RESUMO
The Bone Morphogenetic Protein 4 (BMP4) regulates multiple biological processes, including vascular development and angiogenesis. Here, we investigated the role of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in mediating the angiogenic activity of BMP4. BMP4 induces a rapid relocation and phosphorylation of VEGFR2 on the endothelial cell membrane. These effects occur in the absence of a direct interaction of BMP4 and/or BMP receptors with VEGFR2. At variance, BMP4, by interacting with the BMPRI-II hetero-complex, induces c-Src phosphorylation which, in turn, activates VEGFR2, leading to an angiogenic response. Accordingly, the BMPR inhibitor dorsomorphin prevents c-Src activation and specific inhibition of c-Src significantly reduces downstream VEGFR2 phosphorylation and the angiogenic activity exerted by BMP4 in a chick embryo chorioallantoic membrane assay. Together, our data indicate that the pro-angiogenic activity exerted by BMP4 in endothelial cells is mediated by a BMPR-mediated intracellular transactivation of VEGFR2 via c-Src.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Bovinos , Embrião de Galinha , Humanos , Ativação TranscricionalRESUMO
Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Proteína C-Reativa/farmacologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Componente Amiloide P Sérico/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mitógenos/antagonistas & inibidores , Neovascularização Fisiológica/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Recombinantes/farmacologiaRESUMO
OBJECTIVE: Angiogenesis is regulated by the balance between pro- and antiangiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor 2 (FGF2) is a major proangiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally coexpressed with its ligand tumor necrosis factor-stimulated gene-6 (TSG-6), a secreted glycoprotein that cooperates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis. METHODS AND RESULTS: Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine subcutaneous Matrigel plug assay, the inhibition being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals. CONCLUSION: TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the coexpression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2.
Assuntos
Proteína C-Reativa/metabolismo , Moléculas de Adesão Celular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Componente Amiloide P Sérico/metabolismo , Animais , Ligação Competitiva , Proteína C-Reativa/deficiência , Proteína C-Reativa/genética , Células CHO , Bovinos , Moléculas de Adesão Celular/genética , Embrião de Galinha , Cricetinae , Cricetulus , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Células HEK293 , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Componente Amiloide P Sérico/genética , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
Vascular endothelial growth factor receptor-2 (VEGFR2) is an endothelial cell receptor that plays a pivotal role in physiologic and pathologic angiogenesis and is a therapeutic target for angiogenesis-dependent diseases, including cancer. By leveraging on a dedicated nanomechanical biosensor, we investigated the nanoscale mechanical phenomena intertwined with VEGFR2 surface recognition by its prototypic ligand VEGF-A and its noncanonical ligand gremlin. We found that the two ligands bind the immobilized extracellular domain of VEGFR2 (sVEGFR2) with comparable binding affinity. Nevertheless, they interact with sVEGFR2 with different binding kinetics and drive different in-plane piconewton intermolecular forces, suggesting that the binding of VEGF-A or gremlin induces different conformational changes in sVEGFR2. These behaviors can be effectively described in terms of a different "nanomechanical affinity" of the two ligands for sVEGFR2, about 16-fold higher for VEGF-A with respect to gremlin. Such nanomechanical differences affect the biological activity driven by the two angiogenic factors in endothelial cells, as evidenced by a more rapid VEGFR2 clustering and a more potent mitogenic response triggered by VEGF-A in respect to gremlin. Together, these data point to surface intermolecular interactions on cell membrane between activated receptors as a key modulator of the intracellular signaling cascade.
Assuntos
Fenômenos Mecânicos , Nanotecnologia/métodos , Neovascularização Fisiológica , Ressonância de Plasmônio de Superfície/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Citocinas , Transferência Ressonante de Energia de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The bone morphogenic protein antagonist gremlin is expressed during embryonic development and under different pathologic conditions, including cancer. Gremlin is a proangiogenic protein belonging to the cystine-knot superfamily that includes transforming growth factor-ß proteins and the angiogenic vascular endothelial growth factors (VEGFs). Here, we demonstrate that gremlin binds VEGF receptor-2 (VEGFR2), the main transducer of VEGF-mediated angiogenic signals, in a bone morphogenic protein-independent manner. Similar to VEGF-A, gremlin activates VEGFR2 in endothelial cells, leading to VEGFR2-dependent angiogenic responses in vitro and in vivo. Gremlin thus represents a novel proangiogenic VEGFR2 agonist distinct from the VEGF family ligands with implications in vascular development, angiogenesis-dependent diseases, and tumor neovascularization.
