Comportamento de células endoteliais submetidas a um modelo de hipertensão arterial in vitro
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Data
2019-02-22
Autores
Pinto, Thais Silva [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Mudanças nas forças tensionais do shear-stress estão associadas a um repertório de cascatas de sinalização celular, as quais modulam em conjunto o fenótipo vascular tornando o tecido endotelial susceptível a variações patofisiológica e, portanto, compreensão do repertório molecular neste cenário é necessária. Com este propósito, nós submetemos células endoteliais de veia umbilical humana (HUVEC) a um circuito de diferentes forças tensionais in vitro, considerando os grupos seguintes: 1. condição de fluxo de shear-stress fisiológico (nomeado Normo); 2. fluxo de shear-stress hipertenso (nomeado Hyper), e 3. células do grupo 2 foram retornadas para a condição Normo (nomeado Return). As amostras foram apropriadamente coletadas para seguir em diferentes metodologias. Nossos resultados mostraram um forte envolvimento de c-Src no controle da cascata de mecanotransdução modulando sinalização necessária para o fenótipo de adesão, sobrevivência (PI3K/AKT) e proliferação celulares. Além disso, c-Src parece desenvolver importante papel durante o remodelamento da Matriz Extracelular (MEC), cujo performance de matriz metaloproteinases (MMPs) mostrou mudanças significativas. Além disso, através de análise proteômica, mostramos um forte envolvimento de Heat Shock Protein 70 (HSP70) nas células estressadas de modo Hyper, reduzindo significativamente no grupo Return. Esse resultado levou-nos a investigar o proteassoma 20S como uma alternativa proteolítica intracelular para promover o turnover dessas proteínas. Surpreendentemente, nossos dados revelaram um significante aumento na expressão de genes proteasome subunit α-type (PSMA) e β-type (PSMB), genes que codificam proteínas relacionadas com o core catalítico do complexo proteassômico. Adicionalmente, mostramos que vias intracelulares clássicas (Wnt/β- Catenina, Bax e BCl-2) e genes envolvidos com o metabolismo energético sofreram significante modulação nas diferentes tensões de cisalhamento aplicadas. Em conjunto, nossos dados reportaram que um modelo in vitro de hipertensão é capaz de trazer informações importantes sobre a mecanossensibilidade de células endoteliais. Como padrões de cisalhamento estão associados com mudanças patofisiológicas, como aterosclerose e hipertensão, esses resultados criam novos caminhos para compreender o mecanismo molecular responsável pela mecanotransdução no endotélio e, se forem passíveis de ação medicamentosa, muitos desses alvos devem ser considerados para pesquisas futuras de fase pré-clínica.
Shear-stress changes are associated with a repertory of signaling cascade, modulating vascular phenotype. As shear stress-related tensional forces might be associated with pathophysiological susceptibility, a more comprehensive molecular map needs to be addressed. Thus, we subjected human umbilical vein endothelial cells (HUVECs) to a circuit of different tensional forces in vitro considering the following three groups: one in a physiological blood flow shear-stress condition (named Normo), another in which these cells followed to a hypertensive blood flow shear-stress (named Hyper), and finally one that these hyper-stressed cells were returned to Normo condition (named Return). The samples were properly collected to allow different methodologies analysis. Our data showed a pivotal involvement of c-Src on driving the mechanotransduction cascade by modulating signaling related with adhesion, survival (PI3K/Akt) and proliferative phenotype. Moreover, c-Src seems to develop important role during Extracellular Matrix (ECM) remodeling, which showed significative changes. Additionally, proteomic analysis showed strong involvement of Heat Shock Protein 70 (HSP70) in the hypertensive-stressed cells; it being significantly decreased in Return phenotype. This result prompted us to investigate 20S proteasome as an intracellular proteolytic alternative to promote the turnover of those proteins. Surprisingly, our data reveled significant over expression of sets of proteasome subunit α-type (PSMA) and proteasome subunit β-type (PSMB) genes (encoding proteins related with catalytic core of proteasome). Besides, constituents of classical intracellular pathways - for example Wnt/βCatenina, Bax and BCl-2, including genes involved with energetic metabolism, also suffered strong modulation on different model pressure. Altogether, our data reported that an in vitro hypertension model can bring a lot of insights about the mechanosensitivity in endothelial cells. Since shear patterns are associated with pathophysiologic changes, as well atherosclerosis and hypertension, these results paved new roads to understand the molecular mechanism in mechanotransduction driving in endothelium, and if drugable, many of these targets should be considered for future pre-clinical research.
Shear-stress changes are associated with a repertory of signaling cascade, modulating vascular phenotype. As shear stress-related tensional forces might be associated with pathophysiological susceptibility, a more comprehensive molecular map needs to be addressed. Thus, we subjected human umbilical vein endothelial cells (HUVECs) to a circuit of different tensional forces in vitro considering the following three groups: one in a physiological blood flow shear-stress condition (named Normo), another in which these cells followed to a hypertensive blood flow shear-stress (named Hyper), and finally one that these hyper-stressed cells were returned to Normo condition (named Return). The samples were properly collected to allow different methodologies analysis. Our data showed a pivotal involvement of c-Src on driving the mechanotransduction cascade by modulating signaling related with adhesion, survival (PI3K/Akt) and proliferative phenotype. Moreover, c-Src seems to develop important role during Extracellular Matrix (ECM) remodeling, which showed significative changes. Additionally, proteomic analysis showed strong involvement of Heat Shock Protein 70 (HSP70) in the hypertensive-stressed cells; it being significantly decreased in Return phenotype. This result prompted us to investigate 20S proteasome as an intracellular proteolytic alternative to promote the turnover of those proteins. Surprisingly, our data reveled significant over expression of sets of proteasome subunit α-type (PSMA) and proteasome subunit β-type (PSMB) genes (encoding proteins related with catalytic core of proteasome). Besides, constituents of classical intracellular pathways - for example Wnt/βCatenina, Bax and BCl-2, including genes involved with energetic metabolism, also suffered strong modulation on different model pressure. Altogether, our data reported that an in vitro hypertension model can bring a lot of insights about the mechanosensitivity in endothelial cells. Since shear patterns are associated with pathophysiologic changes, as well atherosclerosis and hypertension, these results paved new roads to understand the molecular mechanism in mechanotransduction driving in endothelium, and if drugable, many of these targets should be considered for future pre-clinical research.
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Palavras-chave
Shear-stress, Célula endotelial, Mecanotransdução, Hipertensão, Shear-stress, Endothelial cell, Mechanotransduction, Hypertension