info:eu-repo/semantics/doctoralThesis
Role of extracellular vesicles in hypoxia-induced hepatic injury in non-alcoholic fatty liver disease
Autor
Hernández Villanueva, Alejandra Andrea
Institución
Resumen
Background: Nonalcoholic fatty liver disease (NAFLD) is considered the most common liver disease worldwide. Transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in NAFLD. Observations in patients with obstructive sleep apnea syndrome (OSAS), which is characterized by occurrence of intermittent hypoxia (IH), suggest that hypoxia contributes to develop NASH. Among other mechanisms, release of extracellular vesicles (EV) by injured hepatocytes has been implicated in NAFLD progression. Aim: In this thesis we aimed to investigate the role of hypoxia condition in modulation of steatosis and liver injury in both in vitro and in vivo models of NAFLD. Also we evaluated the cellular crosstalk between hypoxic and steatotic hepatocytes and non-parenchymal cells through EVs. Methods: Primary rat hepatocytes and hepatoma cell line HepG2 treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, oxidative stress, cell death rates, pro-inflammatory and pro-fibrotic cytokines, inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) and human stellate cell (LX-2) were treated with conditioned medium (CM) and EVs from hypoxic fat-laden hepatocytes. The choline deficient L-amino acid defined (CDAA)-fed mice model used to assess the effects of IH on experimental NAFLD. Results: Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased, oxidative stress and mRNA levels of pro-inflammatory, pro-fibrotic cytokines and inflammasome-components compared to non- treated hepatocytes. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes and from EVs-hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked pro-inflammatory and pro-fibrotic phenotype in KC and LX-2 respectively. Livers from CDAA-fed mice exposed to IH exhibited increased of steatosis, portal inflammation, fibrosis, mRNA levels of pro-inflammatory, pro- fibrotic and inflammasome genes as well as increased levels of cleaved caspase-1 that correlated with an increase of circulating EV-caspase-1. Conclusion: Our findings in both in vivo and in vitro models of NAFLD/NASH indicate that hypoxia may increase liver injury and promote disease progression through amplification of inflammatory and fibrotic signals including inflammasome/caspase-1 activation. Hypoxia also promotes the release of EVs from hepatocytes contributing to cellular crosstalk with non-parenchymal cells by EVs-caspase-1-related mechanisms. These results suggest EVs and their content (caspase-1) as a potential novel biomarker in NAFLD and OSAS. Further characterization of EVs released under hypoxic conditions will allow the detailed delineation of their role in the promotion of steatosis, inflammation and fibrosis in the context of NAFLD and OSAS.