RESUMO
Recruitment of mural cells (MCs), namely pericytes and smooth muscle cells (SMCs), is essential to improve the maturation of newly formed vessels. Sonic hedgehog (Shh) has been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms of this process have not yet been elucidated. We first identified Shh as a target of platelet-derived growth factor BB (PDGF-BB) and found that SMCs respond to Shh by upregulating extracellular signal-regulated kinase 1/2 and Akt phosphorylation. We next showed that PDGF-BB-induced SMC migration was reduced after inhibition of Shh or its signaling pathway. Moreover, we found that PDGF-BB-induced SMC migration involves Shh-mediated motility. In vivo, in the mouse model of corneal angiogenesis, Shh is expressed by MCs of newly formed blood vessels. PDGF-BB inhibition reduced Shh expression, demonstrating that Shh is a target of PDGF-BB, confirming in vitro experiments. Finally, we found that in vivo inhibition of either PDGF-BB or Shh signaling reduces NG2(+) MC recruitment into neovessels and subsequently reduces neovessel life span. Our findings demonstrate, for the first time, that Shh is involved in PDGF-BB-induced SMC migration and recruitment of MCs into neovessels and elucidate the molecular signaling pathway involved in this process.
Assuntos
Movimento Celular/fisiologia , Proteínas Hedgehog/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Transdução de Sinais/fisiologia , Animais , Becaplermina , Western Blotting , Córnea/irrigação sanguínea , Imuno-Histoquímica , Camundongos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pericitos/citologia , Pericitos/metabolismo , RNA Interferente Pequeno , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
RATIONALE: A better understanding of the mechanism underlying skeletal muscle repair is required to develop therapies that promote tissue regeneration in adults. Hedgehog signaling has been shown previously to be involved in myogenesis and angiogenesis: 2 crucial processes for muscle development and regeneration. OBJECTIVE: The objective of this study was to identify the role of the hedgehog transcription factor Gli3 in the cross-talk between angiogenesis and myogenesis in adults. METHODS AND RESULTS: Using conditional knockout mice, we found that Gli3 deficiency in endothelial cells did not affect ischemic muscle repair, whereas in myocytes, Gli3 deficiency resulted in severely delayed ischemia-induced myogenesis. Moreover, angiogenesis was also significantly impaired in HSA-Cre(ERT2); Gli3(Flox/Flox) mice, demonstrating that impaired myogenesis indirectly affects ischemia-induced angiogenesis. The role of Gli3 in myocytes was then further investigated. We found that Gli3 promotes myoblast differentiation through myogenic factor 5 regulation. In addition, we found that Gli3 regulates several proangiogenic factors, including thymidine phosphorylase and angiopoietin-1 both in vitro and in vivo, which indirectly promote endothelial cell proliferation and arteriole formation. In addition, we found that Gli3 is upregulated in proliferating myoblasts by the cell cycle-associated transcription factor E2F1. CONCLUSIONS: This study shows for the first time that Gli3-regulated postnatal myogenesis is necessary for muscle repair-associated angiogenesis. Most importantly, it implies that myogenesis drives angiogenesis in the setting of skeletal muscle repair and identifies Gli3 as a potential target for regenerative medicine.
Assuntos
Isquemia/fisiopatologia , Fatores de Transcrição Kruppel-Like/fisiologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Regeneração/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Fator de Transcrição E2F1/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Proteínas Hedgehog/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Transdução de Sinais/fisiologia , Proteína Gli3 com Dedos de ZincoRESUMO
RATIONALE: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves. OBJECTIVE: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia. METHODS AND RESULTS: We induced hindlimb ischemia in wild-type and Dhh(-/-) mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh(-/-). By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle. CONCLUSIONS: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.
