CXCL4 Integrins Angiogenesis 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 mean6SEM from 3 independent experiments. Error bars represent the mean6SD, #, 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. doi:10.1371/journal.pone.0002657.g007 avb3 interaction. The expression of both integrins avb3 and a5b1 are significantly up-regulated on endothelial during angiogenesis. While a5b1 selectively recognizes primarily a single ECM protein ligand, fibronectin, avb3 can bind several ligands such as vitronectin, fibronectin, fibrinogen, and other matrix proteins (13). The fact that CXCL4 may be a better ligand for avb3 than a5b1, thus blocking the interaction of avb3 integrin to its various ligands, may increase its inhibitory effect on angiogenesis. To study the functional significance of the CXCL4-integrin interaction on angiogenesis, we examined the capacity of CXCL4 to modulate endothelial cell migration. We found that CXCL4 modulates endothelial cell migration, in an integrin-dependent and -specific manners. Cell migration is a vital step in the angiogenesis process and is governed by pro-angiogenic factors, such as VEGF and FGF, and integrins [13,43]. Angiogenic stimulus by growth factors induces an increased expression of integrins in endothelial cells undergoing angiogenesis, thereby leading cells to spread, to migrate and to ultimately form new tubular vessels [13]. Previous studies showed that both CXCL4 and CXCL4/CTF block endothelial cell migration in response to VEGF or FGF growth factors [7,33]. This action is believed to mediate the anti-angiogenic effect of CXCL4, although its receptor mechanism was not characterized. Here we report that soluble CXCL4 or CXCL4/CTF, but not the inactive peptide CXCL4/CTF-S, inhibits endothelial cell migration in avb3 and a5b1 in an integrin-dependent and -specific manners. Similarly, endostatin and thrombospondin-1, two endogenous angiogenesis inhibitors, inhibited endothelial cell migration through b1 integrins [27,44]. Our results suggest that integrins serve as functional receptors for CXCL4 to mediate its inhibition effect on -endothelial cells migration- angiogenesis. On the other hand, other data indicate that VEGF directly activates integrins and enhance cell migration mediated by avb3 and a5b1 [34] Furthermore, FGF-2 stimulated endothelial cell migration medi- ated by avb3 [45]. Further studies are needed to determine the potential significance of integrin-growth factor receptor cross-talk in CXCL4 action. Previous studies showed that CXCL4 and its derived peptide CXCL4/CTF inhibit angiogenesis by interfering with the angiogenic effect of the growth factors FGF and VEGF165, either by directly binding to these angiogenic factors, thereby blocking their interaction with their specific receptors [32,46–47], or by competing with the binding of FGF or VEGF to heparan sulfate proteoglycans on the cell surface [46,48]. Furthermore, CXCL4 was recently shown to bind to CXC3B, a chemokine receptor isoform that is present in some vascular beds [49]. However, the role of CXC3-B receptors in the anti-angiogenic function of CXCL4 remains to be fully established [50]. Our study suggests a novel additional mechanism for the antiangiogenic effects of CXCL4, which involves direct targeting of vessel through integrins. Since integrins are overexpressed in endothelial cells undergoing angiogenesis, this may explain why, in vivo, CXCL4 tends to preferentially target angiogenic blood vessels [19,20]. Furthermore, this targeting mechanism could be useful for selective treatments of cancer by CXCL4. Indeed and as mentioned above, peptides were shown to home selectively in tumor vasculature, through their interaction with integrins, and when coupled to anticancer drugs, they enhanced the in vivo anti- tumor action and reduced the toxicity of anticancer drugs [41]. Finally, integrin-binding of CXCL4 or CXCL4/CTF is also consistent with a heparan sulfate proteoglycan-independent mechanism. Indeed, VEGF121, an endothelial cell mitogen that lacks heparin affinity, is inhibited by CXCL4 [47]. CXCL4 also antagonizes EGF-mediated endothelial cell proliferation indepen- dently from glycosaminoglycans [51]. In summary, a general picture of CXCL4’s antiangiogenic mechanisms is emerging from the results described above. CXCL4 inhibits neovascularization in vivo by several distinct, although not necessarily exclusive, mechanisms including direct integrin binding– reported here –, direct interaction with angiogenic molecules [32], binding to proteoheparan sulfate–which acts as low affinity co-receptors for growth factors, such as FGF–[46,48] or activation of CXCR3-B [49]. These different mechanisms may operate in parallel and in function of the site and/or the type of vessel that undergo angiogenesis. Additional studies will be required to fully understand the significance of integrin binding and other mechanisms involved in CXCL4’s antiangiogenic activity in vivo. Materials and Methods Reagents and Cell Culture Fibronectin from human, plasma Fibrinogen from human plasma, poly-D-lysine and bovine serum albumin (BSA, fraction V) were purchased from Sigma. RGDS and RGES peptides were from Bachem, human purified platelet factor 4/CXCL4 (CXCL4) was obtained from Hyphen BioMed, France. C-terminal peptides of CXCL4, CXCL4/CTF (NGRKICLDLQAPLYKKIIKKLLES) or CXCL4/CTF-S (LGLKPLKQELIAYRDNK IKSICLK) were purchased from ThermoHybaid (Ulm, Germany). Purified integrins and the purified monoclonal anti-human integrin antibodies LM609 (anti-avb3), P1F6 (anti-avb5), JBS5 and HA5 (anti-a5b1), B3A (anti-b3), AV1 (anti-av), CD29 (anti-b1), P1D6 (anti-a5) were purchased from Chemicon. Mouse Ig1 antibody was purchased from Chemicon. Peroxidase and FITC (Fluorescein isothiocyanate) conjugated goat anti-mouse immunoglobulins were purchased from DAKO. Mouse monoclonal anti-human vinculin antibody was from Sigma and Rhodamin-Phalloidin from Molecular probes. The Chinese hamster ovary (CHO) cell line CRL9096, as well as CHO clones expressing human avb3 integrin (avb3-CHO, A06) were established as described (25). Cells lines were grown at 37uC, 5% CO2 in Dublecco’s Modified Eagle Medium (GIBCO) supple- mented with 10% foetal bovine serum, 2 mM glutamine, penicillin and streptomycin (100 UI/ml) and 1% non-essential amino acids. Human umbilical vein endothelial cells (HUVECs, Clonetics) were cultured in EGM medium (Cambrex) supplemented with 10% foetal bovine serum, 2 mM glutamine, and penicillin and streptomycin (100 UI/ml). Experimentation was carried out at cell passage number 4–10. Flow Cytometry Analysis cell-surface integrin expression was performed as described [29]. Cells were suspended in incubation Walsh buffer (137 mM NaCl, 2.7 mM KCL, 3.3 mM NaH2PO4, 3.8 mM HEPES, 1 mM MgCl2, 5.5 mM glucose, and 1 mg/ml BSA, pH 7.4) and 26105 cells incubated for 30 min on ice with a PLoS ONE | www.plosone.org 11 July 2008 | Volume 3 | Issue 7 | e2657