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Lysosome dysfunction and cellular senescence in Atherogenesis: is there a link?
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RESUMO: A disfunção lisossomal e a senescência celular desempenham papéis fundamentais no desenvolvimento da aterosclerose. No entanto, os mecanismos moleculares subjacentes e a potencial ligação entre eles permanecem desconhecidos. Identificámos anteriormente uma nova família de lípidos oxidados, os hemiésteres de colesterol (ChEs), produtos finais da oxidação de ácidos gordos poliinsaturados em ésteres de colesterol (PUFA-CEs) no plasma e nos tecidos de pacientes com doenças cardiovasculares (DCV). Verificou-se que o hemiazelato de colesterol (ChA), o ChE mais prevalente no plasma e nas lesões ateroscleróticas, tem um impacto significativo na função lisossomal das células do musculo liso (CMLs), causando a paragem do ciclo celular (quiescência). No entanto, as CMLs tratadas com ChA nunca atingiram um estado senescente.
Considerando essas informações, pretendíamos construir um modelo celular adequado para estudar a senescência prematura induzida por stress (SIPS) e o mau funcionamento dos lisossomas desencadeado pelas ChEs. Em primeiro lugar, procuramos desenvolver um modelo de senescência utilizando fibroblastos embrionários de ratinho (MEFs) e ChA como insulto patológico. Para estudar o papel da função lisossomal no desenvolvimento da senescência, utilizámos MEFs com dupla knockout (DKO) dos fatores de transcrição TFEB e TFE3, que regulam a função lisossomal e a autofagia, bem como a sobrevivência das células senescentes. Notavelmente, os fibroblastos wild type expostos ao ChA exibiram acumulação perinuclear e lisossomas aumentados cheios de lípidos neutros, mas não apresentaram sinais de senescência. Embora os resultados não tenham apoiado a nossa hipótese inicial de indução da SIPS, eles revelaram outras informações importantes, como a inibição da autofagia, provavelmente através da ativação do mTORC1, resultando em última instância na apoptose dos fibroblastos. No entanto, descobrimos que os fatores de transcrição TFEB/TFE3 são necessários para a internalização do ChA, levando a ausência de fenótipo nas células DKO. Além disso, a passagem em série de células MEF para induzir a senescência replicativa resultou principalmente na morte celular, em vez do estabelecimento de um fenótipo senescente estável. Estas limitações tornaram as células MEF inadequadas para os nossos objetivos experimentais.
Em seguida, mudámos o nosso foco para as CMLs e um membro alternativo da família ChE, o hemiglutarato de colesterol (ChG), para desenvolver um modelo mais robusto de senescência. O ChG é o segundo ChE mais prevalente em lesões ateroscleróticas e, devido à sua estrutura química, é mais difícil de ser hidrolisado nos lisossomas comparativamente ao ChA. As nossas descobertas mostram que a exposição ao ChG prejudica a atividade lisossomal e induz a senescência, nomeadamente um aumento da enzima β-galactosidase associada à senescência, um marcador estabelecido de senescência que liga os dois processos. Essas células permanecem metabolicamente ativas, conforme demonstrado pela ativação de mTORC1/S6K, provavelmente levando a um aumento na secreção de moléculas do fenótipo secretório associado à senescência (SASP). Além disso, a exposição das CMLs a um fármaco lisomotrópico, a cloroquina, induziu resultados semelhantes aos da ChG, sugerindo uma ligação entre a disfunção lisossomal e a senescência. O nosso próximo passo foi investigar a dinâmica da senescência e da disfunção lisossomal em VSMCs
tratadas com o ChG para determinar qual mecanismo ocorre primeiro. No geral, mostramos que o ChG é capaz de induzir disfunção lisossomal e SIPS num modelo murino de VSMC. Usando este modelo, no futuro, poderemos ser capazes de estabelecer se o direcionamento da disfunção lisossomal é uma via adequada para prevenir a senescência no contexto da aterosclerose.
