RESUMO
BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) show remarkable therapeutic potential to repair tissue upon injury via paracrine signaling by secreting diverse trophic factors that promote angiogenesis. However, the mechanisms and signaling pathways that regulate the induction of these specific factors are still mostly unknown. Emerging evidence suggests that Sonic hedgehog (SHH) plays a central role in angiogenesis and tissue maintenance. However, its contribution to the angiogenic potential of MSC has not been fully addressed. The aim of this work was to characterize the expression of the SHH pathway components in WJ-MSC primary cultures and to evaluate their angiogenic responsiveness to SHH signaling. METHODS: Primary cell cultures obtained from human umbilical cords were treated with pharmacological modulators of the SHH pathway. We evaluated the modulation of diverse trophic factors in cell lysates, conditioned medium, and functional in vitro assays. In addition, we determined the angiogenic potential of the SHH pathway in the chicken chorioallantoic membrane, an in vivo model. RESULTS: Our results show that WJ-MSC express components of the canonical SHH pathway and are activated by its signaling. In fact, we provide evidence of basal autocrine/paracrine SHH signaling in WJ-MSC. SHH pathway stimulation promotes the secretion of angiogenic factors such as activin A, angiogenin, angiopoietin 1, granulocyte-macrophage colony-stimulating factor, matrix metallometallopeptidase -9, and urokinase-type plasminogen activator, enhancing the pro-angiogenic capabilities of WJ-MSC both in vitro and in vivo. CONCLUSION: WJ-MSC are a cell population responsive to SHH pathway stimulation. Basal SHH signaling is in part responsible for the angiogenic inductive properties of WJ-MSC. Overall, exogenous activation of the SHH pathway enhances the angiogenic properties of WJ-MSC, making this cell population an ideal target for treating tissue injury.
Assuntos
Proteínas Hedgehog/metabolismo , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Ativinas/genética , Ativinas/metabolismo , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Galinhas , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Hedgehog/genética , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Ativadores de Plasminogênio/genética , Ativadores de Plasminogênio/metabolismo , Transdução de Sinais , Geleia de Wharton/citologiaRESUMO
We successfully replaced the ovalbumin gene of a magnum region in chickens with a human plasminogen activator. We constructed pL-eGFP, pL-tPAGFP and pL-2.8OVtPAGFP vectors and cultured 293FT chicken embryo fibroblasts, chicken primordial germ cells, Hela C127 cells, and oviduct epithelial cells. All vectors were expressed in the transfected cells, except pL-2.8OVtPAGFP vector, which was only expressed in oviduct epithelial cells. A lentivirus with pL-2.8OVtPAGFP was injected in fertilized eggs; 11 chicks hatched in the G0 generation, four of them carried the tPAGFP. Two cockerels from the G0 generation were crossed with four wild-type hens. Three chicks in G1 carried the tPAGFP. We concluded that by using an oviduct-specific vector for transfection, human recombinant plasminogen activator protein can be expressed in the oviducts of laying hens. This character is inherited and can be reproduced with a need for repeated transfection.
Assuntos
Animais Geneticamente Modificados/genética , Galinhas/genética , Engenharia Genética/métodos , Vetores Genéticos/química , Ativadores de Plasminogênio/genética , Transfecção/métodos , Animais , Animais Geneticamente Modificados/metabolismo , Embrião de Galinha , Galinhas/metabolismo , Cruzamentos Genéticos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lentivirus/genética , Masculino , Ovalbumina/genética , Ovalbumina/metabolismo , Oviductos/metabolismo , Ativadores de Plasminogênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ZigotoRESUMO
Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/metabolismo , Bacteroides fragilis/patogenicidade , Ativadores de Plasminogênio/metabolismo , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Infecções por Bacteroides/metabolismo , Bacteroides fragilis/genética , Cromatografia de Afinidade , Clonagem Molecular , DNA Bacteriano/análise , Fibrinolisina/metabolismo , Humanos , Laminina/metabolismo , Espectrometria de Massas , Plasminogênio/metabolismo , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/genética , Análise de Sequência de DNA , VirulênciaRESUMO
We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.
Assuntos
Antígenos Virais/imunologia , Vacinas contra Dengue/administração & dosagem , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/prevenção & controle , Imunização , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/metabolismo , Vacinas contra Dengue/genética , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Ativadores de Plasminogênio/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.
Assuntos
Peptídeo Hidrolases/metabolismo , Ativadores de Plasminogênio/metabolismo , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Glicosilação , Cinética , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Regulation of the extracellular matrix turnover is a crucial process in wound healing and the progress of periodontal disease. It has been proposed that urokinase-type plasminogen activator (uPA), under the control of growth factors or cytokines, provides the proteolytic potential to the accomplishment of these cellular events. Epidermal growth factor (EGF) is one of the growth factors that has been shown to be active in uPA regulation. METHODS: In this study, we have assessed the effect of EGF on uPA expression in primary cultures of human gingival fibroblasts. We also studied the signaling pathways involved in this process and the role of the dietary phytoestrogens curcumin and genistein as potential modulators of this response. RESULTS: Human gingival fibroblasts expressed a basal uPA activity, which was inhibited by genistein, but not by curcumin. After treatment with 10 ng/ml EGF, uPA production was strongly stimulated. Exposure to genistein and curcumin inhibited EGF-stimulated urokinase production, although only genistein showed a statistically significant inhibitory response. Using more specific inhibitors, we found that the mitogen-activated extracellular kinase and c-Jun N-terminal kinase (JNK) inhibitors PD98059 and SP600125 also blocked the EGF-dependent stimulatory effect. On the other hand, SB203580, inhibitor of the p38 member of mitogen-activated protein kinase family, did not alter this response. In accordance to these findings, EGF stimulated a potent activation of JNK and a mild activation of extracellular signal-regulated kinases 1/2. Finally, EGF stimulated the phosphorylation of its receptor and tyrphostin (AG1478), curcumin and genistein were able to inhibit this stimulatory effect. CONCLUSIONS: These results indicate that EGF constitutes a strong stimuli on uPA expression in human gingival fibroblasts. Our data also shows that EGF-stimulated uPA production involves the activation of the extracellular signal-regulated kinases 1/2 and JNK signaling pathways and might be modulated by the natural phytoestrogens curcumin and genistein.
Assuntos
Curcumina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Genisteína/farmacologia , Gengiva/enzimologia , Fitoestrógenos/farmacologia , Ativadores de Plasminogênio/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/enzimologia , Expressão Gênica/efeitos dos fármacos , Gengiva/citologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ativadores de Plasminogênio/antagonistas & inibidores , Ativadores de Plasminogênio/genética , Inibidores de Proteases , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.