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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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Integrin agonists, Mn2+ and PMA, enhance cell spreading, with focal adhesion and stress fiber formation, on immobilized CXCL4 or CXCL4/CTF A, B.a. Phase contrast analysis show an enhance of the number of αvβ3-CHO and HUVECs spreading on immobilized CXCL4 or CXCL4/CTF, upon stimulation with Mn2+ or PMA, b. Confocal analysis show an increase of the number of cell spreading with focal adhesion (Vinculin staining, green) and stress fibers (Rhodamin-Phalloidin staining, Red) formation in αvβ3-CHO and HUVECs spread on immobilized CXCL4 or CXCL4/CTF treated with integrin stimulators, Mn2+ or PMA, as compared with no agonists. C. 100 adherent cells on different substrat were scored for focal adhesions and stress fibers by two independent observers. Data are the means±SEM of 4 separate experiments. Error bars represent the mean±SD, *, P≤0.005 compared to CXCL4 or CXCL4/CTF in the absence of integrin agonists.
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pone-0002657-g006: Integrin agonists, Mn2+ and PMA, enhance cell spreading, with focal adhesion and stress fiber formation, on immobilized CXCL4 or CXCL4/CTF A, B.a. Phase contrast analysis show an enhance of the number of αvβ3-CHO and HUVECs spreading on immobilized CXCL4 or CXCL4/CTF, upon stimulation with Mn2+ or PMA, b. Confocal analysis show an increase of the number of cell spreading with focal adhesion (Vinculin staining, green) and stress fibers (Rhodamin-Phalloidin staining, Red) formation in αvβ3-CHO and HUVECs spread on immobilized CXCL4 or CXCL4/CTF treated with integrin stimulators, Mn2+ or PMA, as compared with no agonists. C. 100 adherent cells on different substrat were scored for focal adhesions and stress fibers by two independent observers. Data are the means±SEM of 4 separate experiments. Error bars represent the mean±SD, *, P≤0.005 compared to CXCL4 or CXCL4/CTF in the absence of integrin agonists.

Mentions: To further examine the postligand events downstream to integrins induced by immobilized CXCL4 or CXCL4/CTF, we examined in more detail cell spreading. As shown above, confocal analysis shows that immobilized CXCL4 or CXCL4/CTF promoted cell spreading, focal adhesion and stress fibers formation (Fig. 1,4). When the number of cells spread on different immobilized substrates was quantified, we found that immobilized CXCL4 or CXCL4/CTF promoted cell spreading by about 50 to 60% in the absence of integrin agonists. When we analyzed the spreaded cells by confocal microscopic we found that around 40% of these cells spread on CXCL4 or CXCL4/CTF (versus 60% with fibronectin, data not shown) presented focal adhesions with stress fiber formation (Fig. 6). These events were significantly increased (from 40% to 60%) when we added two-well known integrin activators, extracellular (Mn2+) and intracellular (Phorbol 12-Mryristate 13 Acetate (PMA) agonists (Fig. 6). These agonists activate αvβ3 integrin (convert αvβ3 to a high-affinity conformation) and stimulate αvβ3 functions [29], such spreading cells, as well as that of other integrins [30], [31]. Incubation of cells with 250 µM of Mn2+ or 100 nM of PMA produced an increase in both the number of attached cells and in cell spreading of about 25% and 30% respectively (Fig. 6). This enhancement was completely blocked by adding EDTA or RGD (data not shown). This finding suggests that the interaction of CXCL4 or CXCL4/CTF to integrins, similar to natural integrin ligands, is subject to integrin function regulation such as affinity modulation (see Discussion).


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

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

Integrin agonists, Mn2+ and PMA, enhance cell spreading, with focal adhesion and stress fiber formation, on immobilized CXCL4 or CXCL4/CTF A, B.a. Phase contrast analysis show an enhance of the number of αvβ3-CHO and HUVECs spreading on immobilized CXCL4 or CXCL4/CTF, upon stimulation with Mn2+ or PMA, b. Confocal analysis show an increase of the number of cell spreading with focal adhesion (Vinculin staining, green) and stress fibers (Rhodamin-Phalloidin staining, Red) formation in αvβ3-CHO and HUVECs spread on immobilized CXCL4 or CXCL4/CTF treated with integrin stimulators, Mn2+ or PMA, as compared with no agonists. C. 100 adherent cells on different substrat were scored for focal adhesions and stress fibers by two independent observers. Data are the means±SEM of 4 separate experiments. Error bars represent the mean±SD, *, P≤0.005 compared to CXCL4 or CXCL4/CTF in the absence of integrin agonists.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481302&req=5

pone-0002657-g006: Integrin agonists, Mn2+ and PMA, enhance cell spreading, with focal adhesion and stress fiber formation, on immobilized CXCL4 or CXCL4/CTF A, B.a. Phase contrast analysis show an enhance of the number of αvβ3-CHO and HUVECs spreading on immobilized CXCL4 or CXCL4/CTF, upon stimulation with Mn2+ or PMA, b. Confocal analysis show an increase of the number of cell spreading with focal adhesion (Vinculin staining, green) and stress fibers (Rhodamin-Phalloidin staining, Red) formation in αvβ3-CHO and HUVECs spread on immobilized CXCL4 or CXCL4/CTF treated with integrin stimulators, Mn2+ or PMA, as compared with no agonists. C. 100 adherent cells on different substrat were scored for focal adhesions and stress fibers by two independent observers. Data are the means±SEM of 4 separate experiments. Error bars represent the mean±SD, *, P≤0.005 compared to CXCL4 or CXCL4/CTF in the absence of integrin agonists.
Mentions: To further examine the postligand events downstream to integrins induced by immobilized CXCL4 or CXCL4/CTF, we examined in more detail cell spreading. As shown above, confocal analysis shows that immobilized CXCL4 or CXCL4/CTF promoted cell spreading, focal adhesion and stress fibers formation (Fig. 1,4). When the number of cells spread on different immobilized substrates was quantified, we found that immobilized CXCL4 or CXCL4/CTF promoted cell spreading by about 50 to 60% in the absence of integrin agonists. When we analyzed the spreaded cells by confocal microscopic we found that around 40% of these cells spread on CXCL4 or CXCL4/CTF (versus 60% with fibronectin, data not shown) presented focal adhesions with stress fiber formation (Fig. 6). These events were significantly increased (from 40% to 60%) when we added two-well known integrin activators, extracellular (Mn2+) and intracellular (Phorbol 12-Mryristate 13 Acetate (PMA) agonists (Fig. 6). These agonists activate αvβ3 integrin (convert αvβ3 to a high-affinity conformation) and stimulate αvβ3 functions [29], such spreading cells, as well as that of other integrins [30], [31]. Incubation of cells with 250 µM of Mn2+ or 100 nM of PMA produced an increase in both the number of attached cells and in cell spreading of about 25% and 30% respectively (Fig. 6). This enhancement was completely blocked by adding EDTA or RGD (data not shown). This finding suggests that the interaction of CXCL4 or CXCL4/CTF to integrins, similar to natural integrin ligands, is subject to integrin function regulation such as affinity modulation (see Discussion).

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