RESUMO
Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (Apc(Min/+)) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.
Assuntos
Carcinogênese , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Mucosa Intestinal/metabolismo , Intestinos/patologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Animais , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Células-Tronco/patologia , Hipóxia Tumoral , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Preoperative radiotherapy (RT) is commonly used to treat localised soft-tissue sarcomas (STS). Hypoxia is an important determinant of radioresistance. Whether antiangiogenic therapy can 'normalise' tumour vasculature, thereby improving oxygenation, remains unknown. METHODS: Two cohorts were prospectively enrolled. Cohort A evaluated the implications of hypoxia in STS, using the hypoxic tracer (18)F-azomycin arabinoside (FAZA-PET). In cohort B, sunitinib was added to preoperative RT in a dose-finding phase 1b/2 design. RESULTS: In cohort A, 13 out of 23 tumours were hypoxic (FAZA-PET), correlating with metabolic activity (r(2)=0.85; P<0.001). Two-year progression-free (PFS) and overall (OS) survival were 61% (95% CI: 0.44-0.84) and 87% (95% CI: 0.74-1.00), respectively. Hypoxia was associated with radioresistance (P=0.012), higher local recurrence (Hazard ratio (HR): 10.2; P=0.02), PFS (HR: 8.4; P=0.02), and OS (HR: 41.4; P<0.04). In Cohort B, seven patients received sunitinib at dose level (DL): 0 (50 mg per day for 2 weeks before RT; 25 mg per day during RT) and two patients received DL: -1 (37.5 mg per day for entire period). Dose-limiting toxicities were observed in 4 out of 7 patients at DL 0 and 2 out of 2 patients at DL -1, resulting in premature study closure. Although there was no difference in PFS or OS, patients receiving sunitinib had higher local failure (HR: 8.1; P=0.004). CONCLUSION: In STS, hypoxia is associated with adverse outcomes. The combination of sunitinib with preoperative RT resulted in unacceptable toxicities, and higher local relapse rates.
Assuntos
Antineoplásicos/administração & dosagem , Indóis/administração & dosagem , Pirróis/administração & dosagem , Sarcoma/tratamento farmacológico , Sarcoma/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Recidiva Local de Neoplasia , Tomografia por Emissão de Pósitrons , Estudos Prospectivos , Radioterapia Adjuvante , SunitinibeRESUMO
The growth and dissemination of tumors rely on an altered vascular network, which supports their survival and expansion and provides accessibility to the vasculature and a route of transport for metastasizing tumor cells. The remodeling of vascular structures through generation of new vessels (for example, via tumor angiogenesis) is a well studied, even if still quite poorly understood, process in human cancer. Antiangiogenic therapies have provided insight into the contribution of angiogenesis to the biology of human tumors, yet have also revealed the ease with which resistance to antiangiogenic drugs can develop, presumably involving alterations to vascular signaling mechanisms. Furthermore, cellular and/or molecular changes to pre-existing vessels could represent subtle pre-metastatic alterations to the vasculature, which are important for cancer progression. These changes, and associated molecular markers, may forecast the behavior of individual tumors and contribute to the early detection, diagnosis and prognosis of cancer. This review, which primarily focuses on the blood vasculature, explores current knowledge of how tumor vessels can be remodeled, and the cellular and molecular events responsible for this process.
