The angiogenic activity of CXC-ELR+ chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-α in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck−/−fgr−/−), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck−/−fgr−/− mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck−/−fgr−/− mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck−/−fgr−/− mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

14 C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-Tat R49/52/53/55/56/57A . Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin.In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value.

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38 -61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-␣ v ␤ 3 antibodies prevent cell adhesion to FGF-2. Also, purified human ␣ v ␤ 3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-␣ v ␤ 3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with ␣ v ␤ 3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells. INTRODUCTIONAngiogenesis, the growth of new blood vessels, plays a key role in different physiological and pathological conditions, including embryonic development, wound repair, inflammation, tumor growth, and angiogenesis-dependent diseases (Folkman, 1995). Neovascularization is a multi-step process. It begins with the degradation of the basement membrane by proteases secreted by activated endothelial cells that will migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space. Then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of new basement membrane (Ausprunk and Folkman, 1977). A close interaction exists among cell-adhesive proteins of the extracellular matrix (ECM), their integrin receptors, and soluble angiogenesic growth factors during each step of the angiogenesis process Folkman, 1989a, 1989b; Davis et al., 1993;Brooks et al., 1994;Plopper et al., 1995).One of the best characterized modulators of angiogenesis is the heparin-binding basic fibroblast growth factor (FGF-2). FGF-2 has been demonstrated to in- (Basilico and Moscatelli, 1992) and to be implicated in the growth of new blood vessels during wound healing and chick embryo development (Broadley et al., 1989;Ribatti et al., 1995). In vitro, FGF-2 induces cell proliferation, migration, and pr...

The data demonstrate that the modality of heparinTat interaction is strongly affected by the size of the saccharide chain. The possibility of establishing multiple interactions increases the affinity of large heparin fragments for Tat protein and the capacity of the glycosaminoglycan to modulate the biological activity of extracellular Tat.

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1–3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1–3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125 I-FGF-2 binds to micelles formed by gangliosides GT 1b , GD 1b , or GM 1 . Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT 1b Ͼ GD 1b Ͼ GM 1 ϭ GM 2 ϭ sulfatide Ͼ GM 3 ϭ galactosyl-ceramide, whereas asialo-GM 1 , neuraminic acid, and Nacetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM 1 to immobilized FGF-2 indicates that FGF-2/GM 1 interaction occurs with a K d equal to 6 M. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130 -155), whereas peptides FGF-2(10 -33), FGF-2(39 -59), , and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125 I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT 1b Ͼ GD 1b Ͼ GM 1 Ͼ sulfatide a ϭ sialo-GM 1 . Accordingly, GT 1b ,GD 1b , GM 1 , and GM 2 , but not GM 3 and asialo-GM 1 , prevent the binding of 125 I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM 1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT 1b was the most effective among the gangliosides tested while asialo-GM 1 , neuraminic acid, N-acetylneuramin-lactose, galactosylceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization. § Corresponding author. E-mail address: presta@med.unibs.it.© 1999 by The American Society for Cell Biology 313 INTRODUCTIONGangliosides are neuraminic acid (NeuAc) 1 -containing glycosphingolipids. Under physiological conditions, gangliosides are mainly associated to the ce...

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Substrate-bound FGF2 promotes endothelial cell adhesion by interacting with a v b 3 integrin. Here, endothelial GM7373 cells spread and organize focal adhesion plaques on immobilized FGF2, ®bronectin (FN), and vitronectin (VN). a v b 3 integrin, paxillin, focal adhesion kinase, vinculin and pp60 src localize in cell-substratum contact sites on FGF2, FN or VN. However, only immobilized FGF2 induces a long-lasting activation of extracellular signal-regulated kinases 1/2 (ERK 1/2 ) and cell proliferation that was inhibited by the ERK 1/2 inhibitor PD 098059 and the tyrosine kinase (TK) inhibitor tyrphostin 23, pointing to the engagement of FGF receptor (FGFR) at the basal side of the cell. To assess this hypothesis, GM7373 cells were transfected with a dominant negative TK 7 -DFGFR1 mutant (GM7373-DFGFR1 cells) or with the full-length receptor (GM7373-FGFR1 cells). Both transfectants adhere and spread on FGF2 but GM7373-DFGFR1 cells do not proliferate. Also, parental and GM7373-FGFR1 cells, but not GM7373-DFGFR1 cells, undergo morphological changes and increased motility on FGF2-coated plastic. Finally, FGFR1, but not TK 7 -DFGFR1, localizes in cell adhesion contacts on immobilized FGF2. In conclusion, substrate-bound FGF2 induces endothelial cell proliferation, motility, and the recruitment of FGFR1 in cell-substratum contacts. This may contribute to the cross talk among intracellular signaling pathways activated by FGFR1 and a v b 3 integrin in endothelial cells.