Perisinusoidal Research Articles

Modelling of FDG metabolism in liver voxels.

Kinetic analysis is a tool used to glean additional information from positron emission tomography (PET) data by exploiting the dynamics of tissue metabolism. The standard irreversible and reversible two compartment models used in kinetic analysis were initially developed to analyse brain PET data. The application of kinetic analysis to PET of the liver presents the opportunity to move beyond the generic standard models and develop physiologically informed pharmacokinetic models that incorporate structural and functional features in particular to the liver. In this paper, we develop a new compartment model, called the tubes model, which is informed by the liver׳s sinusoidal architecture, high fractional blood volume, high perfusion rate, and large hepatocyte surface area facing the space of Disse. The tubes model distributes tracer between the blood and intracellular compartments in more physiologically faithful proportions than the standard model, producing parametric images with improved contrast between healthy and neoplastic tissue.

The uptake mechanism of PEGylated DNA polyplexes by the liver influences gene expression.

Two uptake mechanisms were identified for PEGylated DNA polyplex biodistribution to the liver. At a low polyplex dose, a rapid-uptake mechanism dominates, resulting in 60% capture by liver in 5 min, due to a saturable receptor-mediated process. Rapid-uptake led to the fast metabolism of polyplexes by liver (t1/2 = 2.1 h), correlating with a 1-μg pGL3 polyplex dose losing full transfection competency after 4 h in the liver. Dose escalation of either polyplex or poly(ethylene glycol) (PEG) peptide led to the saturation of rapid-uptake and revealed a delayed-uptake mechanism for polyplexes by liver. Delayed-uptake was characterized by the slower liver accumulation of 40% of the polyplex dose over 40 min, followed by slow metabolism (t1/2 = 15 h) and an extended time (12 h) for a 1-μg pGL3 polyplex dose, remaining fully transfection competent in the liver. The delayed-uptake mechanism is consistent with polyplexes crossing liver fenestrated endothelial cells to reach steady state in the space of Disse. The results describe how to control polyplex biodistribution to liver to avoid rapid-uptake and metabolism, in favor of delayed-uptake, to preserve polyplex transfection competency in the liver for up to 12 h.

Epigenetic regulation during hepatic stellate cell activation

Background Hepatic stellate cells (HSC) have recently been identified as liver-resident mesenchymal stem cells and are thought to contribute to liver repair and fibrogenesis [1]. Quiescent HSC are located in the space of Disse and store vitamin A in characteristic lipid droplets. During their activation HSC lose their vitamin A stores and adopt a myofibroblast-like phenotype. This activation of HSC is necessary to enable hepatic differentiation and is accompanied by changes in the expression of many genes such as extracellular matrix protein encoding genes [2]. Some of these genes like Notch1 and Notch3 are regulated by epigenetic mechanisms. Their expression depends on the promoter DNA methylation and is changed during HSC activation [3]. The aim of the present study is a comprehensive analysis of DNA methylation alterations during HSC activation at a global and gene-specific level to elucidate their impact on hepatic stellate cell activation and to identify basic mechanisms of epigenetic control in adult stem cells during differentiation.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection.

A small-scale anatomical dosimetry model of the liver

Radionuclide therapy is a growing and promising approach for treating and prolonging the lives of patients with cancer. For therapies where high activities are administered, the liver can become a dose-limiting organ; often with a complex, non-uniform activity distribution and resulting non-uniform absorbed-dose distribution. This paper therefore presents a small-scale dosimetry model for various source–target combinations within the human liver microarchitecture. Using Monte Carlo simulations, Medical Internal Radiation Dose formalism-compatible specific absorbed fractions were calculated for monoenergetic electrons; photons; alpha particles; and 125I, 90Y, 211At, 99mTc, 111In, 177Lu, 131I and 18F. S values and the ratio of local absorbed dose to the whole-organ average absorbed dose was calculated, enabling a transformation of dosimetry calculations from macro- to microstructure level. For heterogeneous activity distributions, for example uptake in Kupffer cells of radionuclides emitting low-energy electrons (125I) or high-LET alpha particles (211At) the target absorbed dose for the part of the space of Disse, closest to the source, was more than eight- and five-fold the average absorbed dose to the liver, respectively. With the increasing interest in radionuclide therapy of the liver, the presented model is an applicable tool for small-scale liver dosimetry in order to study detailed dose–effect relationships in the liver.