Assuntos
Vasos Sanguíneos/fisiopatologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Animais , Vasos Sanguíneos/patologia , Carcinogênese , Humanos , Linfonodos/irrigação sanguínea , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
The biological function of inhibin-alpha subunit (INH alpha) in prostate cancer (PCa) is currently unclear. A recent study associated elevated levels of INH alpha in PCa patients with a higher risk of recurrence. This prompted us to use clinical specimens and functional studies to investigate the pro-tumourigenic and pro-metastatic function of INH alpha. We conducted a cross-sectional study to determine a link between INH alpha expression and a number of clinicopathological parameters including Gleason score, surgical margin, extracapsular spread, lymph node status and vascular endothelial growth factor receptor-3 expression, which are well-established prognostic factors of PCa. In addition, using two human PCa cell lines (LNCaP and PC3) representing androgen-dependent and -independent PCa respectively, we investigated the biological function of elevated levels of INH alpha in advanced cancer. Elevated expression of INH alpha in primary PCa tissues showed a higher risk of PCa patients being positive for clinicopathological parameters outlined above. Over-expressing INH alpha in LNCaP and PC3 cells demonstrated two different and cell-type-specific responses. INH alpha-positive LNCaP demonstrated reduced tumour growth whereas INH alpha-positive PC3 cells demonstrated increased tumour growth and metastasis through the process of lymphangiogenesis. This study is the first to demonstrate a pro-tumourigenic and pro-metastatic function for INH alpha associated with androgen-independent stage of metastatic prostate disease. Our results also suggest that INH alpha expression in the primary prostate tumour can be used as a predictive factor for prognosis of PCa.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Inibinas/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Separação Celular , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Inibinas/genética , Masculino , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Estadiamento de Neoplasias , Neoplasias da Próstata/genéticaRESUMO
The recent discovery that members of the vascular endothelial growth factor (VEGF) family of secreted glycoproteins can mediate lymphatic vessel growth (lymphangiogenesis) via cell surface receptor tyrosine kinases expressed on endothelial cells has opened the way for therapeutic intervention for pathologies involving dysregulated lymphatic vessel function. At least two members of this family, VEGF-C and VEGF-D, have been shown to induce lymphangiogenesis in vivo. Lymphatic vessels and their specific growth factors have been directly implicated in a number of significant human pathologies. In cancer, VEGF-C and VEGF-D appear to correlate with tumor metastasis and poor patient outcome in a range of prevalent human cancers. Experimental studies have demonstrated that expression of the lymphangiogenic growth factors in tumor models induces increased lymphangiogenesis and results in spread of tumor cells via the lymphatics. In contrast, conditions such as lymphedema, where lymphatic vessels fail to clear fluid from interstitial spaces, are opportunities for which the application of growth factors to generate new lymphatic vessels may be a viable therapeutic option. The list of molecules that control lymphangiogenesis is now expanding, allowing more opportunities for the development of drugs with which to manipulate the relevant signalling pathways. Modulating these pathways and other molecules with specificity to the lymphatic endothelium could offer alternative treatments for a number of important clinical conditions.
Assuntos
Doença , Linfangiogênese/efeitos dos fármacos , Transdução de Sinais , Humanos , Linfangiogênese/fisiologia , Metástase Linfática , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Recent studies involving animal models of cancer and clinicopathological analyses of human tumours suggest that the growth of lymphatic vessels (lymphangiogenesis) in or nearby tumours is associated with the metastatic spread of cancer. The best validated molecular signalling system for tumour lymphangiogenesis involves the secreted proteins vascular endothelial growth factor-C (VEGF-C) and VEGF-D that induce growth of lymphatic vessels via activation of VEGF receptor-3 (VEGFR-3) localised on the surface of lymphatic endothelial cells. In this review, we discuss the evidence supporting a role for this signalling system in the spread of cancer and potential approaches for blocking this system to prevent tumour metastasis.
Assuntos
Linfangiogênese/efeitos dos fármacos , Neoplasias/terapia , Animais , Humanos , Metástase Linfática/prevenção & controle , Vasos Linfáticos/fisiologia , Neoplasias/metabolismo , Neoplasias/patologia , Fator C de Crescimento do Endotélio Vascular/fisiologia , Fator D de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.
