RESUMO
Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between ß-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-ßH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-ßH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy ß-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.
Assuntos
Efrinas/metabolismo , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Resistência à Insulina , Secreção de Insulina/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Animais , Linhagem Celular , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BLRESUMO
Vascular endothelial growth factor receptor-2 (VEGFR2) is the main pro-angiogenic receptor expressed by endothelial cells (ECs). Using surface plasmon resonance, immunoprecipitation, enzymatic digestion, immunofluorescence and cross-linking experiments with specific sugar-binding lectins, we demonstrated that VEGFR2 bears both α,1-fucose and α(2,6)-linked sialic acid (NeuAc). However, only the latter is required for VEGF binding to VEGFR2 and consequent VEGF-dependent VEGFR2 activation and motogenic response in ECs. Notably, downregulation of ß-galactoside α(2,6)-sialyltransferase expression by short hairpin RNA transduction inhibits VEGFR2 α(2,6) sialylation that is paralleled by an increase of ß-galactoside α(2,3)-sialyltransferase expression. This results in an ex-novo α(2,3)-NeuAc sialylation of the receptor that functionally replaces the lacking α(2,6)-NeuAc, thus allowing VEGF/VEGFR2 interaction. In keeping with the role of VEGFR2 sialylation in angiogenesis, the α(2,6)-NeuAc-binding lectin Sambucus nigra (SNA) prevents VEGF-dependent VEGFR2 autophosphorylation and EC motility, proliferation and motogenesis. In addition, SNA exerts a VEGF-antagonist activity in tridimensional angiogenesis models in vitro and in the chick-embryo chorioallantoic membrane neovascularization assay and mouse matrigel plug assay in vivo. In conclusion, VEGFR2-associated NeuAc plays an important role in modulating VEGF/VEGFR2 interaction, EC pro-angiogenic activation and neovessel formation. VEGFR2 sialylation may represent a target for the treatment of angiogenesis-dependent diseases.
Assuntos
Neovascularização Patológica/metabolismo , Lectinas de Plantas/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Inativadoras de Ribossomos/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide , Regulação para Baixo , Células Endoteliais/metabolismo , Feminino , Imunofluorescência , Galactosídeos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/metabolismo , Neovascularização Fisiológica , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno , Sialiltransferases/genética , Sialiltransferases/metabolismo , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Syndecan-1 is a heparan sulfate proteoglycan (HSPG) commonly upregulated in AIDS-related B lymphoid malignancies. Tat is the main HIV-1 transactivating factor that has a major role in the pathogenesis of AIDS-related lymphomas (ARL) by engaging heparan sulfate proteoglycans (HSPGs), chemokine receptors and integrins at the lymphoid cell (LC) surface. Here B-lymphoid Namalwa cell clones that do not express or overexpress syndecan-1 (EV-Ncs and SYN-Ncs, respectively) were compared for their responsiveness with Tat: in the absence of syndecan-1, Tat induces a limited EV-Nc migration via C-X-C motif chemokine receptor 4 (CXCR4), G-proteins and Rac. Syndecan-1 overexpression increases SYN-Nc responsiveness to Tat and makes this response independent from CXCR4 and G-protein and dependent instead on pp60src phosphorylation. Tat-induced SYN-Nc migration and pp60src phosphorylation require the engagement of αvß3 integrin and consequent pp125FAK phosphorylation. This complex set of Tat-driven activations is orchestrated by the direct interaction of syndecan-1 with pp60src and its simultaneous coupling with αvß3. The Tat/syndecan-1/αvß3 interplay is retained in vivo and is shared also by other syndecan-1+ B-LCs, including BJAB cells, whose responsiveness to Tat is inhibited by syndecan-1 knockdown. In conclusion, overexpression of syndecan-1 confers to B-LCs an increased capacity to migrate in response to Tat, owing to a switch from a CXCR4/G-protein/Rac to a syndecan-1/αvß3/pp60src/pp125FAK signal transduction pathway that depends on the formation of a complex in which syndecan-1 interacts with Tat via its HS-chains, with αvß3 via its core protein ectodomain and with pp60src via its intracellular tail. These findings have implications in ARL progression and may help in identifying new therapeutical targets for the treatment of AIDS-associated neoplasia.
