Avaliação da expressão de pigment epithelium-derived factor (PEDF) em ratos submetidos à hipóxia intermitente
Arquivos
Data
2014
Autores
Julian, Guilherme Silva 
Orientadores
Chagas, Jair Ribeiro
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A apneia obstrutiva do sono (AOS) e caracterizada principalmente por hipoxia intermitente noturna, sendo relacionada com diversas complicacoes e patologias. O modelo de hipoxia intermitente (HI) cronica em roedores e um dos modelos animais mais utilizados para estudo da AOS e, assim como a AOS, causa diversas consequencias como declinio cognitivo e prejuizos neuronais. Pigment-Epithelium Derived Factor (PEDF) e descrito como um fator sensivel a hipoxia, com as atividades neurotrofica, neuroprotetora e anti-angiogenica. O presente estudo teve como objetivo avaliar aprendizado e memoria espacial e as expressoes genica e proteica de PEDF no SNC e no ventriculo esquerdo de ratos submetidos a HI, assim como avaliar os efeitos do retorno a normoxia, apos seis semanas de hipoxia. Alem de avaliar possiveis interacoes de PEDF e enzimas angiogenicas in vitro. Ratos machos Wistar foram submetidos a protocolos de HI (21 a 5% de concentracao de O2, com ciclos de 6 minutos) por seis semanas (grupo HI), e seis semanas de HI seguidas de duas semanas de recuperacao em normoxia (grupo Rb), ambos comparados com grupo controle (CTL). Em seguida, foram realizadas analises de aprendizado e memoria por Labirinto Aquatico de Morris, estabilidade de genes de referencia em diferentes estruturas cerebrais, expressoes genica e proteica de PEDF no hipotalamo, hipocampo e cortices frontal e temporal, alem do ventriculo esquerdo cardiaco. Alem disso, o estudo da interacao de calicreinas com PEDF e com seu fragmento peptidico 388-393 foi realizado atraves da incubacao das enzimas recombinantes com PEDF e seu fragmento 388-393, a atividade foi avaliada utilizando substratos fluorogenicos com supressao interna da fluorescencia. Com excecao de 18S, todos os genes de referencia avaliados se apresentaram estaveis aos protocolos de HI, porem, a classificacao dos genes se apresentou estrutura-dependente. Embora os modelos de HI nao tenham afetado memoria e aprendizado, a expressao genica de PEDF se apresentou aumentada seletivamente no cortex temporal dos grupos HI e Rb. Por outro lado, a expressao proteica de PEDF se manteve inalterada em todas as estruturas estudadas, assim como a expressao genica em hipotalamo, hipocampo, cortex frontal e ventriculo esquerdo. A incubacao realizada in vitro demonstrou que PEDF e seu fragmento peptidico aparentemente nao apresentam nenhuma interacao com as calicreinas tissular e plasmatica. O presente estudo demonstrou que os protocolos de HI afetam a estabilidade de genes de referencia, de maneira estrutura-dependente, no cerebro. Por outro lado, o protocolo de HI nao afetou os processos de aprendizado e memoria espacial, mas afetou a expressao de PEDF no cortex temporal, seletivamente no nivel transcripcional. Alem disso, nao ha indicacoes de que o mecanismo anti-angiogenico de PEDF envolva sua interacao com as calicreinas tissular e plasmatica
Obstructive Sleep Apnea is a syndrome characterized mainly by intermittent hypoxia during sleep and has been already associated with several complications. Chronic intermittent hypoxia is the most studied model of sleep apnea, and causes several cognitive and neuronal impairments. Pigment-Epithelium Derived Factor (PEDF) is has already been described as a hypoxia-sensitive factor acting as neurotrophic, neuroprotector and antiangiogenic factor. Our study aimed to analyze: 1) learning and cognitive spatial memory task; 2) evaluation of housekeeping gene stability in specific brain structures of rats submitted to CIH protocols; 3) analyze PEDF’s gene and protein expression in specific brain structures and heart left ventricle of rats submitted to CIH protocols; 4) evaluate a possible interaction of kallikrein 1 and plasma kallikrein with PEDF and an anti-angiogenic PEDF fragment. Male Wistar rats were submitted CIH protocols (21 to 5% O2 concentration, in a 6 minutes-cycle) for six weeks (HI group) and six weeks followed by two weeks recovery (Rb group), then subjected to learning and memory evaluation by Morris water maze task, housekeeping gene stability evaluation of the different brain