Papel do sindecam-4 no remodelamento da matriz extracelular em células endoteliais resistentes ao anoikis
Data
2022-03-29
Autores
Sousa Onyeisi, Jessica Oyie 
Orientadores
Lopes de Azevedo, Carla Cristina
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Estudar o papel do sindecam-4 no remodelamento da matriz extracelular e esclarecer suas interações com outras moléculas. Métodos: Células endoteliais de aorta de coelho resistentes ao anoikis submetidas ao silenciamento gênico do sindecam-4 (miR-Syn4-Adh-1-EC) foram estudadas comparando-se com células endoteliais de aorta de coelho resistentes ao anoikis parentais (Adh-EC), células endoteliais de aorta de coelho selvagens (EC) e células endoteliais de aorta de coelho transfectadas com o oncogene EJ-ras (EJ-ras EC), em relação a: migração celular, angiogênese, biossíntese de glicosaminoglicanos sulfatados ao longo do ciclo celular, localização, expressão gênica e proteica das macromoléculas da MEC e das enzimas responsáveis pela degradação da MEC. Foi avaliada ainda a atividade das MMPs, a expressão dos TIMPs e da enzima óxido nítrico sintase endotelial (eNOS), bem como a produção de óxido nítrico (NO). A expressão gênica e proteica dos receptores de superfície celular (integrinas α5β1 e αVβ3) e das moléculas de sinalização celular: quinase de adesão focal (FAK) e SRC também foram analisadas. A migração celular foi avaliada pelo ensaio wound healing, a expressão gênica foi avaliada através da técnica de qPCR, a expressão e localização proteica através das técnicas de western blotting e imunofluorescência, respectivamente, a atividade das MMPs foi avaliada pela técnica de zimografia e a produção de NO foi avaliada pela reação de GREISS. Resultados: O silenciamento SDC4 levou à uma diminuição da migração e da capacidade angiogênica das células endoteliais resistentes ao anoikis. Em comparação com as células parentais (Adh1-EC), as células silenciadas SDC4 (miR-Syn-4-1-Adh1-EC e miR-Syn-4-2-Adh1-EC) exibiram um aumento na adesão celular à fibronectina. Além disso, a transfecção com miRNA-SDC4 levou a aumento na síntese de heparam sulfato após 12 horas de estímulo com soro fetal bovino (SFB). Após o silenciamento do SDC4, também observamos um aumento na expressão da fibronectina e do colágeno IV e uma diminuição na expressão das integrinas α5β1 e αVβ3. Além disso, as células silenciadas para o SDC4 apresentam um aumento na expressão de Src e FAK. Em relação à expressão das enzimas responsáveis pela degradação da MEC e seus reguladores, nós observamos uma diminuição na expressão da HPSE, da MMP2 e do TIMP2 após o silenciamento do SDC4. Também observamos que o silenciamento do SDC4 causou uma diminuição na produção de óxido nítrico e na expressão de eNOS. Conclusão: Os resultados apresentados ao longo do presente trabalho sugerem que o SDC4 tem um papel importante no remodelamento da matriz extracelular e também representam um passo importante para a compreensão do mecanismo pelo qual o SDC4 pode reverter o fenótipo transformado das células endoteliais resistentes ao anoikis.
