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1.
J Biol Chem ; 285(35): 26842-26851, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20576609

RESUMO

Heparan sulfates (HSs) modulate various developmental and homeostatic processes by binding to protein ligands. We have evaluated the structural characteristics of porcine HS in cellular signaling induced by basic fibroblast growth factor (FGF2), using CHO745 cells devoid of endogenous glycosaminoglycans as target. Markedly enhanced stimulation of cell signaling, measured as phosphorylation of ERK1/2 and protein kinase B, was only observed with the shortest HS chains isolated from liver, whereas the longer chains from either liver or intestine essentially prolonged duration of signals induced by FGF2 in the absence of polysaccharide. Structural analysis showed that contiguous sulfated domains were most abundant in the shortest HS chains and were more heavily sulfated in HS from liver than in HS from intestine. Moreover, the shortest chains from either source entered into ternary complexes with FGF2 and FGF receptor-1c more efficiently than the corresponding longer chains. In addition to authentic HSs, decasaccharide libraries generated by chemo-enzymatic modification of heparin were probed for effect on FGF2 signaling. Only the most highly sulfated decamers, previously found most efficient in ternary complex formation (Jastrebova, N., Vanwildemeersch, M., Rapraeger, A. C., Giménez-Gallego, G., Lindahl, U., and Spillmann, D. (2006) J. Biol. Chem. 281, 26884-26892), promoted FGF2 cellular signaling as efficiently as short HS chains from liver. Together these results suggest that the effects of HS on FGF2 signaling are determined by both the structure of the highly sulfated domains and by the organization/availability of such domains within the HS chain. These findings underpin the need for regulation of HS biosynthesis in relation to control of growth factor-induced signaling pathways.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Especificidade de Órgãos/fisiologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Suínos
2.
Nature ; 464(7290): 917-21, 2010 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-20228789

RESUMO

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb(-/-) mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.


Assuntos
Endotélio/metabolismo , Ácidos Graxos/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Núcleo Celular/genética , Células Cultivadas , Endotélio/citologia , Proteínas de Transporte de Ácido Graxo/genética , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculos/metabolismo , Miocárdio/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Transdução de Sinais , Transcrição Gênica , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Cardiovasc Res ; 86(1): 122-30, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19955220

RESUMO

AIMS: Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-Rs) are among the most powerful factors regulating vascular growth. However, it has remained unknown whether stimulation of VEGF-R1, VEGF-R2 or both of the receptors produces the best angiogenic responses in myocardium. The aim of this study was to compare the VEGF-R1-specific ligand VEGF-B(186), VEGF-R2-specific ligand VEGF-E and VEGF-A(165,) which stimulates both receptors, regarding their effects on angiogenesis and left ventricular function in mice. METHODS AND RESULTS: High-resolution echocardiography was used to guide the closed-chest injections of adenoviral (Ad) vectors expressing VEGF-B(186,) VEGF-E, and VEGF-A(165) into the anterior wall of the left ventricle in C57Bl/6J mice. Angiogenic and functional effects were analysed using histology, ultrasound and perfusion analyses 6 (D6) and 14 (D14) days after the Ad injection. AdVEGF-A(165) induced a strong angiogenic response seen as an enlargement of myocardial capillaries whereas angiogenesis induced by AdVEGF-B(186) and AdVEGF-E seemed more physiological. The increase in the capillary area was accompanied with an increase in myocardial perfusion at D6 after the gene injection. AdVEGF-A(165) and AdVEGF-E induced endothelial-specific proliferation whereas AdVEGF-B(186) mostly induced proliferation of cardiomyocytes. AdVEGF-A(165) induced more pronounced tissue damage than AdVEGF-B(186) and AdVEGF-E. Left ventricular function measured as ejection fraction did not change during the follow-up. AdVEGF-A(165) increased both VEGF-R1 and VEGF-R2 protein expression whereas AdVEGF-B(186) and AdVEGF-E did not affect endogenous receptor expression levels. CONCLUSION: AdVEGF-B(186) and AdVEGF-E are equally potent in inducing therapeutic angiogenesis in mouse myocardium and produce less side effects than AdVEGF-A(165).


Assuntos
Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular Esquerda/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Circulação Coronária/efeitos dos fármacos , Circulação Coronária/fisiologia , Ecocardiografia , Ensaio de Imunoadsorção Enzimática , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Cardiovasculares , Miocardite/induzido quimicamente , Miocardite/metabolismo , Miocardite/fisiopatologia , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Volume Sistólico/efeitos dos fármacos , Volume Sistólico/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator B de Crescimento do Endotélio Vascular/genética , Fator B de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
4.
Arterioscler Thromb Vasc Biol ; 28(9): 1614-20, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18511699

RESUMO

OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.


