Papel da Caveolina-1 na Capacidade de Migração e Proliferação de Células Estreladas Hepáticas
III Agradecimentos Primeiramente, gostaria de agradecer imensamente aos meus pais maravilhosos Nelson e Adriane por todo o carinho familiar e o apoio aos estudos. Aos meus queridos avós, Arno e Lourdes meus segundos pais por todo o carinho, suporte psicológico e aconselhamentos sobre futuros e profissões. Aos meus queridos avós, Henrique e Walmy por me iniciar no esporte que me faz feliz, o voleibol. Aos meus queridos irmãos Gustavo e Felipe que sempre me encorajaram a enfrentar os mais
... tar os mais difíceis problemas. A toda a minha família, meu porto seguro, que sempre foi muito unida. Ao meu namorado Eduardo Nunes por todo o carinho e amor ao longo destes anos. Ao meu amigo e colega Leo Meira por toda a ajuda com este trabalho. Aos colegas e amigos do laboratório 21 pelas rodas de mate e amizade ao longo dos anos. A minha orientadora, Fátima por sempre me apoiar, orientar, acreditar e me trazer para o caminho da pesquisa. A Florência por me ensinar muitas das técnicas realizadas neste trabalho. Aos demais professores deste PPG pelas excelentes aulas de pós-graduação. Aos suportes financeiros CAPES, CNPq e FAPERGS pelos anos de auxílio à pesquisa realizada neste laboratório. Abstract Liver fibrosis is a common feature of several chronic hepatic diseases and is characterized by the excessive deposition of extracellular matrix in the organ. Usually, this abnormal change of the hepatic parenchyma causes portal hypertension, cirrhosis and liver failure, which can lead to the patient death. Hepatic stellate cells (HSC) participate actively in this process through modifying their quiescent phenotype rich in lipid droplets in the cytoplasm to the activated phenotype in response to the liver injury. GRX line is a model of activated HSC. The caveolae are small invaginations of plasma membrane that reaches 50-100nm of size, rich in glycosphingolipids, cholesterol, and GPI-anchored proteins. These organelles are characterized by the presence of caveolin, a structural and specific protein. These small organelles, which are considered specializations of lipid "rafts" and can be present in several cell types, can act as anchoring platforms for several membrane proteins. These proteins recognize external signals and transmit these signals to modulate the cell activity through regulating or facilitating the transport of fatty acids and lipids, being also responsible for the transport of membrane vesicles. Caveolins are the main structural proteins of caveolae and caveolin-1 (Cav-1) is the most important. Cav-1 is found in all cell types and is related to the oncogenic transformation and tumorigenesis. Previous studies have shown the interaction among caveolae, actin filaments, microtubules, and intermediate filaments. Also, it was found an increase of Cav-1 expression in sinusoidal endothelial cells and HSC of cirrhotic livers, which was suggested to be related to the portal hypertension that accompanies the process of fibrosis. In a previous work, we used the pCav1EGFP plasmid to obtain a permanent cell strain that overexpresses Cav-1 protein and EFGP, which was named GRX EGFP-Cav . In this work we biochemical and morphologically characterized this strain. We also did another cell transfection with an empty pCineoEGFP plasmid to establish a permanent control cell line, which was named GRX EGFPpCineo . Through biochemical analysis, immunocytochemistry, flow cytometry, confocal and electron transmission microscopy, we showed that Cav-1overexpression increased cell proliferation and adhesion, changed cell morphology and cytoskeleton structure, and the cell migration and endocytosis capacity of GRX EGFP-Cav . The actin cytoskeletal changes and the increased cell-cell affinity are indicative of greater cell motility. These results associated to the increase of αSMA and Col-I contents suggest the GRX EGFP-Cav modulation be the activated myofibroblast that characterizes liver damage. As the distribution and the significance of Cav-1 expression in normal and cirrhotic livers are little known and considering the role of HSC in liver diseases, we believe that the permanent GRX EGFP-Cav line can be a very interesting experimental tool for the study of these pathologies.