Mapeamento das catepsinas lisossomais em tecidos oculares humanos e estudo do envolvimento destas enzimas em processos antigênicos
Data
2013
Autores
Coppini, Larissa Pereira 
Orientadores
Carmona, Adriana Karaoglanovic
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O presente trabalho teve como objetivo contribuir para a compreensao do papel das catepsinas da classe das cisteino-peptidases, na modulacao da angiogenese na cornea in vitro e in vivo. Para tanto, foram utilizados 20 olhos humanos, que tiveram suas corneas desprezadas pra transplante. As corneas humanas foram homogenizadas e utilizando o substrato Z-Phe-Arg-MCA (Z = N-benziloxicarbonil; MCA = 7-amino-4-metilcumarina) e inibidor especifico E-64, identificamos a presenca da atividade desta classe de enzimas neste tecido. Alem disso, a analise por PCR em tempo real e western blotting revelou o perfil de expressao das catepsinas B, K, L, S e V. Verificamos ainda que o processamento do plasminogenio (glicoproteina precursora de plasmina e fragmentos angiostaticos) endogeno ocorreu pela acao de cisteino-peptidases presentes na cornea humana. Entretanto, observamos que embora possua elevada expressao genica, a catepsina V nao e a principal responsavel por esta hidrolise, mostrando que outras cisteino-peptidases estao promovendo o processamento da proteina. Por fim, considerando que a manutencao da avascularizacao da cornea e essencial para garantir a transparencia ocular e uma boa visao, realizamos experimentos de inducao de angiogenese na corneas de 20 coelhos, cujo um dos grupos recebeu tratamento com inibidor E-64 in vivo, que comprovou a participacao de cisteino-peptidases na modulacao deste processo. Alem do estudo na cornea, a presente tese apresentou o perfil de distribuicao do plasminogenio, bem como da atividade e expressao genica de cisteino-peptidases nao apenas na cornea, mas tambem na iris, corpo ciliar, retina, coroide, esclera e nervo optico. No entanto, a funcao destas enzimas nestes tecidos oculares humanos, e seu envolvimento na modulacao do plasminogenio e angiogenese, ainda precisam ser elucidados
The aim of this study was to better understand the role of the cysteine peptidases cathepsins in the modulation of corneal angiogenesis in vitro and in vivo. Twenty human eyes, which the cornea couldn’t be transplanted, were used for this strudy. So, using homogenates of human corneas, the substrate Z-Phe-Arg-MC A (Z = N-benzyloxycarbonyl; MCA = 7-amino-4-methylcoumarin) and the specific inhibitor E-64, we identified the activity of this class of enzymes in cornea. Furthermore, real-time PCR and Western blotting analysis revealed the expression profile of cathepsins B, K, L, S and V. We also observed that the processing of plasminogen (a glycoprotein precursor of plasmin and angiostatic fragments) might occur by the action of endogenous cysteine peptidases present in the cornea. However, we observed that although the high gene expression, cathepsin V is not the main responsible for this hydrolysis, showing that other cysteine peptidases are involved. Finally, considering that the maintenance of corneal avascularização is essential to ensure transparency and eye vision, we performed in vivo induction of angiogenesis in rabbit corneas followed by treatment with E-64, which demonstrated the involvement of cysteine peptidases in the modulation of this process. In addition, the current thesis also showed the distribution profile of plasminogen, as well as, activity and gene expression of cysteine peptidases not only in the cornea, but also in the iris, ciliary body, retina, choroid, sclera and optic nerve. However, the role of these enzymes in these human ocular tissues, its involvement in the modulation of angiogenesis, and plasminogen processing, still needs to be elucidated.
The aim of this study was to better understand the role of the cysteine peptidases cathepsins in the modulation of corneal angiogenesis in vitro and in vivo. Twenty human eyes, which the cornea couldn’t be transplanted, were used for this strudy. So, using homogenates of human corneas, the substrate Z-Phe-Arg-MC A (Z = N-benzyloxycarbonyl; MCA = 7-amino-4-methylcoumarin) and the specific inhibitor E-64, we identified the activity of this class of enzymes in cornea. Furthermore, real-time PCR and Western blotting analysis revealed the expression profile of cathepsins B, K, L, S and V. We also observed that the processing of plasminogen (a glycoprotein precursor of plasmin and angiostatic fragments) might occur by the action of endogenous cysteine peptidases present in the cornea. However, we observed that although the high gene expression, cathepsin V is not the main responsible for this hydrolysis, showing that other cysteine peptidases are involved. Finally, considering that the maintenance of corneal avascularização is essential to ensure transparency and eye vision, we performed in vivo induction of angiogenesis in rabbit corneas followed by treatment with E-64, which demonstrated the involvement of cysteine peptidases in the modulation of this process. In addition, the current thesis also showed the distribution profile of plasminogen, as well as, activity and gene expression of cysteine peptidases not only in the cornea, but also in the iris, ciliary body, retina, choroid, sclera and optic nerve. However, the role of these enzymes in these human ocular tissues, its involvement in the modulation of angiogenesis, and plasminogen processing, still needs to be elucidated.
Descrição
Citação
COPPINI, Larissa P. Mapeamento das catepsinas lisossomais em tecidos oculares humanos e estudo do envolvimento destas enzimas em processos antigênicos. 2013. 121f. Tese (Doutorado em Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2013.