Cell Surface Of Hepatocytes Research Articles

Evolutionary analysis of SLC10 family members and insights into function and expression regulation of lamprey NTCP.

The Na ( +)-taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier family 10 (SLC10), which consists of 7 members (SLC10a1-SLC10a7). NTCP is a transporter localized to the basolateral membrane of hepatocytes and is primarily responsible for the absorption of bile acids. Although mammalian NTCP has been extensively studied, little is known about the lamprey NTCP (L-NTCP). Here we show that L-NTCP follows the biological evolutionary history of vertebrates, with conserved domain, motif, and similar tertiary structure to higher vertebrates. L-NTCP is localized to the cell surface of lamprey primary hepatocytes by immunofluorescence analysis. HepG2 cells overexpressing L-NTCP also showed the distribution of L-NTCP on the cell surface. The expression profile of L-NTCP showed that the expression of NTCP is highest in lamprey liver tissue. L-NTCP also has the ability to transport bile acids, consistent with its higher vertebrate orthologs. Finally, using a farnesoid X receptor (FXR) antagonist, RT-qPCR and flow cytometry results showed that L-NTCP is negatively regulated by the nuclear receptor FXR. This study is important for understanding the adaptive mechanisms of bile acid metabolism after lamprey biliary atresia based on understanding the origin, evolution, expression profile, biological function, and expression regulation of L-NTCP.

P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality.

The liver plays a significant role in regulating a wide range of metabolic, homeostatic, and host-defense functions. However, the impact of liver injury on the host's ability to control bacteremia and morbidity in sepsis is not well understood. Leukocyte recruitment and activation lead to cytokine and chemokine release, which, in turn, trigger hepatocellular injury and elevate nucleotide levels in the extracellular milieu. P2Y2 purinergic receptors, G protein-coupled and activated by extracellular ATP/UTP, are expressed at the cell surface of hepatocytes and nonparenchymal cells. We sought to determine whether P2Y2 purinergic receptor function is necessary for the maladaptive host response to bacterial infection and endotoxin-mediated inflammatory liver injury and mortality in mice. We report that P2Y2 purinergic receptor knockout mice (P2Y2-/-) had attenuated inflammation and liver injury, with improved survival in response to LPS/galactosamine (LPS/GalN; inflammatory liver injury) and cecal ligation and puncture (CLP; polymicrobial sepsis). P2Y2-/- livers had attenuated c-Jun NH2-terminal kinase activation, matrix metallopeptidase-9 expression, and hepatocyte apoptosis in response to LPS/GalN and attenuated inducible nitric oxide synthase and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 protein expression in response to CLP. Implicating liver injury in the disruption of amino acid homeostasis, CLP led to lower serum arginine and higher bacterial load and morbidity in the WT mice, whereas serum arginine levels were comparable to sham-operated controls in P2Y2-/- mice, which had attenuated bacteremia and improved survival. Collectively, our studies highlight the pathophysiological relevance of P2Y2 purinergic receptor function in inflammatory liver injury and dysregulation of systemic amino acid homeostasis with implications for sepsis-associated immune dysfunction and morbidity in mice.NEW & NOTEWORTHY Our studies provide experimental evidence for P2Y2 purinergic receptor-mediated potentiation of inflammatory liver injury, morbidity, and mortality, in two well-established animal models of inflammatory liver injury. Our findings highlight the potential to target P2Y2 purinergic signaling to attenuate the induction of "cytokine storm" and prevent its deleterious consequences on liver function, systemic amino acid homeostasis, host response to bacterial infection, and sepsis-associated morbidity and mortality.

ANMCO Position paper: Inclisiran: an innovative therapeutic approach for the clinical management of hypercholesterolemia

Research focused on lipid-lowering treatments has led to the development of new therapeutic options aimed at cardiovascular risk reduction. Gene silencing represents one of the most innovative approaches to reduce low-density lipoprotein cholesterol (LDL-C). Inclisiran is a small interfering RNA that inhibits proprotein convertase subtilisin/kexin type 9 synthesis and promotes LDL-C clearance by enhancing LDL-C receptor expression on hepatocyte cell surface. Several clinical studies have demonstrated inclisiran efficacy in terms of LDL-C reduction (~50%) with a dosage regimen of 300 mg administered twice a year after the first two doses administered at time 0 and after 90 days. Inclisiran use has recently been approved by the European and American drug regulatory agencies as a therapeutic option in addition to the maximum tolerated statin therapy in adults with primary hypercholesterolemia or mixed dyslipidemia who need further LDL-C reduction.

