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Liver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis.

ABSTRACT: Liver fibrosis is the most worrisome feature of non-alcoholic steatohepatitis (NASH). Growing evidence supports a link between hepatocyte apoptosis and liver fibrogenesis. Our aim was to determine the therapeutic efficacy and safety of liver Bid, a key pro-apoptotic molecule, suppression using RNA interference (RNAi) for the treatment of fibrosis.First, we optimized the delivery system for Bid siRNA in mice using ten different stealth RNAi siRNAs and two lipid formulations -Invivofectamine2.0 and a newly developed Invivofectamine3.0 - that have been designed for high efficacy accumulation in the liver, assessed via real-time PCR of Bid mRNA. Next, C57BL/6 mice were placed on a choline-deficient L-amino acid defined (CDAA) diet. After 19weeks of the CDAA diet, a time point that results in severe fibrotic NASH, mice were injected with the selected Bid siRNA-Invivofectamine3.0 biweekly for three weeks. Additionally hepatocyte-specific Bid deficient (Bid(?hep)) mice were placed on CDAA diet for 20weeks.A maximum Bid knockdown was achieved at 1.5mg/kg siRNA with Invivofectamine3.0, whereas it was at 7mg/kg with Invivofectamine2.0. In NASH mice, after 3weeks of treatment, BID protein was reduced to 10% and this was associated with an improvement in liver fibrosis and inflammation associated with a marked reduction in TUNEL positive cells, caspase 3 activation, and a reduction in mitochondrial BAX and BAK. Bid(?hep) mice showed similar protection from fibrotic changes.Our data demonstrate that liver Bid suppression by RNAi technology, as well as hepatocyte-specific Bid deficiency, improves liver fibrosis coupled with a reduction of inflammation in experimental NASH. These findings are consistent with existing evidence that hepatocyte apoptosis triggers hepatic stellate cell activation and liver fibrosis and suggest that Bid inhibition may be useful as an antifibrotic NASH therapy.

SUBMITTER: Eguchi A

PROVIDER: S-EPMC4761314 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Liver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis.

Eguchi Akiko A De Mollerat Du Jeu Xavier X Johnson Casey D CD Nektaria Andronikou A Feldstein Ariel E AE

Journal of hepatology 20151107 3

<h4>Background & aims</h4>Liver fibrosis is the most worrisome feature of non-alcoholic steatohepatitis (NASH). Growing evidence supports a link between hepatocyte apoptosis and liver fibrogenesis. Our aim was to determine the therapeutic efficacy and safety of liver Bid, a key pro-apoptotic molecule, suppression using RNA interference (RNAi) for the treatment of fibrosis.<h4>Methods</h4>First, we optimized the delivery system for Bid siRNA in mice using ten different stealth RNAi siRNAs and two ...[more]