TEMPORARY REMOVAL: CD271 identifies functional human hepatic stellate cells, which localize in peri-sinusoidal and portal areas in liver after partial hepatectomy

Hepatic stellate cells (HSCs) are liver-resident mesenchymal cells involved in essential processes in the liver. However, knowledge concerning these cells in human livers is limited because of the lack of a simple isolation method. We isolated fetal and adult human liver cells by immunomagnetic beads coated with antibodies to a mesenchymal stromal cell marker (CD271) to enrich a population of HSCs. The cells were characterized by cell cultivation, immunocytochemistry, flow cytometry, reverse-transcription polymerase chain reaction and immunohistochemistry. Cells were injected into nude mice after partial hepatectomy to study in vivo localization of the cells. In vitro, CD271(+) cells were lipid-containing cells expressing several HSC markers: the glial fibrillary acidic protein, desmin, vimentin and α-smooth muscle actin but negative for CK8, albumin and hepatocyte antigen. The cells produced several inflammatory cytokines such as interleukin (IL)-6, IL-1A, IL-1B and IL-8 and matrix metalloproteinases MMP-1 and MMP-3 and inhibitors TIMP-1 and TIMP-2. In vivo, fetal CD271(+) cells were found in the peri-sinusoidal space and around portal vessels, whereas adult CD271(+) cells were found mainly in the portal connective tissue and in the walls of the portal vessels, which co-localized withα-smooth muscle actin or desmin. CD271(-) cells did not show this pattern of distribution in the liver parenchyma. The described protocol establishes a method for isolation of mesenchymal cell precursors for hepatic stellate cells, portal fibroblasts and vascular smooth muscle cells. These cells provide a novel culture system to study human hepatic fibrogenesis, gene expression and transcription factors controlling HSC regulation.

Hemostatic effects of microbubble-enhanced low-intensity ultrasound in a liver avulsion injury model.

ObjectivesMicrobubble-enhanced therapeutic ultrasound (MEUS) can block the blood flow in the organs. The aim of this study was to evaluate the hemostatic effect of microbubble-enhanced pulsed, low-intensity ultrasound in a New Zealand White rabbit model of avulsion trauma of the liver. The therapeutic ultrasound (TUS) transducer was operated with the frequency of 1.2 MHz and an acoustic pressure of 3.4 MPa. Microbubble-(MB) enhanced ultrasound (MEUS) (n = 6) was delivered to the distal part of the liver where the avulsion was created. Livers were treated by TUS only (n = 4) or MB only (n = 4) which served as controls. Bleeding rates were measured and contrast enhanced ultrasound (CEUS) was performed to assess the hemostatic effect, and liver hemoperfusion before and after treatment. Generally, bleeding rates decreased more than 10-fold after the treatment with MEUS compared with those of the control group (P<0.05). CEUS showed significant declines in perfusion. The peak intensity value and the area under the curve also decreased after insonation compared with those of the control group (P<0.05). Histological examination showed cloudy and swollen hepatocytes, dilated hepatic sinusoids, perisinusoidal spaces with erythrocyte accumulation in small blood vessels, obvious hemorrhage around portal areas and scattered necrosis in liver tissues within the insonation area of MEUS Group. In addition, necrosis was found in liver tissue 48 h after insonation. We conclude that MEUS might provide an effective hemostatic therapy for serious organ trauma such as liver avulsion injury.

Chicken biliary exosomes enhance CD4+T proliferation and inhibit ALV-J replication in liver

Exosomes, which are small membrane vesicles of endocytic origin, carry lipids, RNA/miRNAs, and proteins and have immune modulatory functions. In this study, we isolated exosomes from the bile of specific pathogen-free chickens, 42-43 days of age, by using an ultracentrifugation and filtration method. The density of the exosomes, isolated by sucrose gradient fractionation, was between 1.13 and 1.19 g/mL. Electron microscopic observation of the liver showed that exosomes were present in the space of Disse and bile canaliculus. Chicken biliary exosomes displayed typical saucer-shaped, rounded morphology. Using liquid chromatography mass spectrum methodology, 196 proteins, including exosomal markers and several unique proteins, were identified and compared with mouse biliary exosomes. Noteworthy, CCCH type zinc finger antiviral protein was found on chicken biliary exosomes never described before. Furthermore, our data show that chicken biliary exosomes promote the proliferation of CD4(+) and CD8(+) T cells and monocytes from liver. In addition, chicken biliary exosomes significantly inhibit avian leukosis virus subgroup J, which is an oncogenic retrovirus, from replicating in the DF-1 cell line. These data indicate that chicken biliary exosomes possess the capacity to influence the immune responses of lymphocytes and inhibit avian leukosis virus subgroup J (ALV-J).

