◽  
Emily N. Price ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Leading Edge ◽  
Cell Junctions ◽  
Endothelial Cell Migration ◽  
Directional Migration ◽  
Cell Contacts ◽  
Angiopoietin 1 ◽  
Cell Cell

Abstract Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis.

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