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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 and soluble CXCL4 or CXCL4/CTF inhibits integrin-dependent endothelial cell migration.A. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin toward VEGF (5 ng/ml). HUVECs migration was determined in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or 25 µg/ml of RGD-, RGE peptides or 10 µg/ml of anti-integrin antibodies (anti-αv; AV1 and anti-α5β1; JBS5) as described in Material and Methods. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.05; *, P≤0.005, **, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides. B. Immobilized CXCL4 or CXCL4/CTF supports HUVECs migration in αvβ3 and α5β1 integrins-dependent manner. Cell migration assay was performed on immobilized CXCL4 or CXCL4/CTF in serum free medium and in the presence or absence of inhibitory anti-integrin antibodies or RGD-, RGE peptides. The results at each point are the mean cell number of 10 randomly selected high magnification microscopic fields. Error bars represent the mean±SD, *, P≤0.005; *, P≤0.001 compared to CXCL4 or CXCL4/CTF in the presence of IgG control antibody; n = 3 independent experiments. C. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin. HUVECs migration was determined in serum free medium and in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or inhibitory anti-integrin antibodies or RGD-, RGE peptides. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.005; *, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides.
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pone-0002657-g007: Immobilized CXCL4 or CXCL4/CTF promotes and soluble CXCL4 or CXCL4/CTF inhibits integrin-dependent endothelial cell migration.A. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin toward VEGF (5 ng/ml). HUVECs migration was determined in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or 25 µg/ml of RGD-, RGE peptides or 10 µg/ml of anti-integrin antibodies (anti-αv; AV1 and anti-α5β1; JBS5) as described in Material and Methods. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.05; *, P≤0.005, **, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides. B. Immobilized CXCL4 or CXCL4/CTF supports HUVECs migration in αvβ3 and α5β1 integrins-dependent manner. Cell migration assay was performed on immobilized CXCL4 or CXCL4/CTF in serum free medium and in the presence or absence of inhibitory anti-integrin antibodies or RGD-, RGE peptides. The results at each point are the mean cell number of 10 randomly selected high magnification microscopic fields. Error bars represent the mean±SD, *, P≤0.005; *, P≤0.001 compared to CXCL4 or CXCL4/CTF in the presence of IgG control antibody; n = 3 independent experiments. C. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin. HUVECs migration was determined in serum free medium and in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or inhibitory anti-integrin antibodies or RGD-, RGE peptides. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.005; *, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides.

Mentions: Cell migration is an essential step in the angiogenesis process [13]. Previous studies showed that CXCL4 or CXCL4/CTF block endothelial cell migration in response to FGF-2 or VEGF in stimulation in vitro [7], [33], a process thought to mediate the anti-angiogenic effect of CXCL4 in vivo. On the other hand, αvβ3 [34], and α5β1 [17] integrins have a crucial role in cell migration during angiogenesis. Taking into account our findings, we determined whether CXCL4 and its antiangiogenic derived peptide modulates endothelial cell migration through αvβ3 and/or α5β1 integrins. Our finding showed that soluble CXCL4 or CXCL4/CTF inhibited HUVECs cells migration through fibronectin a reported αvβ3 and α5β1 ligand [13], toward VEGF, in a dose depending manner (Fig. 7A). This migration is similarly inhibited by adding RGD peptides or by the blocking integrin antibodies, anti-αvβ3 LM609 or anti-α5β1 JBS5 (Fig. 7A). These results are consistent with previous data [7], [33], and suggest that CXCL4 or CXCL4/CTF inhibits endothelial cell migration in an integrin dependent manner.


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 and soluble CXCL4 or CXCL4/CTF inhibits integrin-dependent endothelial cell migration.A. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin toward VEGF (5 ng/ml). HUVECs migration was determined in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or 25 µg/ml of RGD-, RGE peptides or 10 µg/ml of anti-integrin antibodies (anti-αv; AV1 and anti-α5β1; JBS5) as described in Material and Methods. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.05; *, P≤0.005, **, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides. B. Immobilized CXCL4 or CXCL4/CTF supports HUVECs migration in αvβ3 and α5β1 integrins-dependent manner. Cell migration assay was performed on immobilized CXCL4 or CXCL4/CTF in serum free medium and in the presence or absence of inhibitory anti-integrin antibodies or RGD-, RGE peptides. The results at each point are the mean cell number of 10 randomly selected high magnification microscopic fields. Error bars represent the mean±SD, *, P≤0.005; *, P≤0.001 compared to CXCL4 or CXCL4/CTF in the presence of IgG control antibody; n = 3 independent experiments. C. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin. HUVECs migration was determined in serum free medium and in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or inhibitory anti-integrin antibodies or RGD-, RGE peptides. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.005; *, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides.
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Related In: Results  -  Collection

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pone-0002657-g007: Immobilized CXCL4 or CXCL4/CTF promotes and soluble CXCL4 or CXCL4/CTF inhibits integrin-dependent endothelial cell migration.A. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin toward VEGF (5 ng/ml). HUVECs migration was determined in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or 25 µg/ml of RGD-, RGE peptides or 10 µg/ml of anti-integrin antibodies (anti-αv; AV1 and anti-α5β1; JBS5) as described in Material and Methods. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.05; *, P≤0.005, **, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides. B. Immobilized CXCL4 or CXCL4/CTF supports HUVECs migration in αvβ3 and α5β1 integrins-dependent manner. Cell migration assay was performed on immobilized CXCL4 or CXCL4/CTF in serum free medium and in the presence or absence of inhibitory anti-integrin antibodies or RGD-, RGE peptides. The results at each point are the mean cell number of 10 randomly selected high magnification microscopic fields. Error bars represent the mean±SD, *, P≤0.005; *, P≤0.001 compared to CXCL4 or CXCL4/CTF in the presence of IgG control antibody; n = 3 independent experiments. C. Soluble CXCL4 or CXCL4/CTF inhibits migration on fibronectin. HUVECs migration was determined in serum free medium and in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/CTF-S or inhibitory anti-integrin antibodies or RGD-, RGE peptides. Relative cell migration is indicated, each value is a mean±SEM from 3 independent experiments. Error bars represent the mean±SD, #, P≤0.005; *, P≤0.001 compared to the number of cell migrated in the absence of CXCL4 or CXCL4/CTF or anti-integrin antibodies or RGD peptides.
Mentions: Cell migration is an essential step in the angiogenesis process [13]. Previous studies showed that CXCL4 or CXCL4/CTF block endothelial cell migration in response to FGF-2 or VEGF in stimulation in vitro [7], [33], a process thought to mediate the anti-angiogenic effect of CXCL4 in vivo. On the other hand, αvβ3 [34], and α5β1 [17] integrins have a crucial role in cell migration during angiogenesis. Taking into account our findings, we determined whether CXCL4 and its antiangiogenic derived peptide modulates endothelial cell migration through αvβ3 and/or α5β1 integrins. Our finding showed that soluble CXCL4 or CXCL4/CTF inhibited HUVECs cells migration through fibronectin a reported αvβ3 and α5β1 ligand [13], toward VEGF, in a dose depending manner (Fig. 7A). This migration is similarly inhibited by adding RGD peptides or by the blocking integrin antibodies, anti-αvβ3 LM609 or anti-α5β1 JBS5 (Fig. 7A). These results are consistent with previous data [7], [33], and suggest that CXCL4 or CXCL4/CTF inhibits endothelial cell migration in an integrin dependent manner.

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