ABSTRACT: Lysosome dysfunction and cellular senescence play pivotal roles in atherosclerosis development. However, the underlying molecular mechanisms and potential link between them remain elusive. We have previously identified a novel family of oxidized lipids, cholesteryl hemiesters (ChE), the end-products of the oxidation of polyunsaturated fatty acids in cholesteryl esters (PUFA-CEs) in the plasma and tissue of cardiovascular disease (CVD) patients. Cholesteryl hemiazelate (ChA), the most prevalent ChE in plasma and atherosclerotic lesions, has been found to significantly impact lysosomal function in vascular smooth muscle cells (VSMCs) causing cell cycle arrest (quiescence). However, ChA-treated VSMC never reached a senescent state. Considering this information, we intended to build a suitable cell model to study stress-induced premature senescence (SIPS) and lysosome malfunction triggered by ChEs. Firstly, we sought to develop a senescence model using mouse embryonic fibroblasts (MEFs) and ChA as pathological trigger. To study the role of lysosomal function in senescence development, we used MEFs double knockout (DKO) for the transcription factors TFEB and TFE3, which regulate lysosomal function and autophagy, as well as senescent cell survival. Notably, ChA-exposed wild-type fibroblasts exhibited perinuclear accumulation and enlarged lysosomes filled with neutral lipids but failed to trigger senescence. Although the results did not support our initial hypothesis of SIPS induction, they revealed other important insights, such as autophagy inhibition, likely through mTORC1 activation, ultimately resulting in fibroblast apoptosis. Nevertheless, we discovered that the transcription factors TFEB/TFE3 are required for ChA internalization, leading to a lack of phenotype in the DKO cells. In addition, serial passaging of MEF cells to induce replicative senescence resulted primarily in cell death rather than the establishment of a stable senescent phenotype. These limitations rendered MEF cells unsuitable for our experimental goals. We then shifted our focus to VSMCs and an alternative member of the ChE family, cholesteryl hemiglutarate (ChG), to develop a more robust model of senescence. ChG is the second most prevalent ChE in atherosclerotic lesions and due to its chemical structure is more difficult to be hydrolyzed within lysosomes than ChA. Our findings show that ChG exposure impairs lysosomal activity and induces senescence, namely an increase of senescence-associated β-galactosidase enzyme, an established senescence marker linking both processes. These cells remain metabolically active, as shown by mTORC1/S6K activation, likely leading to an increase in the secretion of senescence-associated secretory phenotype (SASP) molecules. Also, the exposure of VSMCs to a lysomotropic drug, chloroquine, induced similar results as ChG, suggesting a link between lysosomal dysfunction and senescence. Our next step was investigating the dynamics of both senescence and lysosomal dysfunction in ChG-treated VSMCs to determine which mechanism occurs first. Overall, we show that ChG is able to induce lysosomal dysfunction and SIPS in a murine VSMC model. Using this model, in the future, we may be able to establish whether targeting lysosomal dysfunction is a suitable avenue to prevent senescence in the context of atherosclerosis.
ABSTRACT: Lysosome dysfunction and cellular senescence play pivotal roles in atherosclerosis development. However, the underlying molecular mechanisms and potential link between them remain elusive. We have previously identified a novel family of oxidized lipids, cholesteryl hemiesters (ChE), the end-products of the oxidation of polyunsaturated fatty acids in cholesteryl esters (PUFA-CEs) in the plasma and tissue of cardiovascular disease (CVD) patients. Cholesteryl hemiazelate (ChA), the most prevalent ChE in plasma and atherosclerotic lesions, has been found to significantly impact lysosomal function in vascular smooth muscle cells (VSMCs) causing cell cycle arrest (quiescence). However, ChA-treated VSMC never reached a senescent state. Considering this information, we intended to build a suitable cell model to study stress-induced premature senescence (SIPS) and lysosome malfunction triggered by ChEs. Firstly, we sought to develop a senescence model using mouse embryonic fibroblasts (MEFs) and ChA as pathological trigger. To study the role of lysosomal function in senescence development, we used MEFs double knockout (DKO) for the transcription factors TFEB and TFE3, which regulate lysosomal function and autophagy, as well as senescent cell survival. Notably, ChA-exposed wild-type fibroblasts exhibited perinuclear accumulation and enlarged lysosomes filled with neutral lipids but failed to trigger senescence. Although the results did not support our initial hypothesis of SIPS induction, they revealed other important insights, such as autophagy inhibition, likely through mTORC1 activation, ultimately resulting in fibroblast apoptosis. Nevertheless, we discovered that the transcription factors TFEB/TFE3 are required for ChA internalization, leading to a lack of phenotype in the DKO cells. In addition, serial passaging of MEF cells to induce replicative senescence resulted primarily in cell death rather than the establishment of a stable senescent phenotype. These limitations rendered MEF cells unsuitable for our experimental goals. We then shifted our focus to VSMCs and an alternative member of the ChE family, cholesteryl hemiglutarate (ChG), to develop a more robust model of senescence. ChG is the second most prevalent ChE in atherosclerotic lesions and due to its chemical structure is more difficult to be hydrolyzed within lysosomes than ChA. Our findings show that ChG exposure impairs lysosomal activity and induces senescence, namely an increase of senescence-associated β-galactosidase enzyme, an established senescence marker linking both processes. These cells remain metabolically active, as shown by mTORC1/S6K activation, likely leading to an increase in the secretion of senescence-associated secretory phenotype (SASP) molecules. Also, the exposure of VSMCs to a lysomotropic drug, chloroquine, induced similar results as ChG, suggesting a link between lysosomal dysfunction and senescence. Our next step was investigating the dynamics of both senescence and lysosomal dysfunction in ChG-treated VSMCs to determine which mechanism occurs first. Overall, we show that ChG is able to induce lysosomal dysfunction and SIPS in a murine VSMC model. Using this model, in the future, we may be able to establish whether targeting lysosomal dysfunction is a suitable avenue to prevent senescence in the context of atherosclerosis.
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Lysosome Atherogenesis