O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos

Detalhes bibliográficos
Autor(a) principal: Lopes, Alejandra Sousa
Data de Publicação: 2018
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da UFMT
Texto Completo: http://ri.ufmt.br/handle/1/2363
Resumo: The Renin Angiotensin System (RAS) plays a fundamental role in the regulation of blood pressure and electrolyte balance. The angiotensin peptide (ANG)-(1-7), bound to RAS, is predominantly generated in the vascular wall under two main pathways: through ANGII by the action of neutral endopeptidases and prolyl-endopeptidases or by ANGI action, by hydrolysis from Angiotensin converting enzyme 2 (ACE2). ANG-(1-7) binds to its receptor Mas coupled to G protein and acts mediating vasodilation, inhibiting cell growth, acting as an important antagonist of ANGII vascular actions (vasoconstriction and inflammation). Recently, studies have shown that ANGII effects can also be attenuated by regulatory cytokines such as Interleukin (IL)-10, which negatively modulates proinflammatory cytokine polysaccharides producers such as tumor necrosis factor-alpha and IL-6 and inhibits the production of reactive oxygen species (ROS), reducing inflammation and favoring vasodilation. Although ANG-(1-7) and IL-10 possess similar functions, in vascular aspects, the association of both factors is still unknown and it seems to be related with other two significant molecules: MAPK, extracellular signal-regulated kinase 1 and 2 (ERK 1/2), which can be stimulated by ANGII and prolongs vasoconstriction and also Phosphatase-1 of mitogen-activated proteins (MKP-1), an important phosphatase that has the primary function of inactivating MAPKs, and it is estimulated by IL-10. The objective of this work is to evaluate if ANG-(1-7) reduces ERK 1/2 activity due to stimulation of MKP-1 and IL-10, resulting in a decrease in vascular contraction. The present study was approved by the UFMT Animal Research Ethics Committee (23108.166477/2016-20). Aortic rings of male C57BL/6J mice, 10-12 weeks of age, were incubated with ANG-(1-7) [10μM] or vehicle for 5 min, to perform ANGII concentration-response curve in the presence or absence of Mas receptor antagonist (A-779, 100μM) and cAMP (Rp-cAMPs, 1μM). Vascular expressions of c-Raf, ERK1/2 and MKP-1 proteins were verified by Western Blot and immunocytochemistry in vascular smooth muscle cell culture (LVC), incubated individually or simultaneously with ANG-(1-7) [10μM] , ANGII [1μM] or vehicle for 5 min. IL-10 knockout animals, infused with ANGII (90ng / min) for 14 days were used to verify IL-10 protector effects. Finally, blood vessel from animals were incubated with ANG-(1-7) [10μM], or vehicle, for 12h, to evaluate indirect expression of IL-10. The results show that ANG-(1-7) reduces vascular contraction induced by ANGII, via activation of Mas receptors and cAMP formation. It was also demonstrated that ANG-(1-7) prevented ERK1/2 activation, possibly through activation of MKP-1 phosphatase, both in aortic tissue and in vascular smooth muscle cell culture. Other result found in this study was that IL-10-lacking animals, ANGII has exacerbated activity, presenting higher vascular contraction, probably due to the increase in ERK 1/2 expression. In addition, it was observed that vessels treated with ANG-(1-7) for 12 hours presented higher expression of STAT3, a crucial element in IL-10 signaling pathway. In conclusion, the results suggest that ANG-(1-7) stimulates the activity of MKP-1 and IL-10 signaling pathway, which inhibits the ERK1/2 phosphorylation and consequently contributes to the reduction of vascular contractile response.
