Surface Of Hepatocytes Research Articles

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using 99mTc-GSA SPECT

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. We aimed to clarify the clinical significance of the regional distribution of ASGPRs in the human liver, especially in chronic viral hepatitis. Eighteen volunteers, 34 patients with chronic hepatitis, and 33 patients with cirrhosis (11/Child-Pugh A, 11/Child-Pugh B, 11/Child-Pugh C) were studied using a newly developed, conventional technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin ( 99mTc-GSA), single photon emission computed tomography (SPECT) method. Using Cantlie's line as a guide, ASGPR dynamics were analyzed separately in the right and left lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and the liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased with increasing progression of chronic viral hepatitis. The LUR for the whole liver correlated well with parameters measuring the hepatic functional reserve and the platelet count. The right LUR correlated particularly well with conventional liver function tests, and comparison of the right LUD with histologic findings showed that it was a good indicator of periportal and/or bridging necrosis and fibrosis. In conclusion, our 99mTc-GSA SPECT method was clinically useful in evaluating regional hepatic function and the progression of chronic viral hepatitis using dynamic changes in ASGPRs. (H epatology 2003;38:1401-1409.)

Hepatocyte behavior on synthetic glycopolymer matrix: inhibitory effect of receptor–ligand binding on hepatocyte spreading

The interaction of carbohydrate-based polymers with asialoglycoprotein receptors (ASGPRs) on the surface of hepatocytes has been used to design hepatocyte adhesion matrices. Therefore, we have characterized the interaction of ASGPR on the surface of hepatocytes with glycopolymer-coated surfaces. Since ASGPRs bound to glycopolymer surfaces escape from internalization and degradation, they were quantified by western blot analysis. The amount of hepatocyte ASGPRs that initially adhered to the glycopolymer surface was proportional to the concentration of the coated glycopolymer. We found that the initial adhesion of hepatocytes to the glycopolymer surface was enhanced by interactions with ASGPR, whereas interactions with ASGPR inhibited the post-adhesion process, a cell adhesion phenomenon that occurs following the initial adhesion. Furthermore, hepatocytes are much more spread on glycopolymer surfaces with lower coating density. Taken together, we suggest that the post-adhesion process triggered hepatocyte spreading on glycopolymer surfaces, and ASGPR–carbohydrate interactions act negatively on the post-adhesion mechanism as well as on hepatocyte spreading on glycopolymer surfaces depending on the density of coated glycopolymers.

Resistance of SHP-null Mice to Bile Acid-induced Liver Damage

The orphan nuclear hormone receptor SHP (gene designation NROB2) is an important component of a negative regulatory cascade by which high levels of bile acids repress bile acid biosynthesis. Short term studies in SHP null animals confirm this function and also reveal the existence of additional pathways for bile acid negative feedback regulation. We have used long term dietary treatments to test the role of SHP in response to chronic elevation of bile acids, cholesterol, or both. In contrast to the increased sensitivity predicted from the loss of negative feedback regulation, the SHP null mice were relatively resistant to the hepatotoxicity associated with a diet containing 0.5% cholic acid and the much more severe effects of a diet containing both 0.5% cholic acid and 2% cholesterol. This was associated with decreased hepatic accumulation of cholesterol and triglycerides in the SHP null mice. There were also alterations in the expression of a number of genes involved in cholesterol and bile acid homeostasis, notably cholesterol 12alpha-hydroxylase (CYP8B1), which was strongly reexpressed in the SHP null mice, but not the wild type mice fed either bile acid containing diet. This contrasts with the strong repression of CYP8B1 observed with short term bile acid feeding, as well as the effects of long term feeding on other bile acid biosynthetic enzymes such as cholesterol 7alpha-hydroxylase (CYP7A1). CYP8B1 expression could contribute to the decreased toxicity of the chronic bile acid treatment by increasing the hydrophilicity of the bile acid pool. These results identify an unexpected role for SHP in hepatotoxicity and suggest new approaches to modulating effects of chronically elevated bile acids in cholestasis.

Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte

Control of cell–matrix interactions is a central principle for the design of biomaterial in tissue engineering. In this study, we evaluated a synthetic glycopolymer, which is recognized by the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes, as an artificial matrix to regulate integrin-mediated signaling. The phosphorylation of focal adhesion kinase was restricted in hepatocytes cultured on the glycopolymer compared with fibronectin. In addition, there was no reorganization of cytoskeleton-related proteins such as actin filaments, microtubules, and vinculin in hepatocytes cultured on the glycopolymer. DNA synthesis and cyclin D1 expression were suppressed in hepatocytes grown on the glycopolymer as compared with those grown on fibronectin and collagen. The data suggest that the glycopolymer will be a good artificial matrix to regulate integrin-mediated signaling and cell growth through the unique ASGPR–carbohydrate interaction.

P.4.020 Efficacy and tolerability of memantine in nursing home patients with moderate to severe dementia of the Alzheimer's type

The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that interacts with the low-density lipoprotein (LDL) receptor at the surface of hepatocytes to regulate circulating LDL cholesterol levels. High circulating PCSK9 levels have been associated with elevated LDL cholesterol. Recently, the Food and Drug Administration of the United States approved new LDL cholesterol–lowering drugs that specifically target the inhibition of PCSK9. Similar to most human proteins, PCSK9 exists in multiple forms as it is the target of posttranslational modifications (PTMs) such as proteolytic cleavage, phosphorylation, and others, which can affect its biological activity. However, commercially available assays, such as enzyme-linked immunosorbent assays, do not discriminate between these forms.To investigate, in 2 patient cohorts, the relationships between circulating levels of multiple forms of PCSK9 and cardiometabolic interventions or treatments known to reduce LDL cholesterol levels.PCSK9 forms were measured in plasma: (1) in 20 patients before and 6 months after bariatric surgery and (2) in 132 patients before and 12 months after daily statin treatment. A series of specific peptides used as surrogates for various PCSK9 forms were quantified by a novel semiautomated proteomic assay termed protein affinity capture coupled to quantitative mass spectrometry.Bariatric surgery resulted in a decrease in the plasma level of PCSK9 prodomain (P < .05), but did not result in a significant change in other measured PCSK9 forms. Statin treatment resulted in an increase in all measured plasma PCSK9 peptides (P < .001), but a 25% decrease in the phosphorylated state of PCSK9 at S688 (P < .05).These unexpected findings indicate that measuring the circulating levels of the various domains and PTMs of PCSK9 provides more in depth information than total PCSK9 and that the prodomain and the phosphorylated state of S688 may represent novel biomarkers to explore in cardiometabolic diseases and response to treatment. In addition, our data generated new hypotheses on the function of PCSK9 PTMs in health and disease.

Modulation of the outcome and severity of hepadnaviral hepatitis in woodchucks by antibodies to hepatic asialoglycoprotein receptor

Viral hepatitis is frequently accompanied by humoral autoimmune responses toward both organ-nonspecific and liver-specific antigens, but contribution of these reactivities to liver injury remains unrecognized. Infection with woodchuck hepatitis virus (WHV) has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediates clearance of desialylated serum proteins. In this study, we applied the WHV-woodchuck model of hepatitis B to examine the effect of experimentally elicited anti-ASGPR on the progression and the severity of WHV hepatitis in initially healthy animals immunized with the receptor and then infected with WHV and in woodchucks with ongoing chronic WHV hepatitis. The results implied that the induction of anti-ASGPR prior to WHV infection tends to modulate acute viral hepatitis toward chronic outcome and, in animals with established chronic WHV infection, exacerbates histologic severity of liver lesions. The findings also suggest that the liver compromised by chronic hepadnavirus infection might be prone to anti-ASGPR-directed complement-mediated hepatocellular injury and that this is associated with formation of the ASGPR-anti-ASGPR immune complexes on hepatocyte surface. In conclusion, the host's immune response mounted against a hepatocyte-specific autoantigen may modulate both the outcome and the severity of liver injury in viral hepatitis. (H epatology 2003;38:629-638.)

