Publicação
Targeting hepatocyte necroptosis to treat liver diseases
| Resumo: | 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. |
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| Autores principais: | Afonso, Marta |
| Assunto: | Teses de doutoramento - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | 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. |
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