Translational Population-Pharmacodynamic Modeling of a Novel Long-Acting siRNA Therapy, Inclisiran, for the Treatment of Hypercholesterolemia.

Inclisiran is a novel N-acetylgalactosamine (GalNAc) conjugated small-interfering ribonucleic acid (siRNA) therapy designed to specifically target proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA in the liver for the treatment of hypercholesterolemia. Inclisiran's GalNAc attachment results in a rapid uptake into the liver, and thus a short plasma half-life, but long duration of effects on PCSK9 inhibition and low-density lipoprotein cholesterol (LDL-C) lowering. The effects on PCSK9 inhibition and consequent LDL-C reduction are sustained for more than 6 months following a single subcutaneous (s.c.) dose, despite inclisiran being detectable in the plasma only for up to 48 hours. A kinetic-pharmacodynamic (K-PD) model was developed to characterize inclisiran's dose-related LDL-C lowering effects and to evaluate the impact of intrinsic and extrinsic factors on LDL-C lowering. To accommodate the long duration of action, the K-PD model incorporated an effect compartment which represents the liver. Inclisiran concentration in the liver leads to decreased production of the PCSK9 protein and allow recycling of more LDL-C receptors on the hepatocyte cell surface, which results in a reduction of circulating LDL-C. The analysis of covariates identified PCSK9 and LDL-C baseline levels as important factors for the effects of LDL-C lowering. Observations and modeling and simulation results demonstrated that PCSK9 and LDL-C reductions are achieved rapidly after dosing and sustained when patients are treated with a 300 mg s.c. dose once every 6 months.

Cargo-Specific Role for Retriever Subunit VPS26C in Hepatocyte Lipoprotein Receptor Recycling to Control Postprandial Triglyceride-Rich Lipoproteins.

The copper metabolism MURR1 domains/coiled-coil domain containing 22/coiled-coil domain containing 93 (CCC) complex is required for the transport of low-density lipoprotein receptor (LDLR) and LRP1 (LDLR-related protein 1) from endosomes to the cell surface of hepatocytes. Impaired functioning of hepatocytic CCC causes hypercholesterolemia in mice, dogs, and humans. Retriever, a protein complex consisting of subunits VPS26C, VPS35L, and VPS29, is associated with CCC, but its role in endosomal lipoprotein receptor transport is unclear. We here investigated the contribution of retriever to hepatocytic lipoprotein receptor recycling and plasma lipids regulation. Using somatic CRISPR/Cas9 gene editing, we generated liver-specific VPS35L or VPS26C-deficient mice. We determined total and surface levels of LDLR and LRP1 and plasma lipids. In addition, we studied the protein levels and composition of CCC and retriever. Hepatocyte VPS35L deficiency reduced VPS26C levels but had minimal impact on CCC composition. VPS35L deletion decreased hepatocytic surface expression of LDLR and LRP1, accompanied by a 21% increase in plasma cholesterol levels. Hepatic VPS26C ablation affected neither levels of VPS35L and CCC subunits, nor plasma lipid concentrations. However, VPS26C deficiency increased hepatic LDLR protein levels by 2-fold, probably compensating for reduced LRP1 functioning, as we showed in VPS26C-deficient hepatoma cells. Upon PCSK9 (proprotein convertase subtilisin/kexin type 9)-mediated LDLR elimination, VPS26C ablation delayed postprandial triglyceride clearance and increased plasma triglyceride levels by 26%. Our study suggests that VPS35L is shared between retriever and CCC to facilitate LDLR and LRP1 transport from endosomes to the cell surface. Conversely, retriever subunit VPS26C selectively transports LRP1, but not LDLR, and thereby may control hepatic uptake of postprandial triglyceride-rich lipoprotein remnants.

Deciphering signal transduction networks in the liver by mechanistic mathematical modelling.

