Protective effect of decellularized lung extracellular matrix hydrogel in idiopathic pulmonary fibrosis models

• Ano de defesa: 2022
• Autor(a) principal: Silva, Daniele Evangelista Leite da
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Decellularization; Descelularização; Extracellular matrix; Fibrose pulmonar idiopática; Hidrogel; Hydrogel; Idiopathic pulmonary fibrosis; Matriz extracelular
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/10/10132/tde-13092022-074914/

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with no cure, characterized by fibroblast proliferation, excessive extracellular matrix (ECM) deposition, and disorganized lung architecture, with costly treatments. Finding cheaper and more efficient therapeutic alternatives is imperative. Therefore, the goal of this project was to produce a cytocompatible decellularized porcine lung ECM- derived hydrogel and evaluate its properties on human lung fibroblasts and rat lungs in both normal and fibrotic conditions. The hypothesis was that ECM-derived hydrogel reduces fibrosis by attenuating oxidative stress, modulating tissue remodeling, preventing epithelial to mesenchymal transition (EMT), and myofibroblast differentiation. Porcine lungs were decellularized and later digested with pepsin to produce the ECM-derived hydrogel. This hydrogel\'s physical and mechanical properties, including its ability to gelate at 37ºC, were tested by scanning electron microscopy and turbidity analysis. Furthermore, the ECM-derived hydrogel peptide composition was analyzed with peptidomics. In vitro studies with primary human lung fibroblasts and A549 lung epithelial cells exposed to transforming growth factor beta- 1 (TGFß1) and treated with ECM-derived hydrogel were protected from myofibroblast differentiation and EMT, respectively, in a dose-dependent matter, as evaluated by immunocytochemistry, western blot, and RT-qPCR. The treatment also attenuated collagen synthesis (visualized by immunocytochemistry and quantified by RT-qPCR), reduced reactive oxygen species (observed by DCFH-DA assay), and altered matrix metalloproteinases remodeling activity (visualized by immunocytochemistry and analyzed by zymography and RT-qPCR). In vivo studies with bleomycin exposed rat lungs confirmed the observations from the in vitro studies and identified stronger inflammatory activity in a higher ECM-derived hydrogel concentration. Proteomic and transcriptomic analysis of rat lung samples identified that the protective effect of the ECM-derived hydrogel in lung fibrosis is caused by: the modulation of inflammatory chemokines, leukocyte chemotaxis, interference in the lung hemostasis, changes in tissue remodeling and cell adhesion pathways, downregulation of TGFβ receptors, interference with key fibrosis-related signaling pathways like Smad and PI3K/AKT. Uncovering the molecular pathways involved with the ECM-derived hydrogel protective effect in an IPF animal model has not been done as extensively as in this study. Moreover, this was also the first work to determine the sequences of all the peptides that compose the ECM-derived hydrogel using peptidomics analysis. These results cover important landmarks for small animal preclinical studies with the ECM- derived hydrogel essential to advance large animal studies. Ultimately, this study points to a promising future for ECM-derived hydrogel treatments in IPF and other diseases that generate fibrosis.