Assuntos
Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/agonistas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Linhagem Celular , Galinhas , Células Endoteliais/citologia , Humanos , CamundongosRESUMO
Malnutrition is common after allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT), and interventions directed to correct nutritional status are warranted to improve transplant outcomes. In this prospective study, an oral polymeric formulation enriched with TGF-ß2 (TE-OPF) was explored to correct malnutrition according to Patient-Generated Subjective Global Assessment (PG-SGA). TE-OPF was proposed to 51 consecutive patients who received transplants at our institution for hematological malignancies, and sufficient dose intake was established per protocol as at least 50% of the prescribed dose of TE-OPF: group A received adequate nutritional support; group B, inadequate. The study met the primary outcomes in terms of safety (no adverse events reported during TE-OPF intake except for its disgusting taste) and malnutrition (PG-SGA C 28 days after transplant): severely malnourished patients (PG-SGA C) accounted for 13% in group A and 88.9% in group B (p = 0.000). At the end of the study, after a median follow-up of 416 days, the estimated median Overall Survival (OS) was 734 days for well or moderately nourished patients (PG-SGA A/B) in comparison to 424 for malnourished patients (p = 0.03). Inadequate TE-OPF intake was associated with an increase in acute gastrointestinal Graft Versus Host Disease (GVHD) cumulative incidence (38% vs. 0% p = 0.006). A higher incidence of pneumonia was reported in group B (p = 0.006). IGF-1 levels at 14 and 28 days after transplant were significantly higher in group A and were associated with a lower incidence of acute GVHD (aGVHD). Higher subsets of B, T, and NK cells were found in group A, and a higher number of CD16+ NK cells was associated with a lower incidence of acute GVHD (p = 0.005) and increased survival at the end of the study (p = 0.023). Artificial neural network analysis suggested that inadequate TE-OPF intake, pneumonia, and sepsis significantly affected malnutrition 28 days after alloHSCT and survival 365 days after alloHSCT (normalized importance 100%, 82%, and 68%, respectively). In this exploratory and preliminary study, the use of TE-OPF appeared to reduce the incidence of malnutrition after alloHSCT, but larger and controlled studies are required.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Desnutrição , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Desnutrição/etiologia , Estudos Prospectivos , Fator de Crescimento Transformador beta2RESUMO
Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.
Assuntos
Proteína C-Reativa/química , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Oligopeptídeos/farmacologia , Componente Amiloide P Sérico/química , Sequência de Aminoácidos , Inibidores da Angiogênese/farmacologia , Animais , Sítios de Ligação/genética , Proteína C-Reativa/metabolismo , Células CHO , Proliferação de Células/efeitos dos fármacos , Transplante de Células/métodos , Embrião de Galinha , Cricetinae , Cricetulus , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Componente Amiloide P Sérico/metabolismo , Transplante Heterólogo , Peixe-ZebraRESUMO
Fibroblast growth factor-2 (FGF2) is a potent angiogenic growth factor. Here, gene expression profiling of FGF2-stimulated microvascular endothelial cells revealed, together with a prominent pro-angiogenic profile, a pro-inflammatory signature characterized by the upregulation of pro-inflammatory cytokine/chemokines and their receptors, endothelial cell adhesion molecules and members of the eicosanoid pathway. Real-time quantitative PCR demonstrated early induction of most of the FGF2-induced, inflammation-related genes. Accordingly, chick embryo chorioallantoic membrane (CAM) and murine Matrigel plug angiogenesis assays demonstrated a significant monocyte/macrophage infiltrate in the areas of FGF2-driven neovascularization. Similar results were obtained when the conditioned medium (CM) of FGF2-stimulated endothelial cells was delivered onto the CAM, suggesting that FGF2-upregulated chemoattractants mediate the inflammatory response. Importantly, FGF2-triggered new blood vessel formation was significantly reduced in phosphatidylinositol 3-kinase-gamma null mice exhibiting defective leucocyte migration or in clodronate liposome-treated, macrophage-depleted mice. Furthermore, the viral pan-chemokine antagonist M3 inhibited the angiogenic and inflammatory responses induced by the CM of FGF2-stimulated endothelial cells and impaired FGF2-driven neovascularization in the CAM assay. These findings point to inflammatory chemokines as early mediators of FGF2-driven angiogenesis and indicate a non-redundant role for inflammatory cells in the neovascularization process elicited by the growth factor.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Sequência de Bases , Embrião de Galinha , Primers do DNA , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologiaRESUMO
