Davide Povero

Redox mechanisms switch on hypoxia-dependent epithelial–mesenchymal transition in cancer cells

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease

Background & Aim Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. Design Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. Results We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192 - two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. Conclusions These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Lipid-Induced Toxicity Stimulates Hepatocytes to Release Angiogenic Microparticles That Require Vanin-1 for Uptake by Endothelial Cells

Fat-overloaded hepatocytes release microparticles that induce angiogenesis and worsening of fatty liver disease. Sending an Angiogenic Message Excess amounts of saturated fatty acids are a potential dietary trigger for the fatty liver disease steatohepatitis, in which the liver develops fat deposits and inflammation. Progression of the disease to more serious forms, which can include scarring and other serious complications, is associated with the formation of new blood vessels, a process called angiogenesis, which requires endothelial cells to migrate and form tubular structures. Povero et al. found that a hepatocyte cell line exposed to excess amounts of saturated fatty acids released membrane-bound microparticles that induced angiogenesis when administered to mice. Microparticles from the blood of mice with diet-induced steatohepatitis originated from the liver and triggered migration and tubular structure formation when applied to an endothelial cell line. The angiogenic effects of microparticles generated by a hepatocyte cell line exposed to saturated fatty acids or of those from mice with diet-induced steatohepatitis involved the uptake of the microparticles by endothelial cells, a process that required Vanin-1, an enzyme located on the surface of the microparticles. Thus, the pathological angiogenesis that can occur in steatohepatitis could be reduced by preventing endothelial cells from internalizing Vanin-1–positive microparticles from hepatocytes. Angiogenesis is a key pathological feature of experimental and human steatohepatitis, a common chronic liver disease that is associated with obesity. We demonstrated that hepatocytes generated a type of membrane-bound vesicle, microparticles, in response to conditions that mimicked the lipid accumulation that occurs in the liver in some forms of steatohepatitis and that these microparticles promoted angiogenesis. When applied to an endothelial cell line, medium conditioned by murine hepatocytes or a human hepatocyte cell line exposed to saturated free fatty acids induced migration and tube formation, two processes required for angiogenesis. Medium from hepatocytes in which caspase 3 was inhibited or medium in which the microparticles were removed by ultracentrifugation lacked proangiogenic activity. Isolated hepatocyte-derived microparticles induced migration and tube formation of an endothelial cell line in vitro and angiogenesis in mice, processes that depended on internalization of microparticles. Microparticle internalization required the interaction of the ectoenzyme Vanin-1 (VNN1), an abundant surface protein on the microparticles, with lipid raft domains of endothelial cells. Large quantities of hepatocyte-derived microparticles were detected in the blood of mice with diet-induced steatohepatitis, and microparticle quantity correlated with disease severity. Genetic ablation of caspase 3 or RNA interference directed against VNN1 protected mice from steatohepatitis-induced pathological angiogenesis in the liver and resulted in a loss of the proangiogenic effects of microparticles. Our data identify hepatocyte-derived microparticles as critical signals that contribute to angiogenesis and liver damage in steatohepatitis and suggest a therapeutic target for this condition.

Intracellular reactive oxygen species are required for directional migration of resident and bone marrow-derived hepatic pro-fibrogenic cells

BACKGROUND & AIMS Liver fibrogenesis is sustained by myofibroblast-like cells originating from hepatic stellate cells (HSC/MFs), portal fibroblasts or bone marrow-derived cells, including mesenchymal stem cells (MSCs). Herein, we investigated the mechanistic role of intracellular generation of reactive oxygen species (ROS) and redox-sensitive signal transduction pathways in mediating chemotaxis, a critical profibrogenic response for human HSC/MFs and for MSC potentially engrafting chronically injured liver. METHODS Intracellular generation of ROS and signal transduction pathways were evaluated by integrating morphological and molecular biology techniques. Chemokinesis and chemotaxis were evaluated by wound healing assay and modified Boydens chamber assay, respectively. Additional in vivo evidence was obtained in human specimens from HCV-related cirrhosis. RESULTS Human MSCs and HSC/MFs migrate in response to a panel of polypeptide chemoattractants and extracellularly generated superoxide anion. All polypeptides induced a NADPH-oxidase-dependent intracellular rise in ROS, resulting in activation of ERK1/2 and JNK1/2. Moreover, menadione or 2,3-dimethoxy-1,4-naphthoquinone, which generate intracellular superoxide anion or hydrogen peroxide, respectively, induced ERK1/2 and JNK1/2 activation and migration. JNK1 activation was predominant for migration as shown by specific silencing. Finally, activation of ERK1/2 and JNK1/2 was found in extracts obtained from HSC/MFs during the course of an oxidative stress-mediated model of liver injury and phosphorylated JNK1/2 isoforms were detected in α-smooth muscle actin-positive myofibroblasts lining fibrotic septa in human cirrhotic livers. CONCLUSIONS Intracellular generation of ROS, through activation of specific signaling pathways, is a critical event for directional migration of HSC/MFs and MSCs.