id UFMT_fdce070b903688e5e95fc35eaf1b6ea8
oai_identifier_str oai:localhost:1/2363
network_acronym_str UFMT
network_name_str Repositório Institucional da UFMT
repository_id_str
spelling O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongosHipertensãoVasoconstriçãoSistema renina-angiotensinaCNPQ::CIENCIAS DA SAUDEHypertensionVasoconstrictionRenin-angiotensin systemThe Renin Angiotensin System (RAS) plays a fundamental role in the regulation of blood pressure and electrolyte balance. The angiotensin peptide (ANG)-(1-7), bound to RAS, is predominantly generated in the vascular wall under two main pathways: through ANGII by the action of neutral endopeptidases and prolyl-endopeptidases or by ANGI action, by hydrolysis from Angiotensin converting enzyme 2 (ACE2). ANG-(1-7) binds to its receptor Mas coupled to G protein and acts mediating vasodilation, inhibiting cell growth, acting as an important antagonist of ANGII vascular actions (vasoconstriction and inflammation). Recently, studies have shown that ANGII effects can also be attenuated by regulatory cytokines such as Interleukin (IL)-10, which negatively modulates proinflammatory cytokine polysaccharides producers such as tumor necrosis factor-alpha and IL-6 and inhibits the production of reactive oxygen species (ROS), reducing inflammation and favoring vasodilation. Although ANG-(1-7) and IL-10 possess similar functions, in vascular aspects, the association of both factors is still unknown and it seems to be related with other two significant molecules: MAPK, extracellular signal-regulated kinase 1 and 2 (ERK 1/2), which can be stimulated by ANGII and prolongs vasoconstriction and also Phosphatase-1 of mitogen-activated proteins (MKP-1), an important phosphatase that has the primary function of inactivating MAPKs, and it is estimulated by IL-10. The objective of this work is to evaluate if ANG-(1-7) reduces ERK 1/2 activity due to stimulation of MKP-1 and IL-10, resulting in a decrease in vascular contraction. The present study was approved by the UFMT Animal Research Ethics Committee (23108.166477/2016-20). Aortic rings of male C57BL/6J mice, 10-12 weeks of age, were incubated with ANG-(1-7) [10μM] or vehicle for 5 min, to perform ANGII concentration-response curve in the presence or absence of Mas receptor antagonist (A-779, 100μM) and cAMP (Rp-cAMPs, 1μM). Vascular expressions of c-Raf, ERK1/2 and MKP-1 proteins were verified by Western Blot and immunocytochemistry in vascular smooth muscle cell culture (LVC), incubated individually or simultaneously with ANG-(1-7) [10μM] , ANGII [1μM] or vehicle for 5 min. IL-10 knockout animals, infused with ANGII (90ng / min) for 14 days were used to verify IL-10 protector effects. Finally, blood vessel from animals were incubated with ANG-(1-7) [10μM], or vehicle, for 12h, to evaluate indirect expression of IL-10. The results show that ANG-(1-7) reduces vascular contraction induced by ANGII, via activation of Mas receptors and cAMP formation. It was also demonstrated that ANG-(1-7) prevented ERK1/2 activation, possibly through activation of MKP-1 phosphatase, both in aortic tissue and in vascular smooth muscle cell culture. Other result found in this study was that IL-10-lacking animals, ANGII has exacerbated activity, presenting higher vascular contraction, probably due to the increase in ERK 1/2 expression. In addition, it was observed that vessels treated with ANG-(1-7) for 12 hours presented higher expression of STAT3, a crucial element in IL-10 signaling pathway. In conclusion, the results suggest that ANG-(1-7) stimulates the activity of MKP-1 and IL-10 signaling pathway, which inhibits the ERK1/2 phosphorylation and consequently contributes to the reduction of vascular contractile response.O Sistema Renina-angiotensina (SRA) exerce