Angiogenesis, the process of new blood vessel formation, is implicated in various physiological/pathological conditions, including embryonic development, inflammation and tumor growth. Fibroblast growth factor-2 (FGF2) is a heparin-binding angiogenic growth factor involved in various physiopathological processes, including tumor neovascularization. Accordingly, FGF2 is considered a target for antiangiogenic therapies. Thus, numerous natural/synthetic compounds have been tested for their capacity to bind and sequester FGF2 in the extracellular environment preventing its interaction with cellular receptors. We have exploited surface plasmon resonance (SPR) technique in search for antiangiogenic FGF2 binders/antagonists. In this review we will summarize our experience in SPR-based angiogenesis research, with the aim to validate SPR as a first line screening for the identification of antiangiogenic compounds.
RESUMO
OBJECTIVE: Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. METHODS AND RESULTS: Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and alpha(v)beta3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/alpha(v)beta3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. CONCLUSIONS: LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/alpha(v)beta3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.
Assuntos
Inibidores da Angiogênese/biossíntese , Escherichia coli/química , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Engenharia Genética/métodos , Heparina de Baixo Peso Molecular/biossíntese , Polissacarídeos Bacterianos/biossíntese , Inibidores da Angiogênese/genética , Animais , Cápsulas Bacterianas , Células CHO/química , Células CHO/metabolismo , Bovinos , Adesão Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Cricetinae , Cricetulus , Células Endoteliais/química , Células Endoteliais/metabolismo , Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/análogos & derivados , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/análogos & derivados , Fatores de Crescimento de Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/análogos & derivados , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina de Baixo Peso Molecular/síntese química , Heparina de Baixo Peso Molecular/genética , Integrina alfaVbeta3/metabolismo , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Polissacarídeos Bacterianos/genéticaRESUMO
In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK-52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK-52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin-activated signalling. These effects appeared to be strictly associated to the Pb-induced arrest of cell cycle at G0/G1 phase also proved in this cell line.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Poluentes Ambientais/toxicidade , Adesões Focais/efeitos dos fármacos , Rim/efeitos dos fármacos , Chumbo/toxicidade , Actinas/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/análise , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Microtúbulos/efeitos dos fármacos , RatosRESUMO
During melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an epithelial-mesenchymal transition (EMT) process associated with the acquisition of an invasive, prometastatic phenotype. The fibroblast growth factor-2 (FGF2)/FGF receptor (FGFR) system plays a pivotal role in melanoma, leading to autocrine/paracrine induction of tumor cell proliferation and angiogenesis. Long pentraxin-3 (PTX3) interacts with FGF2, and other FGF family members, inhibiting FGF-dependent neovascularization and tumor growth. Here, PTX3 protein and the PTX3-derived acetylated pentapeptide Ac-ARPCA-NH2 inhibit FGF2-driven proliferation and downstream FGFR signaling in murine melanoma B16-F10 cells. Moreover, human PTX3-overexpressing hPTX_B16-F10 cells are characterized by the reversed transition from a mesenchymal to an epithelial-like appearance, inhibition of cell proliferation, loss of clonogenic potential, reduced motility and invasive capacity, downregulation of various mesenchymal markers, and upregulation of the epithelial marker E-cadherin. Accordingly, PTX3 affects cell proliferation and EMT transition in human A375 and A2058 melanoma cells. Also, hPTX_B16-F10 cells showed a reduced tumorigenic and metastatic activity in syngeneic C57BL/6 mice. In conclusion, PTX3 inhibits FGF/FGFR-driven EMT in melanoma cells, hampering their tumorigenic and metastatic potential. These data represent the first experimental evidence about a nonredundant role of the FGF/FGFR system in the modulation of the EMT process in melanoma and indicate that PTX3 or its derivatives may represent the basis for the design of novel therapeutic approaches in FGF/FGFR-dependent tumors, including melanoma.
