Modo de ação de uma ramnana sulfatada em células endoteliais e musculares lisas: potencial uso na prevenção da disfunção endotelial
Data
2015-07-31
Autores
Nobre, Leonardo Thiago Duarte Barreto 
Orientadores
Nader, Helena Bonciani
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A disfunção endotelial pode causar aterosclerose, uma doença caracterizada pelo acúmulo de placas contendo lipídeos na camada subendotelial de vasos sanguíneos. O glicocálice endotelial mantém a homeostasia entre o sangue e as células endoteliais e alterações em sua estrutura podem causar doenças. Algas marinhas são fontes de polissacarídeos sulfatados que apresentam diversas propriedades farmacológicas, entre as quais se destacam atividades antioxidante, anti-inflamatória, anticoagulante e antitrombótica. O objetivo deste trabalho foi avaliar os efeitos de uma ramnana sulfatada (RS) obtida da alga Monostroma nitidum em células endoteliais e musculares lisas, bem como em anéis de aorta, assim como, descrever seu provável mecanismo de ação. Foi constatado que RS não afeta a viabilidade dessas células em 24 horas na concentração de até 100 ug/mL e é capaz de aumentar a marcação do glicocálice pela lectina WGA, observada em microscopia confocal, em ambas as linhagens celulares e também no anel de aorta de ratos. Há um aumento na síntese de ácido hialurônico por essas células tratadas com RS e esse ácido hialurônico apresenta alto peso molecular. Através da marcação metabólica com sulfato radioativo foi constatado que células tratadas com RS aumentaram a síntese de glicosaminoglicanos sulfatados com destaque para o heparam sulfato. Esse heparam sulfato teve sua estrutura analisada e foi observado que há um aumento na proporção de dissacarídeo trissulfatado, típico da estrutura de heparinas. Foi observado aumento na expressão das enzimas responsáveis pela síntese e modificação desses glicosaminoglicanos. Utilizando microscopia confocal e ensaio de FRET foi comprovado que RS se liga à fibronectina na matriz extracelular. RS ativa a sinalização celular através da via de FAK-Src-ERK, aumenta os níveis de cálcio intracelular e a produção de óxido nítrico. Por fim foi observado que RS também é capaz de inibir a angiogênese em ensaio de formação de estruturas capilares em matrigel e também no ensaio tridimensional de angiogênese em anel de aorta. Os resultados indicam que RS por aumentar e modificar estruturalmente o glicocálice nas células derivadas de aorta, estimular a síntese do heparam sulfato rico em dissacarídeo trisssulfatado (típico da estrutura de heparinas e com potente efeito antitrombótico), bem como de ácido hialurônico de alto peso molecular (com ação anti-aterogênica e antiangiogênica), aumentar a síntese de óxido nítrico e por si só inibir a angiogênese apresenta-se como um possível agente terapêutico para prevenir o desenvolvimento da aterosclerose e formação de trombos em função dos diversos efeitos benéficos nas células que podem impedir diferentes etapas desses processos patológicos.