papel fundamental na regulação da pressão arterial e equilíbrio hidroeletrolítico. O peptídeo Angiotensina (ANG)-(1-7) é gerado predominantemente na parede vascular a partir da ANGII ou a partir da ANGI. A ANG-(1-7) liga-se ao seu receptor Mas acoplado à proteína G e atua mediando a vasodilatação, inibindo o crescimento celular, portando-se como antagonista das ações vasculares da ANGII. Seus efeitos também podem ser atenuados por citocinas regulatórias, como a Interleucina (IL)-10, que modula negativamente citocinas pró-inflamatórias como o fator de necrose tumoral-alfa (TNF-α), IL-6 e inibe a produção de espécies reativas de oxigênio (EROs), reduzindo a inflamação e favorecendo a vasodilatação. Embora ANG-(1-7) e IL-10 apresentem funções similares, em aspectos vasculares, a associação entre ambas ainda é desconhecida, mas parece estar relacionada com outras duas moléculas: a MAPK, quinase regulada por sinal extracelular 1 e 2 (ERK 1/2), que pode ser estimulada por ANGII e prolongar uma vasoconstrição, bem como a fosfatase-1 de proteínas ativadas por mitógenos (MKP-1), fosfatase que tem por função primaria inativar as MAPKs e é estimulada por IL-10. O objetivo do trabalho foi avaliar se a ANG-(1-7) reduz a atividade da ERK 1/2 pela estimulação da MKP-1 e IL-10, culminando na diminuição da contração vascular. Este estudo foi aprovado pelo Comitê de Ética no Uso de Animais da UFMT com número de protocolo (23108.166477/2016-20). Foram utilizados anéis aórticos de camundongos machos C57BL/6J, com 10-12 semanas de vida, foram incubados com ANG-(1-7) [10µM] ou veículo, por 5 min, para posterior realização de curva concentração-resposta para ANGII, na presença ou ausência do antagonista do receptor Mas (A-779, 100μM) e do AMPc (Rp-cAMPs,1μM). A expressão vascular das proteínas c-Raf, ERK1/2 e MKP-1 foram verificadas por WesternBlot e imunocitoquímica em cultura de células de músculo liso vascular (CMLV), incubadas individualmente, ou simultaneamente, com ANG-(1-7) [10µM], ANGII [1μM] ou veículo, por 5 min. Animais knockouts para IL-10, infundidos com ANGII (90ng/min) por 14 dias foram utilizados para verificar o efeito protetor da IL-10. Por fim, vasos de animais foram incubados com ANG-(1-7) [10µM], ou veículo, durante 12 h, para avaliar a expressão indireta de IL-10. Os resultados mostraram que a ANG-(1-7) reduz a contração vascular induzida por ANGII, via ativação de receptores Mas e formação de AMPc. Foi demonstrado também que a ANG-(1-7) preveniu a ativação da ERK1/2, possivelmente, através da ativação da fosfatase MKP-1, tanto no tecido aórtico como em cultura de células do músculo liso vascular. Animais deficientes de IL-10 tem atividade da ANGII exacerbada, apresentando uma maior contração vascular, provavelmente pelo aumento da expressão de ERK 1/2. Além disso, foi observado que vasos tratados com ANG-(1-7) por 12 horas, apresentam um aumento da expressão de STAT3, elemento crucial na via de sinalização de IL-10. Em conclusão, os resultados sugerem que a ANG-(1-7) estimula a atividade de MKP-1 e da via de sinalização da IL-10, as quais inibem a fosforilação de ERK1/2 que contribui para a redução na resposta vascular contrátil.Universidade Federal de Mato GrossoBrasilInstituto de Ciências Biológicas e da Saúde (ICBS) – AraguaiaUFMT CUA - AraguaiaPrograma de Pós-Graduação em Imunologia e Parasitologia Básicas e AplicadasLima, Victor VitorinoVitorino, Fernanda Regina Casagrande Giachinihttp://lattes.cnpq.br/3100345884689140http://lattes.cnpq.br/7503102443670435Lima, Victor Vitorino005.356.961-08http://lattes.cnpq.br/7503102443670435Américo, Madileine Francely267.529.008-41http://lattes.cnpq.br/4097128727139521005.356.961-08712.962.121-49Santos, Sandra Maria dos002.704.211-14http://lattes.cnpq.br/7483911176076684Lopes, Alejandra Sousa2021-03-10T13:58:11Z2018-12-202021-03-10T13:58:11Z2018-10-23info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisLOPES, Alejandra Sousa. O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos. 2018. 