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Type:	Tese
Title:	Contribution of endothelial cells mechanics properties and their consequences to the development of atherosclerosis: an in vitro and in vivo study
Authors:	Natália Fernanda do Couto
First Advisor:	Luciana de Oliveira Andrade
Abstract:	Atherosclerosis is an inflammatory disease caused by subendothelial retention of oxidized LDL (oxLDL), leading to the formation of a plaque. It preferentially develops at areas of vessel bifurcation or curvature, revealing that hemodynamic forces, such as the shear stress generated by blood flow, also work as local risk factors, likely leading to endothelium mechanical injury. It is known that oxLDL modifies cellular biomechanics, inducing cytoskeleton reorganization in cell cortex. This may compromise membrane trafficking events between cytoplasm and cell surface, which in turn can affect cellular functions such as plasma membrane repair. The latter depends on lysosomal fusion with plasma membrane and the subsequent recycling of vesicles. Using in vitro and in vivo approaches, we evaluated the influence of endothelial cells exposure to oxLDL and hyperlipidemia in cells mechanical modulation, vesicle trafficking, plasma membrane repair and cell susceptibility to injury. Additionally, we evaluated possible contributions of these mechanical alterations together with variations in blood flow shear stress for the development of the atherosclerotic plaque in vivo. Our in vitro data shows that, in a human umbilical vein endothelial cell line (EAhY926), oxLDL exposure led to actin reorganization and de novo polymerization, as well as an increase in cell rigidity and lysosomal exocytosis. Additionally, there was an initial increase in endocytic events rates, likely triggered by the peak of exocytosis induced by both treatments. However, no further endocytic events were observed, suggesting that constitutive endocytosis is blocked upon treatment and that the reorganized cytoskeleton function as a mechanical barrier to membrane traffic. Moreover, the increase in cell rigidity renders cells more prone to mechanical injury. In order to evaluate whether the same was true in vivo and if alterations on shear stress pattern would influence these effects, we surgically implanted a shear stress modifier device (cast) on the right carotid (RC) artery of ApoE-knock out (ApoE-/-) mice, an animal model for atherosclerosis studies. The particular shape of the device causes a gradual stenosis in the vessel, resulting in increased shear stress (HSS) in the vessel segment inside the device, a decrease in blood flow and consequently a low shear stress (LSS) region downstream, and a vortex in the upstream region (oscillatory shear stress, OSS). Wild type C57BL/6 mice, as well as the left carotid (LC) of each animal, were used as controls. The data obtained in the present work indicates a tendency of actin fibers accumulation in areas with higher lipid deposition, corroborating with our in vitro study. Furthermore, shear stress alone was able to increase cell injury in areas of HSS in the RC of both ApoE-/- and C57BL/6 mice. In this analysis, hyperlipidemia was not determinant to vascular cells susceptibility to injury. However, we speculate that exposing cells to excess of lipids can be prejudicial to their ability in repairing from damage, since lysosomal exocytosis, an important step for PMR, is compromised in ApoE-/- mice. Together, these data show that mechanical modulation induced by oxLDL exposure in vitro, as well as hyperlipidemia and variations on shear stress patterns in vivo, not only alters membrane traffic in cells, but also makes them more susceptible to mechanical injury, which may likely contribute to endothelial fragility and thus to the initial steps of atherosclerosis development.
language:	eng
metadata.dc.publisher.country:	Brasil
Publisher:	Universidade Federal de Minas Gerais
Publisher Initials:	UFMG
metadata.dc.publisher.department:	ICB - DEPARTAMENTO DE MORFOLOGIA
metadata.dc.publisher.program:	Programa de Pós-Graduação em Biologia Celular
Rights:	Acesso Restrito
metadata.dc.rights.uri:	http://creativecommons.org/licenses/by-nc-nd/3.0/pt/
URI:	http://hdl.handle.net/1843/35864
Issue Date:	28-Aug-2020
metadata.dc.description.embargo:	28-Aug-2021
Appears in Collections:	Teses de Doutorado

This item is licensed under a Creative Commons License