Assuntos
Quinase 1 de Adesão Focal/genética , Integrina alfaVbeta3/genética , Linfócitos/metabolismo , Neoplasias/genética , Sindecana-1/genética , Adesão Celular/genética , Regulação Neoplásica da Expressão Gênica , HIV-1/genética , Humanos , Linfócitos/patologia , Complexos Multiproteicos/genética , Neoplasias/patologia , Fosforilação , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Receptores CXCR4/genética , Transdução de Sinais/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
INTRODUCTION: Platelet thrombospondin-1 (TSP-1) is a major endogenous regulator of growth factor activity in physiological and pathological processes, including tumor onset, progression and angiogenesis. We previously demonstrated that TSP-1 binds to FGF-2, sequestering the growth factor and inhibiting its angiogenic activity. We also identified a non-peptidic antiangiogenic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of TSP-1. AIM: To identify new small molecule inhibitors of FGF2 that recapitulate the structure and functional properties of the FGF-2-binding site of TSP-1, by investigating the chemical space around SM27. MATERIALS AND METHODS: A similarity-based screening of small molecule libraries has been used to identify candidates, followed by docking calculations, and evaluation of the activity of the resulting compounds in biochemical and biophysical assays, to assess interaction with FGF2, and in experimental models of angiogenesis, to assess biological activity. RESULTS: The used integrated approach allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting FGF2 binding to both heparan sulfate proteoglycans and FGFR1. The compounds inhibited FGF2-induced endothelial cell proliferation, vessel sprouting from aortic rings and angiogenesis in the chorioallantoic membrane assay, with improved potency over SM27. CONCLUSIONS: We have identified new compounds that are valuable as FGF inhibitors for potential therapeutic purposes. Moreover, these compounds are useful chemical tools to identify the minimal stereochemical requirements for FGF2 binding and activity to improve the design of new agents for antineoplastic therapy. ACKNOWLEDGEMENT: Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro).
RESUMO
Angiogenesis and inflammation are closely integrated processes. Fibroblast growth factor-2 (FGF2) is a prototypic angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 exerts its pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. A tight cross-talk exists between FGF2 and the inflammatory response in the modulation of blood vessel growth. Pentraxins act as soluble pattern recognition receptors with a wide range of functions in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with short-pentraxins and possesses a unique N-terminal domain. These structural features indicate that PTX3 may have distinct biological/ligand recognition properties when compared to short-pentraxins. Co-expression of PTX3 and FGF2 has been observed in different inflammation/angiogenesis-dependent diseases. PTX3 binds FGF2 with high affinity and specificity. The interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the angiogenic activity of the growth factor. This suggests that PTX3 may exert a modulatory function by limiting the angiogenic activity of FGF2. An integrated approach that utilized PTX3 fragments, monoclonal antibodies, and surface plasmon resonance analysis has identified the FGF2-binding domain in the unique N-terminal extension of PTX3. On this basis, PTX3-derived synthetic peptides have been designed endowed with a significant antiangiogenic activity in vitro and in vivo. They may provide the basis for the development of novel antiangiogenic FGF2 antagonists.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteína C-Reativa/farmacologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Proteína C-Reativa/química , Humanos , Modelos Moleculares , Conformação Molecular , Neovascularização Patológica/tratamento farmacológico , Proteínas do Tecido Nervoso/química , Peptídeos/químicaRESUMO
Angiogenesis, the process of new blood vessel formation from pre-existing ones, plays a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor growth. The 1980s saw for the first time the identification, purification, and sequencing of the two prototypic heparin-binding angiogenic fibroblast growth factors (FGF) 1 and 2. Since then, 22 structurally-related members of the FGF family and different classes of FGF receptors have been identified. Several experimental evidences point to a role for various FGFs in the neovascularization process that takes place in inflammation, angioproliferative diseases, and tumor growth. Thus, the FGF/FGF receptor system represents a target for the development of anti-angiogenic therapies. Purpose of this review is to summarize the different modalities that have been approached to impair the pro-angiogenic activity of the FGF/FGF receptor system and discuss their possible therapeutic implications.