Normal Sorting but Defective Endocytosis of the Low Density Lipoprotein Receptor in Mice with Autosomal Recessive Hypercholesterolemia

Autosomal recessive hypercholesterolemia (ARH) is a genetic form of hypercholesterolemia that clinically resembles familial hypercholesterolemia (FH). As in FH, the rate of clearance of circulating low density lipoprotein (LDL) by the LDL receptor (LDLR) in the liver is markedly reduced in ARH. Unlike FH, LDL uptake in cultured fibroblasts from ARH patients is normal or only slightly impaired. The gene defective in ARH encodes a putative adaptor protein that has been implicated in linking the LDLR to the endocytic machinery. To determine the role of ARH in the liver, ARH-deficient mice were developed. Plasma levels of LDL-cholesterol were elevated in the chow-fed Arh-/- mice (83 +/- 8 mg/dl versus 68 +/- 8 mg/dl) but were lower than those of mice expressing no LDLR (Ldlr-/-) (197 +/- 8 mg/dl). Cholesterol feeding elevated plasma cholesterol levels in both strains. The fractional clearance rate of radiolabeled LDL was reduced to similar levels in the Arh-/- and Ldlr-/- mice, whereas the rate of removal of alpha2-macroglobulin by the LDLR-related protein, which also interacts with ARH, was unchanged. Immunolocalization studies revealed that a much greater proportion of immunodetectable LDLR, but not LDLR-related protein, was present on the sinusoidal surface of hepatocytes in the Arh-/- mice. Taken together, these results are consistent with ARH playing a critical and specific role in LDLR endocytosis in the liver.

EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.

Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin-tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin-tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.

Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytes

Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes ( e.g., 102% with 1 μmol/L glucagon) and a simultaneous decrease in intracellular membranes ( e.g., 38% with 1 μmol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion. (H epatology 2003;37:1435-1441.)

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes.

Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1. Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

Lead nitrate and gadolinium chloride administration modify hepatocyte cell surfaces.

Modifications of hepatocyte cell surface were determined after single i.v. injection to rats of Pb(NO(3))(2) (known to induce liver hyperplasia followed by apoptosis) or GdCl(3) (known to induce proliferation of parenchymal cells and Kupffer cell depletion) or administration of GdCl(3) 24 h before Pb(NO(3))(2) injection (known to reduce hyperplasia and apoptosis induced in the parenchymal liver cells by the single Pb(NO(3))(2) injection). Rats were sacrificed at fixed times after the treatments (1, 3 and 5 days) and hepatocytes were isolated by enzymatic liver perfusion. In spite of the intracellular target of the heavy metals, signals leading to liver hyperplasia and apoptosis (with rates different for the different experimental conditions) were generated, which in turn were responsible for cell surface alteration. Increment or decrement of phosphatidylserine (PS) expression, asialoglycoprotein receptors (ASGPRs) and sugar residue expression on hepatocyte surfaces was measured in parallel with apoptosis and proliferation. When GdCl(3) was injected 24 h before Pb(NO(3))(2) injection, liver modifications were significantly reduced, thus suggesting that GdCl(3) could prevent and/or reduce liver damage.

Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis.

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.

Circulating Intercellular Adhesion Molecule-1 in Chronic Hepatitis C Patients with Normal or Elevated Aminotransferase before and after Alpha-Interferon Treatment