Endothelial dysfunction can cause atherosclerosis, a disease characterized by accumulation of plaques containing lipids in the subendothelial layer of blood vessels. The endothelial glycocalyx maintains the homeostasis between the blood and the endothelial cells, and changes in their structure can cause disease. Seaweed sources are sulfated polysaccharides that have different pharmacological properties, among which we highlight the antioxidant activity, anti-inflammatory, anticoagulant and antithrombotic. The objective of this study was to evaluate the effects of a sulfated ramnana (RS) obtained from the algae Monostroma nitidum in endothelial and smooth muscle cells as well as in aortic rings, as well as describe its probable mechanism of action. It was found that the RS does not affect cell viability in 24 hours at concentrations up to 100 ug/ml and is able to increase the marking glycocalyx by WGA lectin observed in confocal microscopy for both cell lines and also in the aortic ring mice. There is an increase in hyaluronic acid synthesis by such cells treated with RS and that hyaluronic acid has a high molecular weight. By metabolic labeling with radioactive sulfate was found to RS-treated cells increased the synthesis of sulfated glycosaminoglycans highlighting the heparan sulfate. This heparan sulfate was analyzed and their structure was observed that there is an increase in the proportion of trisulfated disaccharide structure typical of the heparins. It was observed an increase in the expression of enzymes responsible for the synthesis and modification of these glycosaminoglycans. Using confocal microscopy and FRET assay it was proven that RS binds to fibronectin in the extracellular matrix. It was shown that RS actives cell signaling by FAK, Src and ERK pathway increasing intracellular calcium levels and nitric oxide production. Finally it was observed that RS is also capable of inhibiting angiogenesis in assay formation of capillary structures in matrigel and also in the three-dimensional test in aortic ring angiogenesis. The results indicate that RS acts increasing and structurally modifying the glycocalyx in the derived aortic cells, stimulate the synthesis of heparan sulfate rich in trisulfated disaccharide, a typical structure of heparins with potent antithrombotic effect, as well as high molecular weight hyaluronic acid which is known to be anti-atherogenic and anti-angiogenic, increase nitric oxide synthesis and inhibit angiogenesis itself presents itself as a possible therapeutic agent to prevent the development of atherosclerosis and thrombus formation in function of the various beneficial effects on cells that can prevent different stages of these pathological processes.
Endothelial dysfunction can cause atherosclerosis, a disease characterized by accumulation of plaques containing lipids in the subendothelial layer of blood vessels. The endothelial glycocalyx maintains the homeostasis between the blood and the endothelial cells, and changes in their structure can cause disease. Seaweed sources are sulfated polysaccharides that have different pharmacological properties, among which we highlight the antioxidant activity, anti-inflammatory, anticoagulant and antithrombotic. The objective of this study was to evaluate the effects of a sulfated ramnana (RS) obtained from the algae Monostroma nitidum in endothelial and smooth muscle cells as well as in aortic rings, as well as describe its probable mechanism of action. It was found that the RS does not affect cell viability in 24 hours at concentrations up to 100 ug/ml and is able to increase the marking glycocalyx by WGA lectin observed in confocal microscopy for both cell lines and also in the aortic ring mice. There is an increase in hyaluronic acid synthesis by such cells treated with RS and that hyaluronic acid has a high molecular weight. By metabolic labeling with radioactive sulfate was found to RS-treated cells increased the synthesis of sulfated glycosaminoglycans highlighting the heparan sulfate. This heparan sulfate was analyzed and their structure was observed that there is an increase in the proportion of trisulfated disaccharide structure typical of the heparins. It was observed an increase in the expression of enzymes responsible for the synthesis and modification of these glycosaminoglycans. Using confocal microscopy and FRET assay it was proven that RS binds to fibronectin in the extracellular matrix. It was shown that RS actives cell signaling by FAK, Src and ERK pathway increasing intracellular calcium levels and nitric oxide production. Finally it was observed that RS is also capable of inhibiting angiogenesis in assay formation of capillary structures in matrigel and also in the three-dimensional test in aortic ring angiogenesis. The results indicate that RS acts increasing and structurally modifying the glycocalyx in the derived aortic cells, stimulate the synthesis of heparan sulfate rich in trisulfated disaccharide, a typical structure of heparins with potent antithrombotic effect, as well as high molecular weight hyaluronic acid which is known to be anti-atherogenic and anti-angiogenic, increase nitric oxide synthesis and inhibit angiogenesis itself presents itself as a possible therapeutic agent to prevent the development of atherosclerosis and thrombus formation in function of the various beneficial effects on cells that can prevent different stages of these pathological processes.
Descrição
Citação
NOBRE, Leonardo Thiago Duarte Barreto. Modo de ação de uma ramnana sulfatada em células endoteliais e musculares lisas: potencial uso na prevenção da disfunção endotelial. 2015. 122 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.