MiRNAs in Alcohol-Related Liver Diseases and Hepatocellular Carcinoma: A Step toward New Therapeutic Approaches?

Alcohol-related Liver Disease (ALD) is the primary cause of chronic liver disorders and hepatocellular carcinoma (HCC) development in developed countries and thus represents a major public health concern. Unfortunately, few therapeutic options are available for ALD and HCC, except liver transplantation or tumor resection for HCC. Deciphering the molecular mechanisms underlying the development of these diseases is therefore of major importance to identify early biomarkers and to design efficient therapeutic options. Increasing evidence indicate that epigenetic alterations play a central role in the development of ALD and HCC. Among them, microRNA importantly contribute to the development of this disease by controlling the expression of several genes involved in hepatic metabolism, inflammation, fibrosis, and carcinogenesis at the post-transcriptional level. In this review, we discuss the current knowledge about miRNAs' functions in the different stages of ALD and their role in the progression toward carcinogenesis. We highlight that each stage of ALD is associated with deregulated miRNAs involved in hepatic carcinogenesis, and thus represent HCC-priming miRNAs. By using in silico approaches, we have uncovered new miRNAs potentially involved in HCC. Finally, we discuss the therapeutic potential of targeting miRNAs for the treatment of these diseases.

Dipalmitoyl-phosphatidylserine-filled cationic maltodextrin nanoparticles exhibit enhanced efficacy for cell entry and intracellular protein delivery in phagocytic THP-1 cells.

Vaccination through the upper respiratory tract is a promising strategy, and particulate antigens, such as antigens associated with nanoparticles, triggered a stronger immune response than the sole antigens. Cationic maltodextrin-based nanoparticles loaded with phosphatidylglycerol (NPPG) are efficient for intranasal vaccination but non-specific to trigger immune cells. Here we focused on phosphatidylserine (PS) receptors, specifically expressed by immune cells including macrophages, to improve nanoparticle targeting through an efferocytosis-like mechanism. Consequently, the lipids associated with NPPG have been substituted by PS to generate cationic maltodextrin-based nanoparticles with dipalmitoyl-phosphatidylserine (NPPS). Both NPPS and NPPG exhibited similar physical characteristics and intracellular distribution in THP-1 macrophages. NPPS cell entry was faster and higher (two times more) than NPPG. Surprisingly, competition of PS receptors with phospho-L-serine did not alter NPPS cell entry and annexin V did not preferentially interact with NPPS. Although the protein association is similar, NPPS delivered more proteins than NPPG in cells. On the contrary, the proportion of mobile nanoparticles (50%), the movement speed of nanoparticles (3 µm/5 min), and protein degradation kinetics in THP-1 were not affected by lipid substitution. Together, the results indicate that NPPS enter cells and deliver protein better than NPPG, suggesting that modification of the lipids of cationic maltodextrin-based nanoparticles may be a useful strategy to enhance nanoparticle efficacy for mucosal vaccination.

Starch nanoparticles improve curcumin-induced production of anti-inflammatory cytokines in intestinal epithelial cells.

Inflammatory bowel disease (IBD), encompassing Crohn's disease and ulcerative colitis, is a long-term condition resulting from self-sustained intestinal inflammation. Curcumin (Cur), a powerful, naturally occurring antioxidant and anti-inflammatory polyphenol, has been investigated as a therapeutic for IBD, but its poor stability and low bioavailability limits its efficacy. We investigated the use of crosslinked starch nanocarrier (NPL) on the intracellular delivery and the anti-inflammatory efficiency of curcumin. Caco-2 epithelial cells were stimulated with TNFα for 24 h and the anti-inflammatory effects of NPL/Cur formulations were evaluated at the early stages of inflammation (4 h) or later, when fully established (24 h). NPL allowed the intracellular delivery of curcumin, which was enhanced in inflammatory cells, due to a modification of the endocytosis pathways. NPL/Cur decreased the secretion of pro-inflammatory cytokines IL-1ß, IL-6 and IL-8 while increasing the anti-inflammatory cytokine IL-10. Finally, the inflammation-related opening of the tight junctions better allowed NPL/Cur to cross the epithelium by paracellular transport. This was confirmed by ex vivo analysis where NPL/Cur, administered to colonic explants from chemically-induced acute colitis mouse model, delivered curcumin deeper in the epithelium. To conclude, NPL/Cur formulation emphasizes the anti-inflammatory effects of curcumin and could constitute a therapeutic alternative in the management of IBD.