Assuntos
Proteína C-Reativa/metabolismo , Proteína C-Reativa/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Melanoma/metabolismo , Melanoma/patologia , Proteínas Recombinantes/farmacologia , Componente Amiloide P Sérico/metabolismo , Componente Amiloide P Sérico/farmacologia , Animais , Proteína C-Reativa/genética , Embrião de Galinha , Transição Epitelial-Mesenquimal/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Humanos , Melanoma/genética , Melanoma Experimental , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Componente Amiloide P Sérico/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
Fibroblast growth factor-8b (FGF8b) exerts nonredundant autocrine/paracrine functions in steroid hormone-regulated tumors. Previous observations had shown that the soluble pattern recognition receptor long pentraxin-3 (PTX3) is a natural selective antagonist for a restricted number of FGF family members, inhibiting FGF2 but not FGF1 and FGF4 activity. Here, we assessed the capacity of PTX3 to antagonize FGF8b and to inhibit the vascularization and growth of steroid hormone-regulated tumors. Surface plasmon resonance analysis shows that PTX3 binds FGF8b with high affinity (K(d) = 30-90 nmol/L). As a consequence, PTX3 prevents the binding of FGF8b to its receptors, inhibits FGF8b-driven ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells, and suppresses FGF8b-induced neovascularization in vivo. Also, PTX3 inhibits dihydrotestosterone (DHT)- and FGF8b-driven proliferation of androgen-regulated Shionogi 115 (S115) mouse breast tumor cells. Furthermore, DHT-treated, PTX3 overexpressing hPTX3_S115 cell transfectants show a reduced proliferation rate in vitro and a limited angiogenic activity in the chick embryo chorioallantoic membrane and murine s.c. Matrigel plug assays. Accordingly, hPTX3_S115 cells show a dramatic decrease of their tumorigenic activity when grafted in immunodeficient male mice. These results identify PTX3 as a novel FGF8b antagonist endowed with antiangiogenic and antineoplastic activity with possible implications for the therapy of hormonal tumors.
Assuntos
Androgênios/fisiologia , Inibidores da Angiogênese/farmacologia , Proteína C-Reativa/farmacologia , Fator 8 de Crescimento de Fibroblasto/antagonistas & inibidores , Componente Amiloide P Sérico/farmacologia , Inibidores da Angiogênese/metabolismo , Animais , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Células CHO , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Cricetinae , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Ligação Proteica , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/metabolismoRESUMO
Any molecular recognition reaction supported by a solid phase drives a specific change of the solid-solution interfacial tension. Sessile contact angle (CA) experiments can be readily used to track this thermodynamic parameter, prompting this well-known technique to be reinvented as an alternative, easy-access and label-free way to probe and study molecular recognition events. Here we deploy this technique, renamed for this application CONAMORE (CONtact Angle MOlecular REcognition), to study the interaction of the tumor-derived pro-angiogenic vascular endothelial growth factor-A (VEGF-A) with the extracellular domain of its receptor VEGFR2. We show that CONAMORE recognizes the high affinity binding of VEGF-A at nanomolar concentrations to surface-immobilized VEGFR2 regardless of the presence of a ten-fold excess of a non-specific interacting protein, and that it further proofs its specificity and reliability on competitive binding experiments involving neutralizing anti-VEGF-A antibodies. Finally, CONAMORE shows the outstanding capability to detect the specific interaction between VEGFR2 and low molecular weight ligands, such as Cyclo-VEGI, a VEGFR2 antagonist cyclo-peptide, that weighs about 2 kDa.