The biphasic nature of hypoxia-induced directional migration of activated human hepatic stellate cells.

Liver fibrogenesis is sustained by pro‐fibrogenic myofibroblast‐like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia‐dependent angiogenesis. Human HSC/MFs were reported to express vascular‐endothelial growth factor (VEGF) and VEGF‐receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia‐induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non‐oriented and oriented migration were evaluated by using wound healing assay and the modified Boydens chamber assay, respectively. The data indicate that hypoxia‐induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria‐related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF‐1α‐dependent increase in VEGF expression (facilitated by ROS) and its progressive, time‐dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV‐related fibrotic/cirrhotic livers revealed HIF‐2α and haem‐oxygenase‐1 positivity in hepatocytes and α‐SMA‐positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia‐induced migration of HSC/MFs involves an early, mitochondrial‐dependent ROS‐mediated activation of ERK and JNK, followed by a delayed‐ and HIF‐1α‐dependent up‐regulation and release of VEGF. Copyright

Dissection of the Biphasic Nature of Hypoxia-Induced Motogenic Action in Bone Marrow-Derived Human Mesenchymal Stem Cells†‡§

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony‐forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia‐dependent migration of bone marrow‐derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boydens chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia‐dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c‐Jun N‐terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor‐1 (HIF‐1)–dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia‐dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF‐1‐dependent expression and release of VEGF. STEM CELLS 2011;29:952–963

Lipid-Induced Hepatocyte-Derived Extracellular Vesicles Regulate Hepatic Stellate Cells via MicroRNA Targeting Peroxisome Proliferator-Activated Receptor-γ

Background & Aims Hepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The trigger for HSC activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype. Methods EVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSC activation, migration, and proliferation were assessed. Loss- and gain-of-function studies were performed to explore the potential role of peroxisome proliferator-activated receptor-γ (PPAR-γ)-targeting microRNAs (miRNAs) carried by EVs into HSC. Results Hepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of profibrogenic genes (collagen-I, α-smooth muscle actin, and tissue inhibitor of metalloproteinases-2), proliferation, chemotaxis, and wound-healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSCs. The hepatocyte-derived EV miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression, with miR-128-3p being the most efficiently transferred. Furthermore, loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation. Conclusions Our findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel antifibrotic targets for NAFLD and other fibrotic diseases.

Arginase 2 deficiency results in spontaneous steatohepatitis: A novel link between innate immune activation and hepatic de novo lipogenesis

BACKGROUND & AIMS Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favours progression from isolated hepatic steatosis, induced by high fat feeding, to steatohepatitis. METHODS Arginase 2-knockout (Arg2(-/-)) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2(-/-) mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS Unexpectedly, Arg2(-/-) mice showed profound changes in their livers at baseline, characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked mRNA level increases of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2(-/-) mice. CONCLUSIONS This study identifies arginase 2 as a novel link between innate immune responses, hepatic lipid deposition, and liver injury.

Microparticles release by adipocytes act as "find-me" signals to promote macrophage migration.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical “find-me” signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel “find me” signals that contributes to macrophage infiltration associated with obesity.

Redox nanoparticles as a novel treatment approach for inflammation and fibrosis associated with nonalcoholic steatohepatitis

AIM Oxidative stress (OS) is largely thought to be a central mechanism responsible for liver damage, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Our aim was to investigate whether suppression of OS in the liver via redox nanoparticles (RNPs) reduces liver damage in a mouse model of NASH. MATERIALS & METHODS RNPs were prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals and were orally administered by daily gavage for 4 weeks. RESULTS The redox polymer was delivered to the liver after disintegration of nanoparticle in the stomach. RNP treatment in NASH mice via gavage led to a reduction of liver OS, improvement of fibrosis, and significant reduction of inflammation. CONCLUSION These findings uncover RNP as a novel potential NASH therapy.