76 f. Dissertação (Mestrado em Imunologia e Parasitologia Básicas e Aplicadas) - Universidade Federal de Mato Grosso, Instituto de Ciências Biológicas e da Saúde, Barra do Garças, 2018.http://ri.ufmt.br/handle/1/2363porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFMTinstname:Universidade Federal de Mato Grosso (UFMT)instacron:UFMT2021-03-11T07:01:22Zoai:localhost:1/2363Repositório InstitucionalPUBhttp://ri.ufmt.br/oai/requestjordanbiblio@gmail.comopendoar:2021-03-11T07:01:22Repositório Institucional da UFMT - Universidade Federal de Mato Grosso (UFMT)false
dc.title.none.fl_str_mv O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
title O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
spellingShingle O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
Lopes, Alejandra Sousa
Hipertensão
Vasoconstrição
Sistema renina-angiotensina
CNPQ::CIENCIAS DA SAUDE
Hypertension
Vasoconstriction
Renin-angiotensin system
title_short O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
title_full O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
title_fullStr O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
title_full_unstemmed O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
title_sort O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos
author Lopes, Alejandra Sousa
author_facet Lopes, Alejandra Sousa
author_role author
dc.contributor.none.fl_str_mv Lima, Victor Vitorino
Vitorino, Fernanda Regina Casagrande Giachini
http://lattes.cnpq.br/3100345884689140
http://lattes.cnpq.br/7503102443670435
Lima, Victor Vitorino
005.356.961-08
http://lattes.cnpq.br/7503102443670435
Américo, Madileine Francely
267.529.008-41
http://lattes.cnpq.br/4097128727139521
005.356.961-08
712.962.121-49
Santos, Sandra Maria dos
002.704.211-14
http://lattes.cnpq.br/7483911176076684
dc.contributor.author.fl_str_mv Lopes, Alejandra Sousa
dc.subject.por.fl_str_mv Hipertensão
Vasoconstrição
Sistema renina-angiotensina
CNPQ::CIENCIAS DA SAUDE
Hypertension
Vasoconstriction
Renin-angiotensin system
topic Hipertensão
Vasoconstrição
Sistema renina-angiotensina
CNPQ::CIENCIAS DA SAUDE
Hypertension
Vasoconstriction
Renin-angiotensin system
description The Renin Angiotensin System (RAS) plays a fundamental role in the regulation of blood pressure and electrolyte balance. The angiotensin peptide (ANG)-(1-7), bound to RAS, is predominantly generated in the vascular wall under two main pathways: through ANGII by the action of neutral endopeptidases and prolyl-endopeptidases or by ANGI action, by hydrolysis from Angiotensin converting enzyme 2 (ACE2). ANG-(1-7) binds to its receptor Mas coupled to G protein and acts mediating vasodilation, inhibiting cell growth, acting as an important antagonist of ANGII vascular actions (vasoconstriction and inflammation). Recently, studies have shown that ANGII effects can also be attenuated by regulatory cytokines such as Interleukin (IL)-10, which negatively modulates proinflammatory cytokine polysaccharides producers such as tumor necrosis factor-alpha and IL-6 and inhibits the production of reactive oxygen species (ROS), reducing inflammation and favoring vasodilation. Although ANG-(1-7) and IL-10 possess similar functions, in vascular aspects, the association of both factors is still unknown and it seems to be related with other two significant molecules: MAPK, extracellular signal-regulated kinase 1 and 2 (ERK 1/2), which can be stimulated by ANGII and prolongs vasoconstriction and also Phosphatase-1 of mitogen-activated proteins (MKP-1), an important phosphatase that has the primary function