Assuntos
Proteínas Angiogênicas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Ressonância de Plasmônio de Superfície/métodos , Fatores de Crescimento Endotelial/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Imobilizadas , Técnicas In Vitro , Ligantes , Peptídeos Cíclicos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Termodinâmica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Fibroblast growth factor receptor-1 (FGFR-1) transduces proangiogenic and proliferative signals in human cancers. Thus, FGFR-1 may represent a target for the development of antiangiogenic/antineoplastic therapies. We screened a human single-chain fragment variable (scFv) antibody phage display library against the extracellular domain of the FGFR-1-IIIc isoform that harbors the FGF binding site. Several phages were isolated and tested for specificity and sensitivity, and the most promising antibody fragment RR-C2 was characterized for its biochemical and biological properties. ScFv RR-C2 specifically recognizes FGFR-1α and FGFR-1ß isoforms in ELISA, Western blotting, and surface plasmon resonance analysis with a K(d) value of 300 and 144 nmol/L for the 2 receptor isoforms, respectively. The antibody fragment also recognizes FGFR-1 when the receptor is exposed on the cell surface, thus preventing the formation of the ternary complex among FGFR-1, its ligand FGF2, and cell surface heparan sulfate proteoglycans. Accordingly, scFv RR-C2 specifically inhibits FGF2-mediated mitogenic activity in endothelial cells of human, bovine, and murine origin in a nanomolar range of concentrations. Also, the antibody fragment prevents FGF2-triggered sprouting of both human umbilical vein endothelial cell spheroids and of murine endothelium from aortic rings. Finally, the antibody fragment hampers the angiogenic activity exerted both by FGF2 in the chick embryo chorioallantoic membrane assay and by S115 mouse mammary tumor cells in the Matrigel plug assay. Taken together, the data show that scFv RR-C2 recognizes and neutralizes FGFR-1 activity in different animal species, including humans, thus representing a novel tool for the development of antiangiogenic/antineoplastic therapies.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Neutralizantes/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Anticorpos de Cadeia Única/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Neutralizantes/uso terapêutico , Especificidade de Anticorpos/efeitos dos fármacos , Bovinos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Feminino , Humanos , Técnicas In Vitro , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Neovascularização Patológica/tratamento farmacológico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
Angiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature, plays a pivotal role in tumor growth, progression and metastasis. Over the last 30 years, numerous pro- and antiangiogenic molecules, their ligands, and intracellular signaling pathways have been identified, and significant efforts have been undertaken to develop antiangiogenic strategies for cancer therapy. Agents that selectively target vascular endothelial growth factor (VEGF) and its receptors have shown promising activity in clinical trials and have been approved for use in selected cancer indications. However, patients may ultimately develop resistance to these drugs. One proposed mechanism of tumor escape from anti-VEGF therapy is the up-regulation of fibroblast growth factor-2 (FGF2). FGF2 is a pleiotropic, angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 is expressed by numerous tumor types and exerts its proangiogenic activity by interacting with tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins expressed on the endothelial cell surface. Experimental evidence suggests that targeting FGF2, in addition to VEGF, might provide synergistic effects in the treatment of angiogenesis-related diseases, including cancer. Several FGF2 inhibitors, with different chemical structure and mechanism of action, have been identified. Recent observations have shown the ability of the soluble pattern recognition receptor long-pentraxin-3 (PTX3) to bind FGF2, thus acting as a FGF2 antagonist. PTX3 binds FGF2 with high affinity and specificity. This interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the angiogenic activity of the growth factor. Further, preliminary observations support the hypothesis that PTX3 may inhibit FGF2-mediated tumor angiogenesis and growth. The identification of the FGF2-binding domain in the unique N-terminal extension of PTX3 has allowed the design of PTX3-derived synthetic peptides endowed with significant antiangiogenic activity in vitro and in vivo. These findings may provide the basis for the development of novel antiangiogenic FGF2 antagonists, with potential implications for cancer therapy.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteína C-Reativa/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Componente Amiloide P Sérico/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/uso terapêutico , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Ligação Proteica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis and inflammation are closely integrated processes in a number of physiological and pathological conditions, including wound healing, psoriasis, diabetic retinopathy, rheumatoid arthritis, arteriosclerosis, and cancer. Fibroblast growth factor-2 (FGF2) belongs 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. Elevated levels of FGF2 have been implicated in the pathogenesis of several diseases characterized by a deregulated angiogenic/inflammatory response. FGF2 induces the expression of a wide repertoire of inflammation-related genes in endothelial cells, including pro-inflammatory cytokines/chemokines and their receptors, endothelial cell adhesion molecules, and components of the prostaglandin pathway. Consistent with this pro-inflammatory signature, in vivo evidence points to a non-redundant role for chemokines and infiltrating monocytes/macrophages in FGF2-driven neovascularization. This review will focus on the cross-talk between FGF2 and the inflammatory response in the modulation of blood vessel growth.
