Chiara Urbinati

Cutting Edge: Extracellular High Mobility Group Box-1 Protein Is a Proangiogenic Cytokine

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists.

Heparin binds extracellular HIV-1 Tat protein and modulates its HIV long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d’Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313–11320). On this basis, the glutathioneS-transferase (GST)-TatR49/52/53/55/56/57Amutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat for its capacity to bind immobilized heparin. Dissociation of the GST-TatR49/52/53/55/56/57A·heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat·heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-TatR49/52/53/55/56/57A with a dissociation constant equal to 0.3 and 1.0 μm, respectively. Also, the synthetic basic domain Tat-(41–60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction,125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative14C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-TatR49/52/53/55/56/57A. 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.

Cutting Edge: Proangiogenic Properties of Alternatively Activated Dendritic Cells

Angiogenesis plays an important role in tissue remodeling and repair during the late phase of inflammation. In the present study, we show that human dendritic cells (DC) that matured in the presence of anti-inflammatory molecules such as calcitriol, PGE2, or IL-10 (alternatively activated DC) selectively secrete the potent angiogenic cytokine vascular endothelial growth factor (VEGF) isoforms VEGF165 and VEGF121. No VEGF production was observed in immature or classically activated DC. Also, the capacity to produce VEGF was restricted to the myeloid DC subset. When implanted in the chick embryo chorioallantoic membrane, alternatively activated DC elicit a marked angiogenic response, which is inhibited by neutralizing anti-VEGF Abs and by the VEGFR-2 inhibitor SU5416. Therefore, alternatively activated DC may contribute to the resolution of the inflammatory reaction by promoting VEGF-induced angiogenesis.

Cell membrane GM1 ganglioside is a functional coreceptor for fibroblast growth factor 2.

Free gangliosides bind fibroblast growth factor 2 (FGF2), thus preventing cell interaction and biological activity of the growth factor in endothelial cells. Here we investigated the role of cell-associated gangliosides in mediating the biological activity of FGF2. Treatment of endothelial cells of different origin with the ganglioside biosynthesis inhibitors fumonisin B1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or D-1-threo-1-phenyl-2-hexa-decanoylamino-3-pyrrolidino-1-propanol-HCl, impairs their capacity to proliferate when exposed to FGF2. Also, the mitogenic activity of FGF2 is inhibited by the GM1-binding cholera toxin B subunit (CTB). Conversely, overloading of endothelial GM 7373 cell membranes with exogenous GM1 causes a 10-fold increase of the mitogenic potency of FGF2. 125I-FGF2 binds to cell membrane GM1 (Kd = 3 nM) in complex ganglioside/heparan sulfate-deficient Chinese hamster ovary (CHO)-K1-pgsA745 cell mutants that were overloaded with exogenous GM1. Moreover, FGF2 competes with FITC-CTB for the binding to cell membrane GM1 in different CHO cell lines independently of their capacity to express heparan sulfate proteoglycans. Conversely, CTB inhibits cell proliferation triggered by FGF2 in CHO cells overexpressing the tyrosine kinase FGF receptor 1. Finally, GM1-overloading confers to FGF receptor 1-transfected, complex ganglioside-deficient CHO-K1 cell mutants the capacity to proliferate when stimulated by FGF2. This proliferation is inhibited by CTB. Cell proliferation triggered by serum or by phorbol 12-myristate 13-acetate is instead independent of the cell membrane ganglioside milieu. In conclusion, cell membrane GM1 binds FGF2 and is required for the mitogenic activity of the growth factor. Our data indicate that cell-associated gangliosides may act as functional FGF2 co-receptors in different cell types.

Biological activity of substrate-bound basic fibroblast growth factor (FGF2): Recruitment of FGF receptor-1 in endothelial cell adhesion contacts

Substrate-bound FGF2 promotes endothelial cell adhesion by interacting with αvβ3 integrin. Here, endothelial GM7373 cells spread and organize focal adhesion plaques on immobilized FGF2, fibronectin (FN), and vitronectin (VN). αvβ3 integrin, paxillin, focal adhesion kinase, vinculin and pp60src localize in cell-substratum contact sites on FGF2, FN or VN. However, only immobilized FGF2 induces a long-lasting activation of extracellular signal-regulated kinases1/2 (ERK1/2) and cell proliferation that was inhibited by the ERK1/2 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−-ΔFGFR1 mutant (GM7373-ΔFGFR1 cells) or with the full-length receptor (GM7373-FGFR1 cells). Both transfectants adhere and spread on FGF2 but GM7373-ΔFGFR1 cells do not proliferate. Also, parental and GM7373-FGFR1 cells, but not GM7373-ΔFGFR1 cells, undergo morphological changes and increased motility on FGF2-coated plastic. Finally, FGFR1, but not TK−-ΔFGFR1, 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 αvβ3 integrin in endothelial cells.

EXPRESSION OF BASIC FIBROBLAST GROWTH FACTOR AND ITS RECEPTORS IN HUMAN FETAL MICROGLIA CELLS

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long‐term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA‐stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross‐reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell‐associated Mr 18,000 protein as well as trace amounts of the Mr 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus.

Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists

The HIV‐1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat‐antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O‐sulfated K5 with high sulfation degree (K5‐OS(H)) and N,O‐sulfated K5 with high (K5‐N,OS(H)) or low (K5‐N,OS(L)) sulfation degree, but not unmodified K5, N‐sulfated K5, and O‐sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV‐LTR‐transactivation. Also, K5‐OS(H) and K5‐N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor‐2 on endothelial cell (EC) surface. Finally, K5‐OS(H) inhibits αvβ3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5‐OS(H) and K5‐N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti‐AIDS therapies.

αvβ3-integrin-dependent activation of focal adhesion kinase mediates NF-κB activation and motogenic activity by HIV-1 Tat in endothelial cells

Once in the extracellular environment, the transactivator protein HIV-1 Tat exerts several pleiotropic effects by interacting with different cellular receptors, including integrin αvβ3. Real-time surface plasmon resonance analysis reveals that Tat/αVβ3 interaction occurs with rapid kinetics (association and dissociation rates equal to 1.16×107 M-1 s-1 and 3.78×10-1 s-1, respectively) and high affinity (dissociation constant = 32 nM). Through this interaction, substratum-immobilized Tat promotes adhesion and motogenic activity in endothelial cells. Also, αvβ3/Tat interaction triggers the activation of focal adhesion kinase, RhoA and pp60src. Overexpression of the dominant negative form of focal adhesion kinase, but not of an inactive Leu1034Ser substitution mutant isoform, impairs the activation of focal adhesion kinase and RhoA, but not that of pp60src, without affecting endothelial cell adhesion and spreading. αvβ3/Tat interaction triggers the activation of NF-κB in endothelial cells in a focal adhesion kinase-, RhoA- and pp60src-dependent manner, as shown in dominant negative focal adhesion kinase transfectants or using specific pharmacological inhibitors. Finally, the activation of focal adhesion kinase, RhoA, NF-κB and pp60src are required to mediate the motogenic activity of Tat in endothelial cells. Since Tat accumulates in an immobilized form in the extracellular matrix, these results provide new biochemical and biological insights about αvβ3/Tat interaction exploitable for the design of anti-Tat strategies.

Heparin/Heparan Sulfate Proteoglycans Glycomic Interactome in Angiogenesis: Biological Implications and Therapeutical Use

Angiogenesis, the process of formation of new blood vessel from pre-existing ones, is involved in various intertwined pathological processes including virus infection, inflammation and oncogenesis, making it a promising target for the development of novel strategies for various interventions. To induce angiogenesis, angiogenic growth factors (AGFs) must interact with pro-angiogenic receptors to induce proliferation, protease production and migration of endothelial cells (ECs). The action of AGFs is counteracted by antiangiogenic modulators whose main mechanism of action is to bind (thus sequestering or masking) AGFs or their receptors. Many sugars, either free or associated to proteins, are involved in these interactions, thus exerting a tight regulation of the neovascularization process. Heparin and heparan sulfate proteoglycans undoubtedly play a pivotal role in this context since they bind to almost all the known AGFs, to several pro-angiogenic receptors and even to angiogenic inhibitors, originating an intricate network of interaction, the so called “angiogenesis glycomic interactome”. The decoding of the angiogenesis glycomic interactome, achievable by a systematic study of the interactions occurring among angiogenic modulators and sugars, may help to design novel antiangiogenic therapies with implications in the cure of angiogenesis-dependent diseases.

Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of human immunodeficiency virus type 1 tat and gp120 proteins

ABSTRACT Human immunodeficiency virus (HIV) Tat and gp120 intriguingly share the feature of being basic peptides that, once released by HIV+ cells, bind to polyanionic heparan sulfate proteoglycans (HSPGs) on target uninfected cells, contributing to the onset of AIDS-associated pathologies. To identify multitarget anti-HIV prodrugs, we investigated the gp120 and Tat antagonist potentials of a series of polyanionic synthetic sulfonic acid polymers (SSAPs). Surface plasmon resonance revealed that SSAPs inhibit with a competitive mechanism of action the binding of Tat and gp120 to surface-immobilized heparin, an experimental condition that resembles binding to cellular HSPGs. Accordingly, SSAPs inhibited HSPG-dependent cell internalization and the transactivating activity of Tat. Little is known about the binding of free gp120 to target cells. Here, we identified two classes of gp120 receptors expressed on endothelial cells, one of which was consistent with an HSPG-binding, low-affinity/high-capacity receptor that is inhibited by free heparin. SSAPs inhibited the binding of free gp120 to endothelial cells, as well as its capacity to induce apoptosis in the same cells. In all the assays, poly(4-styrenesulfonic acid) (PSS) proved to be the most potent antagonist of Tat and gp120. Accordingly, PSS bound both proteins with high affinity. In conclusion, SSAPs represent an interesting class of compounds that bind both gp120 and Tat and inhibit their HSPG-dependent cell surface binding and pathological effects. As these activities contribute to both AIDS progression and associated pathologies, SSAPs can be considered prototypic molecules for the development of multitarget drugs for the treatment of HIV infection and AIDS-associated pathologies.