PMID: 26555271

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Project description:ObjectiveClinical diagnosis and approval of new medications for non-alcoholic steatohepatitis (NASH) require invasive liver biopsies. The aim of our study was to identify non-invasive biomarkers of NASH and/or liver fibrosis.DesignThis multicentre study includes 250 patients (discovery cohort, n=100 subjects (Bariatric Surgery Versus Non-alcoholic Steato-hepatitis - BRAVES trial); validation cohort, n=150 (Liquid Biopsy for NASH and Liver Fibrosis - LIBRA trial)) with histologically proven non-alcoholic fatty liver (NAFL) or NASH with or without fibrosis. Proteomics was performed in monocytes and hepatic stellate cells (HSCs) with iTRAQ-nano- Liquid Chromatography - Mass Spectrometry/Mass Spectrometry (LC-MS/MS), while flow cytometry measured perilipin-2 (PLIN2) and RAB14 in peripheral blood CD14+CD16- monocytes. Neural network classifiers were used to predict presence/absence of NASH and NASH stages. Logistic bootstrap-based regression was used to measure the accuracy of predicting liver fibrosis.ResultsThe algorithm for NASH using PLIN2 mean florescence intensity (MFI) combined with waist circumference, triglyceride, alanine aminotransferase (ALT) and presence/absence of diabetes as covariates had an accuracy of 93% in the discovery cohort and of 92% in the validation cohort. Sensitivity and specificity were 95% and 90% in the discovery cohort and 88% and 100% in the validation cohort, respectively.The area under the receiver operating characteristic (AUROC) for NAS level prediction ranged from 83.7% (CI 75.6% to 91.8%) in the discovery cohort to 97.8% (CI 95.8% to 99.8%) in the validation cohort.The algorithm including RAB14 MFI, age, waist circumference, high-density lipoprotein cholesterol, plasma glucose and ALT levels as covariates to predict the presence of liver fibrosis yielded an AUROC of 95.9% (CI 87.9% to 100%) in the discovery cohort and 99.3% (CI 98.1% to 100%) in the validation cohort, respectively. Accuracy was 99.25%, sensitivity 100% and specificity 95.8% in the discovery cohort and 97.6%, 99% and 89.6% in the validation cohort. This novel biomarker was superior to currently used FIB4, non-alcoholic fatty liver disease fibrosis score and aspartate aminotransferase (AST)-to-platelet ratio and was comparable to ultrasound two-dimensional shear wave elastography.ConclusionsThe proposed novel liquid biopsy is accurate, sensitive and specific in diagnosing the presence and severity of NASH or liver fibrosis and is more reliable than currently used biomarkers.Clinical trialsDiscovery multicentre cohort: Bariatric Surgery versus Non-Alcoholic Steatohepatitis, BRAVES, ClinicalTrials.gov identifier: NCT03524365.Validation multicentre cohort: Liquid Biopsy for NASH and Fibrosis, LIBRA, ClinicalTrials.gov identifier: NCT04677101.

Project description:The fatty acid translocase, also known as CD36, is a well-established scavenger receptor for fatty acid (FA) uptake and is abundantly expressed in many metabolically active tissues. In the liver, CD36 is known to contribute to the progression of non-alcoholic fatty liver disease and to the more severe non-alcoholic steatohepatitis, by promoting triglyceride accumulation and subsequent lipid-induced endoplasmic reticulum (ER) stress. Given the recent discovery that the hepatocyte-secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) blocks CD36 expression, we sought to investigate the role of PCSK9 in liver fat accumulation and injury in response to saturated FAs and in a mouse model of diet-induced hepatic steatosis. Methods:In this study, we investigated the role of PCSK9 on the uptake and accumulation of FAs, as well as FA-induced toxicity, in a variety of cultured hepatocytes. Diet-induced hepatic steatosis and liver injury were also assessed in Pcsk9 -/- mice. Results:Our results indicate that PCSK9 deficiency in cultured hepatocytes increased the uptake and accumulation of saturated and unsaturated FAs. In the presence of saturated FAs, PCSK9 also protected cultured hepatocytes from ER stress and cytotoxicity. In line with these findings, a metabolic challenge using a high-fat diet caused severe hepatic steatosis, ER stress inflammation and fibrosis in the livers of Pcsk9 -/- mice compared to controls. Given that inhibition of CD36 ablated the observed accumulation of lipid in vitro and in vivo, our findings also highlight CD36 as a strong contributor to steatosis and liver injury in the context of PCSK9 deficiency. Conclusions:Collectively, our findings demonstrate that PCSK9 regulates hepatic triglyceride content in a manner dependent on CD36. In the presence of excess dietary fats, PCSK9 can also protect against hepatic steatosis and liver injury. Lay summary:The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein known to reduce the abundance of receptors on the surface of liver cells charged with the task of lipid uptake from the circulation. Although PCSK9 deficiency is known to cause lipid accumulation in mice and in cultured cells, the toxicological implications of this observation have not yet been reported. In this study, we demonstrate that PCSK9 can protect against cytotoxicity in cultured liver cells treated with a saturated fatty acid and we also show that Pcsk9 knockout mice develop increased liver injury in response to a high-fat diet.

Dataset Information

Liver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis.

Publications

Liver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis.

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