Modelo in vitro da barreira hematoencefálica para investigação da ação dos produtos finais de glicação avançada relacionados à diabetes mellitus no risco da doença de Alzheimer
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Data
2023-12-13
Autores
Story, Amanda Martins [UNIFESP]
Orientadores
Porcionatto, Marimélia Aparecida [UNIFESP]
Tipo
Trabalho de conclusão de curso de graduação
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A diabetes mellitus (DM) é uma doença metabólica cada vez mais associada a danos cognitivos e demência, aumentando o risco de neuropatias, especialmente a doença de Alzheimer (DA). Um dos efeitos da hiperglicemia é o acúmulo de produtos finais de glicação avançada (AGEs), cujas ações têm se destacado como um forte elo entre as doenças. Para compreender os mecanismos associados e desencadeados pelos AGEs, torna-se necessário desenvolver modelos in vitro que capturem a complexidade da composição e do funcionamento da BHE. Com isso, objetivamos desenvolver um modelo celular da BHE onde pudéssemos mimetizar uma condição hiperglicêmica semelhante à da DM para investigarmos a ação dos AGEs nessa estrutura, com foco nos neurônios. O modelo foi organizado no sistema transwell em tri-cultura com células endoteliais e astrócitos derivados de células-tronco pluripotentes induzidas humanas (hiPSC-EC e hiPSC-AST), organizados na porção apical e basolateral do inserto, respectivamente, e neurônios derivados da linhagem de neuroblastoma humano SH-SY5Y, presentes no fundo do poço da placa. Monoculturas em 2D das hiPSC-EC e células SH-SY5Y receberam diferentes contrações de soroalbumina bovina ligada a produtos finais de glicação avançada (AGE-BSA) e em concentrações normal e de alta de glicose. Como resultado, as SH-SY5Y não diferenciadas apresentaram maior estresse oxidativo e menores índices de morte celular em comparação com as diferenciadas. As hiPSC-EC apresentaram redução na viabilidade quando cultivadas na presença de AGE-BSA 100 µg/mL. Assim, essa concentração foi utilizada no tratamento das células cultivadas em transwell por 48 h. A análise da integridade da BHE, através da medida de resistência elétrica transendotelial (TEER) e permeabilidade ao FITC-Dextran, mostraram que não houve danos significativos à BHE. Já a análise qualitativa da imunofluorescência dos neurônios sugeriu um aumento nos níveis de proteína precursora amilóide (APP), receptores de AGEs (RAGE) e oligômeros de beta-amilóide (AβO), além de alterações morfológicas. Apesar dos nossos dados anteriores mostrarem que não houve mudanças significativas na permeabilidade da BHE, houve uma comunicação entre as células e os neurônios responderam com alterações metabólicas que podem estar presentes na patogênese da DA.
Diabetes mellitus (DM) is a metabolic disease that has been increasingly associated with cognitive impairment and dementia, increasing the risk of neuropathies, especially Alzheimer's disease (AD). One of the effects of hyperglycemia is the accumulation of advanced glycation end products (AGEs), whose actions have been highlighted as a strong link between these diseases. To understand the mechanisms associated with and triggered by AGEs, it is found necessary the development of in vitro models that capture the complexity of the composition and functioning of the blood-brain barrier (BBB). Therefore, we aimed to develop a cellular model of the BBB to mimic a hyperglycemic condition similar to DM and investigate the action of AGEs in this structure, focusing on the neurons. The model was organized in a tri-culture transwell system with endothelial cells and astrocytes derived from human induced pluripotent stem cells (hiPSC-EC and hiPSC-AST), located in the apical and basolateral portion of the insert, respectively, and neurons derived from the SH-SY5Y human neuroblastoma cell line, present at the bottom of the well. The 2D monocultures of hiPSC-EC and SH-SY5Y cells received different concentrations of advanced glycation end products bovine serum albumin (AGE-BSA) and at normal and high glucose concentrations. As a result, non-differentiated SH-SY5Y showed greater oxidative stress and lower rates of cell death compared to the differentiated cells. hiPSC-EC showed reduced viability when cultured in the presence of AGE-BSA 100 µg/mL. Therefore, this concentration was used in cells cultured in the transwell system for 48 h. Analysis of the integrity of the BBB, through measurement of transendothelial electrical resistance (TEER) and permeability to FITC-Dextran, revealed no significant damage to the BBB. Qualitative analysis of neurons’ immunofluorescence suggested an increase in amyloid protein precursor (APP), AGE receptors (RAGE) and beta-amyloid oligomers (AβO), in addition to morphological changes. Despite our data showed no significant changes on BBB permeability, cell-cell communication occurred and neurons responded with metabolic changes that may be present in the pathogenesis of AD.
Diabetes mellitus (DM) is a metabolic disease that has been increasingly associated with cognitive impairment and dementia, increasing the risk of neuropathies, especially Alzheimer's disease (AD). One of the effects of hyperglycemia is the accumulation of advanced glycation end products (AGEs), whose actions have been highlighted as a strong link between these diseases. To understand the mechanisms associated with and triggered by AGEs, it is found necessary the development of in vitro models that capture the complexity of the composition and functioning of the blood-brain barrier (BBB). Therefore, we aimed to develop a cellular model of the BBB to mimic a hyperglycemic condition similar to DM and investigate the action of AGEs in this structure, focusing on the neurons. The model was organized in a tri-culture transwell system with endothelial cells and astrocytes derived from human induced pluripotent stem cells (hiPSC-EC and hiPSC-AST), located in the apical and basolateral portion of the insert, respectively, and neurons derived from the SH-SY5Y human neuroblastoma cell line, present at the bottom of the well. The 2D monocultures of hiPSC-EC and SH-SY5Y cells received different concentrations of advanced glycation end products bovine serum albumin (AGE-BSA) and at normal and high glucose concentrations. As a result, non-differentiated SH-SY5Y showed greater oxidative stress and lower rates of cell death compared to the differentiated cells. hiPSC-EC showed reduced viability when cultured in the presence of AGE-BSA 100 µg/mL. Therefore, this concentration was used in cells cultured in the transwell system for 48 h. Analysis of the integrity of the BBB, through measurement of transendothelial electrical resistance (TEER) and permeability to FITC-Dextran, revealed no significant damage to the BBB. Qualitative analysis of neurons’ immunofluorescence suggested an increase in amyloid protein precursor (APP), AGE receptors (RAGE) and beta-amyloid oligomers (AβO), in addition to morphological changes. Despite our data showed no significant changes on BBB permeability, cell-cell communication occurred and neurons responded with metabolic changes that may be present in the pathogenesis of AD.
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Citação
STORY, Amanda Martins. Modelo in vitro da barreira hematoencefálica para investigação da ação dos produtos finais de glicação avançada relacionados à diabetes mellitus no risco da doença de Alzheimer. 2023. 57 f. Trabalho de Conclusão de Curso (Graduação em Biomedicina) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2023.