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Doctoral Thesis
DOI
Document
Author
Full name
William Salibe Filho
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Terra Filho, Mario (President)
Arakaki, Jaquelina Sonoe Ota
Fernandes, Caio Julio Cesar dos Santos
Laurindo, Francisco Rafael Martins
Title in Portuguese
Alterações na proteostase de células endoteliais pulmonares em pacientes com hipertensão pulmonar tromboembólica crônica
Keywords in Portuguese
Células endoteliais
Deficiências na proteostase
Embolia pulmonar
Endarterectomia
Hipertensão pulmonar
Homeostase
Abstract in Portuguese
Introdução: A hipertensão pulmonar tromboembólica crônica (HPTEC) está incluída no grupo 4 da Classificação Internacional de Hipertensão Pulmonar (HP). É caracterizada pela persistência de obstrução por trombos sanguíneos na circulação pulmonar, associada à presença de HP, após três meses de anticoagulação efetiva. O tratamento de escolha é a cirurgia de tromboendarterectomia pulmonar (TEAP), mas alguns dos mecanismos fisiopatológicos envolvidos nesta forma de hipertensão ainda permanecem incertos. O redirecionamento dos fluxos sanguíneos pulmonares e a hipóxia exercem papel importante na HPTEC, como também em casos de hipertensão pulmonar residual, após a cirurgia de TEAP. Entretanto, existem poucos dados sobre as respostas das células endoteliais pulmonares a essas mudanças de fluxo e de oxigenação, surgindo a necessidade do estudo da proteostase celular nesta doença. Objetivo: (A) Caracterização morfológica das células em culturas provenientes de artéria pulmonar de pacientes com HPTEC submetidos à TEAP. (B) Avaliação da resposta das células endoteliais, a partir da análise de proteínas envolvidas na proteostase celular, quando submetidas a diferentes níveis de stress mecânico e à hipóxia. Método: Trombos extraídos por TEAP foram processados, as células retiradas foram cultivadas, marcadas com CD31 e submetidas a stress mecânico por vinte e quatro horas, constituindo o grupo HPTEC. A proteostase celular foi avaliada pela medida de proteínas expressas por essas células, tanto em culturas quanto pela análise imuno-histoquímica do tecido vascular pulmonar. Como grupo controle foram utilizadas células endoteliais pulmonares humanas de linhagem (CE) e tecido de artérias pulmonares de doadores de transplante de pulmão. As culturas de ambos os grupos também foram colocadas em hipóxia e analisada a expressão indireta de óxido nítrico (NO) por meio da medida de nitrato. Resultado: as células do grupo HPTEC com morfologia endotelial foram marcadas positivamente com CD31 e apresentaram características semelhantes às do grupo CE. Em relação ao stress mecânico, na condição estática as células HPTEC expressaram menor quantidade de óxido nítrico sintase endotelial (eNOS). Quando submetidas a stress de alta intensidade (shear stress >= 15 dynes/cm2), as reduções ficaram ainda mais evidentes, sinalizando uma disfunção endotelial. Na análise de outras proteínas, como GRP94, GRP78, HSP70, as respostas também foram menores no alto fluxo. Na avaliação imunohistoquímica da camada íntima do vaso pulmonar, a HSP70 apresentava-se diminuída, corroborando os achados das culturas. Os valores de NO foram inferiores no grupo HPTEC quando se comparam hipóxia e normóxia. Conclusão: (A) A avaliação morfológica mostrou que as culturas de células HPTEC eram endoteliais. (B) A análise funcional revelou que estas células apresentaram redução de resposta, o que caracteriza alteração da proteostase, que se tornou mais evidente quando foram submetidas a shear stress de alta magnitude. A hipóxia reduziu a produção de NO, entretanto sem diferenciar os grupos celulares estudados
Title in English
Alterations in proteostasis of endothelial cells in patients with chronic thromboembolic pulmonary hypertension
Keywords in English
Endarterectomy
Endothelial cells
Homeostasis
Hypertension pulmonary
Proteostasis deficiencies
Pulmonary embolism
Abstract in English
Introduction: Chronic Thromboembolic Pulmonary Hypertension (CTEPH) is included in group 4 of the International Classification of Pulmonary Hypertension (PH). It is characterized by persistent obstruction by blood clots in the pulmonary circulation, associated with the presence of PH, after 3 months of effective anticoagulation. The treatment of choice is pulmonary endarterectomy (PEA). However, some of the pathophysiological mechanisms involved in this form of hypertension still remain uncertain. The redirection of pulmonary blood flow and hypoxia play an important role in CTEPH, and also, in cases of residual pulmonary hypertension after PEA surgery. Nevertheless, there is insufficient data from the pulmonary endothelial cell responses to this flow and oxygenation changes, reflecting the need to further study of cellular proteostasis in this disease. Objective: (A) Morphological characterization of cells in cultures from the pulmonary artery of CTEPH patients submitted to PEA. (B) Evaluation of the response of endothelial cells, through the analysis of proteins involved in cellular proteostasis, when submitted to different levels of mechanical stress and hypoxia. Method: Thrombus extracted by PEA were processed and the cells removed were cultured, marked with CD31 and submitted to mechanical stress for 24 hours and constituted the group CTEPH. Cellular proteostasis was measured by the quantification of the proteins expressed in cultures and in pulmonary vascular tissue by immunohistochemistry analysis. As a control group, the human pulmonary endothelial cells (EC) and pulmonary artery tissue from lung transplant donors were used. Cultures of both groups were also placed in hypoxia and the indirect expression of nitric oxide (NO) was analyzed by nitrate measurement. Results: The cells with endothelial morphology from the CTEPH group were positively marked with CD31 and presented similar characteristics as the EC group. Regarding mechanical stress, in the static condition, the CTEPH cells expressed a lesser amount of endothelial nitric oxide synthase (eNOS). When submitted to high flow (shear stress > 15 dynes / cm2) the reductions became even more evident, signaling an endothelial dysfunction. In the analysis of other proteins, such as GRP94, GRP78, HSP70, responses were also lower in high shear stress. In the immunohistochemistry analysis of the intimal layer of the pulmonary vessel HSP70 was diminished, corroborating with the findings of the cultures. The NO values were lower in the CTEPH group when compared hypoxia and normoxia. Conclusion: (A) Morphological evaluation showed that cultures of CTEPH cells were endothelial. (B) Functional analysis revealed that these cells had reduced response, which characterizes proteostasis alterations, which became more evident when they underwent shear stress of high magnitude. Hypoxia reduced NO production, however without differentiating the cell groups studied
 
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Publishing Date
2019-05-23
 
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