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The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.

Aidoudi S, Bujakowska K, Kieffer N, Bikfalvi A - PLoS ONE (2008)

Bottom Line: We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner.Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration.As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U920, Talence, France. salouaaidoudi@yahoo.fr

ABSTRACT
The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

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Immobilized CXCL4 or CXCL4/CTF promotes human umbilical vein endothelial cells (HUVECs) spreading, focal adhesion and stress fiber formation.(A) Analysis of spreading, focal adhesion and stress fibers. HUVECs were plated for 4h on coverslips that had been coated with (25 µg/ml) CXCL4, (25 µg/ml) CXCL4/CTF, (100 µg/ml) fibrinogen, (25 µg/ml), CXCL4/CTF-S and (100 µg/ml) poly-D-lysine. After washing, the cells are fixed. The degree of cell spreading is seen from the phase contrast micrographs (left side) Then the cells were permeabilized and stained to visualize focal adhesion (vinculin staining, green), and actin stress fibers (Rhodamin-Phalloidin staining, red) by confocal microscopy (right side). (B) Analysis of tyrosine phosphorylation of FAK. Effect of HUVEC cell adhesion to CXCL4 or CXCL4/CTF on tyrosine phosphorylation of FAK. HUVECs were maintained in suspension for 60 min (BSA, lane 1) or allowed to attach to integrins ligand (fibrinogen, lane 4) or CXCL4 (lane 2) or CXCL4/CTF (lane 3). Cells lysates containing equal amounts of protein were immunoprecipitated with anti-FAK antibody. One half of immunoprecipitates was subjected to immunoblotting with anti-phosphotyrosine mAbs, 4G10, and PY20, and the other half was probed with mAb anti-FAK.
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pone-0002657-g001: Immobilized CXCL4 or CXCL4/CTF promotes human umbilical vein endothelial cells (HUVECs) spreading, focal adhesion and stress fiber formation.(A) Analysis of spreading, focal adhesion and stress fibers. HUVECs were plated for 4h on coverslips that had been coated with (25 µg/ml) CXCL4, (25 µg/ml) CXCL4/CTF, (100 µg/ml) fibrinogen, (25 µg/ml), CXCL4/CTF-S and (100 µg/ml) poly-D-lysine. After washing, the cells are fixed. The degree of cell spreading is seen from the phase contrast micrographs (left side) Then the cells were permeabilized and stained to visualize focal adhesion (vinculin staining, green), and actin stress fibers (Rhodamin-Phalloidin staining, red) by confocal microscopy (right side). (B) Analysis of tyrosine phosphorylation of FAK. Effect of HUVEC cell adhesion to CXCL4 or CXCL4/CTF on tyrosine phosphorylation of FAK. HUVECs were maintained in suspension for 60 min (BSA, lane 1) or allowed to attach to integrins ligand (fibrinogen, lane 4) or CXCL4 (lane 2) or CXCL4/CTF (lane 3). Cells lysates containing equal amounts of protein were immunoprecipitated with anti-FAK antibody. One half of immunoprecipitates was subjected to immunoblotting with anti-phosphotyrosine mAbs, 4G10, and PY20, and the other half was probed with mAb anti-FAK.

Mentions: Integrin-mediated cell attachment on cognate integrin ligands, such as ECM proteins, results in cell spreading, focal adhesion formation, and tyrosine phosphorylation of intracellular proteins [21]. When integrin inhibitors such as antibodies are immobilized on a substrate, they act as agonist and similarly activate intracellular events [22], [23]. To examine whether immobilized CXCL4 would function as an integrin agonist, HUVECs were plated on coverslips that had been coated with CXCL4. As shown in Fig. 1, immobilized CXCL4 similar to natural integrin ligands fibrinogen or fibronectin, promoted endothelial cells spreading, focal adhesion and stress fibers formation. Furthermore, to determine whether the C-terminus of CXCL4 exhibited similar effects, we used a synthetic peptide encompassing amino-acid sequence 47–70 (CXCL4/CTF). Previous data showed that the peptide retains full anti-angiogenic activity of CXCL4 [5]–[7]. As shown in Fig. 1, CXCL4/CTF demonstrated similar effects on endothelial cell spreading, focal adhesion and stress fibers formation as full length CXCL4. Furthermore, when HUVECs are plated on a –scrambled peptide containing amino acides derived from CXCL4/CTF (CXCL4/CTF-S) (that does not exhibit anti-angiogenic activity), or on polylysine (to which cells adhere in an integrin-independent manner), they remained round, and failed to spread and to induce focal adhesion formation (Fig. 1).


The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.