Effective Nanoparticle-Based Nasal Vaccine Against Latent and Congenital Toxoplasmosis in Sheep.

Toxoplasma gondii is a parasitic protozoan of worldwide distribution, able to infect all warm-blooded animals, but particularly sheep. Primary infection in pregnant sheep leads to millions of abortions and significant economic losses for the livestock industry. Moreover, infected animals constitute the main parasitic reservoir for humans. Therefore, the development of a One-health vaccine seems the best prevention strategy. Following earlier work, a vaccine constituted of total extract of Toxoplasma gondii proteins (TE) associated with maltodextrin nanoparticles (DGNP) was developed in rodents. In this study we evaluated the ability of this vaccine candidate to protect against latent and congenital toxoplasmosis in sheep. After two immunizations by either intranasal or intradermal route, DGNP/TE vaccine generated specific Th1-cellular immune response, mediated by APC-secretion of IFN-γ and IL-12. Secretion of IL-10 appeared to regulate this Th1 response for intradermally vaccinated sheep but was absent in intranasally-vaccinated animals. Finally, protection against latent toxoplasmosis and transplacental transmission were explored. Intranasal vaccination led to a marked decrease of brain cysts compared with the non-vaccinated group. This DGNP/TE vaccine administered intranasally conferred a high level of protection against latent toxoplasmosis and its transplacental transmission in sheep, highlighting the potential for development of such a vaccine for studies in other species.

Prevention of influenza virus infection and transmission by intranasal administration of a porous maltodextrin nanoparticle-formulated vaccine.

Influenza vaccines administered intramuscularly exhibit poor mucosal immune responses in the respiratory tract which is the prime site of the infection. Intranasal vaccination is a potential route for vaccine delivery which has been demonstrated effective in inducing protective immune responses in both systemic and mucosal compartments. For this purpose, nanoparticles have been used as antigen delivery systems to improve antigen capture by immune cells. In this paper we demonstrate efficient delivery of viral antigens to airway epithelial cells, macrophages and dendritic cells, using polysaccharide nanoparticles (NPL), leading to a strong protection against influenza virus infection. A formulation combining split Udorn virus antigens with NPL and the mucosal protein adjuvant CTA1-DD was administered intranasally and resulted in an enhanced specific humoral immune response. Furthermore, NPL carrying split Udorn, with or without CTA1-DD, inhibited virus transmission from infected to uninfected naive mice. These results demonstrate that an intranasal delivery system combining NPL, mucosal adjuvant CTA1-DD and split virus antigens confers robust protection against influenza infection and inhibits virus transmission.

Importance of the Phospholipid Core for Mucin Hydrogel Penetration and Mucosal Cell Uptake of Maltodextrin Nanoparticles.

Nanoparticles (NPs) used as mucosal antigen delivery systems are a promising way to vaccinate. However, NPs have to cross the mucus gel and penetrate into mucosal cells to deliver antigens, and a mismatch exists between mucopenetrating NPs, rarely able to interact with cells, and NPs designed to deliver antigens into cells, but often described as mucoadhesives. Here, we compared the ability of cationic maltodextrin-based NPs, either without (NP+) or with an anionic phospholipid core (NPL), to interact with mucins and airway epithelial cells. We showed that their lipid core increased the NPL's mobility in mucin hydrogel by reducing interactions with mucins. Similarly, the uptake and protein delivery by NPLs into airway epithelial cells were not hindered by mucins. This highlights the importance of anionic lipids in the NPL, which are more efficient in crossing the mucin hydrogel while retaining the ability to interact with epithelial cells, an intermediate behavior between mucoadhesive and mucopenetrating NPs.

Protein delivery by porous cationic maltodextrin-based nanoparticles into nasal mucosal cells: Comparison with cationic or anionic nanoparticles.