Assuntos
Quimiocinas/fisiologia , Células Endoteliais/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Inflamação/patologia , Macrófagos/fisiologia , Monócitos/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Moléculas de Adesão Celular/metabolismo , Quimiotaxia , Embrião de Galinha , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/fisiologia , Prostaglandinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
The extracellular matrix protein osteopontin (OPN) plays a nonredundant role in atherosclerosis and restenosis. Here we investigated the impact of OPN up-regulation in an in vitro model of re-endothelialization after mechanical injury of the endothelial cell monolayer. Murine aortic endothelial (MAE) cells interact via alpha(v) integrins with the integrin-binding Arg-Gly-Asp OPN sequence and adhere to immobilized OPN. On this basis, MAE cells were stably transfected with a wild-type OPN cDNA (OPN-MAE cells), with an OPN mutant lacking the Arg-Gly-Asp sequence (DeltaRGD-OPN-MAE cells), or with vector alone (mock-MAE cells). When compared with mock-MAE and DeltaRGD-OPN-MAE cells, OPN-MAE cells showed a reduced sprouting activity in fibrin gel, a reduced motility in a Boyden chamber assay, and a reduced capacity to repair the wounded monolayer. Accordingly, OPN-MAE cells at the edge of the wound were unable to form membrane ruffles, to reorganize their cytoskeleton, and to activate the focal adhesion kinase and the small GTPase Rac1, key regulators of the cell entry into the first phase of the cell migration cycle. Accordingly, wounded OPN-MAE cells failed to activate the intracellular signals RhoA and ERK1/2, involved in the later phases of the cell migration cycle. Also, parental MAE cells showed reduced re-endothelialization after wounding when seeded on immobilized OPN and exhibited increased adhesiveness to OPN-enriched extracellular matrix. In conclusion, OPN up-regulation impairs re-endothelialization by inhibiting the first phase of the cell migration cycle via alpha(v) integrin engagement by the extracellular matrix-immobilized protein. This may contribute to the adverse effects exerted by OPN in restenosis and atherosclerosis.
Assuntos
Movimento Celular , Células Endoteliais/metabolismo , Integrina alfaV/metabolismo , Sistema de Sinalização das MAP Quinases , Osteopontina , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Linhagem Celular Transformada , Células Endoteliais/patologia , Matriz Extracelular/metabolismo , Fibrina , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Neuropeptídeos/metabolismo , Osteopontina/genética , Proteínas Recombinantes/genética , Transfecção , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Inflammation plays an important role in the onset of angiogenesis. In the present study, we show that osteopontin (OPN), a proinflammatory mediator involved in tissue repair, induces IL-1beta up-regulation in human monocytes. This was accompanied by the enhanced production of TNF-alpha, IL-8, and IL-6, a decreased release of IL-10, and increased p38 phosphorylation. The supernatants of OPN-treated monocytes were highly angiogenic when delivered on the chick embryo chorioallantoic membrane. The angiogenic response was completely abrogated by a neutralizing anti-IL-1 Ab, thus indicating that this cytokine represents the major proangiogenic factor expressed by OPN-activated monocytes. Accordingly, rIL-1beta mimicked the proangiogenic activity of OPN-treated monocyte supernatants, and IL-1R (type I) was found to be expressed in the chorioallantoic membrane. In conclusion, OPN-activated monocytes may contribute to the onset of angiogenesis through a mechanism mediated by IL-1beta.
Assuntos
Interleucina-1/metabolismo , Monócitos/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Western Blotting , Movimento Celular/fisiologia , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Interleucina-10/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Camundongos , Monócitos/imunologia , Neovascularização Patológica , Osteopontina , Fosforilação , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-DeltaRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial alpha(v)beta(3) integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-DeltaRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-alpha and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.