Assuntos
Proteínas Hedgehog/fisiologia , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Nervos Periféricos/fisiologia , Angiopoietina-1/metabolismo , Animais , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Denervação Muscular , Músculo Esquelético/inervação , Fatores de Crescimento Neural/metabolismo , Nervos Periféricos/citologia , Células de Schwann/citologia , Células de Schwann/fisiologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: The purpose of this study is to further document alteration of signal transduction pathways, more particularly of hedgehog (Hh) signaling, causing impaired ischemic muscle repair in old mice. APPROACH AND RESULTS: We used 12-week-old (young mice) and 20- to 24-month-old C57BL/6 mice (old mice) to investigate the activity of Hh signaling in the setting of hindlimb ischemia-induced angiogenesis and skeletal muscle repair. In this model, delayed ischemic muscle repair observed in old mice was associated with an impaired upregulation of Gli1. Sonic Hh expression was not different in old mice compared with young mice, whereas desert Hh (Dhh) expression was downregulated in the skeletal muscle of old mice both in healthy and ischemic conditions. The rescue of Dhh expression by gene therapy in old mice promoted ischemia-induced angiogenesis and increased nerve density; nevertheless, it failed to promote myogenesis or to increase Gli1 mRNA expression. After further investigation, we found that, in addition to Dhh, smoothened expression was significantly downregulated in old mice. We used smoothened haploinsufficient mice to demonstrate that smoothened knockdown by 50% is sufficient to impair activation of Hh signaling and ischemia-induced muscle repair. CONCLUSIONS: The present study demonstrates that Hh signaling is impaired in aged mice because of Dhh and smoothened downregulation. Moreover, it shows that hegdehog-dependent regulation of angiogenesis and myogenesis involves distinct mechanisms.
Assuntos
Envelhecimento/metabolismo , Proteínas Hedgehog/metabolismo , Isquemia/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Neovascularização Fisiológica , Fatores Etários , Envelhecimento/genética , Animais , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Regulação da Expressão Gênica , Terapia Genética , Proteínas Hedgehog/genética , Membro Posterior , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Isquemia/terapia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regeneração , Transdução de Sinais , Receptor Smoothened , Transfecção , Proteína GLI1 em Dedos de ZincoRESUMO
Bronchoalveolar lavage fluid (BALF) provides a source of mucosal CD4(+) T cells. We investigated the physiological properties of T lymphocytes from BALF and blood and their role on the dynamic of HIV-1 replication among AIDS patients with active lung infections. Pulmonary CD4(+) T cells consist mainly of effector memory cells (CD45RO(+) and CCR7(-)) with increased expression of activation markers (HLA-DR(+) and CD69(+)) when compared to the blood counterpart. We observed a high frequency of BALF cells capable of secreting HIV-1-Ags suggesting that the local lung environment may support favorable conditions for CD4(+) T lymphocytes harboring HIV-1 DNA to initiate the viral cycle. Nevertheless, the high number of IFN-γ-producing cells and the predominance of Th1 immune response in the lung could limit the secretion of HIV-1 RNA. In conclusion, the capacity of activated CD4(+) T cells to produce HIV-1 is driven by both the level and quality of cellular activation in the lung.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , HIV-1/fisiologia , Pulmão/virologia , Ativação Linfocitária/imunologia , Infecções Respiratórias/imunologia , Replicação Viral/imunologia , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/imunologia , Antígenos CD/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Cicloeximida/farmacologia , DNA Viral/metabolismo , Antígenos HIV/metabolismo , HIV-1/isolamento & purificação , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Pulmão/imunologia , Pulmão/metabolismo , RNA Viral/metabolismo , Receptores CCR5 , Receptores CXCR4 , Infecções Respiratórias/etiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/virologia , Carga Viral/imunologia , Tropismo Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
The Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes latency in resting memory B lymphocytes, and is involved in oncogenesis through poorly understood mechanisms. The EBV lytic cycle is initiated during plasma cell differentiation by mRNAs transcripts encoded by BZLF1, which induce the synthesis of EBV proteins such as the immediate-early antigen ZEBRA and the late membrane antigen gp350. Therefore, we assessed the capacity of circulating EBV-infected B lymphocytes from healthy EBV-seropositive subjects to enter and complete the EBV lytic cycle. Purified B lymphocytes were polyclonally stimulated and BZLF1- or gp350-secreting cells (BZLF1-SCs or gp350-SCs) were enumerated by ELISpot assays. The number of BZLF1-SCs ranged from 50 to 480/107 lymphocytes (median, 80; 25th-75th percentiles, 70-150) and gp350-SCs from 10 to 40/107 lymphocytes (median, 17; 25th-75th percentiles, 10-20). gp350-SCs represented only 7.7% to 28.6% of BZLF1-SCs (median, 15%; 25th-75th percentiles, 10.5%-20%). This EBV functional reservoir was preferentially restricted to plasma cells derived from CD27(+) IgD(-) memory B lymphocytes. In 9 of 13 subjects, EBV DNA quantification in B-cell culture supernatants gave evidence of completion of EBV lytic cycle. These results demonstrate that EBV proteins can be secreted by EBV-infected B lymphocytes from healthy carriers, a majority generating an abortive EBV lytic cycle and a minority completing the cycle.