Different types of biodegradable nanoparticles (NPs) have been studied as delivery systems for proteins into nasal mucosal cells, especially for vaccine applications. Such a nanocarrier must have the ability to be loaded with proteins and to transport this payload into mucosal cells. However, comparative data on nanoparticles' capacity for protein loading, efficiency of subsequent endocytosis and the quantity of nanocarriers used are either lacking or contradictory, making comparisons and the choice of a best candidate difficult. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: the NPL (cationic maltodextrin NP with an anionic lipid core), cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP, and cationic and anionic liposomes. We first quantified the protein association efficiency and NPL associated the largest amount of ovalbumin, used as a model protein. In vitro, the delivery of fluorescently-labeled ovalbumin into mucosal cells (airway epithelial cells, dendritic cells and macrophages) was assessed by flow cytometry and revealed that the NPL delivered protein to the greatest extent in all 3 different cell lines. Taken together, these data underlined the potential of the porous and cationic maltodextrin-based NPL as efficient protein delivery systems to mucosal cells.

Nucleotide-binding oligomerization domain 1 (NOD1) modulates liver ischemia reperfusion through the expression adhesion molecules.

BACKGROUND & AIMS: In liver transplantation, organ shortage leads to the use of marginal grafts that are more susceptible to ischemia-reperfusion (IR) injury. We identified nucleotide-binding oligomerization domain 1 (NOD1) as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in IR. Herein, we aimed to elucidate the role of NOD1 in IR injury, particularly focusing on its effects on the endothelium and hepatocytes. METHOD: Nod1 WT and KO mice were treated with NOD1 agonists and subjected to liver IR. Expression of adhesion molecules was analyzed in total liver, isolated hepatocytes and endothelial cells. Interactions between PMNs and hepatocytes were studied in an ex vivo co-culture model using electron microscopy and lactate dehydrogenase levels. We generated NOD1 antagonist-loaded nanoparticles (np ALINO). RESULTS: NOD1 agonist treatment increased liver injury, PMN tissue infiltration and upregulated ICAM-1 and VCAM-1 expression 20 hours after reperfusion. NOD1 agonist treatment without IR increased expression of adhesion molecules (ICAM-1, VCAM-1) in total liver and more particularly in WT hepatocytes, but not in Nod1 KO hepatocytes. This induction is dependent of p38 and ERK signaling pathways. Compared to untreated hepatocytes, a NOD1 agonist markedly increased hepatocyte lysis in co-culture with PMNs as shown by the increase of lactate dehydrogenase in supernatants. Interaction between hepatocytes and PMNs was confirmed by electron microscopy. In a mouse model of liver IR, treatment with np ALINO significantly reduced the area of necrosis, aminotransferase levels and ICAM-1 expression. CONCLUSION: NOD1 regulates liver IR injury through induction of adhesion molecules and modulation of hepatocyte-PMN interactions. NOD1 antagonist-loaded nanoparticles reduced liver IR injury and provide a potential approach to prevent IR, especially in the context of liver transplantation. LAY SUMMARY: Nucleotide-binding oligomerization domain 1 (NOD1) is as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in ischemia-reperfusion. Here, we show that the NOD1 pathway targets liver adhesion molecule expression on the endothelium and on hepatocytes through p38 and ERK signaling pathways. The early increase of adhesion molecule expression after reperfusion emphasizes the importance of adhesion molecules in liver injury. In this study we generated nanoparticles loaded with NOD1 antagonist. These nanoparticles reduced liver necrosis by reducing PMN liver infiltration and adhesion molecule expression.

Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections.