Objectives: Intercellular adhesion molecule-1 (ICAM-1) plays a fundamental role during liver inflammation. In fact, weak ICAM-1 expression is physiologically restricted to the endothelium of portal vessels and to sinusoidal lining cells, but it becomes markedly evident on sinusoidal lining cells and at the surface of hepatocytes during inflammatory liver diseases. The aim of this study was to evaluate the behaviour of soluble ICAM-1 (sICAM-1) in chronic hepatitis C (CH-C) patients with persistently normal aminotransferase in comparison with patients with CH-C and elevated aminotransferase, and its changes during α-interferon (IFN) therapy. Immunohistochemical localization of ICAM-1 was also performed on liver tissue specimens of both groups. Methods: Sixty subjects were divided into 3 groups: group A included 19 patients with CH-C and persistently normal aminotransferase; group B included 21 patients with CH-C and persistently elevated aminotransferase levels, and group C included 20 healthy subjects representing the control group. The first two groups were treated with recombinant α-IFN 2b at a dose of 6 MU 3 times a week for 3 months and followed up with 3 MU 3 times a week for another 3 months. Results: Baseline values of serum ICAM-1 in groups A and B were significantly higher than those in group C (p < 0.0001). The median baseline value of sICAM-1 in group A (525.0 ng/ml) was lower than that of group B (561.0 ng/ml), but the difference was not statistically significant. Furthermore, there was a trend toward higher ICAM-1 values as histological severity increased (Mantel-Haenszel χ² 8.8, p < 0.003). Post-treatment sICAM-1 serum values showed a marked decrease in both groups, but only among responder patients, while ICAM-1 levels were unchanged in non-responders. Immunohistochemical localization showed no staining for ICAM-1 in normal liver specimens, while there was a quite similar staining for ICAM-1 in the two groups of patients, consistent with an inflammatory process. Conclusions: This study shows that circulating ICAM-1 levels in most patients with CH-C and persistently normal aminotransferase are higher than those in a control group and the fact that ICAM-1 molecules are also expressed on the hepatocyte membrane suggests that they could play an important role, in association with other molecules, in the intercellular adhesion processes during the induction and maintenance phases of the immune response. In both groups, only patients responding to α-IFN therapy showed a marked decrease in serum ICAM-1 below baseline values.

Mice lacking endothelial ACE: normal blood pressure with elevated angiotensin II.

Recently, the concept of local renin-angiotensin systems (RAS) capable of generating angiotensin II apart from the circulation has received considerable attention. To investigate this, we generated ACE 1/3 mice in which one allele of ACE is null and the second allele was engineered to express ACE on the surface of hepatocytes. ACE 1/3 mice express no endothelial ACE and lack ACE within the lungs. Their kidneys contain <7.8% the enzyme levels present in control mice. Plasma conversion of angiotensin I to angiotensin II was 43.3% normal. The baseline blood pressure and renal function of the ACE 1/3 mice were normal, probably as a function of a marked increase of both plasma angiotensin I and angiotensin II. When exposed to 2 weeks of a salt-free diet (a stress diet stimulating the RAS), blood pressure in ACE 1/3 mice decreased to 92.3+/-2.0 mm Hg, a level significantly lower than that of wild-type control mice. The ACE 1/3 mice demonstrate the plasticity of the RAS and show that significant compensation is required to maintain normal, basal blood pressure in a mouse with an impaired local vascular and renal RAS.

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity.

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.

The expression of Fas and Fas ligand, and the effects of interferon in chronic liver diseases with hepatitis C virus

In viral hepatitis, binding of Fas ligand (FasL) with Fas expressed on the surfaces of infected hepatocytes induces apoptosis, removing hepatitis virus along with infected hepatocytes. We measured serum concentrations of soluble Fas (sFas) and FasL (sFasL), expression of membrane-bound FasL, and expression of FasL–mRNA in patients with chronic hepatitis C without cirrhosis (CH-C) and chronic hepatitis C with liver cirrhosis (LC-C). In CH-C, sFasL concentrations were lower and FasL–mRNA expression was significantly less than in volunteers. In LC-C, sFas concentrations were significantly greater than in healthy volunteers, while sFasL, membrane-bound FasL expression, and FasL–mRNA expression did not show significant differences. We also examined these variables over 24 h following the first interferon (IFN) treatment in patients with CH-C. Serum concentrations of sFas and sFasL, and FasL–mRNA expression increased markedly beyond amounts present before IFN injection until 12 h after IFN injection. However, membrane-bound FasL expression decreased until 6 h, followed by an increase until 24 h. Our findings suggest that the ratio of membrane-bound FasL to sFasL may be regulated to remove virally infected cells in CH-C. In addition, apoptosis mediated by the Fas/FasL system may be influenced by IFN injection for treatment of CH-C.

