Meio condicionado de explante cardíaco humano : fatores solúveis e efeito cardiomiogênico nas células tronco mesenquimais

Abstract

Resumo

Resumo: Neste trabalho o meio condicionado derivado de explante cardiaco humano foi analisado para identificar fatores soluveis liberados pelos explantes e determinar o potencial cardiomiogenicas sobre celulas tronco mesenquimais (CTMs) derivadas da medula ossea. Atraves da utilizacao de arranjos de anticorpos foi possivel identificar muitos fatores de crescimento e citocinas liberados pelos explantes cardiacos. Dentre os fatores liberados pelos explantes cardiacos foram identificados citocinas pro-inflamatorias e anti-inflamatorias, bFGF, IGFBP-1 e IGFBP-2. Alem disso tambem foram realizados geis bidimensionais e espectometria de massas para determinar algumas da proteinas que sao liberadas ao meio de cultura. Muitas proteinas estruturais foram identificadas provavelmente devido ao dano do tecido e apoptose. Uma grande parte dos fatores liberados pelos explantes parecem importantes na citoprotecao, diferenciacao do tecido cardiaco e reparo. Nos hipotetizamos que os explantes cardiacos humanos talvez funcionem como uma fonte de fatores soluveis que talvez tenham influencia na inducao das CTMs a celulas semelhantes a cardiomiocitos, ou talvez, a celulas semelhantes ao endotelio. Assim, a influencia do meio condicionado nas CTMs foi analisado atraves da expressao de genes selecionados por RT-PCR e por imunofluorescencia indireta (IFI). Niveis aumentados do mRNA de VEGF, conexina-43, troponina I e troponina T foram observados nas amostras tratadas quando comparadas com o controle. O mRNA de Nkx 2,5, um importante fator transcricional envolvido na cardiomiogenese, foi detectado em duas amostras. As analises da expressao de proteinas e localizacao por IFI mostram que as CTMs tratadas com meio condicionado tem uma grande quantidade de celulas positivas para troponina T cardiaca, troponina I cardiaca e ƒ¿-actinina. Essas celulas apresentam uma morfologia e nucleo distintos. Embora os niveis de mRNA de conexina-43 parecam aumentadas nas culturas tratadas, as analises de IFI nao mostram diferencas evidentes entre as celulas tratadas e nao tratadas. Nossos resultados sugerem que o meio condicionado de explantes cardiacos humanos talvez funcionem como um modelo in vitro para estudar fatores de diferenciacao cardiaca, alem de futuramente esses fatores serem usados em aplicacoes clinicas.

Abstract: In the present work conditioned medium by human cardiac explant was analyzed to identify soluble factors released by the explants and to determine the cardiomyogenic potential on bone marrow derived mesenchymal stem cells (BM-MSCs). By using antibody arrays, it was possible to identify several growth factors and cytokines produced by the human explants. Among the several factors released by the cardiac explants we identified pro-inflammatory and anti-inflammatory cytokines, bFGF, IGFBP-1 e IGFBP-2. Besides, two-dimensional gels and mass spectrometry were carried out to determine some of the abundant proteins that are released to the culture medium. Several structural proteins were detected probably due to tissue damage and apoptosis. A great deal of the factors released from the explant seems to be important in cytoprotection, cardiac tissue differentiation and repair. We hypothesized that human cardiac explants may function as a source of soluble factor that might commission MSC differentiation to cardiomyocyte-like and, perhaps, tondothelial-like cells. Thus, the influence of the conditioned medium on BM-MSC was analyzed through the expression of selected genes by RT-PCR/qPCR and by indirect immunofluorescence (IFI). Increased levels of VEGF, Connexin 43, Troponin T and Troponin I mRNAs were observed in the treated samples when compared with the controls. Nkx2.5 mRNA, an important transcription factor involved in cardiomyogenesis, was detected in only two treated sample. The analysis of protein expression and localization by IFI showed that BM-MSCs treated with cardiac explant- conditioned medium had a high number of positive cells for cardiac Troponin T, Troponin I and á-actinin. These cells presented a distinct morphology and nucleus. Even though Connexin 43 mRNA seems to have increased level in the treated cultures, IFI analyses do not show evident differences between treated and not treated cells. Our results suggest that conditioned medium with human cardiac explant might function as an in vitro model for studying cardiac differentiation factors; moreover, it might have future clinical applications.