Due to the high risk of an outbreak of pandemic influenza, the development of a broadly protective universal influenza vaccine is highly warranted. The design of such a vaccine has attracted attention and much focus has been given to nanoparticle-based influenza vaccines which can be administered intranasally. This is particularly interesting since, contrary to injectable vaccines, mucosal vaccines elicit local IgA and lung resident T cell immunity, which have been found to correlate with stronger protection in experimental models of influenza virus infections. Also, studies in human volunteers have indicated that pre-existing CD4+ T cells correlate well to increased resistance against infection. We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. To improve the protective ability of the self-adjuvanting fusion protein, CTA1-3M2e-DD, we incorporated it into porous maltodextrin nanoparticles (NPLs). This proof-of-principle study demonstrates that the combined vaccine vector given intranasally enhanced immune protection against a live challenge infection and reduced the risk of virus transmission between immunized and unimmunized individuals. Most importantly, immune responses to NPLs that also contained recombinant hemagglutinin (HA) were strongly enhanced in a CTA1-enzyme dependent manner and we achieved broadly protective immunity against a lethal infection with heterosubtypic influenza virus. Immune protection was mediated by enhanced levels of lung resident CD4+ T cells as well as anti-HA and -M2e serum IgG and local IgA antibodies.

Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: Comparison with anionic and cationic nanoparticles.

Different types of biodegradable nanoparticles (NP) have been studied as nasal mucosa cell delivery systems. These nanoparticles need to strongly interact with mucosa cells to deliver their payload. However, only a few simultaneous comparisons have been made and it is therefore difficult to determine the best candidate. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: cationic and anionic liposomes, cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP and porous and cationic maltodextrin NP (cationic surface with an anionic lipid core: NPL). We first quantified their nasal residence time after nasal administration in mice using in vivo live imaging and NPL showed the longest residence time. In vitro endocytosis on mucosal cells (airway epithelial cells, macrophages and dendritic cells) using labeled nanoparticles were performed by flow cytometry and confocal microscopy. Among the 5 nanoparticles, NPL were taken up to the greatest extent by the 3 different cell lines and the endocytosis mechanisms were characterized. Taken together, we observed that the nanoparticles' cationic surface charge is insufficient to improve mucosal residence time and cellular uptake and that the NPL are the best candidates to interact with airway mucosal cells.

Vectorization by nanoparticles decreases the overall toxicity of airborne pollutants.

Atmospheric pollution is mainly composed of volatile pollutants and particulate matter that strongly interact. However, their specific roles in the induction of cellular toxicity, in particular the impact of the vectorization of atmospheric pollutants by ultrafine particles, remains to be fully elucidated. For this purpose, non-toxic poly-lactic co-glycolic acid (PLGA) nanoparticles were synthesized and three pollutants (benzo(a)pyrene, naphthalene and di-ethyl-hexyl-phthalate) were adsorbed on the surface of the nanoparticles in order to evaluate the toxicity (cytotoxicity, genotoxicity and ROS induction) of these complexes to a human airway epithelial cell line. The adsorption of the pollutants onto the nanoparticles was confirmed by HPLC analysis. Interestingly, the cytotoxicity assays (MTT, LDH and CellTox Green) clearly demonstrated that the vectorization by nanoparticles decreases the toxicity of the adsorbed pollutants. Genotoxicity was assessed by the micronucleus test and the comet assay and showed no increase in primary DNA damage or in chromosomal aberrations of nanoparticle vectorized pollutants. Neither cytotoxicity nor genotoxicity was correlated with ROS induction. To conclude, our results indicate that the vectorization of pollutants by nanoparticles does not potentiate the toxicity of the pollutants studied and that, on the contrary, adsorption onto nanoparticles could protect cells against pollutants' toxicity.

Development and validation of a reversed-phase HPLC method for the quantification of paclitaxel in different PLGA nanocarriers.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantification of paclitaxel encapsulated in biodegradable poly(lactic-co-glycolic) (PLGA) copolymer nanoparticles. This simple (isocratic mode, without additive) and rapid (retention time of the paclitaxel under 4 min) methodology permits the detection of low quantities of paclitaxel in nanoparticulate formulations and the determination of the encapsulation efficiency (EE). Analysis was achieved on an octadecyl stationary phase. The isocratic mobile phase consisted of acetonitrile:water 80:20 (v/v) (flow rate = 0.8 mL/min). Stability of free paclitaxel was preliminary studied in those chromatographic conditions. The calibration curve was linear in the concentration range of 2-10 µg/mL (R2  = 0.9994). The method was specific with valuable trueness, repeatability (intra-day precision) and intermediate precision (inter-day precision) based on relative standard deviation (RSD) values (less than 2%). The limits of detection (LOD) and quantification (LOQ) were 0.56 and 1.85 ng/mL, respectively. This developed method was successfully employed for quantifying paclitaxel in PLGA 50:50 co-polymer nanoparticles. The accurate knowledge of the encapsulated paclitaxel concentration is essential to define the quantities of PLGA nanoparticles necessary to achieve the in vitro cell viability study.