Assuntos
Inibidores da Angiogênese/farmacologia , Desenho de Fármacos , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neovascularização Fisiológica , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 - a synthetic peptide derived from it and bearing amino acids 1-20 of the phage p8 protein - interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin beta and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100 microM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.
Assuntos
Bacteriófago M13/química , Proteínas do Capsídeo/metabolismo , Produtos do Gene tat/metabolismo , HIV-1/química , Fármacos Anti-HIV/metabolismo , HIV-1/fisiologia , Células HeLa , Humanos , Sinais de Localização Nuclear/fisiologia , Peptídeos Cíclicos/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot growth as a mass above few mm(3) unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.
Assuntos
Inibidores da Angiogênese/farmacologia , Heparina/análogos & derivados , Heparina/farmacologia , Neovascularização Patológica/tratamento farmacológico , Indutores da Angiogênese/antagonistas & inibidores , Animais , Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Proteoglicanas de Heparan Sulfato/metabolismo , Proteoglicanas de Heparan Sulfato/fisiologia , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/fisiopatologiaRESUMO
The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Escherichia coli/metabolismo , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Polissacarídeos/farmacologia , Animais , Células CHO , Sequência de Carboidratos , Bovinos , Células Cultivadas , Embrião de Galinha , Cricetinae , Endotélio Vascular/citologia , Humanos , Polissacarídeos/química , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
Extracellular Tat protein, the transactivating factor of the human immunodeficiency virus type 1 (HIV-1), modulates gene expression, growth, and angiogenic activity in endothelial cells by interacting with the vascular endothelial growth factor (VEGF) receptor-2 (Flk-1/KDR). Recombinant Tat protein, produced as glutathione-S-transferase chimera (GST-Tat), activates mitogen-activated protein kinase (MAPK) ERK(1/2) in human, murine, and bovine endothelial cells whereas GST is ineffective. In bovine aortic endothelial cells, GST-Tat and the 165 amino acid VEGF isoform (VEGF165) induce transient ERK(1/2) phosphorylation with similar potency and kinetics. The synthetic peptide Tat(41-60), but not peptides Tat(1-21) and Tat(71-86), causes ERK(1/2) phosphorylation, thus implicating Tat/KDR interaction in the activation of this signalling pathway. Accordingly, GST-Tat induces ERK(1/2) phosphorylation in KDR-transfected porcine aortic endothelial cells but not in parental cells. MAPK kinase inhibitors PD098059 and U0126 prevent ERK(1/2) phosphorylation by Tat. However, they do not affect the angiogenic activity exerted by Tat in the murine Matrigel plug and chick embryo chorioallantoic membrane assays. Blocking of MAPK kinase activity impairs instead the angiogenic response to VEGF165 and to fibroblast growth factor-2 (FGF-2). Our data demonstrate that ERK(1/2) activation following the interaction of HIV-1 Tat protein with endothelial cell Flk-1/KDR receptor does not represent an absolute requirement for a full angiogenic response to this growth factor that appears to utilize mechanism(s) at least in part distinct from those triggered by other prototypic angiogenic growth factors.
Assuntos
Endotélio Vascular/fisiologia , Produtos do Gene tat/metabolismo , HIV-1/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Fisiológica/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Animais , Butadienos/farmacologia , Bovinos , Células Cultivadas , Colágeno , Combinação de Medicamentos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Produtos do Gene tat/farmacologia , Humanos , Cinética , Laminina , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Nitrilas/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosforilação , Proteoglicanas , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 Tat protein, released from HIV-infected cells, may act as a pleiotropic heparin-binding growth factor. From this observation, extracellular Tat has been implicated in the pathogenesis of AIDS and of AIDS-associated pathologies. Here we demonstrate that the heparin analog pentosan polysulfate (PPS) inhibits the interaction of glutathione S-transferase (GST)-Tat protein with heparin immobilized to a BIAcore sensor chip. Competition experiments showed that Tat-PPS interaction occurs with high affinity (K(d) = 9.0 nm). Also, GST.Tat prevents the binding of [(3)H]heparin to GST.Tat immobilized to glutathione-agarose beads. In vitro, PPS inhibits GST.Tat internalization and, consequently, HIV-1 long terminal repeat transactivation in HL3T1 cells. Also, PPS inhibits cell surface interaction and mitogenic activity of GST.Tat in murine adenocarcinoma T53 Tat-less cells. In all assays, PPS exerts its Tat antagonist activity with an ID(50) equal to approximately 1.0 nm. In vivo, PPS inhibits the neovascularization induced by GST.Tat or by Tat-overexpressing T53 cells in the chick embryo chorioallantoic membrane. In conclusion, PPS binds Tat protein and inhibits its cell surface interaction, internalization, and biological activity in vitro and in vivo. PPS may represent a prototypic molecule for the development of novel Tat antagonists with therapeutic implications in AIDS and AIDS-associated pathologies, including Kaposi's sarcoma.
