Avaliação da atividade anti-angiogênica do secretoma de Aspergillus fumigatus e de drogas antifúngicas

• Ano de defesa: 2016
• Autor(a) principal: Xafranski, Hemílio [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Aspergillus fumigatus; Antifungal drugs; Angiogenesis; Fungal secretome; Biological activity
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=4051423; http://repositorio.unifesp.br/handle/11600/46264

Introduction: Aspergillus fumigatus is the most commonly isolated agent causing aspergillosis. Invasive pulmonary aspergillosis (IA) is an important cause of morbidity and mortality in immunocompromised patients. Chronic cavitary pulmonary aspergillosis (CCPA) affects immunocompromised patients with previous pulmonary lesion. Angiogenesis is an important physiological response to both clinical presentations of aspergillosis. Thus, patients presenting IA develop necrosis and tissue hypoxia, indicating angiogenesis inhibition at the infected lung region, meanwhile CCPA patients develop major hemoptysis, indicating that angiogenesis is not inhibited at the infection site. Fungi, specially Aspergillus sp., can produce a wide array of secondary metabolites, as well as molecules of pharmaceutical and biotechnological interest. In this context, the study of angiogenesis modulating molecules produced by A. fumigatus is promising to deepen the understanding of aspergillosis pathogenesis and to prospect new molecules with pharmacological potential. Objectives: The objective of this study was to evaluate the angiogenesis modulation by the secretome of A. fumigatus clinical isolates and antifungal drugs used in the treatment of pulmonary aspergillosis. Methods: Five A. fumigatus isolates from IA and CCPA patients were selected for secretome harvesting, anti-angiogenic activity evaluation, and secretome separation into fractions and sub-fractions, aiming to identify anti-angiogenic secretome fractions. Three pilot methods for the growth and harvest of A. fumigatus secretome were standardized and the secretomes obtained by the most suitable method (pilot 3) were analysed by LC-MS. The angiogenesis modulation activity of the secretomes was evaluated in RAEC cell culture, through capillary-like formation assays using Geltrex®. The most active secretome was separated into fractions and sub-fractions which were tested for anti-angiogenic activity. Additionally, the angiogenesis modulation by antifungal drugs was evaluated through RAEC cell proliferation, migration and capillary-like tube formation assays. Results: We successfully standardized three reproducible methods for the production of A. fumigatus secretome. From the 3 tested methods, the pilot 3 method was the most suitable for the prospection of new biologically active molecules. Despite the low number of isolates, we observed that secretomes from IA isolates mostly inhibited angiogenesis, while the secretomes from CCPA did not inhibited angiogenesis. Using this methodology we could successfully isolate an A. fumigatus secretome fraction bearing anti-angiogenic activity and not containing gliotoxin or fumagillin, fungi produced molecules previously described to present anti-angiogenic activity. We also observed inhibition of capillary-like structures, migration and proliferation in RAEC by the antifungal drug itraconazole and inhibition of proliferation in RAEC by the antifungal drug posaconazole. Our findings highlight the potential of the investigation of molecules secreted by Aspergillus fumigatus to contribute to the understanding of the pathogenesis of aspergillosis and to isolate new biologically active molecules.