In health and disease, liver cells are continuously exposed to cytokines and growth factors. While individual signal transduction pathways induced by these factors were studied in great detail, the cellular responses induced by repeated or combined stimulations are complex and less understood. Growth factor receptors on the cell surface of hepatocytes were shown to be regulated by receptor interactions, receptor trafficking and feedback regulation. Here, we exemplify how mechanistic mathematical modelling based on quantitative data can be employed to disentangle these interactions at the molecular level. Crucial is the analysis at a mechanistic level based on quantitative longitudinal data within a mathematical framework. In such multi-layered information, step-wise mathematical modelling using submodules is of advantage, which is fostered by sharing of standardized experimental data and mathematical models. Integration of signal transduction with metabolic regulation in the liver and mechanistic links to translational approaches promise to provide predictive tools for biology and personalized medicine.

Constitutive oxidants from hepatocytes of male iPLA2β-null mice increases the externalization of phosphatidylethanolamine on plasma membrane

We have found that group VIA calcium-independent phospholipase A2 (iPLA2β) has specificity for hydrolysis of phosphatidylethanolamine (PE) in mouse livers. Phospholipids (PLs) are transported to plasma membrane and some PLs including PE are externalized to maintain membrane PL asymmetry. Here we demonstrated that hepatocytes of iPLA2β-null (KO) mice showed an increase in PE containing palmitate and oleate. We aimed to examine whether externalization of PE on the outer leaflets could be affected by iPLA2β deficiency and its modulation by reactive oxygen species (ROS) or apoptosis. As duramycin has high affinity to PE, we used duramycin conjugated with biotin (DLB) and streptavidin 488 as a probe for detection of externalized PE. Compared to WT, naïve KO hepatocytes showed an increase in both PE externalization and ROS generation. These events were observed in male but not in female KO mice. Hydrogen peroxide or menadione treatment enhanced PE externalization to the same extent for both male/female WT and KO hepatocytes. By indirect immunofluorescence, DLB-streptavidin staining was observed as small punctuated spots on the cell surface of menadione-treated KO hepatocytes. Unlike the reported PS externalization, CD95/FasL treatment did not lead to any increase in PE externalization, and iPLA2β deficiency-dependent PE externalization was also not correlated with apoptosis. Thus, constitutive (but not induced) ROS generation in iPLA2β-deficient hepatocytes leads to PE externalization observed only in male mice. Such PE externalization may imply detrimental effects regarding further oxidation of PE fatty acids and the binding with pathogens on the outer leaflets of hepatocyte plasma membrane.

A Paradigm Shift in the Management of Atherosclerosis: Protective Role of Sirtuins in Atherosclerosis.

Facing the rise of an aging population and age-related pathologies such as atherosclerosis will continue to be some of the biggest challenges encountering health care. Regardless of considerable advancements in management and prevention to deal with atherosclerosis and other related pathologies. The current guidelines for preventing and managing atherosclerotic diseases are lifestyle changes, blood pressure control, blood glucose control, and lipid control. There has been an increase in pre-clinical studies regarding the effects of sirtuins on atherosclerosis and this review aims to highlight the benefits of sirtuins in atherosclerosis. We did an extensive search using the PubMed database with the medical subject headings (MeSH) keywords “sirtuin'' and “atherosclerosis.” The reviewed literature reported that sirtuins prevent and ameliorate atherosclerosis by halting inflammation, apoptosis, oxidative stress, and regulating low-density lipoprotein (LDL) cholesterol. Sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) inhibit the RELA component of NF-kB, thus suppressing inflammation, SIRT1 inhibits p53 by deacetylation, and the latter stabilize telomeres thus preventing apoptosis and cell death. Sirtuin 3 (SIRT3) inhibits oxidative stress by driving the production of reduced glutathione. Sirtuin 2 (SIRT2) regulates LDL cholesterol by inhibiting pcsk9, increasing LDL receptors on the cell surface of hepatocytes. A combination of these effects of sirtuins in the endothelial cells suggests sirtuins are anti-atherogenic and could revolutionize the standards for the management of atherosclerosis. This article also emphasizes the need for future research on human cells or subjects rather than animal subjects.

PD-L1 is induced on the hepatocyte surface via CKLF-like MARVEL transmembrane domain-containing protein 6 up-regulation by the anti-HBV drug Entecavir.