Assuntos
Vasos Coronários/metabolismo , Isquemia/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Neovascularização Fisiológica , Fator B de Crescimento do Endotélio Vascular/metabolismo , Indutores da Angiogênese/metabolismo , Animais , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/métodos , Membro Posterior , Isquemia/patologia , Isquemia/fisiopatologia , Isquemia/terapia , Pulmão/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Músculo Esquelético/irrigação sanguínea , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/terapia , Miocárdio/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Vasos Retinianos/metabolismo , Pele/irrigação sanguínea , Regulação para Cima , Fator B de Crescimento do Endotélio Vascular/administração & dosagem , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
J Biol Chem ; 281(37): 26884-92, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16807244

RESUMO

Biosynthesis of heparan sulfate (HS) is strictly regulated to yield products with cell/tissue-specific composition. Interactions between HS and a variety of proteins, including growth factors and morphogens, are essential for embryonic development and for homeostasis in the adult. Fibroblast growth factors (FGFs) and their various receptors (FRs) form ternary complexes with HS, as required for receptor signaling. Libraries of HS-related, radiolabeled oligosaccharides were generated by chemo-enzymatic modification of heparin and tested for affinity to immobilized FR ectodomains in the presence of FGF1 or FGF2. Experiments were designed to enable assessment of N-sulfated 8- and 10-mers with defined numbers of iduronic acid 2-O-sulfate and glucosamine 6-O-sulfate groups. FGF1 and FGF2 were found to require similar oligosaccharides in complex formation with FR1c-3c, FGF2 affording somewhat more efficient oligosaccharide recruitment than FGF1. FR4, contrary to FR1c-3c, bound oligosaccharides at physiological ionic conditions even in the absence of FGFs, and this interaction was further promoted by FGF1 but not by FGF2. In all systems studied, the stability of FGF-oligosaccharide-FR complexes correlated with the overall level of saccharide O-sulfation rather than on the precise distribution of sulfate groups.


Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/química , Heparitina Sulfato/química , Oligossacarídeos/química , Animais , Células COS , Chlorocebus aethiops , Cromatografia , Humanos , Modelos Químicos , Ligação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/química , Sulfatos/química
6.
J Biol Chem ; 281(15): 10298-304, 2006 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-16436387

RESUMO

The plasma protein histidine-rich glycoprotein (HRGP), which has been identified as an angiogenesis inhibitor, binds to heparan sulfate (HS) in a Zn(2+)-dependent manner. We wished to test whether this interaction is mechanistically important in mediation of the anti-angiogenic effect of HRGP. Inhibition of angiogenesis by HRGP is exerted through its central His/Pro-rich domain, which is proteolytically released. A 35-amino-acid residue synthetic peptide, HRGP330, derived from the His/Pro-rich domain retains the inhibitory effect on blood vessel formation in vitro and in vivo, an effect dependent on the presence of Zn(2+). We now show that HRGP330 binds heparin/HS with the same capacity as full-length HRGP, and the binding is Zn(2+)-dependent. Peptides derived from the His/Pro-rich domain of HRGP downstream of HRGP330 fail to inhibit endothelial cell migration and display a significantly reduced heparin-binding capacity. An even shorter peptide, HRGP335, covering a 26-amino-acid sequence within HRGP330 retains full heparin/HS-binding capacity. Characterization of the HS interaction shows that there is a tissue-specific HS pattern recognized by HRGP335 and that the minimal length of heparin/HS required for binding to HRGP335 is an 8-mer oligosaccharide. Saturation of the HS binding sites in HRGP330 by pre-incubation with heparin abrogates the HRGP330-induced rearrangement of endothelial cell focal adhesions, suggesting that interaction with cell surface HS is needed for HRGP330 to exert its anti-angiogenic effect.


Assuntos
Células Endoteliais/metabolismo , Heparitina Sulfato/química , Histidina/química , Prolina/química , Zinco/química , Animais , Biotinilação , Cátions , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Quimiotaxia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Heparina/química , Humanos , Cinética , Oligossacarídeos/química , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Distribuição Tecidual
7.
EMBO J ; 21(23): 6303-11, 2002 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-12456637

RESUMO

The anti-angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of >/=15 kDa bound quantitatively to ES whereas N-sulfated HS decasaccharides, with affinity for several fibroblast growth factor (FGF) species, failed to bind. Instead, ES-binding oligosaccharides composed of mixed N-sulfated and N-acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES-binding epitope was identified, with two N-sulfated regions separated by at least one N-acetylated glucosamine unit (SAS-domain). Cleavage at the N-acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial growth factor-induced endothelial cell migration was blocked by the ES-binding SAS-domains and by heparin oligosaccharides (12mers) similar in length to the ES-binding SAS-domains, but not by 6mers capable of FGF binding. We propose that SAS-domains modulate the biological activities of ES and other protein ligands with extended HS-binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.


Assuntos
Colágeno/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/fisiologia , Heparitina Sulfato/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Colágeno/farmacologia , Endostatinas , Heparina/metabolismo , Heparitina Sulfato/química , Oligossacarídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Suínos
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