Assuntos
Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/virologia , Memória Imunológica , Plasmócitos/virologia , Ativação Viral/fisiologia , Adulto , Linfócitos B/metabolismo , Células Cultivadas , DNA Viral/análise , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Citometria de Fluxo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Plasmócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Latência Viral/fisiologiaRESUMO
Immunostimulatory ODN CpGs have extensively been tested as adjuvants and immunotherapeutics and hold a lot of promise for human use. In our studies we took advantage of their negative charge to study their biological activities after being complexed with carbon nanotubes, a novel vector for vaccine delivery and Tat protein of HIV, a target protein for therapeutic or prophylactic intervention. In the case of carbon nanotubes, ODN CpGs were able to form stable complexes based on charge interaction and exert increased immunostimulatory activity in vitro. With regard to the Tat protein, ODN CpGs were shown to bind effectively through the basic domain of the protein representing residues 44-61. Moreover, using surface Plasmon Resonance Technology and an in vitro cellular system, ODN CpGs were shown to inhibit the interaction of Tat protein with the transactivation responsive element, a bulged RNA hairpin structure. However, when ODN CpGs were complexed with Tat they readily increased the apoptotic properties of this protein as studied in CD3-stimulated Jurkat cells. Overall, our findings together with published data support the view that for harnessing the beneficial effects of ODN CpGs a careful consideration has to be given depending on the target intervention.
Assuntos
Ilhas de CpG/imunologia , Fatores Imunológicos/imunologia , Nanotubos de Carbono , Oligodesoxirribonucleotídeos/imunologia , Poliaminas/imunologia , Animais , Humanos , Fatores Imunológicos/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Poliaminas/administração & dosagem , Polieletrólitos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologiaRESUMO
The transcription factor cAMP responsive element-binding protein (CREB) has been found to be involved in arterial smooth muscle cell (SMC) migration. We previously demonstrated that osteopontin (OPN) expression is a key step for UTP-mediated migration of arterial SMCs and that activator protein (AP)-1, nuclear factor kappaB, and upstream stimulatory transcription factors are involved in this OPN expression. The present study aims to determine the role of CREB in UTP-induced migration and OPN expression in cultured SMCs. We found that CREB is activated by UTP via extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways but not by protein kinase A. Both overexpression of a dominant negative CREB and CREB small interfering RNA treatment suppressed UTP-induced OPN expression and SMC migration. Gel-shift and chromatin immunoprecipitation assays revealed that CREB binds 2 AP-1 sites (-1870 and -76) and a cAMP responsive element-like site (-1403) on the OPN promoter. Mutations of these sites showed that only the 2 AP-1 sites were required for UTP-induced OPN expression. Moreover, gel-supershift and sequential chromatin immunoprecipitation assays suggested that CREB was associated with c-Fos on the AP-1 sites of the OPN promoter. These results demonstrate that CREB participates in the induction of UTP-activated OPN expression via its binding to 2 AP-1 sites and is thus involved in UTP-mediated SMC migration.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Osteopontina/genética , Fator de Transcrição AP-1/metabolismo , Uridina Trifosfato/farmacologia , Movimento Celular , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Elementos de RespostaRESUMO
OBJECTIVE: Estradiol (E(2)) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ERalpha(-/-) mice have previously shown that E(2) acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E(2) to mediate endothelial repair are still poorly understood. METHODS AND RESULTS: We show here that after endovascular carotid artery injury, E(2)-enhanced endothelial repair is lost in osteopontin-deficient mice (OPN(-/-)). Transplantation of OPN(-/-) bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E(2). In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E(2)-enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E(2) enhances bone marrow-derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin. CONCLUSIONS: We demonstrate here that E(2) acceleration of the endothelial repair requires osteopontin, both for bone marrow-derived cell recruitment and for endothelial cell migration and proliferation.