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.

Hepatic lipase:structure/function relationship, synthesis,and regulation

Hepatic lipase (HL) is a lipolytic enzyme, synthesized by hepatocytes and found localized at the surface of liver sinusoid capillaries. In humans, the enzyme is mostly bound onto heparan-sulfate proteoglycans at the surface of hepatocytes and also of sinusoid endothelial cells. HL shares a number of functional domains with lipoprotein lipase and with other members of the lipase gene family. It is a secreted glycoprotein, and remodelling of the N-linked oligosaccharides appears to be crucial for the secretion process, rather than for the acquisition of the catalytic activity. HL is also present in adrenals and ovaries, where it might promote delivery of lipoprotein cholesterol for steroidogenesis. However, evidence of a local synthesis is still controversial. HL activity is fairly regulated according to the cell cholesterol content and to the hormonal status. Coordinate regulations have been reported for both HL and the scavenger-receptor B-I, suggesting complementary roles in cholesterol metabolism. However, genetic variants largely contribute to HL variability and their possible impact in the development of a dyslipidemic phenotype, or in a context of insulin-resistance, is discussed.

Identification of a target antigen recognized by a mouse monoclonal antibody to the bile canalicular surface of rat hepatocytes with a random phage display library.

We developed a monoclonal antibody (MoAb) (clone 5E8) against an antigen on the bile canalicular membrane of rat hepatocyte. By immunoblotting, MoAb 5E8 detected a band of 110 kD. In this study, we used the phage display technique to identify the target antigen recognized by MoAb 5E8. We screened a random phage display library expressing 12-mer peptide sequences and identified a peptide sequence, FHFNPYTGHPLT, as an epitope. We compared this peptide sequence with those of dipeptidyl peptidase IV (DPP IV, E.C.3.4.14.5) and Cell-CAM105, which proteins were located by a database search based on the information of tissue localization and approximate molecular weight of the MoAb 5E8 antigen, and sequence similarity with a region in DPP IV (amino acids 225-233) but not with Cell-CAM105 was found. In addition, we immunohistochemically stained various tissues (liver, small intestine, and kidney) of Japanese Fischer 344 rats, known to be deficient for DPP IV, with MoAb 5E8 and showed that the expression of MoAb 5E8 antigen was negligible or weak. In contrast, tissues sampled from the same organs of Sprague-Dawley rats, known to express DPP IV, were positively stained. These findings suggest that the antigen recognized by MoAb 5E8 is DDPIV and its major epitope is located in amino acids at positions 225-233.

BIOARTIFICIAL LIVER DEVELOPMENT WITH TISSUE ENGINEERING

Adhesiveness between hepatocytes or hepatocyte and their surrounding tissue may be altered by shear stress(SS), possibly affecting the expression of liver-specific function. In the present study, hepatocytes were seeded over 3-dimensional structures, followed by SS and then the dynamics of the hepatocytes and their adhesiveness to the scaffold were observed. Methods: In order to apply SS, We used the rotating radial flow bioreactor (RRFB) (Able, Japan). In the RRFB, hepatocytes attached to the matrix rotated above the stirrer and a centrifugal force was applied. Hepatocytes (4 × 106/cm2) were seeded on a piece of gauze having no collagen coat. While SS was altered by RRFB, the cells were incubated in W-E medium for 3 hours, and then the yield of albumin was measured. Hepatocytes in SS culture were collected over time. After that, actin fibers were stained with FITC. Results: The most sufficient yield of albumin was observed at 30 rpm. Hepatocytes did not adhere to the gauze without SS, but the cells adhered to the gauze under SS, showing spheroid-like aggregation. In a culture with SS, hepatocytes spread actin fibers over the gauze and adhered to the gauze. Conclusions: In culture without SS, hepatocytes could not adhere to the gauze without collagen coating. However, it was demonstrated that actin fibers increased on the hepatocyte surface by loading SS to an adequate extent and adhered to a scaffold, and hepatocytes could also aggregate one another. This adhesion between hepatocytes seemed to essentially require polymerization of actin fibers.