CXCL4 Integrins Angiogenesis Figure 6. 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 avb3-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 avb3-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 means6SEM of 4 separate experiments. Error bars represent the mean6SD, *, P#0.005 compared to CXCL4 or CXCL4/CTF in the absence of integrin agonists. doi:10.1371/journal.pone.0002657.g006 CXCL4/CTF inhibits endothelial cell migration in an integrin- dependent and -specific manners. Discussion We sought to assess whether CXCL4, a CXC chemokine that exhibit potent anti-angiogenic activities, and its C-terminus derived peptide CXCL4/CTF, would function as ligands for the integrin receptors on the surface of endothelial cells. We report here (1) that CXCL4 or CXCL4/CTF binds to avb3 and to some extent to avb5 and a5b1 integrins. (2) CXCL4 or CXCL4/CTF binding to integrins on the surface of avb3-CHO and HUVECs is blocked by RGD peptide but not by RGE peptide. (3) The inactive peptide derived from CXCL4/CTF (CXCL4/CTF-S), fails to interact with integrins on the surface of avb3-CHO or HUVECs. (4) Immobilized CXCL4 or CXCL4/CTF promotes avb3-CHO and HUVEC cells spreading, focal adhesion and stress fibers formation and FAK phosphorylation (5) Functional studies show that immobilized CXCL4- or CXCL4/CTF support endothelial cells migration in an integrin-dependent manner and soluble CXCL4 or CXCL4/CTF, in turn, inhibits integrin- dependent endothelial cell migration. Thus, the present study is the first to show that CXCL4 interacts with integrins. This interaction may constitute a novel mechanism for the inhibitory effects of CXCL4 on angiogenic blood vessels. Several lines of cell biological and biochemical evidence demonstrated the interaction between CXCL4 and integrins in our study. Thus, inhibitory anti-integrin antibodies prevented avb3-CHO and HUVEC cell attachment whereas CXCL4 interacted directly with purified avb3 integrin in a specific manner in a solid-phase binding assay. Despite the fact that neither CXCL4 nor CXCL4/CTF contain an RGD sequence, the interaction of CXCL4 with integrins is divalent cation-dependent and is blocked by RGD peptide. Interestingly, a number of endogenous angiogenesis inhibitors such as endostatin [27], arresten [35] canstatin [36] and tumstatin [37,38] also bind directly to integrin, despite the fact that they lack the RGD- binding site. For example, the interaction of endostatin with a5b1 integrin is inhibited by RGD peptide, however tumstatin binds to avb3 in an RGD-independent manner. The fact that the interaction of CXCL4 with integrins is blocked by the RGD peptide indicates that CXCL4 interacts with the same or an overlapping RGD-binding site in integrin. Alternatively, it is possible that other amino acid sequences within CXCL4 also interact with the RGD binding site. Interestingly, CXCL4 and CXCL4/CTF both contain the NGR sequence that is known to interact with integrins, such as avb3, avb5 and a5b1 [39,40]. The NGR motif homes to the tumor vasculature, but not in normal endothelium, through its interaction with integrins. As a consequence, when coupled to anticancer drugs, the NGR peptide was shown to enhance their in vivo anti-tumor action and to reduce their toxicity [41]. Additional studies are required to determine the sequence motif of CXCL4 that integrins recognize. Our results show that immobilized CXCL4 or CXCL4/CTF supported cell spreading, focal adhesion and stress fibers formation and tyrosine phosphorylation (i.e., activation) of the focal adhesion kinase (FAK). These events are specific to integrin signaling [21]. This indicates that immobilized CXCL4 or CXCL4/CTF activates postligand binding events downstream of integrins. The addition of two well-known integrin activators, Mn2+ -acting as integrin activator from outside the cells (referred to as outside/in signaling)- and PMA -activating integrins from inside the cells via activation of PKC (referred to as inside/out signaling)-, produced a strong increase in both the number of attached cells and cell spreading on immobilized CXCL4 or CXCL4/CTF, comparable to fibrinogen. Thus, PF4 seems to bind to avb3 integrins as a natural ligand of avb3, such as fibrinogen, which binds with low affinity to unstimulated avb3 and binds with high affinity to activated avb3 upon stimulation by agonists, such as Mn2+ or PMA [29]. Thus, our finding suggests that interaction of CXCL4 or CXCL4/CTF to integrins is subject to integrin function regulation (i.e. affinity modulation). These results may be relevant to the antiangiogenic activity of CXCL4 in some pathophysiolog- ical circumstances in vivo. Indeed, it was shown that the basal activation state of avb3 varies with the cell type and that an increase in the number of active avb3 with high affinity for their ligands is correlated with the acquisition of cell malignancy [29] and with the metastasis of some cancer, such as in human breast cancer [42]. Considering that the binding of CXCL4 to integrins depends on the integrin state (active or not), our results suggest that the binding of CXCL4 to endothelial cells should increase during pathological angiogenesis, where affinity modulation of integrins occurs. Our results show that soluble CXCL4 or CXCL4/CTF inhibited endothelial cell adhesion to immobilized matrix proteins, as fibronectin or vitronectin. These results suggest that soluble CXCL4 or CXCL4/CTF act as antagonist for integrins avb3 and a5b1, to mediate its inhibition effect on -endothelial cells adhesion- angiogenesis. Theoretically, it is possible that CXCL4 could also act on attached endothelial cells by further promoting spreading. However, we clearly demonstrated that surface immobilized but not soluble CXCL4 promoted spreading. This finding argues against a similar effect on already attached cells. In addition, it is unlikely that spreading of already attached endothelial cells is inhibited by CXCL4, because of the strong binding of integrins to matrix proteins. Although a5b1 is the most abundant integrin on HUVECS (Fig. 5A), adhesion experiments on immobilized CXCL4 in the presence of different blocking antibodies against avb3, a5b1, b1, a5, av, b3 may suggest a greater interaction of CXCL4 to avb3 integrin (Fig. 5C). However this conclusion must be interpreted with caution because these differences may also be due to more efficiency antibody binding to avb3 than to a5b1. Also, a quantitative analysis comparing affinities is required to ascertain the potentially better interaction of CXCL4 to avb3. Nevertheless, assuming that CXCL4 interacts better with avb3 than a5b1, we may speculate that CXCL4 contains additional sequences for PLoS ONE | www.plosone.org 9 July 2008 | Volume 3 | Issue 7 | e2657