Assuntos
Lesões das Artérias Carótidas/fisiopatologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Estradiol/farmacologia , Osteopontina/fisiologia , Animais , Transplante de Medula Óssea , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Feminino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteopontina/deficiência , Osteopontina/genética , Regeneração/efeitos dos fármacos , Regeneração/fisiologiaRESUMO
AIMS: beta-adrenoceptor (beta-AR)-mediated relaxation was characterized in pulmonary arteries from normoxic and hypoxic (as model of pulmonary hypertension) mice. The endothelial NO synthase (eNOS) pathway was especially investigated because of its potential vasculoprotective effects. METHODS: Pulmonary arteries from control or hypoxic (0.5 atm for 21 days) wild-type or eNOS-/- mice were used for pharmacological characterization of beta-AR-mediated relaxation in myograph, and for immunohistochemistry using anti-beta-AR antibodies. RESULTS: In pulmonary arteries from normoxic mice, isoproterenol (beta-AR agonist) and procaterol (selective beta2-AR agonist) elicited relaxation, while cyanopindolol and CL316243 (beta3-AR agonists) were ineffective. The effect of isoproterenol was antagonized by CGP20712A and ICI118551 (beta1- or beta2-AR antagonists, respectively) and also partially inhibited by N omega-nitro-L-arginine methylester (L-NAME, a NOS inhibitor), endothelium denudation, or eNOS gene deletion. Relaxation to procaterol was abolished by L-NAME or endothelium removal. In eNOS-/- mice, procaterol-induced relaxation was decreased but was insensitive to L-NAME, this residual effect involving other endothelium-dependent relaxant factors as compensatory mechanisms. Immunostaining for beta2-AR was observed in the endothelial layer, but not the medial layer of pulmonary arteries. Pulmonary arteries from hypoxic mice exhibited decreased endothelial NO-dependent relaxation to acetylcholine. However, in these arteries, relaxation to procaterol was either unaffected (extralobar segments) or even increased (intralobar segments) and was still abolished by L-NAME or endothelium removal. CONCLUSION: beta1- and beta2-AR, but not beta3-AR, mediate relaxation of mice pulmonary arteries. The beta2-AR component is dependent on eNOS activity and is preserved following chronic hypoxia. These data highlight the role of the beta2-AR as a pharmacological target to induce/restore endothelial NO-dependent protective effects in pulmonary circulation.