Assuntos
Produtos do Gene tat/antagonistas & inibidores , HIV-1/metabolismo , Poliéster Sulfúrico de Pentosana/farmacologia , Animais , Linhagem Celular , Embrião de Galinha , Endocitose , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV/genética , Humanos , Poliéster Sulfúrico de Pentosana/metabolismo , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Ativação Transcricional/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Tat, the transactivator protein of human immunodeficiency virus-1, has the unusual capacity of being internalized by cells when present in the extracellular milieu. This property can be exploited for the cellular delivery of heterologous proteins fused to Tat both in cell culture and in living animals. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate (HS) proteoglycans act as receptors for extracellular Tat uptake. Cells genetically defective in the biosynthesis of fully sulfated HS are selectively impaired in the internalization of recombinant Tat fused to the green fluorescent protein, as evaluated by both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell surface HS proteoglycans also mediate physiological internalization of Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells and cells genetically impaired in proteoglycan synthesis are equally proficient in the extracellular release of Tat, thus indicating that proteoglycans are not required for this process. The ubiquitous distribution of HS proteoglycans is consistent with the efficient intracellular delivery of heterologous proteins fused with Tat to different mammalian cell types.
Assuntos
Endocitose/fisiologia , Produtos do Gene tat/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Animais , Células CHO , Cricetinae , Células HeLa , Humanos , Cinética , Ativação Transcricional/fisiologiaRESUMO
Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.
Assuntos
Produtos do Gene tat/metabolismo , HIV-1 , Trombospondina 1/metabolismo , Adenocarcinoma/irrigação sanguínea , Animais , Antineoplásicos/metabolismo , Disponibilidade Biológica , Embrião de Galinha , Produtos do Gene tat/genética , Repetição Terminal Longa de HIV , Células HeLa , Heparina/farmacologia , Humanos , Camundongos , Camundongos Transgênicos , Mitógenos/metabolismo , Neovascularização Patológica , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumour neovascularization. Several growth factors and cytokines have been shown to stimulate endothelial cell proliferation in vitro and in vivo and among them FGF2 was one of the first to be characterised. FGF2 is a Mr 18,000 heparin-binding cationic polypeptide that induces proliferation, migration, and protease production in endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells through two distinct classes of receptors, the high affinity tyrosine-kinase receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix. Besides experimental evidence for paracrine mode of action for FGF2, some observations raise the hypothesis that FGF2 may also play an autocrine role in endothelial cells. FGF2 may therefore represent a target for anti-angiogenic therapies. In order to assess the angiostatic potential of different classes of compounds, novel experimental models have been developed based on the autocrine and/or the paracrine capacity of FGF2.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Neovascularização Patológica/metabolismo , Inibidores da Angiogênese , Animais , Endotélio Vascular/citologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Hemangioma/metabolismo , Hemangioma/patologia , Humanos , Camundongos , Modelos Biológicos , Polímeros , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Ácidos Sulfônicos , Transfecção , Células Tumorais CultivadasRESUMO