Aidoudi S, Bujakowska K, Kieffer N, Bikfalvi A - PLoS ONE (2008)

Immobilized CXCL4 or CXCL4/CTF promotes human umbilical vein endothelial cells (HUVECs) spreading, focal adhesion and stress fiber formation.(A) Analysis of spreading, focal adhesion and stress fibers. HUVECs were plated for 4h on coverslips that had been coated with (25 µg/ml) CXCL4, (25 µg/ml) CXCL4/CTF, (100 µg/ml) fibrinogen, (25 µg/ml), CXCL4/CTF-S and (100 µg/ml) poly-D-lysine. After washing, the cells are fixed. The degree of cell spreading is seen from the phase contrast micrographs (left side) Then the cells were permeabilized and stained to visualize focal adhesion (vinculin staining, green), and actin stress fibers (Rhodamin-Phalloidin staining, red) by confocal microscopy (right side). (B) Analysis of tyrosine phosphorylation of FAK. Effect of HUVEC cell adhesion to CXCL4 or CXCL4/CTF on tyrosine phosphorylation of FAK. HUVECs were maintained in suspension for 60 min (BSA, lane 1) or allowed to attach to integrins ligand (fibrinogen, lane 4) or CXCL4 (lane 2) or CXCL4/CTF (lane 3). Cells lysates containing equal amounts of protein were immunoprecipitated with anti-FAK antibody. One half of immunoprecipitates was subjected to immunoblotting with anti-phosphotyrosine mAbs, 4G10, and PY20, and the other half was probed with mAb anti-FAK.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481302&req=5

pone-0002657-g001: Immobilized CXCL4 or CXCL4/CTF promotes human umbilical vein endothelial cells (HUVECs) spreading, focal adhesion and stress fiber formation.(A) Analysis of spreading, focal adhesion and stress fibers. HUVECs were plated for 4h on coverslips that had been coated with (25 µg/ml) CXCL4, (25 µg/ml) CXCL4/CTF, (100 µg/ml) fibrinogen, (25 µg/ml), CXCL4/CTF-S and (100 µg/ml) poly-D-lysine. After washing, the cells are fixed. The degree of cell spreading is seen from the phase contrast micrographs (left side) Then the cells were permeabilized and stained to visualize focal adhesion (vinculin staining, green), and actin stress fibers (Rhodamin-Phalloidin staining, red) by confocal microscopy (right side). (B) Analysis of tyrosine phosphorylation of FAK. Effect of HUVEC cell adhesion to CXCL4 or CXCL4/CTF on tyrosine phosphorylation of FAK. HUVECs were maintained in suspension for 60 min (BSA, lane 1) or allowed to attach to integrins ligand (fibrinogen, lane 4) or CXCL4 (lane 2) or CXCL4/CTF (lane 3). Cells lysates containing equal amounts of protein were immunoprecipitated with anti-FAK antibody. One half of immunoprecipitates was subjected to immunoblotting with anti-phosphotyrosine mAbs, 4G10, and PY20, and the other half was probed with mAb anti-FAK.
Mentions: Integrin-mediated cell attachment on cognate integrin ligands, such as ECM proteins, results in cell spreading, focal adhesion formation, and tyrosine phosphorylation of intracellular proteins [21]. When integrin inhibitors such as antibodies are immobilized on a substrate, they act as agonist and similarly activate intracellular events [22], [23]. To examine whether immobilized CXCL4 would function as an integrin agonist, HUVECs were plated on coverslips that had been coated with CXCL4. As shown in Fig. 1, immobilized CXCL4 similar to natural integrin ligands fibrinogen or fibronectin, promoted endothelial cells spreading, focal adhesion and stress fibers formation. Furthermore, to determine whether the C-terminus of CXCL4 exhibited similar effects, we used a synthetic peptide encompassing amino-acid sequence 47–70 (CXCL4/CTF). Previous data showed that the peptide retains full anti-angiogenic activity of CXCL4 [5]–[7]. As shown in Fig. 1, CXCL4/CTF demonstrated similar effects on endothelial cell spreading, focal adhesion and stress fibers formation as full length CXCL4. Furthermore, when HUVECs are plated on a –scrambled peptide containing amino acides derived from CXCL4/CTF (CXCL4/CTF-S) (that does not exhibit anti-angiogenic activity), or on polylysine (to which cells adhere in an integrin-independent manner), they remained round, and failed to spread and to induce focal adhesion formation (Fig. 1).

Bottom Line: We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner.Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration.As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U920, Talence, France. salouaaidoudi@yahoo.fr

ABSTRACT
The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

Show MeSH
Related in: MedlinePlus