Pathology of the liver sinusoids

The hepatic sinusoids comprise a complex of vascular conduits to transport blood from the porta hepatis to the inferior vena cava through the liver. Under normal conditions, portal venous and hepatic artery pressures are equalized within the sinusoids, oxygen and nutrients from the systemic circulation are delivered to the parenchymal cells and differentially distributed throughout the liver acini, and proteins of liver derivation are carried into the cardiac/systemic circulation. Liver sinusoid structures are lined by endothelial cells unique to their location, and Kupffer cells. Multifunctional hepatic stellate cells and various immune active cells are localized within the space of Disse between the sinusoid and the adjacent hepatocytes. Flow within the sinusoids can be compromised by physical or pressure blockage in their lumina as well as obstructive processes within the space of Disse. The intimate relationship of the liver sinusoids to neighbouring hepatocytes is a significant factor affecting the health of hepatocytes, or transmission of the effects of injury within the sinusoidal space. Pathologists should recognize several patterns of injury involving the sinusoids and surrounding hepatocytes. In this review, injury, alterations and accumulations within the liver sinusoids are illustrated and discussed.

Extrahepatic bile duct ligation in broiler chickens: ultrastructural study of Ito cell

The Ito cell (fat-storing cell) is a cell lying in perisinusoidal space of liver. The function of Ito cell is expanding from a site of fat-storing site to a center of extracellular matrix metabolism and mediator production in the liver. This study was performed in order to evaluate the Ito cells in cholestatic condition. The artificial cholestatic was conducted by ligation of extrahepatic bile ducts (bile duct ligation = BDL) in broilers. The results showed that BDL induced bile congestion, fibrosis, proliferation of Ito cells and intrahepatic bile ductules. Immunohistochemistry demonstrated that Ito cells were scattered throughout the fibrotic areas, and larger in size with more extensive immunoreactivity than those in normal livers. Ultrastructural study demonstrated that Ito cells were closely associated with the production of extracellular collagen fibers. Ito cells actively react against hepatocytic injuries, especially in fibrogenesis of cholestatic livers. Key words: Bile duct, ito cell, broiler

Effect of Caloric Restriction on Hepatic Sinusoidal System and Stellate Cells in Mice

Aging associated changes in liver include reduced hepatic blood flow, increased number of stellate cells, and collagen deposits in perisinusoidal space. We tested the possibility of mitigating these changes with caloric restriction. Two-month-old mice were subjected to 30 percent caloric restriction for 12 months and then examined for the effect of caloric restriction on the sinusoidal network, collagen deposition, and the number of stellate cells. Using intravital fluorescence microscopy, assessments were made on sinusoidal diameter, density, volumetric flow, perfusion index, and autofluorescence of vitamin A that was primarily stored with lipid droplets in stellate cells. A significant effect was observed in the vitamin A autofluorescence of stellate cells; stellate cell associated fluorescence was diminished in terms of number and size of fluorescent spots. Caloric restriction reduced collagen deposits in liver sections and lowered the gene expression of α1-(I) collagen but not α-smooth muscle actin. No differences were detected in sinusoidal dimension measurements. Our results showed that caloric restriction was effective in ameliorating the increase in stellate cells and the mild fibrosis in old mice. However, caloric restriction had no impact on stellate cell activity level as indicated by the unaffected α-smooth muscle actin expression.