Chronic hepatitis B is now controllable when treated with nucleoside reverse transcriptase inhibitors (NRTIs), which inhibit hepatitis B virus (HBV) replication. However, once the NRTIs are discontinued, most patients relapse, necessitating lifelong NRTIs treatment. HBV infection relapse is assumed to be caused by the persistent existence of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. The mechanism by which cccDNA-positive hepatocytes escape immune surveillance during NRTIs treatment remains elusive. Entecavir (ETV), a commonly used NRTI, post-transcriptionally up-regulates programmed cell death-ligand 1 (PD-L1), an immune checkpoint molecule, on the cell surface of hepatocytes regardless of HBV infection. Up-regulation by ETV depends on up-regulation of CKLF-like MARVEL transmembrane domain-containing 6, a newly identified potent regulator of PD-L1 expression on the cell surface. ETV-treated hepatic cells suppressed the activity of primary CD3 T cells and programmed cell death protein-1 (PD-1)-over-expressed Jurkat cells. Finally, ETV induces PD-L1 in primary hepatocytes infected by HBV. These results provide evidence that ETV considerably up-regulates PD-L1 on the cell surface of infected hepatocytes, which may be one of the mechanisms by which infected hepatocytes subvert immune surveillance.

Rationale and Design of a Phase 3 Study to Evaluate the Efficacy and Safety of ION-682884 in Patients with Transthyretin-Mediated Amyloid Cardiomyopathy (ATTR-CM)

Background: Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is a life-threatening, irreversible condition, which can lead to heart failure (HF) and, ultimately, heart transplant or death. Despite the recent approval in United States of a TTR stabilizer (VYNDAQEL®-tafamidis meglumine;VYNDAMAX™-tafamidis) for the treatment of ATTR-CM, disease progression still occurs. This study aims to determine if treatment with AKCEA-TTR-LRx (ION-682884), an antisense oligonucleotide (ASO), is safe and superior to placebo in reducing the risk of cardiovascular (CV) death or CV clinical events in patients with hereditary (hATTR-CM) or wild-type ATTR-CM (wtATTR-CM). Study Design and Methods: AKCEA-TTR-LRx (ION-682884) is a follow-on compound that incorporates the Ligand-Conjugated Antisense (LICA) technology; in this case, a triantennary N-acetyl galactosamine (GalNAc) moiety which targets the asialoglycoprotein receptors (ASGPR) expressed abundantly on the hepatocyte cell surface. In comparison to inotersen, its parent compound, ION-682884 requires a lower dose and frequency of administration (27-fold smaller; 45mg SC Q4W) to achieve a similar reduction in ATTR, providing greater patient convenience. ION-682884-CS2 (EudraCT No: 2019-002835-27) is a Phase 3 global, double-blind, randomized, placebo-controlled study assessing the efficacy and safety of AKCEA-TTR-LRx (ION-682884) in hATTR-CM or wtATTR-CM patients receiving available background standard of care (SoC) therapy. Approximately 750 patients with a history of HF due to ATTR-CM will be randomized 1:1 to receive AKCEA-TTR-LRx (ION-682884) or placebo administered by subcutaneous injection once every 4 weeks. The main inclusion criteria include confirmed diagnosis of ATTR-CM by tissue biopsy or positive PYP/DPD scan, end-diastolic interventricular septum thickness of >12mm, NT-proBNP >600 pg/mL, NYHA class I-III and 6-minute walk distance (6MWD) >150 m. The main exclusion criteria include estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73m2, platelet count below the low limit of normality and urine protein/creatinine ratio (UPCR) ≥ 1000 mg/g. Patients are allowed to concomitantly receive tafamidis/tafamidis meglumine as SoC for ATTR-CM, if locally approved and available, per physician's discretion. The study consists of a 120-week Treatment Period and a 20-week Post-Treatment Evaluation Period. During each study visit, subjects will undergo laboratory tests, cardiac assessments (echocardiography), and functional evaluations. Patient-reported outcomes (PRO) will also be collected. Primary efficacy endpoint is the composite of CV mortality and frequency of CV clinical events (HF-related urgent visits requiring administration of IV diuretics and/or CV-related hospitalizations) at Week 120 study visit, analyzed by the Finkelstein-Shoenfeld method. This test is based on the principle of each patient in the study being compared with every other patient in a pairwise manner in hierarchical fashion. Secondary endpoints include the change from baseline in the 6MWD, Kansas City Cardiomyopathy Questionnaire score, rate of CV mortality, CV clinical events, and all-cause of mortality at Week 120. Additional exploratory endpoints include a change from baseline in cardiac imaging parameters, renal function, biomarkers, and PROs questionnaires and disease scores. An interim analysis on change from baseline in 6MWD is also planned at Week 60. All deaths and CV clinical events will be adjudicated by an independent, blinded Clinical Adjudication Committee, using predefined endpoint criteria. Conclusions: Despite recent advances, there is still a need for more efficacious, safe and convenient treatment options for ATTR-CM. The ION-682884-CS2 is a large Phase 3 trial designed to evaluate the clinical efficacy and safety of AKCEA-TTR-LRx (ION-682884) compared to placebo for the treatment of ATTR-CM. Figure Disclosures Falk: Ionis Pharmaceuticals: Consultancy. Gertz:Ionis/Akcea: Consultancy; Alnylam: Consultancy; Proclara: Membership on an entity's Board of Directors or advisory committees; Prothena Biosciences Inc: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Spectrum: Consultancy, Research Funding; Annexon: Consultancy; Appellis: Consultancy; Amgen: Consultancy; Medscape: Consultancy, Speakers Bureau; Physicians Education Resource: Consultancy; Abbvie: Other: personal fees for Data Safety Monitoring board; Research to Practice: Consultancy; Teva: Speakers Bureau; Johnson and Johnson: Speakers Bureau; DAVA oncology: Speakers Bureau; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; i3Health: Other: Development of educational programs and materials; Springer Publishing: Patents & Royalties; Amyloidosis Foundation: Research Funding; International Waldenstrom Foundation: Research Funding. Benson:Ionis Pharmaceuticals: Research Funding. Buchele:Ionis Pharmaceuticals: Employment. Brambatti:Ionis Pharmaceuticals: Employment. Tsimikas:Ionis Pharmaceuticals: Employment. Viney:Ionis Pharmaceuticals: Employment. Tai:Ionis Pharmaceuticals: Employment. Monteiro:Ionis Pharmaceuticals: Employment. Yang:Ionis Pharmaceuticals: Employment. O'Dea:Akcea Therapeutics: Employment. Karwatowska-Prokopczuk:Akcea Therapeutics: Employment. Schneider:Ionis Pharmaceuticals: Employment. Geary:Ionis Pharmaceuticals: Employment. Monia:Ionis Pharmaceuticals: Employment.