Synthetic parasites: a successful mucosal nanoparticle vaccine against Toxoplasma congenital infection in mice.

AIM: Development of protein vaccine to prevent congenital infection is a major public health priority. Our goal is the design of mucosal synthetic pathogen inducing protective immune responses against congenital toxoplasmosis. MATERIALS & METHODS: Mice were immunized intranasally, establishing pregnancy and challenging orally. Placental immune response, congenital infection, pup growth, parasitic load rates were studied. RESULTS: Pups born to vaccinated infected dams had significantly fewer brain cysts, no intraocular inflammation and normal growth. Protection was associated with a placental cellular Th1 response downregulated by IL-6 and correlated with persistence of vaccine for few hours in the nose before being totally eliminated. CONCLUSION: Our vaccine conferred high protection against congenital toxoplasmosis. These results provide support for future studies of other congenital vaccine.

Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and endocytosis.

With the ongoing commercialization of nanotechnology products, human exposure to nanoparticles (NPs) is set to increase dramatically and an evaluation of their potential adverse effects is essential. Surface charge, among other physico-chemicals parameters, is a key criterion that should be considered when using a definition for nanomaterials in a regulatory context. It has recently been recognized as an important factor in determining the toxicity of NPs; however, a complete understanding of the mechanisms involved is still lacking. In this context, the aim of the present study was to investigate the influence of the surface charge modification of NPs on in vitro toxicity assays. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles bearing different surface charges, positive(+), neutral(n) or negative(-), were synthesized. In vitro genotoxicity assays (micronucleus and comet assays) coupled with an assessment of cytotoxicity, were performed in different cell lines (L5178Y mouse lymphoma cells, TK6 human B-lymphoblastoid cells and 16HBE14o- human bronchial epithelial cells). Reactive oxygen species (ROS) production and endocytosis studies were also performed. Our results showed that PLGA(+) NPs were cytotoxic. They are endocytosed by the clathrin pathway and induced ROS in the three cell lines. They led to chromosomal aberrations without primary DNA damage in 16HBE14o- cells, suggesting that aneuploidy may be considered as an important biomarker when assessing the genotoxic potential of NPs. Moreover, 16HBE14o- cells seem to be more suitable for the in vitro screening of inhaled NPs than the regulatory L5178Y and TK6 cells.

Evolution of availability of curcumin inside poly-lactic-co-glycolic acid nanoparticles: impact on antioxidant and antinitrosant properties.

PURPOSE: Curcumin exhibits antioxidant properties potentially beneficial for human health; however, its use in clinical applications is limited by its poor solubility and relative instability. Nanoparticles exhibit interesting features for the efficient distribution and delivery of curcumin into cells, and could also increase curcumin stability in biological systems. There is a paucity of information regarding the evolution of the antioxidant properties of nanoparticle-encapsulated curcumin. METHOD: We described a simple method of curcumin encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles without the use of detergent. We assessed, in epithelial cells and in an acellular model, the evolution of direct antioxidant and antinitrosant properties of free versus PLGA-encapsulated curcumin after storage under different conditions (light vs darkness, 4°C vs 25°C vs 37°C). RESULTS: In epithelial cells, endocytosis and efflux pump inhibitors showed that the increased antioxidant activity of PLGA-encapsulated curcumin relied on bypassing the efflux pump system. Acellular assays showed that the antioxidant effect of curcumin was greater when loaded in PLGA nanoparticles. Furthermore, we observed that light decreased, though heat restored, antioxidant activity of PLGA-encapsulated curcumin, probably by modulating the accessibility of curcumin to reactive oxygen species, an observation supported by results from quenching experiments. Moreover, we demonstrated a direct antinitrosant activity of curcumin, enhanced by PLGA encapsulation, which was increased by light exposure. CONCLUSION: These results suggest that the antioxidant and antinitrosant activities of encapsulated curcumin are light sensitive and that nanoparticle modifications over time and with temperature may facilitate curcumin contact with reactive oxygen species. These results highlight the importance of understanding effects of nanoparticle maturation on an encapsulated drug's activity.

