Comportamento de células endoteliais e muscular submetidas ao shear stress: um panorama celular e bioquímico
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Data
2019-02-22
Autores
Gomes, Anderson Moreira
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Universidade Estadual Paulista (Unesp)
Resumo
As células endoteliais (ECs) e células musculares lisas (AoSMCs) são os principais componentes celulares do endotélio. As interações entre estes tipos celulares desempenham funções na homeostase e na estrutura vascular. Como uma interface entre o sangue e a parede do vaso, as ECs ocupam um local único diretamente exposto ao shear stress (SS), a força mecânica de atrito lateral produzido pelo fluxo de sangue na membrana apical da célula endotelial, que pode influenciar o comportamento de ambas ECs e AoSMCs. Geralmente, AoSMCs não sofrem diretamente às forcas de cisalhamento, no entanto, estas são diretamente expostas ao fluxo sanguíneo quando ocorre alguma injúria vascular, como por exemplo em algumas lesões ateroscleróticas ou por técnicas invasivas, como a angioplastia. As forças hemodinâmicas influenciam as propriedades funcionais do endotélio, porém estas não são profundamente compreendidas quanto aos mecanismos bioquímicos de respostas de células endoteliais e de musculatura lisa. Assim, a proposta desta dissertação foi estabelecer um modelo de cultivo in vitro que mimetize as forças tensionais de cisalhamento (shear stress), buscando compreender mecanismos celulares, bioquímicos e epigenéticos. Cultura de células primárias endoteliais e de musculatura lisa humanas foram obtidas da empresa LONZA e mantidas conforme recomendações do fabricante. Estas células foram mantidas rotineiramente em condições convencionais em incubadora de CO2. Para mimetizar o fluxo sanguíneo, estas células, separadamente, foram semeadas em anel periférico de uma placa de petri (100mm de diâmetro), modificada de acordo estudos anteriores, e mantidas sob fluxo laminar por 72 horas, quando as células foram coletadas e encaminhadas para as diferentes metodologias. Nossos resultados estão apresentados em 2 capítulos nessa dissertação, sendo o Cap 2 dedicado aos mecanismos envolvidos na adaptação de células endoteliais ao shear stress, e o Cap. 3, dedicado aos mecanismos desencadeados em células musculares lisa. De um modo geral, nossos resultados mostram respostas diferenciais entre células endoteliais e de musculatura lisa ao shear-stress, as quais são regidas por mecanismos epigenéticos no controle da expressão gênica desencadeados pelo remodelamento da matriz extracelular, avaliada aqui como um mecanismo importante de adaptação destas células às forças de cisalhamento.
Endothelial cells (ECs) and smooth muscle cells (AoSMCs) are the major cellular components of the endothelium. Interactions between these cell types play roles in homeostasis and vascular structure. As an interface between the blood and the vessel wall, ECs occupy a single site directly exposed to shear stress (SS), the mechanical lateral friction force produced by blood flow on the apical membrane of the endothelial cell, which can influence behavior of both ECs and AoSMCs. Generally, AoSMCs do not directly undergo shear forces, however, they are directly exposed to blood flow when vascular injury occurs, for example in some atherosclerotic lesions or by invasive techniques such as angioplasty or surgical endarterectomy. Hemodynamic forces influence the functional properties of the endothelium, but these are not deeply understood as to the biochemical mechanisms of endothelial cell and smooth muscle responses. Thus, the purpose of this dissertation was to establish an in vitro culture model that mimics the shear forces, seeking to understand cellular, biochemical and epigenetic mechanisms. Culture of human endothelial and smooth muscle cells were obtained from the company LONZA and maintained according to the manufacturer's recommendations. These cells were routinely maintained under standard conditions in CO2 incubator. To mimic blood flow, these cells were separately seeded in a peripheral ring of a petri dish (100mm diameter), modified according to previous studies, and maintained under laminar flow for 72 hours, when the cells were collected and sent to the different methodologies. Our results are presented in 2 chapters in this dissertation, with Cap. 2 dedicated to the mechanisms involved in the adaptation of endothelial cells to shear stress, and Cap. 3, dedicated to the mechanisms triggered in smooth muscle cells. Overall, our results show differential responses between endothelial and smooth muscle cells at shear stress, which are governed by epigenetic mechanisms in the control of gene expression triggered by the remodeling of the extracellular matrix, evaluated here as an important mechanism of adaptation of these cells to shear forces.
Endothelial cells (ECs) and smooth muscle cells (AoSMCs) are the major cellular components of the endothelium. Interactions between these cell types play roles in homeostasis and vascular structure. As an interface between the blood and the vessel wall, ECs occupy a single site directly exposed to shear stress (SS), the mechanical lateral friction force produced by blood flow on the apical membrane of the endothelial cell, which can influence behavior of both ECs and AoSMCs. Generally, AoSMCs do not directly undergo shear forces, however, they are directly exposed to blood flow when vascular injury occurs, for example in some atherosclerotic lesions or by invasive techniques such as angioplasty or surgical endarterectomy. Hemodynamic forces influence the functional properties of the endothelium, but these are not deeply understood as to the biochemical mechanisms of endothelial cell and smooth muscle responses. Thus, the purpose of this dissertation was to establish an in vitro culture model that mimics the shear forces, seeking to understand cellular, biochemical and epigenetic mechanisms. Culture of human endothelial and smooth muscle cells were obtained from the company LONZA and maintained according to the manufacturer's recommendations. These cells were routinely maintained under standard conditions in CO2 incubator. To mimic blood flow, these cells were separately seeded in a peripheral ring of a petri dish (100mm diameter), modified according to previous studies, and maintained under laminar flow for 72 hours, when the cells were collected and sent to the different methodologies. Our results are presented in 2 chapters in this dissertation, with Cap. 2 dedicated to the mechanisms involved in the adaptation of endothelial cells to shear stress, and Cap. 3, dedicated to the mechanisms triggered in smooth muscle cells. Overall, our results show differential responses between endothelial and smooth muscle cells at shear stress, which are governed by epigenetic mechanisms in the control of gene expression triggered by the remodeling of the extracellular matrix, evaluated here as an important mechanism of adaptation of these cells to shear forces.
Descrição
Palavras-chave
Células endoteliais, Célula de musculatura lisa, Epigenética, Shear stress, Endothelial cells, Smooth muscle cell, Epigenetics, Shear-stress