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AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

ABSTRACT: 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.

SUBMITTER: Ruozi G

PROVIDER: S-EPMC4477044 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

Ruozi Giulia G Bortolotti Francesca F Falcione Antonella A Dal Ferro Matteo M Ukovich Laura L Macedo Antero A Zentilin Lorena L Filigheddu Nicoletta N Gortan Cappellari Gianluca G Baldini Giovanna G Zweyer Marina M Barazzoni Rocco R Graziani Andrea A Zacchigna Serena S Giacca Mauro M

Nature communications 20150611

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 peptid ...[more]

PMID: 26066847

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Project description:Background and aimsAlthough type 2 innate lymphoid cells (ILC2s) were originally found to be liver-resident lymphocytes, the role and importance of ILC2 in liver injury remains poorly understood. In the current study, we sought to determine whether ILC2 is an important regulator of hepatic ischaemia/reperfusion injury (IRI).MethodsILC2-deficient mice (ICOS-T or NSG) and genetically modified ILC2s were used to investigate the role of ILC2s in murine hepatic IRI. Interactions between ILC2s and eosinophils or macrophages were studied in coculture. The role of human ILC2s was assessed in an immunocompromised mouse model of hepatic IRI.ResultsAdministration of IL-33 prevented hepatic IRI in association with reduction of neutrophil infiltration and inflammatory mediators in the liver. IL-33-treated mice had elevated numbers of ILC2s, eosinophils, and regulatory T cells. Eosinophils, but not regulatory T cells, were required for IL-33-mediated hepatoprotection in IRI mice. Depletion of ILC2s substantially abolished the protective effect of IL-33 in hepatic IRI, indicating that ILC2s play critical roles in IL-33-mediated liver protection. Adoptive transfer of ex vivo-expanded ILC2s improved liver function and attenuated histologic damage in mice subjected to IRI. Mechanistic studies combining genetic and adoptive transfer approaches identified a protective role of ILC2s through promoting IL-13-dependent induction of anti-inflammatory macrophages and IL-5-dependent elevation of eosinophils in IRI. Furthermore, in vivo expansion of human ILC2s by IL-33 or transfer of ex vivo-expanded human ILC2s ameliorated hepatic IRI in an immunocompromised mouse model of hepatic IRI.ConclusionsThis study provides insight into the mechanisms of ILC2-mediated liver protection that could serve as therapeutic targets to treat acute liver injury.Impact and implicationsWe report that type 2 innate lymphoid cells (ILC2s) are important regulators in a mouse model of liver ischaemia/reperfusion injury (IRI). Through manipulation of macrophage and eosinophil phenotypes, ILC2s mitigate liver inflammation and injury during liver IRI. We propose that ILC2s have the potential to serve as a therapeutic tool for protecting against acute liver injury and lay the foundation for translation of ILC2 therapy to human liver disease.

Project description:Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (syn.: Vernonia polyanthes Less) is popularly known as "assa-peixe" and its leaves are used in folk medicine mainly to treat respiratory diseases. In this study, we evaluated the cytogenotoxic and anticytogenotoxic potential of the V. polyanthes leaf aqueous extract (VpLAE) and its n-butanol fraction (n-BF) in the presence or absence of doxorubicin (DXR) (pre-, co-, and post-treatments) on a murine model for 24 h or 120 h. The micronucleus test (MN) and the comet assay were used to assess the cytogenotoxic and anticytogenotoxic potential of VpLAE and n-BF (250, 500, and 1000 mg/kg) administered via gavage to Swiss Webster mice. The chemical profiles of VpLAE and n-BF were assessed by liquid chromatography coupled to mass spectrometry, and their metabolites were putatively identified. Lastly, the possible biological activities related to the (anti) cytogenotoxicity of the compounds were predicted using the PASS online webserver. The in vivo results showed that different doses of VpLAE and n-BF did not present cytotoxic activity; however, the MN test revealed a slight mutagenic activity for the 24 h treatments. Moderate genotoxic effects were demonstrated for all treatments in the comet assay. Regarding anticytotoxicity and antimutagenicity, VpLAE and n-BF presented a high cytoprotective potential against DXR toxic effects. In the co-treatment, VpLAE reduced the DXR genotoxicity by ~27%, and n-BF did not demonstrate antigenotoxic potential. In contrast, an antigenotoxic effect was observed for both VpLAE and n-BF in the pre- and post-treatments, reducing DXR genotoxicity by ~41% and ~47%, respectively. Chemical analysis of VpLAE and n-BF showed the presence of eight phenolic compounds, including seven chlorogenic acids and a flavonoid. The PASS online tool predicted antimutagenic, anticancer, antineoplastic, chemoprotective, antioxidant, and radical scavenging activities for all constituents identified in VpLAE and n-BF. V. polyanthes leaves presented a protective effect against DXR cytogenotoxicity. In general, VpLAE and n-BF showed a greater antigenotoxic potential in the pre- and post-treatments. The metabolites putatively identified in VpLAE and n-BF exhibited antioxidant and chemoprotective potential according to computational prediction analysis. Altogether, our results highlight the potential application of V. polyanthes to protect against toxic manifestations induced by DXR.

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AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

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AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

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