Porous nanoparticles as delivery system of complex antigens for an effective vaccine against acute and chronic Toxoplasma gondii infection.

Development of sub-unit mucosal vaccines requires the use of specific delivery systems or immune-modulators such as adjuvants to improve antigen immunogenicity. Nasal route for vaccine delivery by nanoparticles has attracted much interest but mechanisms triggering effective mucosal and systemic immune response are still poorly understood. Here we study the loading of porous nanoparticles (DGNP) with a total extract of Toxoplasma gondii antigens (TE), the delivery of TE by DGNP into airway epithelial, macrophage and dendritic cells, and the subsequent cellular activation. In vitro, DGNP are able to load complex antigens in a stable and quantitative manner. The outstanding amount of antigen association by DGNP is used to deliver TE in airway mucosa cells to induce a cellular maturation with an increased secretion of pro-inflammatory cytokines. Evaluation of nasal vaccine efficiency is performed in vivo on acute and chronic toxoplasmosis mouse models. A specific Th1/Th17 response is observed in vivo after vaccination with DGNP/TE. This is associated with high protection against toxoplasmosis regarding survival and parasite burden, correlated with an increased delivery of antigens by DGNP in airway mucosa cells. This study provides evidence of the potential of DGNP for the development of new vaccines against a range of pathogens.

Influence of surface charge and inner composition of nanoparticles on intracellular delivery of proteins in airway epithelial cells.

The delivery of protein in the airway using nanoparticles (NP) is an emerging strategy that shows encouraging results in vivo for several applications. However, the mechanisms by which NP deliver proteins to the inside of cells remain poorly understood. In this study, we investigated the intracellular delivery of ovalbumin (OVA) in human airway cells by two porous cationic polysaccharides nanoparticles. These NP have the same surface charge density but differ in that their inner core contains either cationic or anionic charges (respectively: NP(+) and DGNP(+)). Confocal microscopy showed a rapid uptake of both NP by human airway cells, followed by a significant accumulation in clathrin vesicles and early endosomes. Both NP were found to associate OVA in a quantitative manner, and this association was stable even in presence of serum proteins. We observed that the two NP greatly increased OVA uptake by human airway cells, meanwhile FRET studies using FITC-labelled NP and TRITC-labelled OVA showed a gradual release of OVA from NP within cells, and this was much faster with DGNP(+) than NP(+). These results were confirmed using OVA-DQ to follow OVA degradation fragments within cells. Both NP increased intracellular proteolysis of OVA, however DGNP(+) facilitated OVA escape from endosomes. Studies with trypsin and pepsin at different pH strongly suggested that both NP can protect (in the extracellular medium) or promote (in acidic endosomes) protein proteolysis, depending on the environment. Interestingly, the mechanisms involved could be explained as a function of protein global charge at different pH. All these results confirm the importance of not only the surface charge but also the inner composition of NP in determining their efficacy as tools for the delivery of proteins to different cellular compartments.

Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice.