Tat protein, a transactivating factor of the human immunodeficiency virus type I, acts also as an extracellular molecule. Heparin affects the bioavailability and biological activity of extracellular Tat (Rusnati, M., Coltrini, D., Oreste, P., Zoppetti, G., Albini, A., Noonan, D., D'Adda di Fagagna, F., Giacca, M., and Presta, M. (1997) J. Biol. Chem. 272, 11313-11320). Here, a series of homogeneously sized, (3)H-labeled heparin fragments were evaluated for their capacity to bind to free glutathione S-transferase (GST)-Tat protein and to immobilized GST-Tat. Hexasaccharides represent the minimum sized heparin fragments able to interact with GST-Tat at physiological ionic strength. Also, the affinity of binding increases with increasing the molecular size of the oligosaccharides, with large fragments (>/=18 saccharides) approaching the affinity of full-size heparin. 6-Mer heparin binds GST-Tat with a dissociation constant (K(d)) equal to 0.7 +/- 0.4 microM and a molar oligosaccharide:GST-Tat ratio of about 1:1. Interaction of GST-Tat with 22-mer or full-size heparin is consistent instead with two-component binding. At subsaturating concentrations, a single molecule of heparin interacts with 4-6 molecules of GST-Tat with high affinity (K(d) values in the nanomolar range of concentration); at saturating concentrations, heparin binds GST-Tat with lower affinity (K(d) values in the micromolar range of concentration) and a molar oligosaccharide:GST-Tat ratio of about 1:1. In agreement with the binding data, a positive correlation exists between the size of heparin oligosaccharides and their capacity to inhibit cell internalization, long terminal repeat-transactivating activity of extracellular Tat in HL3T1 cells, and its mitogenic activity in murine adenocarcinoma T53 Tat-less cells. The data demonstrate that the modality of heparin-Tat 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.
Assuntos
Produtos do Gene tat/metabolismo , HIV-1/metabolismo , Heparina/metabolismo , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade , Primers do DNA , Heparina/química , Heparina/isolamento & purificação , Humanos , Peso Molecular , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth.
Assuntos
Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Compostos de Enxofre/farmacologia , Animais , Ligação Competitiva , Células CHO , Bovinos , Células Cultivadas , Simulação por Computador , Cricetinae , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Heparina/química , Heparina/farmacologia , Modelos Moleculares , Mimetismo Molecular , Polímeros/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácidos Sulfônicos/farmacologia , Compostos de Enxofre/química , Tripsina/metabolismoRESUMO
Kaposi's sarcoma (KS), a neoplasm often associated with iatrogenic and acquired immunosuppression, is characterized by prominent angiogenesis. Angiogenic factors released by both KS and host cells, as well as HHV-8 and HIV viral products, have been implicated in the pathogenesis of this lesion. Angiogenesis is the result of imbalance among angiogenesis promoters and inhibitors, which disrupts homeostasis. The aim of this study was to investigate the expression and mechanism of KS control of thrombospondin-1 (TSP), a physiological inhibitor of angiogenesis. Immunohistochemical analysis of four KS lesions showed only spotty reactivity for TSP in the stroma and in less than 10 per cent of lesional blood vessels. In addition, the typical KS spindle cells were not stained. In agreement with these findings, decreased levels of TSP were measured with an ELISA assay in the supernatants of cultured KS cells, compared with endothelial cells. In vitro, TSP inhibited the endothelial cell proliferation and motility induced by KS cell supernatants. TSP also prevented endothelial cell motility induced by Tat, a product of HIV-1 endowed with angiogenic potential and implicated in the pathogenesis of AIDS-KS. In vivo, TSP inhibited the angiogenic activity exerted by Tat in the Matrigel sponge model. These results suggest that TSP down-regulation might be permissive for the development of KS-associated angiogenesis.