of inactivating MAPKs, and it is estimulated by IL-10. The objective of this work is to evaluate if ANG-(1-7) reduces ERK 1/2 activity due to stimulation of MKP-1 and IL-10, resulting in a decrease in vascular contraction. The present study was approved by the UFMT Animal Research Ethics Committee (23108.166477/2016-20). Aortic rings of male C57BL/6J mice, 10-12 weeks of age, were incubated with ANG-(1-7) [10μM] or vehicle for 5 min, to perform ANGII concentration-response curve in the presence or absence of Mas receptor antagonist (A-779, 100μM) and cAMP (Rp-cAMPs, 1μM). Vascular expressions of c-Raf, ERK1/2 and MKP-1 proteins were verified by Western Blot and immunocytochemistry in vascular smooth muscle cell culture (LVC), incubated individually or simultaneously with ANG-(1-7) [10μM] , ANGII [1μM] or vehicle for 5 min. IL-10 knockout animals, infused with ANGII (90ng / min) for 14 days were used to verify IL-10 protector effects. Finally, blood vessel from animals were incubated with ANG-(1-7) [10μM], or vehicle, for 12h, to evaluate indirect expression of IL-10. The results show that ANG-(1-7) reduces vascular contraction induced by ANGII, via activation of Mas receptors and cAMP formation. It was also demonstrated that ANG-(1-7) prevented ERK1/2 activation, possibly through activation of MKP-1 phosphatase, both in aortic tissue and in vascular smooth muscle cell culture. Other result found in this study was that IL-10-lacking animals, ANGII has exacerbated activity, presenting higher vascular contraction, probably due to the increase in ERK 1/2 expression. In addition, it was observed that vessels treated with ANG-(1-7) for 12 hours presented higher expression of STAT3, a crucial element in IL-10 signaling pathway. In conclusion, the results suggest that ANG-(1-7) stimulates the activity of MKP-1 and IL-10 signaling pathway, which inhibits the ERK1/2 phosphorylation and consequently contributes to the reduction of vascular contractile response.
publishDate 2018
dc.date.none.fl_str_mv 2018-12-20
2018-10-23
2021-03-10T13:58:11Z
2021-03-10T13:58:11Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv LOPES, Alejandra Sousa. O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos. 2018. 76 f. Dissertação (Mestrado em Imunologia e Parasitologia Básicas e Aplicadas) - Universidade Federal de Mato Grosso, Instituto de Ciências Biológicas e da Saúde, Barra do Garças, 2018.
http://ri.ufmt.br/handle/1/2363
identifier_str_mv LOPES, Alejandra Sousa. O papel da ANG-(1-7) na inativação de ERK 1/2 modulada por MKP-1 e IL-10 em aorta de camundongos. 2018. 76 f. Dissertação (Mestrado em Imunologia e Parasitologia Básicas e Aplicadas) - Universidade Federal de Mato Grosso, Instituto de Ciências Biológicas e da Saúde, Barra do Garças, 2018.
url http://ri.ufmt.br/handle/1/2363
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Universidade Federal de Mato Grosso
Brasil
Instituto de Ciências Biológicas e da Saúde (ICBS) – Araguaia
UFMT CUA - Araguaia
Programa de Pós-Graduação em Imunologia e Parasitologia Básicas e Aplicadas
publisher.none.fl_str_mv Universidade Federal de Mato Grosso
Brasil
Instituto de Ciências Biológicas e da Saúde (ICBS) – Araguaia
UFMT CUA - Araguaia
Programa de Pós-Graduação em Imunologia e Parasitologia Básicas e Aplicadas
dc.source.none.fl_str_mv reponame:Repositório Institucional da UFMT
instname:Universidade Federal de Mato Grosso (UFMT)
instacron:UFMT
instname_str Universidade Federal de Mato Grosso (UFMT)
instacron_str UFMT
institution UFMT
reponame_str Repositório Institucional da UFMT
collection Repositório Institucional da UFMT
repository.name.fl_str_mv Repositório Institucional da UFMT - Universidade Federal de Mato Grosso (UFMT)
repository.mail.fl_str_mv jordanbiblio@gmail.com
_version_ 1804648505315164160