Assuntos
Endotélio Vascular/fisiologia , Hipertensão Pulmonar/fisiopatologia , Óxido Nítrico/fisiologia , Artéria Pulmonar/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Vasodilatação , Animais , Doença Crônica , Hipertensão Pulmonar/prevenção & controle , Hipóxia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Adrenérgicos beta/classificaçãoRESUMO
OBJECTIVE: Migration of smooth muscle cells (SMCs) from the media to the intima of arteries is involved in intimal thickening. The platelet-derived growth factor (PDGF) BB is recognized as a major migratory factor for arterial SMCs both in vitro and during neointima formation. Since PDGF acts in synergy with the matrix protein osteopontin (OPN) and also induces its expression, the present study was conceived to explore the role of the OPN produced in an autocrine fashion by PDGF-stimulated SMCs in the migration process and to define regulatory mechanisms of OPN expression. METHODS AND RESULTS: PDGF stimulation of quiescent rat aortic SMCs induced their migration (transfilter assays) and the increase of OPN expression (mRNA and protein assays). Blockade of either OPN expression by a specific short interference RNA (siRNA) or of its function by a blocking antibody decreased the PDGF-stimulated migration by about 70%, demonstrating that autocrine production and excretion of OPN are integral to the PDGF-induced SMC migration. In parallel, SMC stimulation by PDGF also activated the transcription factor CREB essentially through mitogen-activated protein kinase (MAPK) 1/2 and protein kinase A (PKA) pathways. Inhibition of either CREB expression (via siRNA) or function (via dominant-negative CREB) decreased both PDGF-induced SMC migration and OPN expression. SMC transfection with OPN promoter reporter constructs demonstrated that PDGF-induced OPN transcription is mediated by CREB binding to two functional sites of the OPN promoter: a CRE site located at -1403 and an AP-1 site located at -76. CONCLUSION: The present study demonstrates that the autocrine expression of OPN plays a major role in PDGF-induced SMC migration. It further shows that the transcription factor CREB, activated in PDGF-stimulated SMCs, plays a key role in PDGF-induced SMC migration, probably by regulating OPN expression.
Assuntos
Comunicação Autócrina/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Osteopontina/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Túnica Íntima/patologia , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Becaplermina , Calcinose/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Osteopontina/análise , Osteopontina/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Interferência de RNA , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Estimulação Química , Transcrição Gênica/efeitos dos fármacos , Transfecção/métodosRESUMO
Beta2-integrins are a family of dimeric adhesion molecules expressed on leukocytes. Their capacity to bind ligand is regulated by their state of activation. CD11b, an alphaMbeta2 integrin, is implicated in a number of physiological and pathological events such as inflammation, thrombosis, or atherosclerosis. The GTPase Rap1 is essential for its activation and could therefore play a strategic role in the regulation of leukocyte functioning. Because low levels of circulating TGF-beta have been linked with severe atherosclerosis, we have assessed the role of this cytokine in the regulation of Rap1 and CD11b activation in differentiated U937 cells and in human peripheral blood monocytes. TGF-beta1 caused a significant reduction in the expression of CD11b but not in the expression of other integrins tested. More importantly, TGF-beta1 greatly reduced the capacity of PMA or chemokines to activate CD11b and Rap1, a phenomenon paralleled by a loss of the Epac transcript and a reduction in 8-pCPT-2'-O-Me-cAMP-mediated activation of Rap1. This inhibition diminished the capacity of monocytes to migrate across a monolayer of endothelial cells. The inhibitory effect of TGF-beta1 on Rap1 activity may exert a general protective influence against aberrant transendothelial migration, thereby holding inflammatory responses in check.
Assuntos
Antígeno CD11b/fisiologia , Movimento Celular/fisiologia , Células Endoteliais , Leucócitos/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Antígeno CD11b/análise , Adesão Celular , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Quimiocina CCL4 , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Integrina alfa4/análise , Integrinas/análise , Receptores de Lipopolissacarídeos/análise , Proteínas Inflamatórias de Macrófagos/farmacologia , Masculino , Monócitos/fisiologia , RNA Mensageiro/análise , Rosa Bengala , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fator de Crescimento Transformador beta1 , Células U937 , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Many factors have been shown to be involved in the development of hyperplasic lesions of vessels, but the role of extracellular nucleotides remains largely unknown. The presence of P2Y and P2X nucleotide receptors on arterial endothelial and smooth muscle cells suggests a potential role for nucleotides in the vessel pathophysiology. Although the role of P2X in physiology of vessels is well documented, that of P2Y is not completely understood. We recently demonstrated that extracellular nucleotides, and particularly UTP, induced migration of cultured arterial smooth muscle cells (ASMCs). This migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. An important question is to determine the specific role of the different P2Y receptors of rat ASMCs in the UTP-induced migration process. Therefore, we first quantified mRNA levels of P2Y(2), P2Y(4), and P2Y(6) nucleotide receptors in cultured rat ASMCs by a competitive RT-PCR approach and demonstrated that P2Y(2) is the most highly expressed among these receptors potentially involved in the UTP-mediated response. In addition to UTP, UDP also induced ASMC migration even when UTP regeneration was inhibited, suggesting the involvement of UDP receptor P2Y(6). Moreover, suramin, a specific antagonist of rat P2Y(2) receptor, acted as an inhibitor of UTP-induced migration. Taken together, these results suggest a prominent role for the UTP receptor, P2Y(2), and for the UDP receptor, P2Y(6), in UTP-induced rat ASMC migration.
