RESUMO
The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.
Assuntos
Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular , Processamento Alternativo , Sequência de Aminoácidos , Indutores da Angiogênese/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Permeabilidade Capilar/genética , Permeabilidade Capilar/fisiologia , Quimiotaxia/efeitos dos fármacos , Clonagem Molecular , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Cobaias , Células HEK293 , Humanos , Camundongos , Mitógenos/farmacologia , Mitose/efeitos dos fármacos , Mitose/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Neuropilina-1/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.
Assuntos
Neovascularização de Coroide/tratamento farmacológico , Proteoglicanas de Heparan Sulfato/farmacologia , Degeneração Macular/tratamento farmacológico , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Inibidores da Angiogênese/química , Animais , Proliferação de Células/efeitos dos fármacos , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Cristalografia por Raios X , Células Endoteliais/efeitos dos fármacos , Olho/efeitos dos fármacos , Olho/patologia , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/imunologia , Heparina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/ultraestrutura , Injeções Intravítreas , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/ultraestrutura , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Corpo Vítreo/efeitos dos fármacosRESUMO
We tested cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/-KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.
Assuntos
Neoplasias Colorretais/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Indutores da Angiogênese/metabolismo , Animais , Anticorpos/imunologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Células Mieloides/metabolismo , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.
Assuntos
Hormônios Gastrointestinais/metabolismo , Células Mieloides/metabolismo , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Neuropeptídeos/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Hormônios Gastrointestinais/antagonistas & inibidores , Hormônios Gastrointestinais/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Camundongos , Camundongos Nus , Células Mieloides/efeitos dos fármacos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
PURPOSE: Bv8, also known as prokineticin 2, has been recently shown to be a mediator of myeloid cell-dependent tumor angiogenesis in mouse models. We wished to determine whether these findings might be potentially relevant to human disease. EXPERIMENTAL DESIGN: We characterized Bv8 expression in human blood cells in vitro and in vivo, and did Bv8 immunohistochemistry in human tumor sections. We also partially purified Bv8 from human neutrophils and tested its bioactivity. RESULTS: We found that Bv8 expression is regulated by several cytokines in a cell type-specific fashion. Both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor induced Bv8 expression in neutrophils and bone marrow cells, whereas interleukin 10 up-regulated Bv8 expression in monocytes and lymphocytes. Bv8 potently promoted neutrophil chemotaxis. Bv8 protein isolated from human neutrophils was found to be biologically active. Of the two receptors for Bv8 [prokineticin receptor 1(PKR1)/endocrine gland-derived vascular endothelial growth factor receptor 1 (EG-VEGFR1) and PKR2/EG-VEGFR2], only PKR2/EG-VEGFR2 was detectable in human neutrophils. Also, we found a marked up-regulation of Bv8 mRNA and protein in peripheral blood mononuclear cells from G-CSF-treated donors compared with those from untreated individuals, verifying our in vitro observations. Finally, immunohistochemistry showed Bv8 expression in neutrophils infiltrating human tumors. CONCLUSIONS: These results provide the basis for further investigation of the pathophysiologic role of Bv8 in human tumors and inflammatory disorders and, potentially, for therapeutic application of Bv8 inhibitors.
Assuntos
Hormônios Gastrointestinais/sangue , Monócitos/metabolismo , Neuropeptídeos/sangue , Neutrófilos/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Hormônios Gastrointestinais/metabolismo , Hormônios Gastrointestinais/farmacologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Neutrófilos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.
Assuntos
Antineoplásicos/administração & dosagem , Antígeno CD11b/metabolismo , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Receptores de Quimiocinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.
Assuntos
Neovascularização Fisiológica , Proteínas/metabolismo , Estresse Fisiológico , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/efeitos dos fármacos , Feminino , Glicosilação/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Hexosaminas/farmacologia , Membro Posterior/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microvasos/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pele/patologia , Estresse Fisiológico/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Quimiocina CXCL12/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Cricetinae , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Mapeamento de Epitopos , Feminino , Humanos , Camundongos , Modelos Moleculares , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Conformação Proteica , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1(+) myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1(+) cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1(+) cells. CONCLUSIONS/SIGNIFICANCE: We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1(+) myeloid cells to facilitate growth and/or angiogenesis.
Assuntos
Antígeno CD11b/fisiologia , Quimiocina CXCL12/fisiologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Neoplasias Experimentais/fisiopatologia , Proteína Wnt1/fisiologia , Animais , Camundongos , Neoplasias Experimentais/virologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Tumor- or cancer-associated fibroblasts (TAFs or CAFs) from different tumors exhibit distinct angiogenic and tumorigenic properties. Unlike normal skin fibroblasts or TAFs from TIB6 tumors that are sensitive to anti-VEGF treatment (TAF-TIB6), TAFs from resistant EL4 tumors (TAF-EL4) can stimulate TIB6 tumor growth even when VEGF is inhibited. We show that platelet-derived growth factor C (PDGF-C) is upregulated in TAFs from resistant tumors. PDGF-C-neutralizing antibodies blocked the angiogenesis induced by such TAFs in vivo, slowed the growth of EL4 and admixture (TAF-EL4 + TIB6) tumors, and exhibited additive effects with anti-VEGF-A antibodies. Hence, our data reveal an additional mechanism for TAF-mediated tumorigenesis and suggest that some tumors may overcome inhibition of VEGF-mediated angiogenesis through upregulation of PDGF-C.
Assuntos
Anticorpos/uso terapêutico , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Fibroblastos/metabolismo , Linfocinas/metabolismo , Neovascularização Patológica/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos/imunologia , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Separação Celular , Transformação Celular Neoplásica/imunologia , Células Cultivadas , Quimiocina CXCL12/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Imunoterapia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Regulação para CimaRESUMO
Cells of the innate immune system have a key role in maintaining homeostasis by providing the first line of defense against many pathogens. Innate immunity can also modulate the activity of acquired immunity by several mechanisms. However, subsets of myeloid cells can facilitate tumor growth, because these cells produce angiogenic factors and can also prevent the immune system from attacking tumor cells. Recent studies also emphasize the role of myeloid cells in mediating refractoriness to anti-VEGF treatments. This function of myeloid cells occurs through a proangiogenic pathway that is, at least in part, driven by the secreted protein Bv8. This review summarizes recent findings on the complex role of bone marrow-derived cells in tumor growth.