The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Development and characterization of L-HSA conjugated PLGA nanoparticle for hepatocyte targeted delivery of antiviral drug

The present study demonstrates preparation and characterization of polymeric nanoparticles (NPs) conjugated with Lactosaminated-Human Serum Albumin (L-HSA) peptide. Ligand L-HSA was targeted to Asialoglycoprotein-Receptors (ASGP-R) overexpressed on parenchymal cell surface of hepatocytes. Poly (lactic-co-glycolic acid) PLGA NPs surface functionalized with maleimide group were formulated using Interfacial Activity Assisted Surface Functionalization (IAASF) technique. The hydrophilic antiretroviral drug lamivudine was encapsulated into PLGA NP using double emulsion solvent evaporation technique for liver specificity. A Central Composite Design (CCD) was applied to optimize maleimide functionalized PLGA NP formulation parameters and studied its effect on particle size and encapsulation efficiency. The optimized batch was conjugated with L-HSA. Inclusion of maleimide group was confirmed by FTIR, 1H-NMR, and conjugation of L-HSA to the PLGA NP was confirmed by SDS-PAGE, in vitro cell uptake, in vivo pharmacokinetic and biodistribution studies. L-HSA conjugation resulted in 2.17 fold increase in cell uptake and 3.84 fold longer retention in HepG2 cells. The in vivo pharmacokinetic and biodistribution study revealed higher AUC and efficient target ability of L-HSA conjugated NP in mice liver compared to other organs. These results concluded that proposed L-HSA conjugated maleimide functionalized PLGA NP can be used as promising hepatocyte targeted drug delivery system for Lamivudine.

Transcriptome analysis reveals TMPRSS6 isoforms with distinct functionalities.