The protective effects of dexmedetomidine on liver injury- -induced myocardial ischemia reperfusion

The aim of this study was to evaluate the effect of dexmedetomidine (100 µg/kg-ip) on liver injury-induced myocardial ischemia and reperfusion (IR) in rats. Twenty-four Wistar Albino rats were separated into four groups. There were four experimental groups (Group C (Control; n = 6), Group IR (ischemia-reperfusion, n = 6), Group D (Dexmedetomidine; n = 6) that underwent left thoracotomy and received ip dexmedetomidine without IR administered via 100 µg/kg ip route 30 minutes before ligating the left coronary artery, and Group IR-D (IR-Dexmedetomidine; n = 6). A small plastic snare was threaded through the ligature and placed in contact with the heart. To produce IR, a branch of the left coronary artery was occluded for 30 min followed by two hours of reperfusion. However, after the above procedure, the coronary artery was not occluded or reperfused in the control rats. At the end of the study, liver tissue was obtained for histochemical and immunohistochemical determination.Some part of tissue samples were stained with Masson-trichrome for the evaluation of ultrastructural changes and inducible nitric oxide synthase (iNOS) expression was evaluated in other part of samples for immunohistochemical examination. Histopathological changes were detected in Group IR when compared with Group C. iNOS expression was found to be increased and stronger particularly in the vascular wall, perisinusoidal space and hepatocytes around vena centralis in this group compared to the control group. Perivascular oedema was detected to be decreased in Group IR-D compared to Group IR. It was also observed that the impairment in the radial arrangement of hepatocytes significantly recovered in Group IR-D. The immunoreactivity was found to be significantly decreased in the assessment of iNOS expression in the same group when compared with Group IR. Administration of dexmedetomidine ameliorates liver injury induced by myocardial ischemia and reperfusion (Fig. 8, Ref. 33).

Pediatric non-alcoholic steatohepatitis: the first report on the ultrastructure of hepatocyte mitochondria.

To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH). Ultrastructural investigations were conducted on biopsy liver specimens obtained from 10 children (6 boys and 4 girls) aged 2-14 years with previously clinicopathologically diagnosed NASH. The disease was diagnosed if liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, or focal necrosis, varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver diseases, or celiac disease. For ultrastructural analysis, fresh small liver blocks (1 mm(3) volume) were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer. The specimens were postfixed in osmium tetroxide, subsequently dehydrated through a graded series of ethanols and propylene oxide, and embedded in Epon 812. The material was sectioned on a Reichert ultramicrotome to obtain semithin sections, which were stained with methylene blue in sodium borate. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined using an Opton EM 900 transmission electron microscope. Ultrastructural analysis of bioptates obtained from children with non-alcoholic steatohepatitis revealed characteristic repetitive mitochondrial abnormalities within hepatocytes; mainly mitochondrial polymorphisms such as megamitochondria, loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of an increased electron density. The crystalline inclusions were particularly evident within megamitochondria (MMC), which seemed to be distributed randomly both within the hepatic parenchymal cell and the zones of hepatic lobule, without special variations in abundance. The inclusions appeared as bundles viewed longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles. Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease.

The Transcriptomic Response of Rat Hepatic Stellate Cells to Endotoxin: Implications for Hepatic Inflammation and Immune Regulation

With their location in the perisinusoidal space of Disse, hepatic stellate cells (HSCs) communicate with all of the liver cell types both by physical association (cell body as well as cytosolic processes penetrating into sinusoids through the endothelial fenestrations) and by producing several cytokines and chemokines. Bacterial lipopolysaccharide (LPS), circulating levels of which are elevated in liver diseases and transplantation, stimulates HSCs to produce increased amounts of cytokines and chemokines. Although recent research provides strong evidence for the role of HSCs in hepatic inflammation and immune regulation, the number of HSC-elaborated inflammatory and immune regulatory molecules may be much greater then known at the present time. Here we report time-dependent changes in the gene expression profile of inflammatory and immune-regulatory molecules in LPS-stimulated rat HSCs, and their validation by biochemical analyses. LPS strongly up-regulated LPS-response elements (TLR2 and TLR7) but did not affect TLR4 and down-regulated TLR9. LPS also up-regulated genes in the MAPK, NFκB, STAT, SOCS, IRAK and interferon signaling pathways, numerous CC and CXC chemokines and IL17F. Interestingly, LPS modulated genes related to TGFβ and HSC activation in a manner that would limit their activation and fibrogenic activity. The data indicate that LPS-stimulated HSCs become a major cell type in regulating hepatic inflammatory and immunological responses by altering expression of numerous relevant genes, and thus play a prominent role in hepatic pathophysiology including liver diseases and transplantation.