Assuntos
Movimento Celular/fisiologia , Músculo Liso Vascular/fisiologia , Receptores Purinérgicos P2/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Músculo Liso Vascular/citologia , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suramina/farmacologia , Difosfato de Uridina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
The conformational and immunological properties of different analogues corresponding to the 600-612 disulfide loop of the human immunodeficiency virus (HIV) gp41 glycoprotein envelope were studied. Fourteen analogues were designed and synthesised; namely, a series of seven analogues in which the disulfide bond was replaced by a lactam bridge and a series of seven analogues in which one residue of each analogue at a time, was replaced by its corresponding homologised alpha-amino acid (beta(3)-amino acid). In the case of the lactam analogues, the influence of the two possible CO-NH and NH-CO orientations of the lactam bridge as well as the size of the lactam ring was explored. The analogues were tested in ELISA with monoclonal antibodies raised against the 600-612 cyclic parent peptide as well as with sera from HIV-1 infected patients. A structural analysis of the parent and analogue peptides was carried out in dimethyl sulfoxide (DMSO-d(6)) using two-dimensional NMR techniques and molecular dynamics simulations. Comparison of the own conformation of the cyclic analogues with their either strong or weak reactivity with the antibodies reveals structural features that may be correlated with the antibody reactivity. Thus, a close structural similarity, particularly a characteristic orientation of the side-chains of residues Lys606, Leu607 and Ile608 in the loop, was found in certain beta(3)-analogues that were better recognised than the parent peptide by anti-peptide mouse monoclonal antibodies and patients' antibodies.
Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Peptídeos/química , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Estrutura Secundária de ProteínaRESUMO
OBJECTIVE: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. METHODS: SMC cultures derived from the rabbit aorta were exposed to 25 microM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. RESULTS: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65% at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. CONCLUSION: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.
Assuntos
Citocromos c/metabolismo , Cetocolesteróis/farmacologia , Músculo Liso Vascular/enzimologia , Animais , Aorta , Apoptose , Transporte Biológico , Biomarcadores/análise , Western Blotting/métodos , Células Cultivadas , Citocromos c/análise , Citosol/química , Citosol/metabolismo , Fragmentação do DNA , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Dilatação Mitocondrial , Músculo Liso Vascular/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Coelhos , Proteína X Associada a bcl-2RESUMO
The purpose of this study was to investigate the mechanism of the vascular relaxation produced by polyphenolic substances from red wine, with a particular focus on the possible involvement of purinoceptors. With this aim, relaxing responses induced by procyanidin from grape seeds (GSP), anthocyanins, catechin and epicatechin were assessed in rat isolated aortic rings left intact (+E) or endothelium-denuded (-E). In preparations precontracted with noradrenaline, incubation with NG-nitro-L-arginine methyl ester (100 microM, 30 min) fully inhibited the GSP-induced relaxations. Concentration-effect curves to these substances (from 10(-7) to 10(-1) g/L) were determined in depolarized (60 mM KCl) preparations in control condition, after incubation with reactive blue 2 (an antagonist of P2Y purinoceptors, 30 microM), with apyrase (an enzyme which hydrolyses ATP and ADP, 0.8 U/mL) or with alpha,beta-methylene ATP (an inhibitor of ecto ATPases, 10 microM). In (+E) rings, relaxations (expressed as percentage of initial contraction) were 41 +/- 2 and 37 +/- 3 for GSP and anthocyanins, respectively. Only modest relaxations (ca. 10%) were observed in (-E) rings, as it was the case for catechin and epicatechin in (+/- E) rings. Reactive blue 2 or apyrase inhibited the GSP- and anthocyanin-induced relaxations in (+E) rings, while alpha,beta-methylene ATP shifted to the left the relaxation curves obtained with GSP. These data confirm that modest relaxations observed with catechin and epicatechin are not endothelium-dependent but that GSP and anthocyanins induce a relaxing effect, which is related to the integrity of the endothelium and the synthesis and release of nitric oxide (NO). Furthermore, the inhibition by apyrase and the increase by ecto-ATPase inhibition of the GSP- and anthocyanin-induced relaxation suggest that these substances could act via an initial release of nucleotides, which in turn could activate P2Y1 and/or P2Y2 purinoceptors of endothelial cells, trigger the synthesis and release of NO and then lead to relaxation.
