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1.
Angiogenesis ; 15(4): 609-22, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22869002

RESUMO

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1ß expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1ß expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.


Assuntos
Neovascularização de Coroide/prevenção & controle , Endotélio Vascular/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Macrófagos Peritoneais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Western Blotting , Proteínas de Ligação ao Cálcio , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
Am J Pathol ; 178(4): 1861-9, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21406174

RESUMO

A close relationship between tumor angiogenesis, growth, and carcinomatosis has been observed. Netrin-4 (NT-4) has been shown to regulate angiogenic responses. We aimed to examine the effects of NT-4 on colon tumor angiogenesis, growth, and carcinomatosis. We showed that NT-4 was expressed in human colon cancer cells (LS174). A 20-fold increase in NT-4 expression was stably induced by NT-4 pcDNA in LS174 cells. In vivo, a Matrigel angiogenesis assay showed that NT-4 overexpression altered vascular endothelial growth factor (VEGF)/basic fibroblast growth factor-induced angiogenesis. In nude mice with LS174 xenografts, NT-4 overexpression inhibited tumor angiogenesis and growth. In addition, these NT-4-involved inhibitory effects were associated with decreased tumor cell proliferation and increased tumor cell apoptosis. Using an orthotopic peritoneal carcinomatosis model, we demonstrated that NT-4 overexpression decreased colorectal cancer carcinomatosis. Moreover, carcinomatosis-related ascites formation was significantly decreased in mice transplanted with NT-4 LS174 cells versus control LS174 cells. The antiangiogenic activity of NT-4 was probably mediated by binding to its receptor neogenin. Netrin-4 had a direct effect on neither in vitro apoptosis and proliferation of cultured LS174 cells nor the VEGF-induced acute increase in vascular permeability in vivo. We propose that NT-4 overexpression decreases tumor growth and carcinomatosis, probably via an antiangiogenic effect, underlying the potential therapeutic interest in NT-4 in the treatment of colorectal cancer growth and carcinomatosis.


Assuntos
Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neovascularização Patológica , Fatores de Crescimento Neural/fisiologia , Inibidores da Angiogênese/farmacologia , Animais , Carcinoma/patologia , Linhagem Celular Tumoral , Colágeno/química , Progressão da Doença , Combinação de Medicamentos , Feminino , Perfilação da Expressão Gênica , Humanos , Laminina/química , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fatores de Crescimento Neural/biossíntese , Netrinas , Proteoglicanas/química
3.
Proc Natl Acad Sci U S A ; 105(34): 12491-6, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18719102

RESUMO

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF- or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.


Assuntos
Células Endoteliais/citologia , Proteínas de Membrana/metabolismo , Neovascularização Patológica , Fatores de Crescimento Neural/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Quimiotaxia , Feminino , Humanos , Lasers/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/irrigação sanguínea , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Netrinas , Neoplasias da Próstata/patologia , Ligação Proteica/fisiologia , Proteínas Recombinantes/farmacologia , Transplante Heterólogo , Regulação para Cima/genética
4.
Vasc Cell ; 6(1): 1, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24472220

RESUMO

Netrins are secreted molecules involved in axon guidance and angiogenesis. We previously showed that Netrin-4 acts as an anti-angiogenic factor by inhibiting endothelial cell (EC) functions. In this study, we investigated the effects of Netrin-4 on vascular smooth muscle cell (VSMC) activity in vitro and in vivo. We show that exogenous Netrin-4 stimulated VSMC adhesion and migration, and increased their coverage on EC tubes (grown on a Matrigel substrate). siRNA knock-down of endogenous Netrin-4 expression in VSMC decreased their recruitment to EC tubes. VSMC expressed Netrin-4 and three of the six Netrin-1 cognate receptors: DCC, Neogenin, and Unc5B. Silencing of these receptors reduced Netrin-4 adhesion to VSMC, strongly suggesting that these receptors were involved in the recruitment process. We previously showed that Netrin-4 overexpression in PC3 cancer cells delayed tumor growth in a model of subcutaneous xenograft by reducing tumor vessel density. Here, we show that Netrin-4 overexpression improved tumor blood vessel structure and increased VSMC coverage. Thus, Netrin-4 induced mural cell recruitment may play a role in the inhibition of tumor growth. Our data suggest that Netrin-4 is important for blood vessel normalization through the regulation of both endothelial and perivascular cells.