α-smooth muscle actin-positive fibroblasts correlate with poor survival in hepatocellular carcinoma

The current case-control study was conducted to assess the value of activated fibroblasts with high α-smooth muscle actin (α-SMA) expression as an indicator of survival in hepatocellular carcinoma (HCC) patients. In total, 47 patients diagnosed with HCC who underwent a liver biopsy at the Veterans Affairs Medical Center (Memphis, TN, USA) between January 2000 and December 2009, and 10 age- and gender-matched controls with normal liver biopsy samples were included in the study. The immunohistochemical staining for α-SMA was performed and classified qualitatively and quantitatively within tumor stroma and perisinusoidal spaces. HCC patients with high qualitative and quantitative expression of α-SMA revealed a statistically significant negative correlation with 3-year [odds ratio (OR), 0.021 and 0.111; P=0.001 and 0.0302, respectively] and 1.5-year (OR, 0.040 and 0.051; P=0.0330 and 0.0492, respectively) survival rates in comparison with low expression. The results of the present study demonstrated a predictive role of high qualitative and quantitative expression in activated fibroblasts for poor survival in patients with HCC.

Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid

This paper presents a novel liver model that mimics the liver sinusoid where most liver activities occur. A key aspect of our current liver model is a layered co-culture of primary rat hepatocytes (PRHs) and primary rat liver sinusoidal endothelial cells (LSECs) or bovine aortic endothelial cells (BAECs) on a transwell membrane. When a layered co-culture was attempted with a thin Matrigel layer placed between hepatocytes and endothelial cells to mimic the space of Disse, the cells did not form completely separated monolayers. However, when hepatocytes and endothelial cells were cultured on the opposite sides of a transwell membrane, PRHs co-cultured with LSECs or BAECs maintained their viability and normal morphology for 39 and 57 days, respectively. We assessed the presence of hepatocyte-specific differentiation markers to verify that PRHs remained differentiated in the long-term co-culture and analyzed hepatocyte function by monitoring urea synthesis. We also noted that the expression of cytochrome P-450 remained similar in the co-cultured system from day 1 to day 48. Thus, our novel liver model system demonstrated that primary hepatocytes can be cultured for extended times and retain their hepatocyte-specific functions when layered with endothelial cells.

Increased Expression Of NGF In Hepatocytes Is An Early Event In Iron Overloaded Mouse By Transcriptome Analysis

The liver plays a central role in iron metabolism by storing and sensing the amounts of iron in the body. The dietary iron from duodenum and recycled-iron by reticuloendothelial system are the main source of body iron. When excess iron enters the liver, hepatocytes secrete hepcidin, an anti-microbial peptide which negatively regulates iron excretion from enterocytes and macrophages, and stores the excess iron as ferritin-bound iron. A dysfunction of this regulatory system causes iron overload in the liver. Aberrant iron accumulation in the liver is found in hereditary hemochromatosis and chronic liver disease, and this is considered to be an exacerbating factor in liver cirrhosis and hepatocellular carcinoma. It is therefore important to understand the precise molecular events that take place as a result of iron accumulation during the early stages of iron overload.In the present study, we performed transcriptome analysis on the liver of dietary iron overloaded mice. Transcriptome analysis using a high throughput sequencer is capable of comprehensive analysis with high sensitivity. We hypothesized that this method will be suitable in detecting the changes in gene expression induced by iron overload, even in slightly expressed genes. C57B1/6 mice were fed a normal diet, and a 2.5% iron diet for 8 weeks. Serum and liver tissue samples were then collected, and histological analysis showed the features of early stage iron overload without significant hepatic damage in the iron-fed mice.From the results of the transcriptome analysis, we found that nerve growth factor (NGF) was significantly expressed in the slightly iron overloaded liver. This observation was also confirmed by real time RT-PCR, Western blotting and immunohistochemistry. Similarly, NGF upregulation was induced in mice primary hepatocytes cultured in conditioned iron overloaded medium (with high concentration of holo-transferrin or ferric ammonium citrate). Furthermore, immunohistochemical analysis showed that TrkA, a high affinity NGF receptor, was expressed in liver sinusoidal endothelial cells (LSECs). Using scanning electron microscopy, we sought to examine any morphological changes in the sinusoids of the iron overloaded liver and observed that although sieve plate structures (so-called ‘fenestrae') were found in the LSECs of mice fed a normal diet, they were not visible in the iron-fed mice. The loss of fenestrae was also observed in the LSECs of mice that received intraperitoneal injections of NGF. In cultured isolated primary LSECs, treatment with NGF, or conditioned medium from iron overloaded primary hepatocytes reduced the fenestrae while the anti-NGF neutralization antibody or TrkA inhibitor K252a cancelled this effect. In addition, a fresh iron overloaded medium did not reduce the fenestrae in primary LSECs, indicating that iron itself has no direct effects on the fenestrae in LSECs.LSECs constitute the sinusoidal wall in the liver and can be regarded as unique capillaries which differ from other capillaries in the body due to the presence of fenestrae which lack a diaphragm, and are therefore open connections between the lumen of the sinusoid and the space of Disse. The fenestrae in LSECs therefore play an important role in the exchange of solutes between the lumen of the sinusoid and hepatocytes.The results of this study indicate that iron accumulation induces the expression of NGF in hepatoctyes, which in turn leads to the loss of fenestrae in LSECs via TrkA. This phenomenon may therefore contribute to the defensive machinery against iron accumulation in hepatocytes in the early stages of iron overload. These data further suggest a novel function of NGF in the regulation of iron transport. Disclosures:No relevant conflicts of interest to declare.