Assuntos
Aorta Torácica/fisiologia , Biflavonoides , Flavonoides/farmacologia , Fenóis/farmacologia , Proantocianidinas , Receptores Purinérgicos P2/fisiologia , Vitis/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Antocianinas/antagonistas & inibidores , Antocianinas/isolamento & purificação , Antocianinas/farmacologia , Aorta Torácica/lesões , Apirase/farmacologia , Catequina/antagonistas & inibidores , Catequina/isolamento & purificação , Catequina/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/lesões , Endotélio Vascular/fisiologia , Flavonoides/isolamento & purificação , Ginsenosídeos/farmacologia , Masculino , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Norepinefrina/antagonistas & inibidores , Norepinefrina/farmacologia , Fenóis/isolamento & purificação , Polifenóis , Ratos , Ratos Wistar , Receptores Purinérgicos P2/efeitos dos fármacos , Sementes/química , Triazinas/farmacologia , Vasodilatação/efeitos dos fármacos , VinhoRESUMO
BACKGROUND: Inflammatory processes play a critical role in myocarditis, dilated cardiomyopathy, and heart failure. The expression of the inflammatory chemokine osteopontin (OPN) is dramatically increased in cardiomyocytes and inflammatory cells during myocarditis and heart failure in human and animals. However, its role in the development of heart diseases is not known. METHODS AND RESULTS: To understand whether OPN is involved in cardiomyopathies, we generated a transgenic mouse (MHC-OPN) that specifically overexpresses OPN in cardiomyocytes with cardiac-specific promoter-directed OPN expression. Young MHC-OPN mice were phenotypically indistinguishable from their control littermates, but most of them died prematurely with a half-life of 12 weeks of age. Electrocardiography revealed conduction defects. Echocardiography showed left ventricular dilation and systolic dysfunction. Histological analysis revealed cardiomyocyte loss, severe fibrosis, and inflammatory cell infiltration. Most of these inflammatory cells were activated T cells with Th1 polarization and cytotoxic activity. Autoantibodies against OPN, cardiac myosin, or troponin I, were not found in the serum of MHC-OPN mice. CONCLUSIONS: These data show that OPN expression in the heart induces in vivo T-cell recruitment and activation leading to chronic myocarditis, the consequence of which is myocyte destruction and hence, dilated cardiomyopathy. Thus, OPN might therefore constitute a potential therapeutic target to limit heart failure.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Miócitos Cardíacos/metabolismo , Osteopontina/metabolismo , Animais , Cardiomiopatia Dilatada/etiologia , Modelos Animais de Doenças , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Insuficiência Cardíaca/etiologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Miocardite/etiologia , Miocardite/metabolismo , Miocardite/patologia , Infiltração de NeutrófilosRESUMO
The Epstein-Barr virus (EBV) generally latently infects its target cells with expression of genes conferring resistance to apoptosis. However, the modulation of apoptotic signals during lytic cycle remains poorly understood. We show here that resulting from viral reactivation in the EBV-positive Mutu-I and Akata Burkitt's lymphoma cell lines, a two steps proteasome-dependent downregulation of expression of the proapoptotic protein BimEL occurs. The first drop might be EBV-independent, is ERK 1/2 dependent, and BimEL is phosphorylated on Ser69. A second dramatic drop of the BimEL level observed during the lytic cycle is dependent of EBV-late-gene expression, ERK 1/2 independent, and no further phosphorylation of BimEL on Ser69 occurred. These results demonstrate for the first time, that the lytic cycle contributes to downregulation of BimEL and then could add to protection against apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Regulação para Baixo , Herpesvirus Humano 4/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ativação Viral , Apoptose , Proteína 11 Semelhante a Bcl-2 , Ácidos Borônicos/farmacologia , Linfoma de Burkitt , Butadienos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/genética , Humanos , Nitrilas/farmacologia , FosforilaçãoRESUMO