5.
J Biol Chem ; 279(45): 47272-7, 2004 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-15322073

RESUMO

Dorsoventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Dorsal and ventral aspects of the eye are distinct at early stages of development; cVax homeodomain protein expression is confined to the ventral optic cup, whereas Tbx5 (T-box transcription factor) expression domain becomes restricted to the dorsal region. Misexpression of cVax or Tbx5 induces profound defects in eye morphology and abnormal visual projections. In the Pax6-/- mutant Tbx5 fails to be expressed, and Vax1 and -2 are abnormally present in the entire optic vesicle. During eye development Pax6 becomes expressed in a gradient at the optic cup stage due to the specific activation of a highly conserved intronic alpha enhancer in the Pax6 locus. We observed that the highest level of Pax6 in the optic cup corresponds to the boundary between non-overlapping cVax and Tbx5 territories. To further investigate how these transcription factors control the patterning of the eye, we overexpressed Pax6 in the chick optic cup (E2) using in ovo electroporation. We observed that overexpression of Pax6 extends the Tbx5 and Bmp4 domains but reduces the cVax expression domains in the E3 chick eye. This results in an abnormal eye phenotype at E4. In addition, we showed that cVax and Tbx5 interact with Pax6 and modulate in an opposite manner the activity of the Pax6 alpha enhancer. Moreover, the Pax6/cVax interaction inhibits the transactivation properties of Pax6. These results demonstrate that Pax6 together with cVax and Tbx5 mediate dorsoventral patterning of the eye.


Assuntos
Proteínas Aviárias/metabolismo , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Padronização Corporal , Linhagem Celular , Embrião de Galinha , Cricetinae , DNA/química , DNA Complementar/metabolismo , Eletroporação , Elementos Facilitadores Genéticos , Proteínas do Olho , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Camundongos , Modelos Biológicos , Mutação , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Repressoras , Transcrição Gênica , Ativação Transcricional , Transfecção , Transgenes
6.
J Biol Chem ; 279(40): 41911-7, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15277526

RESUMO

Mitf encodes a basic helix-loop-helix transcription factor that plays an essential role in the differentiation of the retinal pigmented epithelium (RPE) and neural crest-derived melanocytes. As cells containing melanogenic enzymes (TRP2) are found in Mitf mouse mutants, it is not clear whether Mitf is a downstream factor or a master regulator of melanocyte differentiation. To further study the role of Mitf in committing cells to the melanocyte lineage, we express Mitf in the cultured quail neuroretina cells. This leads to the induction of two types of pigmented cells: neural crest-derived melanocytes, according to their dendritic morphology, physiology, and gene expression pattern are observed together with pigmented epithelial RPE-like cells. The expression of Mitf is lower in pigmented epithelial RPE-like cells than in neural crest-derived melanocytes. Accordingly, overexpression of Mitf in cultured quail RPE causes cells to develop into neural crest-like pigmented cells. Thus, Mitf is sufficient for the proper differentiation of crest-like pigmented cells from retinal cells and its expression level may determine the type of pigment cell induced.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Melanócitos/citologia , Crista Neural/citologia , Epitélio Pigmentado Ocular/citologia , Retina/citologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Embrião não Mamífero , Camundongos , Fator de Transcrição Associado à Microftalmia , Fenótipo , Codorniz , Fatores de Transcrição/genética , Transfecção
7.
J Biol Chem ; 278(24): 21721-31, 2003 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-12663655

RESUMO

The retina pigment epithelium (RPE) is fundamental for the development and function of the vertebrate eye. Molecularly, the presumptive RPE can be identified by the early expression of two transcription factors, Mitf and Otx. In mice deficient for either gene, RPE development is impaired with loss of melanogenic gene expression, raising the possibility that in the eye OTX proteins operate either in a feedback loop or in cooperation with MITF for the control of RPE-specific gene expression. Here we show that Otx2 induces a pigmented phenotype when overexpressed in avian neural retina cells. In addition, OTX2 binds specifically to a bicoid motif present in the promoter regions of three Mitf target genes, QNR71, TRP-1, and tyrosinase, leading to their transactivation. OTX2 and MITF co-localize in the nuclei of RPE cells and physically interact, and their co-expression results in a cooperative activation of QNR71 and tyrosinase promoters. Collectively, these data suggest that both transcription factors operate at the same hierarchical level to establish the identity of the RPE.


Assuntos
Proteínas de Homeodomínio , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso/fisiologia , Oxirredutases , Epitélio Pigmentado Ocular/citologia , Transativadores/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Hibridização In Situ , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia , Microscopia de Fluorescência , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx , Fenótipo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Codorniz , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção
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