RESUMO
The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.
Assuntos
Neovascularização Fisiológica/genética , Sindecana-2/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Camundongos , Domínios Proteicos , Retina/crescimento & desenvolvimento , Análise de Sequência de Proteína , Sindecana-2/genética , Sindecana-2/metabolismo , Sindecana-4/genética , Sindecana-4/metabolismo , Sindecana-4/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
PURPOSE: To develop an interventional hindlimb ischemic model and compare its angiogenic effect versus surgical ligation (SL) and excision of the femoral artery in rats treated with transplantation of bone marrow mononuclear cells (MNCs) as an angiogenic stimulator. MATERIALS AND METHODS: Forty-eight Lewis rats randomly received interventional embolization (IE) with hydrogel wire or SL and excision of the right femoral artery. Rodents were intraarterially transplanted with 1.5 × 10(7) MNCs in 500 µL medium from 24 isogenic donor rats. Functional and structural recovery was evaluated by laser Doppler imaging (LDI), cytokine/chemokine assay, and histologic staining. RESULTS: In vivo microscopic images showed significantly dilated vasa vasorum around the embolized segment of the right femoral artery at 3 days compared with disorganized tissue structure in the SL group. However, the LDI index was significantly higher in the SL group at 3 days compared with the IE group. LDI did not significantly differ between the two groups at 2 weeks after transplantation. Cytokine assay showed higher levels of interleukin (IL)-1α and IL-18 in the SL group; the IE group had higher levels of interferon-γ, IL-6, IL-13, and granulocyte colony-stimulating factor. Histologic examination demonstrated inflammatory infiltration near the incision within nerve fibers with dilated capillaries, showing nerve degeneration in the SL group. At 2 weeks, histologic analysis demonstrated massive scarring under the skin spreading into the musculature in the SL group. CONCLUSIONS: A minimally invasive hindlimb ischemia model has been successfully developed that preserves tissue integrity and minimizes inflammation and confounding factors in the early stages of angiogenesis and arteriogenesis.
Assuntos
Transplante de Medula Óssea , Embolização Terapêutica , Artéria Femoral/cirurgia , Isquemia/cirurgia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Animais , Velocidade do Fluxo Sanguíneo , Quimiocinas/sangue , Citocinas/sangue , Modelos Animais de Doenças , Membro Posterior , Mediadores da Inflamação/sangue , Isquemia/sangue , Isquemia/etiologia , Isquemia/patologia , Isquemia/fisiopatologia , Fluxometria por Laser-Doppler , Ligadura , Masculino , Ratos , Ratos Endogâmicos Lew , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Fatores de TempoRESUMO
Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter's binding to RhoGDI1, which in turn enhances RhoGDI1's affinity for RhoG. S4 clustering activates PKCalpha, which phosphorylates RhoGDI1 at Ser(96). This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4-synectin-RhoGDI1 protein complex and activation via PKCalpha.
Assuntos
Proteínas de Transporte/metabolismo , GTP Fosfo-Hidrolases/antagonistas & inibidores , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Proteína Quinase C-alfa/metabolismo , Sindecana-4/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Análise por Conglomerados , Ativação Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ratos , Proteínas rho de Ligação ao GTP , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Cell-associated proteoglycans provide highly complex and sophisticated systems to control interactions of extracellular cell matrix components and soluble ligands with the cell surface. Syndecans, a conserved family of heparan- and chondroitin-sulfate carrying transmembrane proteins, are emerging as central players in these interactions. Recent studies have demonstrated the essential role of syndecans in modulating cellular signaling in embryonic development, tumorigenesis, and angiogenesis. In this review, we focus on new advances in our understanding of syndecan-mediated cell signaling.
Assuntos
Glicoproteínas de Membrana/fisiologia , Proteoglicanas/fisiologia , Transdução de Sinais/fisiologia , Animais , Movimento Celular/fisiologia , Sulfatos de Condroitina/química , Sulfatos de Condroitina/fisiologia , Citoesqueleto/fisiologia , Desenvolvimento Embrionário/fisiologia , Endocitose/fisiologia , Matriz Extracelular/fisiologia , Regulação da Expressão Gênica , Substâncias de Crescimento/química , Substâncias de Crescimento/fisiologia , Heparitina Sulfato/química , Heparitina Sulfato/fisiologia , Humanos , Glicoproteínas de Membrana/química , Camundongos , Morfogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Ligação Proteica , Conformação Proteica , Proteína Quinase C/fisiologia , Proteína Quinase C-alfa , Estrutura Terciária de Proteína , Proteoglicanas/química , Relação Estrutura-AtividadeRESUMO
Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.