Osteopontin (OPN) is an important chemokinetic agent for several cell types. Our earlier studies have shown that its expression is essential for uridine triphosphate (UTP)-mediated migration of vascular smooth muscle cells. We demonstrated previously that the activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat OPN promoter is involved in the induction of OPN expression. In this work, using a luciferase promoter deletion assay, we identified a new region of the rat OPN promoter (-1837 to -1757) that is responsive to UTP. This region contains an NFkappaB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFkappaB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IkappaB kinase and USF transcription factors, we confirmed that NFkappaB and USF-1/USF-2 are involved in the UTP-mediated expression of OPN. Using a pharmacological approach, we demonstrated that USF proteins are regulated by the extracellular signal-regulated kinase (ERK)1/2 pathway, just as the earlier discovered AP-1 complex, whereas NFkappaB is up-regulated through PKCdelta signals. Finally, our work suggests that the UTP-stimulated OPN expression involves a coordinate regulation of PKCdelta-NFkappaB, ERK1/2-USF, and ERK1/2/NAD(P)H oxidase AP-1 signaling pathways.
Assuntos
Artérias/patologia , Proteínas de Ligação a DNA/metabolismo , Músculo Liso/citologia , NF-kappa B/metabolismo , Sialoglicoproteínas/biossíntese , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Uridina Trifosfato/metabolismo , Animais , Sítios de Ligação , Western Blotting , Movimento Celular , Células Cultivadas , DNA/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes Dominantes , Luciferases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Mutação , NADPH Oxidases/metabolismo , Osteopontina , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Wistar , Sialoglicoproteínas/genética , Transdução de Sinais , Transcrição Gênica , Regulação para Cima , Fatores Estimuladores UpstreamRESUMO
In this study we examined the hypothesis that the binding affinity of two double-stranded (ds) RNA motifs to HIV-1 Tat protein might affect transactivation and the type of anti-Tat immune responses. Using surface plasmon resonance technology we demonstrated the capacity of the poly(A):poly(U) (pA:pU) motif to bind with high affinity to a totally synthetic Tat protein and to inhibit more efficiently the Tat/transactivation response element (TAR) RNA interaction as compared to the poly(I):poly(C) (pI:pC) motif. Furthermore, the pA:pU motif was tenfold more effective in inhibiting Tat-driven transactivation than the pI:pC motif. Following intranasal immunization of mice, both dsRNA motifs enhanced the antibody (serum and mucosal) and cellular responses to Tat. However, only the serum samples of mice immunized with Tat + pI:pC inhibited Tat-driven transactivation. The profile of serum antibody subclasses together with the secreted cytokines by Tat-stimulated splenocyte cultures indicated that both dsRNA motifs favored the induction of a balanced Th1 and Th2 immune response. The demonstration in this study that two dsRNA motifs had a marked effect on Tat/TAR RNA interaction and on the neutralizing capacity of anti-Tat specific antibody responses highlights their potential for biological applications and the importance of selecting the appropriate motif as an adjuvant for vaccine design.