TMPRSS6 (matriptase‐2) is a type II transmembrane serine protease involved in iron homoeostasis. At the cell surface of hepatocytes, TMPRSS6 cleaves haemojuvelin (HJV) and regulates the BMP/SMAD signalling pathway leading to production of hepcidin, a key regulator of iron absorption. Although four TMPRSS6 human isoforms and three mice Tmprss6 isoforms are annotated in databases (Ensembl and RefSeq), their relative expression or activity has not been studied. Analyses of RNA‐seq data and RT‐PCR from human tissues reveal that TMPRSS6 isoform 1 (TMPRSS6‐1) and 3 are mostly expressed in human testis while TMPRSS6‐2 and TMPRSS6‐4 are the main transcripts expressed in human liver, testis and pituitary. Furthermore, we confirm the existence and analyse the relative expression of three annotated mice Tmprss6 isoforms. Using heterologous expression in HEK293 and Hep3B cells, we show that all human TMPRSS6 isoforms reach the cell surface but only TMPRSS6‐1 undergoes internalization. Moreover, truncated TMPRSS6‐3 or catalytically altered TMPRSS6‐4 interact with HJV and prevent its cleavage by TMPRSS6‐2, suggesting their potential role as dominant negative isoforms. Taken together, our results highlight the importance of understanding the precise function of each TMPRSS6 isoforms both in human and in mouse.

Solute carrier NTCP, a key actor in liver infections by hepatitis viruses and a promising antiviral target

Liver chronic infections by hepatotropic viruses remain a major health problem worldwide. Hepatitis B, C, and D viruses (HBV, HCV, HDV) are responsible for severe liver damages. In spite of different viral structures and life cycles, they all exclusively infect the liver, suggesting the presence of hepatocyte-specific factors allowing viral infection. If viral life cycles are already well described, the molecular interactions between liver host factors and viral structures remain elusive. Host factors represent promising targets for the development of new antiviral strategies. Recently, the bile acid transporter NTCP, mainly expressed at the cell surface of hepatocytes, was described as the first bona-fide receptor for both HBV and HDV. Moreover, this solute carrier was shown to be involved in the modulation of HCV entry, confirming its importance in liver infection by hepatic viruses. This review summarizes the interactions between NTCP and these three major viruses.

Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake.

Human hepatic lipase (hHL) is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL) is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT). In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included β-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL)-mediated free fatty acid (FFA) lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL.

Evolocumab

Evolocumab

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes

Ecto-F1-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F1-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F1-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes.This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.

PCSK9 inhibition in the management of hyperlipidemia: focus on evolocumab.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases low-density lipoprotein cholesterol (LDL-C) concentrations through interference with normal physiologic hepatic LDL receptor (LDLR) recycling. Inhibiting PCSK9 results in improved LDLR recycling, increased LDLR availability on hepatocyte cell surfaces, and reduced blood LDL-C levels, making PCSK9 inhibition a novel therapeutic strategy for managing hypercholesterolemia. Monoclonal antibodies directed against PCSK9 have been developed for this purpose. A large number of clinical trials have demonstrated that monoclonal antibodies against PCSK9 yield substantial reductions in LDL-C when administered as monotherapy or in combination with statins to patients with nonfamilial and familial forms of hypercholesterolemia. Data from long-term trials demonstrate that the LDL-C-lowering effect of PCSK9 inhibitors is durable. These agents are generally well tolerated, and few patients discontinue treatment due to adverse events. Moreover, PCSK9 inhibitors do not appear to elicit the hepatic and muscle-related side effects associated with statin use. The ultimate value of PCSK9 inhibitors will be measured by their effect on clinical outcomes. Early evidence of a reduction in cardiovascular events after 1 year of treatment was shown in a prospective exploratory analysis of two ongoing long-term open-label extension evolocumab trials. Similarly, cardiovascular events were reduced in another exploratory analysis after >1 year of therapy with alirocumab. For the primary care physician, PCSK9 inhibitors represent a welcome additional option for lowering LDL-C in patients with familial forms of hypercholesterolemia and those with clinical atherosclerotic cardiovascular disease who are on maximally tolerated statin therapy.

One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1.

Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B, Type I (SR-BI)

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.