Mitochondrial Role on Cellular Apoptosis, Autophagy, and Senescence during Osteoarthritis Pathogenesis.

Authors have demonstrated that apoptosis activation is a pathway related to cartilage degradation characteristics of the OA process. Autophagy is an adaptive response to protect cells from various environmental changes, and defects in autophagy are linked to cell death. In this sense, decreased autophagy of chondrocytes has been observed in OA articular cartilage. The aim of this work was to study the role of OA mitochondria in apoptosis, autophagy, and senescence, using OA and Normal (N) transmitochondrial cybrids. Results: OA cybrids incubated with menadione showed a higher percentage of late apoptosis and necrosis than N cybrids. Stimulation of cybrids with staurosporine and IL-1ß showed that OA cybrids were more susceptible to undergoing apoptosis than N cybrids. An analysis of the antioxidant response using menadione on gene expression revealed a lower expression of nuclear factor erythroid 2-like 2 and superoxide dismutase 2 in OA than N cybrids. Activation of microtubule-associated protein 1A/1B-light chain 3 was reduced in OA compared to N cybrids. However, the percentage of senescent cells was higher in OA than N cybrids. Conclusion: This work suggests that mitochondria from OA patients could be involved in the apoptosis, autophagy, and senescence of chondrocytes described in OA cartilage.

Inhibition of acyl-CoA binding protein (ACBP) by means of a GABAARγ2-derived peptide.

Acyl-CoA binding protein (ACBP) encoded by diazepam binding inhibitor (DBI) is an extracellular inhibitor of autophagy acting on the gamma-aminobutyric acid A receptor (GABAAR) γ2 subunit (GABAARγ2). Here, we show that lipoanabolic diets cause an upregulation of GABAARγ2 protein in liver hepatocytes but not in other major organs. ACBP/DBI inhibition by systemically injected antibodies has been demonstrated to mediate anorexigenic and organ-protective, autophagy-dependent effects. Here, we set out to develop a new strategy for developing ACBP/DBI antagonists. For this, we built a molecular model of the interaction of ACBP/DBI with peptides derived from GABAARγ2. We then validated the interaction between recombinant and native ACBP/DBI protein and a GABAARγ2-derived eicosapeptide (but not its F77I mutant) by pull down experiments or surface plasmon resonance. The GABAARγ2-derived eicosapeptide inhibited the metabolic activation of hepatocytes by recombinant ACBP/DBI protein in vitro. Moreover, the GABAARγ2-derived eicosapeptide (but not its F77I-mutated control) blocked appetite stimulation by recombinant ACBP/DBI in vivo, induced autophagy in the liver, and protected mice against the hepatotoxin concanavalin A. We conclude that peptidomimetics disrupting the interaction between ACBP/DBI and GABAARγ2 might be used as ACBP/DBI antagonists. This strategy might lead to the future development of clinically relevant small molecules of the ACBP/DBI system.

Orthotopic Model of Hepatocellular Carcinoma in Mice.

Orthotopic models of hepatocellular carcinoma (HCC) consist in the implantation of tumor cells into the liver by direct intrahepatic injection. In this model, tumorigenesis is triggered within the hepatic microenvironment, thus mimicking the metastatic behavior of HCC. Herein, we detail a surgically mediated methodology that allows the reproducible and effective induction of liver-sessile tumors in mice. We enumerate the steps to be followed before and after the surgical procedure, including HCC cell preparation, the quantity of cancer cells to be injected, presurgical preparation of the mice, and finally, postoperative care. The surgical procedure involves laparotomy to expose the liver, injection of cells into the left-lateral hepatic lobe, and closure of the incision with sutures followed by wound clips. We also provide information concerning the subsequent tumor growth follow-up, as well as the application of bioluminescence imaging to monitor tumor development.

A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetrachloride.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD). Obesity is a known risk factor of NASH, which, in turn, increases the risk of developing cirrhosis (liver scarring) and hepatocellular carcinoma (HCC). In addition to being a potentially life-threatening condition, public health concerns surrounding NASH are amplified by the lack of FDA-approved treatments. Although various preclinical models reflecting both the histopathology and the pathophysiological progression of human NASH exist, most of these models are diet-based and require 6-13 months for NASH symptom manifestation. Here, we describe a simple and rapid-progression model of NASH and NASH-driven HCC in mice. Mice received a western diet equivalent (WD; i.e., a high-fat, high-fructose, and high-cholesterol diet), high-sugar water (23.1 g/L fructose and 18.9 g/L glucose), and weekly intraperitoneal injections of carbon tetrachloride (CCl4) at a dose of 0.2 µL/g of body weight. The resulting phenotype, consisting in liver fibrosis and HCC, appeared within 24 weeks of diet/treatment initiation and presented similar histological and transcriptomic features as human NASH and NASH-driven HCC, thereby supporting the adequacy of this preclinical model for the development and evaluation of drugs that can prevent or reverse these diseases.