BACKGROUND & AIMS: Self-renewal of mature hepatocytes promotes homeostasis and regeneration of adult liver. However, recent studies have indicated that liver progenitor cells (LPC) could give rise to hepatic epithelial cells during normal turnover of the liver and after acute injury. We investigated the capacity of LPC to differentiate into hepatocytes in vivo and contribute to liver regeneration. METHODS: We performed lineage tracing experiments, using mice that express tamoxifen-inducible Cre recombinase under control of osteopontin regulatory region crossed with yelow fluorescent protein reporter mice, to follow the fate of LPC and biliary cells. Adult mice received partial (two-thirds) hepatectomy, acute or chronic administration of carbon tetrachloride (CCl(4)), choline-deficient diet supplemented with ethionine, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet. RESULTS: LPC and/or biliary cells generated 0.78% and 2.45% of hepatocytes during and upon recovery of mice from liver injury, respectively. Repopulation efficiency by LPC and/or biliary cells increased when extracellular matrix and laminin deposition were reduced. The newly formed hepatocytes integrated into hepatic cords, formed biliary canaliculi, expressed hepato-specific enzymes, accumulated glycogen, and proliferated in response to partial hepatectomy, as neighboring native hepatocytes. By contrast, LPC did not contribute to hepatocyte regeneration during normal liver homeostasis, in response to surgical or toxic loss of liver mass, during chronic liver injury (CCl(4)-induced), or during ductular reactions. CONCLUSIONS: LPC or biliary cells terminally differentiate into functional hepatocytes in mice with liver injury.

The nuclear orphan receptor Nur77 is a lipotoxicity sensor regulating glucose-induced insulin secretion in pancreatic ß-cells.

The NR4A orphan nuclear receptors Nur77, Nurr1, and Nor1 exert multiple cellular and metabolic functions. These transcriptional regulators are activated in response to extracellular stresses, including lipotoxic fatty acids (FA) and proinflammatory cytokines. The contribution of NR4As to ß-cell pathophysiology is, however, unknown. We have therefore examined the role of NR4As as downstream contributors to FA-induced ß-cell dysfunctions. Human pancreatic islets and insulinoma ß-cells were used to determine transcriptional programs elicited by NR4A, which were compared to those triggered by palmitate treatment. Functional studies evaluated the consequence of an increased NR4A expression on insulin biosynthesis and secretion and cell viability in insulinoma ß-cells. FA and cytokine treatment increased NR4A expression in pancreatic ß-cells, with Nur77 being most highly inducible in murine ß-cells. Nur77, Nurr1, or Nor1 modulated common and distinct clusters of genes involved notably in cation homeostasis and insulin gene transcription. By altering zinc homeostasis, insulin gene transcription, and secretion, Nur77 was found to be a major transcriptional mediator of part of FA-induced ß-cell dysfunctions. The repressive role of Nur77 in insulin gene regulation was tracked down to protein-protein interaction with FoxO1, a pivotal integrator of the insulin gene regulatory network. The present study identifies a member of the NR4A nuclear receptor subclass, Nur77/NR4A1, as a modulator of pancreatic ß-cell biology. Together with its previously documented role in liver and muscle, its role in ß-cells establishes Nur77 as an important integrator of glucose metabolism.

Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.

UNLABELLED: BACKGROUND& AIMS: Embryonic biliary precursor cells form a periportal sheet called the ductal plate, which is progressively remodeled to generate intrahepatic bile ducts. A limited number of ductal plate cells participate in duct formation; those not involved in duct development are believed to involute by apoptosis. Moreover, cells that express the SRY-related HMG box transcription factor 9 (SOX9), which include the embryonic ductal plate cells, were proposed to continuously supply the liver with hepatic cells. We investigated the role of the ductal plate in hepatic morphogenesis. METHODS: Apoptosis and proliferation were investigated by immunostaining of mouse and human fetal liver tissue. The postnatal progeny of SOX9-expressing ductal plate cells was analyzed after genetic labeling, at the ductal plate stage, by Cre-mediated recombination of a ROSA26RYFP reporter allele. Inducible Cre expression was induced by SOX9 regulatory regions, inserted in a bacterial artificial chromosome. Livers were studied from mice under normal conditions and during diet-induced regeneration. RESULTS: Ductal plate cells did not undergo apoptosis and showed limited proliferation. They generated cholangiocytes lining interlobular bile ducts, bile ductules, and canals of Hering, as well as periportal hepatocytes. Oval cells that appeared during regeneration also derived from the ductal plate. We did not find that liver homeostasis required a continuous supply of cells from SOX9-expressing progenitors. CONCLUSIONS: The ductal plate gives rise to cholangiocytes lining the intrahepatic bile ducts, including its most proximal segments. It also generates periportal hepatocytes and adult hepatic progenitor cells.