A carregar...
Projeto de investigação
Sem título
Financiador
Autores
Publicações
Targeting hepatocyte necroptosis to treat liver diseases
Publication . Afonso, Marta; Rodrigues, Cecília M. P., 1968-; Castro, Rui Eduardo Mota, 1978-
Chronic liver diseases are considered a major public health concern, and their prevalence and incidence are increasing with serious long-term implications, such as cirrhosis, hepatocellular carcinoma (HCC) and, ultimately, premature death. In particular, non-alcoholic fatty liver disease (NAFLD) and chronic cholestatic liver disease are two distinct pathologic conditions sharing a progressive nature and lacking effective medical therapies. Persistent cell death represents a dominant trigger for chronic inflammation, fibrosis and compensatory cell proliferation, increasing the risk of cancer. Of note, necroptosis or regulated necrosis, which depends on kinase activity of receptor-interacting protein 3 (RIP3), was recently described as novel immunogenic type of cell death that may pathologically impinge on inflammation-driven liver diseases. The main goal of the work presented in this thesis was to evaluate the role of necroptosis in human and experimental models of NAFLD and cholestatic liver disease, and to test whether its inhibition could provide potential therapeutic benefits. First, the role of necroptosis in human and experimental NAFLD was evaluated. We showed that hepatic necroptosis is triggered in NAFLD patients and in two dietary models of non-alcoholic steatohepatitis (NASH), playing a role in disease progression. Indeed, inhibition of necroptosis through genetic ablation of Rip3 attenuated liver injury, steatosis, inflammation, fibrosis and oxidative stress in mice fed a methionine- and choline-deficient NASH-inducing diet. In turn, tumor necrosis factor-α (TNF-α) was shown to trigger RIP3-dependent oxidative stress during hepatocyte necroptosis. We next explored the role of RIP3-dependent signaling in NAFLD-associated HCC using the choline-deficient L amino acid-defined (CDAA) diet murine model. Remarkably, despite associating with increased insulin resistance and hepatic steatosis, absence of RIP3 decreased long-term inflammation and fibrosis, compensatory proliferation of hepatocytes, oxidative stress, genetic cell death resistance in dysplastic hepatocytes, and tissue microenvironment alterations closely associated with NAFLD-driven hepatocarcinogenesis. In parallel, we evaluated the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Our results showed that necroptosis is triggered in the liver of primary biliary cholangitis patients (PBC), mediating hepatic necro-inflammation induced by common bile duct ligation (BDL) in mice. However, deletion of Rip3 failed to prevent BDL-induced secondary biliary fibrosis and was associated with enhanced chronic cholestasis and hepatic iron accumulation, through up-regulation of heme oxygenase-1. As an alternative putative therapeutic approach, we next evaluated whether ablation of microRNA-21 (miR-21) could simultaneously prevent necroptosis and fibrosis in BDL mice. Functional studies established the association between miR-21, its target cyclin dependent kinase 2 associated protein 1 (CDK2AP1) and necroptosis. Further, miR-21 was found increased in the liver of PBC patients and BDL mice, whereas deletion of miR-21 attenuated necroptosis, liver damage, oxidative stress and expression of pro-fibrogenic genes in BDL mice. miR-21 ablation further improved BDL-induced adaptive responses in bile acid homeostasis. Taken together, our studies highlight that inhibition of necroptosis may, at least partially, inhibit NAFLD and NAFLD-associated HCC, as well as cholestatic liver disease pathogenesis. miR-21 inhibition reduces necroptosis and may also afford complementary protective effects. A broader understanding of necroptosis and its intricate role in liver pathophysiology is likely to provide a rationale for the development of therapeutic strategies for NAFLD and cholestatic liver disease based on the targeting of necroptosis-associated signaling pathways.
Activation of necroptosis in human and experimental cholestasis
Publication . Afonso, Marta; Rodrigues, Pedro; Simão, André; Ofengeim, Dimitry; Carvalho, Tânia; Amaral, Joana D.; Gaspar, Maria Manuela; Cortez-Pinto, Helena; Castro, Rui E.; Yuan, Junying; Rodrigues, Cecília M. P.
Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3-/-) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro. We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3-/- mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
SFRH
Número da atribuição
SFRH/BD/91119/2012