Objective: To study the role of syndecan-4 in extracellular matrix remodeling and to clarify its interactions with other molecules. Methods: Anoikis-resistant rabbit aortic endothelial cells submitted to syndecan-4 gene silencing (miR-Syn4-Adh-1-EC) will be studied by comparing with parental anoikis-resistant rabbit aortic endothelial cells (Adh-EC), wild rabbit aortic endothelial cells (EC) and EJ-ras oncogene transfected rabbit aortic endothelial cells (EJ-ras EC) in relation to: cell migration, angiogenesis, biosynthesis of sulfated glycosaminoglycans in the cell cycle, localization, gene and protein expression of extracellular matrix macromolecules as well as enzymes responsible for their regulation. The activity of MMPs, the expression of TIMPs and the endothelial nitric oxide synthase (eNOS) enzyme, as well as the production of nitric oxide (NO) were evaluated. Gene and protein expression of cell surface receptors (α5β1 and αVβ3 integrins) and cell signaling molecules: focal adhesion kinase (FAK) and SRC were also analyzed. Cell migration was assessed by the wound healing assay, gene expression was assessed by the qPCR, protein expression and localization by the western blotting and immunofluorescence techniques, respectively, the activity of MMPs was assessed by the zymography and NO production was assessed by the reaction of GREISS. Results: SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells. Compared to parental cells (Adh1-EC), SDC4 silenced cells (miR-Syn-4-1-Adh1-EC and miR-Syn-4-2-Adh1-EC) exhibited an increase in adhesion to fibronectin. Moreover, transfection with miRNA-SDC4 caused an increase in the heparan sulfate synthesis after 12 h of stimulus with fetal calf serum (FCS). Furthermore, after SDC4 silencing, we also observed an increase in the expression of fibronectin and collagen IV and a decrease in the expression of α5β1 and αVβ3 integrins. In addition, SDC4 silenced cells show an increase in Src and FAK expression. In relation to expression of enzymes responsible for the degradation of ECM and its regulators, we observed a decrease in the expression of HPSE, MMP2 and TIMP2 after SDC4 silencing. We also observed that SDC4 silencing caused a decrease in nitric oxide production and eNOS expression. Conclusion: The results presented throughout this work suggest that SDC4 plays an important role in extracellular matrix remodeling. In addition, our results represent an important step towards understanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.
Objective: To study the role of syndecan-4 in extracellular matrix remodeling and to clarify its interactions with other molecules. Methods: Anoikis-resistant rabbit aortic endothelial cells submitted to syndecan-4 gene silencing (miR-Syn4-Adh-1-EC) will be studied by comparing with parental anoikis-resistant rabbit aortic endothelial cells (Adh-EC), wild rabbit aortic endothelial cells (EC) and EJ-ras oncogene transfected rabbit aortic endothelial cells (EJ-ras EC) in relation to: cell migration, angiogenesis, biosynthesis of sulfated glycosaminoglycans in the cell cycle, localization, gene and protein expression of extracellular matrix macromolecules as well as enzymes responsible for their regulation. The activity of MMPs, the expression of TIMPs and the endothelial nitric oxide synthase (eNOS) enzyme, as well as the production of nitric oxide (NO) were evaluated. Gene and protein expression of cell surface receptors (α5β1 and αVβ3 integrins) and cell signaling molecules: focal adhesion kinase (FAK) and SRC were also analyzed. Cell migration was assessed by the wound healing assay, gene expression was assessed by the qPCR, protein expression and localization by the western blotting and immunofluorescence techniques, respectively, the activity of MMPs was assessed by the zymography and NO production was assessed by the reaction of GREISS. Results: SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells. Compared to parental cells (Adh1-EC), SDC4 silenced cells (miR-Syn-4-1-Adh1-EC and miR-Syn-4-2-Adh1-EC) exhibited an increase in adhesion to fibronectin. Moreover, transfection with miRNA-SDC4 caused an increase in the heparan sulfate synthesis after 12 h of stimulus with fetal calf serum (FCS). Furthermore, after SDC4 silencing, we also observed an increase in the expression of fibronectin and collagen IV and a decrease in the expression of α5β1 and αVβ3 integrins. In addition, SDC4 silenced cells show an increase in Src and FAK expression. In relation to expression of enzymes responsible for the degradation of ECM and its regulators, we observed a decrease in the expression of HPSE, MMP2 and TIMP2 after SDC4 silencing. We also observed that SDC4 silencing caused a decrease in nitric oxide production and eNOS expression. Conclusion: The results presented throughout this work suggest that SDC4 plays an important role in extracellular matrix remodeling. In addition, our results represent an important step towards understanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.
Descrição
Citação
ONYEISI, J O S. Papel do sindecam-4 no remodelamento da matriz extracelular em células endoteliais resistentes ao anoikis. São Paulo, 2022. 111 f. Tese (Doutorado em Ciências Biológicas (Biologia Molecular) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2022.