Assuntos
Adenoviridae/genética , Doenças da Aorta/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Transdução Genética/métodos , Fator D de Crescimento do Endotélio Vascular/genética , Animais , Aorta , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Cateterismo , Constrição Patológica/terapia , Neovascularização Patológica , Óxido Nítrico/metabolismo , Coelhos , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Fator D de Crescimento do Endotélio Vascular/metabolismoRESUMO
The dysfunction or proliferation of lymphatic vessels (lymphangiogenesis) is linked to a number of pathological conditions including lymphedema and cancer. The recent discovery and characterisation of the lymphangiogenic growth factors vascular endothelial growth factor-C (VEGF-C) and VEGF-D and of their receptor on lymphatic endothelial cells, VEGFR-3, has provided an understanding of the molecular mechanisms controlling the growth of lymphatic vessels. In addition, other genes and protein markers have been identified with specificity for lymphatic endothelium that have enhanced the characterization and isolation of lymphatic endothelial cells. Our growing understanding of the molecules that control lymphangiogenesis allows us to design more specific drugs with which to manipulate the relevant signalling pathways. Modulating these pathways and other molecules with specificity to the lymphatic system could offer alternative treatments for a number of important clinical conditions.
Assuntos
Vasos Linfáticos/patologia , Animais , Humanos , Linfangiogênese , Vasos Linfáticos/metabolismo , Vasos Linfáticos/fisiopatologia , Linfedema/metabolismo , Linfedema/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/fisiologia , Fator D de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.
Assuntos
Apoptose/fisiologia , Fatores de Crescimento Endotelial/farmacologia , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/fisiologia , Endotélio/fisiologia , Sistema Linfático/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Apoptose/efeitos dos fármacos , Técnicas Biossensoriais , Capilares/embriologia , Capilares/fisiologia , Divisão Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Endotélio/citologia , Endotélio Vascular/citologia , Ativação Enzimática , Humanos , Cinética , Sistema Linfático/citologia , Microcirculação/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Pele/irrigação sanguínea , Veias Umbilicais , Fator C de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.
Assuntos
Fatores de Crescimento Endotelial/genética , Isoformas de Proteínas/genética , Splicing de RNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Crescimento Endotelial/química , Fatores de Crescimento Endotelial/metabolismo , Glicosilação , Humanos , Hidrólise , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator D de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Sistema Linfático/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Pele/metabolismo , Animais , Meia-Vida , Hiperplasia , Sistema Linfático/crescimento & desenvolvimento , Sistema Linfático/patologia , Camundongos , Camundongos Transgênicos , Comunicação Parácrina , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Pele/irrigação sanguínea , Pele/crescimento & desenvolvimento , Pele/patologia , Fator A de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bioensaio , Técnicas Biossensoriais , Western Blotting , Reagentes de Ligações Cruzadas/farmacologia , Eletroforese em Gel de Poliacrilamida , Embrião de Mamíferos/metabolismo , Fatores de Crescimento Endotelial/biossíntese , Endotélio Vascular/metabolismo , Epiderme/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Cinética , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Homologia de Sequência de Aminoácidos , Pele/embriologia , Pele/metabolismo , Fatores de Tempo , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Neovascularização Patológica , Animais , Linhagem Celular Transformada , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Feminino , Humanos , Metástase Linfática , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/patologia , Neoplasias/fisiopatologia , Fator D de Crescimento do Endotélio VascularRESUMO
Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti-VEGF antibodies indicated that other angiogenic factors may be involved. VEGF-D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor-2 (VEGFR-2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor-binding domain of VEGF-D and the use of these antibodies to localize VEGF-D in malignant melanoma. VEGF-D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF-D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF-D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR-2 and VEGFR-3. As VEGF receptors can be up-regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF-D in smooth muscle of the blood vessels suggest that VEGF-D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Melanoma/metabolismo , Neovascularização Patológica/metabolismo , Animais , Anticorpos Monoclonais/fisiologia , Colo/irrigação sanguínea , Fatores de Crescimento Endotelial/metabolismo , Feminino , Humanos , Melanoma/irrigação sanguínea , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso Vascular/metabolismo , Receptores de Fatores de Crescimento/fisiologia , Fator D de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is a hypervascularized and locally infiltrating brain tumor of astroglial origin with a very poor prognosis. An X-linked c-fos oncogene-inducible mitogenic, morphogenic, and angiogenic factor, endothelial growth factor-D (VEGF-D), is the newest mammalian member of VEGF family. We analyzed VEGF-D in GBM because of its high angiogenic potential and its linkage to the X chromosome. MATERIALS AND METHODS: Nonmalignant brain and GBM tissue sections as well as GBM cell lines were analyzed by immunofluorescence for the expression of VEGF-D, factor VIII (endothelial cell marker), glial-fibrillary acidic protein (GFAP) (astrocytic cell lineage cytoplasmic marker), and several Fos family transcription factors, including c-Fos and Fra-1. The proteins were also detected by Western blots. The differences between genotypes of normal brain and GBM cells were examined by cDNA microarrays. RESULTS AND CONCLUSIONS: GBM expressed ubiquitously VEGF-D, which colocalized with GFAP. Contrary to our expectations, low levels of c-Fos were detected in GBM cells. However, we identified another Fos family member, Fra-1, together with its transcriptional activation partner, c-Jun, as being stably up-regulated in GBM cells. Furthermore, we demonstrated that a fra-1 transgene induced VEGF-D expression in cultured cells and GBM cell stimulation evoked a sustained increase in both Fra-1 and VEGF-D levels. This study reveals that an up-regulation of AP-1 factors may be a hallmark of GBM. Because VEGF-D activates VEGF receptor 2 and 3, receptors important for tumor angiogenesis, it may represent an X-linked/AP-1-regulated onco-angiogen in human GBM. The VEGF-D system and AP-1 activity appear to be very attractive targets for new molecular diagnostics and rational molecular anti-cancer therapies.
Assuntos
Encéfalo/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Glioblastoma/metabolismo , Neovascularização Patológica , Cromossomo X/genética , Animais , Encéfalo/patologia , Fatores de Crescimento Endotelial/genética , Ligação Genética , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Células Tumorais Cultivadas , Fator D de Crescimento do Endotélio VascularRESUMO
Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced.
Assuntos
Ectima Contagioso/fisiopatologia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Vírus do Orf/fisiologia , Proteínas Virais/metabolismo , Animais , Células Cultivadas , Ectima Contagioso/virologia , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Deleção de Genes , Humanos , Imuno-Histoquímica , Linfocinas/genética , Neovascularização Patológica , Vírus do Orf/genética , Recombinação Genética , Ovinos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteínas Virais/genética , Replicação ViralAssuntos
Fatores de Crescimento Endotelial , Sistema Linfático/embriologia , Animais , Hibridização In Situ , Camundongos , Processamento de Proteína Pós-Traducional , Receptores Proteína Tirosina Quinases , Receptores de Superfície Celular , Fator C de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the alpha cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries.
Assuntos
Vasos Sanguíneos/química , Fatores de Crescimento Endotelial/isolamento & purificação , Endotélio Vascular/química , Sistemas Neurossecretores/química , Receptores Proteína Tirosina Quinases/isolamento & purificação , Receptores de Superfície Celular/isolamento & purificação , Adulto , Fatores Etários , Especificidade de Anticorpos , Vasos Sanguíneos/anatomia & histologia , Glândulas Endócrinas/metabolismo , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/imunologia , Endotélio Vascular/anatomia & histologia , Feminino , Feto , Humanos , Imuno-Histoquímica , Sistema Linfático/química , Gravidez , Receptores Proteína Tirosina Quinases/genética , Receptores de Superfície Celular/genética , Distribuição Tecidual , Fator C de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.
Assuntos
Anticorpos Monoclonais/imunologia , Fatores de Crescimento Endotelial/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Afinidade de Anticorpos , Especificidade de Anticorpos , Sequência de Bases , Divisão Celular/imunologia , Células Cultivadas , Primers do DNA , Fatores de Crescimento Endotelial/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Mapeamento de Epitopos , Humanos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.