A Mouse Model of Hepatocellular Carcinoma Induced by Streptozotocin and High-Fat Diet.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the second most common cause of cancer-related death. HCC is associated to chronic diseases such as viral hepatitis, alcoholic, and non-alcoholic fatty liver disease (NAFLD), diabetes mellitus, and obesity, among others. Although pre-clinical models have been investigated to mimic the transition from NAFLD to HCC, they do not accurately reproduce the phenotypic evolution from simple steatosis to steatohepatitis, fibrosis/cirrhosis, and HCC. Hence, these models have failed to demonstrate the influence of diabetes on hepatic carcinogenesis. Here, we report a novel mouse model of HCC triggered by fast-developing diabetes and NAFLD. The first step consists in a single intraperitoneal injection of a low dose of streptozotocin into neonatal C57BL/6J mice to induce type 2 diabetes. In a second step, mice are fed with high-fat diet to accelerate the development of simple steatosis. Continuous high-fat diet exacerbates hepatic fat deposition with increased lobular inflammation (by activation of foam cell-like macrophages) and fibrosis (by activating hepatic stellate cells), two representative pathological traits of steatohepatitis/fibrosis. After 20 weeks, all mice developed multiple HCCs. This model of hepatic carcinogenesis triggered by diabetes mellitus and NAFLD offers the advantage of being rapid and accurately recapitulates the pathogenesis of human HCC without the need of administering hepatic carcinogens.

Targeted Analysis of Glycerophospholipids and Mono-, Di-, or Tri-Acylglycerides in Liver Cancer.

The metabolic rearrangements of hepatic metabolism associated with liver cancer are still incompletely understood. There is an ongoing need to identify novel and more efficient diagnostic biomarkers and therapeutic targets based on the metabolic mechanisms of these diseases. In comparison to traditional diagnostic biomarkers, metabolomics is a comprehensive technique for discovering chemical signatures for liver cancer screening, prediction, and earlier diagnosis. Lipids are a large and diverse group of complex biomolecules that are at the heart of liver physiology and play an important role in the development and progression of cancer. In this chapter, we described two detailed protocols for targeted lipids analysis: glycerophospholipids and mono, di, tri-acylglycerides, both by Flow Injection Analysis (FIA) HPLC coupled to a SelexIon/QTRAP 6500+ system. These approaches provide a targeted lipidomic metabolomic signature of dissimilar metabolic disorders affecting liver cancers.

Biomarker Identification in Liver Cancers Using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) Imaging: An Approach for Spatially Resolved Metabolomics.

Liver cancers are characterized by interindividual and intratumoral heterogeneity, which makes early diagnosis and the development of therapies challenging. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging is a potent and sensitive MS ionization technique for direct, unaltered 2D and 3D imaging of metabolites in complex biological samples. Indeed, DESI gently desorbs and ionizes analyte molecules from the sample surface using an electrospray source of highly charged aqueous spray droplets in ambient conditions. DESI-MS imaging of biological samples allows untargeted analysis and characterization of metabolites in liver cancers to identify new biomarkers of malignancy. In this chapter, we described a detailed protocol using liver cancer samples collected and stored for histopathology examination, either as frozen or as formalin-fixed, paraffin-embedded specimens. Such hepatocellular carcinoma samples can be subjected to DESI-MS analyses, illustrating the capacity of spatially resolved metabolomics to distinguish malignant lesions from adjacent normal liver tissue.

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

Fibrates as drugs with senolytic and autophagic activity for osteoarthritis therapy.

BACKGROUND: Ageing-related failure of homeostasis mechanisms contributes to articular cartilage degeneration and osteoarthritis (OA), for which disease-modifying treatments are not available. Our objective was to identify molecules to prevent OA by regulating chondrocyte senescence and autophagy. METHODS: Human chondrocytes with IL-6 induced senescence and autophagy suppression and SA-ß-gal as a reporter of senescence and LC3 as reporter of autophagic flux were used to screen the Prestwick Chemical Library of approved drugs. Preclinical cellular, tissue and blood from OA and blood from OA and ageing models were used to test the efficacy and relevance of activating PPARα related to cartilage degeneration. FINDINGS: Senotherapeutic molecules with pro-autophagic activity were identified. Fenofibrate (FN), a PPARα agonist used for dyslipidaemias in humans, reduced the number of senescent cells via apoptosis, increased autophagic flux, and protected against cartilage degradation. FN reduced both senescence and inflammation and increased autophagy in both ageing human and OA chondrocytes whereas PPARα knockdown conferred the opposite effect. Moreover, PPARα expression was reduced through both ageing and OA in mice and also in blood and cartilage from knees of OA patients. Remarkably, in a retrospective study, fibrate treatment improved OA clinical conditions in human patients from the Osteoarthritis Initiative (OAI) Cohort. INTERPRETATION: These results demonstrate that FDA-approved fibrate drugs targeting lipid metabolism protect against cartilage degeneration seen with ageing and OA. Thus, these drugs could have immediate clinically utility for age-related cartilage degeneration and OA treatment. FUND: This study was supported by Instituto de Salud Carlos III- Ministerio de Ciencia, Innovación y Universidades, Spain, Plan Estatal 2013-2016 and Fondo Europeo de Desarrollo Regional (FEDER), "Una manera de hacer Europa", PI14/01324 and PI17/02059, by Innopharma Pharmacogenomics platform applied to the validation of targets and discovery of drugs candidates to preclinical phases, Ministerio de Economía y Competitividad, by grants of the National Instiutes of Health to PDR (P01 AG043376 and U19 AG056278). We thank FOREUM Foundation for Research in Rheumatology for their support.