Assuntos
Produtos do Gene tat/farmacologia , Neovascularização Patológica/metabolismo , Sarcoma de Kaposi/metabolismo , Trombospondina 1/análise , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neovascularização Patológica/etiologia , Sarcoma de Kaposi/patologia , Trombospondina 1/farmacologia , Células Tumorais CultivadasRESUMO
Angiogenesis has been identified as an important target for antineoplastic therapy. The use of purine analogue antimetabolites in combination chemotherapy of solid tumors has been proposed. To assess the possibility that selected purine analogues may affect tumor neovascularization, 6-methylmercaptopurine riboside (6-MMPR), 6-methylmercaptopurine, 2-aminopurine, and adenosine were evaluated for the capacity to inhibit angiogenesis in vitro and in vivo. 6-MMPR inhibited fibroblast growth factor-2 (FGF2)-induced proliferation and delayed the repair of mechanically wounded monolayer in endothelial GM 7373 cell cultures. 6-MMPR also inhibited the formation of solid sprouts within fibrin gel by FGF2-treated murine brain microvascular endothelial cells and the formation of capillary-like structures on Matrigel by murine aortic endothelial cells transfected with FGF2 cDNA. 6-MMPR affected FGF2-induced intracellular signaling in murine aortic endothelial cells by inhibiting the phosphorylation of extracellular signal-regulated kinase-2. The other molecules were ineffective in all of the assays. In vivo, 6-MMPR inhibited vascularization in the chick embryo chorioallantoic membrane and prevented blood vessel formation induced by human endometrial adenocarcinoma specimens grafted onto the chorioallantoic membrane. Also, topical administration of 6-MMPR caused the regression of newly formed blood vessels in the rabbit cornea. Thus, 6-MMPR specifically inhibits both the early and the late phases of the angiogenesis process in vitro and exerts a potent anti-angiogenic activity in vivo. These results provide a new rationale for the use of selected purine analogues in combination therapy of solid cancer.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Metiltioinosina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , 2-Aminopurina/farmacologia , Adenocarcinoma/irrigação sanguínea , Adenosina/farmacologia , Alantoide/irrigação sanguínea , Alantoide/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Aorta/citologia , Encéfalo/irrigação sanguínea , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Bovinos , Células Cultivadas , Embrião de Galinha , Córion/irrigação sanguínea , Córion/efeitos dos fármacos , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Depressão Química , Neoplasias do Endométrio/irrigação sanguínea , Endotélio Vascular/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Mercaptopurina/análogos & derivados , Mercaptopurina/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , Transplante de Neoplasias , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Coelhos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
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, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. 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), FGF-2(86-96), 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 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 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, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, 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.
Assuntos
Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Gangliosídeos/metabolismo , Animais , Sítios de Ligação , Sequência de Carboidratos , Bovinos , Linhagem Celular Transformada , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Gangliosídeos/química , Gangliosídeos/farmacologia , Humanos , Cinética , Dados de Sequência Molecular , Neovascularização Patológica/etiologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
A close relationship exists between angiogenesis and the formation of vascular lesions. The development of the vascular system in the chick embryo chorioallantoic membrane (CAM) may thus represent a model to study the effects of the deregulation of endothelial cell behaviour. Alterations of the developing vascular tree of the CAM were observed after exposure to murine aortic endothelial (MAE) cells overexpressing human fibroblast growth factor-2 (FGF2) cDNA (pZipFGF2 MAE cells), or to their conditioned medium (CM). pZipFGF2 MAE cells injected into the allantoic sac or applied on to the CAM of day 8-9 chick embryos induce neovascularization and the appearance of haemangioma-like lesions. This activity was not prevented by anti-FGF2 antibodies. The CM from pZipFGF2 MAE cells was also active when adsorbed into a gelatin sponge and applied on to the CAM, both in the absence and in the presence of anti-FGF2 antibodies. No effects on vessel development were exerted by parental MAE cells, FGF2-transfected NIH 3T3 fibroblasts, or their conditioned media. In vitro, pZipFGF2 MAE cell CM caused parental MAE cells to invade fibrin gels and to undergo morphogenesis on Matrigel. This activity was not mimicked by recombinant FGF2 nor affected by anti-FGF2 antibodies, and depended on a M (r) approximately 45 000 heat-labile heparin-binding factor. Size exclusion chromatography of pZipFGF2 MAE cell CM demonstrated that the in vitro activity co-purified with an in vivo angiogenic capacity. Thus, FGF2 overexpression in mouse endothelial cells induces the production of an angiogenic activity distinct from FGF2, which may contribute to the genesis of angioproliferative lesions.