Immunohistochemical Staining of rFVIIIFc and rFVIII in Liver Cells Differentiates Between a VWF-Dependent and Independent Clearance Pathway in Mice

IntroductionrFVIIIFc is a fully recombinant fusion protein consisting of a single B domain-deleted human FVIII covalently attached to the dimeric Fc domain of human IgG1. rFVIIIFc has a 1.5-fold extended half-life and decreased clearance compared to rFVIII in patients with hemophilia A (Powell, Blood 2012). The Fc region of rFVIIIFc binds to the neonatal Fc receptor (FcRn), which is part of a naturally occurring pathway that cycles IgG back into circulation, delaying lysosomal degradation. Our previous studies with FcRn-chimeric mice showed that the decreased clearance of rFVIIIFc is mediated by FcRn expressed in somatic cells and not in hematopoietic cells. Biodistribution studies with 125I-rFVIIIFc identified liver as the major organ for rFVIIIFc disposition. Furthermore, qPCR studies showed that three different cell types in liver all express FcRn: hepatocytes (HC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). In primary liver cell co-cultures, rFVIIIFc and rFVIII were both internalized by somatic LSEC and HC, but not KC, in the absence of von Willebrand factor (VWF). It has been reported that in vivo, 95% of FVIII circulates as a non-covalent complex with VWF (Lenting, JTH 2007) and we found that VWF delays liver uptake of FVIII and improves the circulating half-life of rFVIIIFc in mice. AimCompare the cellular localization of rFVIII and rFVIIIFc in the murine liver, in the presence and absence of VWF, in order to identify the cells responsible for the prolonged half-life of rFVIIIFc. MethodsFVIII KO (HemA) and FVIII/VWF DKO mice were dosed with rFVIII, rFVIIIFc or the mutants rFVIIIFc-IHH and rFVIIIFc-N297A that are deficient in interacting with FcRn and FcgR, respectively. The cellular localization in the liver of rFVIII, rFVIIIFc and mutants was investigated by immunohistochemistry, using a panel of anti-human FVIII monoclonal antibodies or anti-human-IgG (Fc) for detection, along with markers for LSEC, KC, and VWF. ResultsIn HemA mice with circulating endogenous VWF, the majority of both rFVIII and rFVIIIFc signal is found in KC, which co-stain for VWF. In contrast, neither rFVIII nor rFVIIIFc is detected in the endothelial cells of the large vessels that stained for VWF in the Weibel-Palade bodies. Closer examination shows faint vesicular staining by rFVIII in HC in contrast to a diffuse staining by rFVIIIFc in the liver sinusoid. However in FVIII/VWF DKO mice lacking VWF, neither rFVIII nor rFVIIIFc is detected in KC consistent with the notion that internalization of FVIII by KC is mediated by VWF. The majority of rFVIII is found in HC, whereas rFVIIIFc again appears as a diffused liver sinusoidal staining pattern that is more intense than that observed in the HemA mice expressing VWF. These findings suggest free rFVIII is internalized and cleared by HC, while rFVIIIFc is cycled out of the HC and localizes in the Space of Disse, hence its sinusoidal localization. Alternately, in the absence of VWF, rFVIIIFc may cycle through LSEC rather than HC. In order to distinguish these two pathways, an rFVIIIFc variant, rFVIIIFc-IHH, which is not competent to bind FcRn, was tested. In a similar manner as rFVIII, the rFVIIIFc-IHH mutant localizes into HC in DKO mice and is found predominantly in KC in HemA with endogenous VWF. In contrast, rFVIIIFc-N297A, which is not competent to bind FcgR, localizes similarly to rFVIIIFc in DKO mice, excluding a role for FcgR. ConclusionsOur current immunohistochemical studies and previous biodistribution and PK studies in mice indicate that there are two parallel, linked clearance pathways for rFVIII and rFVIIIFc. rFVIII or rFVIIIFc complexed with endogenous VWF is internalized predominantly by macrophages, including KC. However, because the VWF-FVIII complex is in constant equilibrium, a fraction of free rFVIII or rFVIIIFc is available for clearance by HC. We propose that this free fraction of rFVIIIFc entering HC is then cycled by FcRn back into the liver sinusoid and into circulation, in contrast to the free rFVIII entering the HC. Staining of the rFVIIIFc-IHH mutant in HC suggests that these cells play a dominant role in vivo, however LSEC may also contribute to cycling of VWF-free rFVIIIFc. The fate of the VWF-bound rFVIIIFc fraction internalized by KC is less clear, however data using FcRn-chimeric mice suggest that FcRn expressed in hematopoietic cells, including KC contributes only marginally to the delayed clearance of rFVIIIFc. Disclosures:van der Flier:Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership.