structures, PEDF gene and protein expression in the heart left ventricle, hypothalamus, hippocampus, frontal and temporal cortices. PEDF and its the peptidic fragment were studied by incubation with recombinant kallikrein 1 and plasma kallikrein, to evaluate any interference in the enzyme´s kinetic profile. With exception of 18S, all evaluated genes were stable to effects of CIH. Nonetheless, gene stability ranking was dependent on the brain structure, analyzed and varied according to the used software. Although CIH models did not present effect on learning and cognition, PEDF curiously presented an increase in gene expression on the temporal cortex of animals submitted to CIH and CIH followed by a 2 weeks recovery in normoxia, though, PEDF protein levels were maintained unaltered. mRNA and protein levels were kept unaltered on the heart left ventricle, frontal cortex and hippocampus. Incubation performed in vitro demonstrated that PEDF and the anti-angiogenic PEDF fragment (P1) did not present any influence on kallikrein 1 and plasma kallikrein´s activity. This study demonstrated that CIH affects gene expression stability differently in brain structures. On the other hand, CIH did not affect learning and cognition task, but did affect PEDF only in the temporal cortex, selectively in transcriptional level. In addition, PEDF anti-angiogenic mechanisms do not seem to involve kallikrein 1 and plasma kallikrein.
Obstructive Sleep Apnea is a syndrome characterized mainly by intermittent hypoxia during sleep and has been already associated with several complications. Chronic intermittent hypoxia is the most studied model of sleep apnea, and causes several cognitive and neuronal impairments. Pigment-Epithelium Derived Factor (PEDF) is has already been described as a hypoxia-sensitive factor acting as neurotrophic, neuroprotector and antiangiogenic factor. Our study aimed to analyze: 1) learning and cognitive spatial memory task; 2) evaluation of housekeeping gene stability in specific brain structures of rats submitted to CIH protocols; 3) analyze PEDF’s gene and protein expression in specific brain structures and heart left ventricle of rats submitted to CIH protocols; 4) evaluate a possible interaction of kallikrein 1 and plasma kallikrein with PEDF and an anti-angiogenic PEDF fragment. Male Wistar rats were submitted CIH protocols (21 to 5% O2 concentration, in a 6 minutes-cycle) for six weeks (HI group) and six weeks followed by two weeks recovery (Rb group), then subjected to learning and memory evaluation by Morris water maze task, housekeeping gene stability evaluation of the different brain structures, PEDF gene and protein expression in the heart left ventricle, hypothalamus, hippocampus, frontal and temporal cortices. PEDF and its the peptidic fragment were studied by incubation with recombinant kallikrein 1 and plasma kallikrein, to evaluate any interference in the enzyme´s kinetic profile. With exception of 18S, all evaluated genes were stable to effects of CIH. Nonetheless, gene stability ranking was dependent on the brain structure, analyzed and varied according to the used software. Although CIH models did not present effect on learning and cognition, PEDF curiously presented an increase in gene expression on the temporal cortex of animals submitted to CIH and CIH followed by a 2 weeks recovery in normoxia, though, PEDF protein levels were maintained unaltered. mRNA and protein levels were kept unaltered on the heart left ventricle, frontal cortex and hippocampus. Incubation performed in vitro demonstrated that PEDF and the anti-angiogenic PEDF fragment (P1) did not present any influence on kallikrein 1 and plasma kallikrein´s activity. This study demonstrated that CIH affects gene expression stability differently in brain structures. On the other hand, CIH did not affect learning and cognition task, but did affect PEDF only in the temporal cortex, selectively in transcriptional level. In addition, PEDF anti-angiogenic mechanisms do not seem to involve kallikrein 1 and plasma kallikrein.
Descrição
Citação
JULIAN, Guilherme Silva. Avaliação da expressão de Pigment Epithelium-Derived Factor (PEDF) em ratos submetidos à hipóxia intermitente: Consequências cognitivas e bioquímicas. 2014. 183 f. Dissertação (Mestrado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2014.