RESUMO
Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy.
Assuntos
Apoptose/genética , Autofagia/genética , Grelina/genética , Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/genética , Animais , Animais Recém-Nascidos , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Dependovirus , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Biblioteca Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Membro Posterior/irrigação sanguínea , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Isquemia/genética , Isquemia/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias Cardíacas/ultraestrutura , Músculo Esquelético/irrigação sanguínea , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ultrassonografia , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
Over the last several years, mesenchymal stromal cells (MSCs) have been isolated from different tissues following a variety of different procedures. Here, we comparatively assess the ex vivo and in vivo properties of MSCs isolated from either adipose tissue or bone marrow by different purification protocols. After MSC transplantation into a mouse model of hindlimb ischemia, clinical and histological analysis revealed that bone marrow MSCs purified on adhesive substrates exerted the best therapeutic activity, preserving tissue viability and promoting formation of new arterioles without directly transdifferentiating into vascular cells. In keeping with these observations, these cells abundantly expressed cytokines involved in vessel maturation and cell retention. These findings indicate that the choice of MSC source and purification protocol is critical in determining the therapeutic potential of these cells and warrant the standardization of an optimal MSC isolation procedure in order to select the best conditions to move forward to more effective clinical experimentation.