Hepatic fibrogenesis and transforming growth factor/Smad signaling activation in rats chronically exposed to low doses of lead.

Lead is an important heavy metal pollutant in the environment. The nervous system, kidney and liver are the most susceptible organs to lead deposition, showing that this pollutant has no single target system. To examine the cellular and molecular mechanisms involved in their pathobiology of chronic lead at low-dose exposure in the liver, male Wistar rats were exposed to 0.06% lead acetate in drinking water every day for 4 months. At the end of the study, hepatic metal accumulation, morphology and function were examined. Immunochemical staining and Western blot analysis were performed to detect extracellular matrix proteins, α-smooth muscle actin and transforming growth factor (TGF)β1/Smad pathway expression. Results showed increased laminin, collagen IV and fibronectin, located at the perisinusoidal space. Phenotypic transformation of hepatic stellate cells into myofibroblast-like cells was evidenced at the ultrastructural level and a significant expression of α-smooth muscle actin in Disse’s space was observed. These findings were associated with a marked increase in TGFβ1/Smad2/3 signaling. Our data suggest that, chronically, exposure to low levels of lead could trigger the onset of a hepatic fibrogenic process through upregulated TGFβ1/Smad signaling.

Kupffer cell structure in the juvenile Nile crocodile,Crocodylus niloticus

The morphology of Kupffer cells was examined in the liver of the juvenile Nile crocodile using light microscopy and transmission electron microscopy. Pleomorphic Kupffer cells were located in the sinusoids, in the space of Disse, in the hepatic parenchyma and often connected adjacent sinusoids. The cell surfaces were irregular due to the presence of filopodia and lamelliapodia with phagocytosis of white blood cells, red blood cells and thrombocytes being evident. The cells were in close contact with endothelial cells and pit cells in the sinusoidal lumen and with stellate cells in the space of Disse. The cytoplasm contained large phagosomes comprising a combination of ceroid pigment, melanosomes and siderosomes. The nuclei were often indented and eccentrically placed due to the presence of the phagosomes. Conspicuous clusters of membrane-bound tubular organelles with a filamentous or crystalline interior were observed in the cytoplasm. The clusters were sometimes separated into smaller groups around phagosomes. A clear zone existed between the limiting membrane and the interior of these tubular organelles with the electron-dense interior profiles being, respectively, circular, angular or divided. The tubular organelles have not previously been described in Kupffer cells and possibly represent lysosomes with specialized functions. Mitochondria, microtubules, Golgi profiles, granular and smooth endoplasmic reticulum, and a few cytoplasmic lipid droplets were also present. The presence of the tubular organelles and the occurrence of the Kupffer cells in different locations in the liver of the juvenile Nile crocodile are indicative of particularly active and mobile cells.