The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. Conclusion: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs. (HEPATOLOGY 2007;45:1454-1461

Purpose To investigate the correlation between MRE assessed spleen stiffness and direct portal vein pressure gradient (D-HVPG) measurements in a large animal model of portal hypertension. Materials and Methods Cholestatic liver disease was established in adult canines by common bile duct ligation. A spin echo based EPI MRE sequence was used to acquire 3-D/3-axis abdominal MRE data at baseline, four weeks, and eight weeks. Liver biopsies, blood samples, and D-HVPG measurements were obtained simultaneously. Results Animals developed portal hypertension (D-HVPG: 11.0±5.1 mmHg) with only F1 fibrosis after four weeks. F3 fibrosis was confirmed after eight weeks despite no further rise in portal hypertension (D-HVPG: 11.3±3.2 mmHg). Mean stiffnesses of the spleen increased over two-fold from baseline (1.72±0.33 kPa) to four weeks (3.54±0.31 kPa), and stabilized at eight weeks (3.38±0.06 kPa) in a pattern consistent with changes in portal pressure. A positive correlation was observed between spleen stiffness and D-HVPG (r2 = 0.86, p<0.01). Conclusion These findings indicate a temporal relationship between portal hypertension and the development of liver fibrosis in a large animal model of cholestatic liver disease. The observed direct correlation between spleen stiffness and D-HVPG suggest a non-invasive MRE approach to diagnose and screen for portal hypertension.

Background: Increased intracranial pressure (ICP) worsens the outcome of acute liver failure (ALF). This study investigates the underlying pathophysiological mechanisms and evaluates the therapeutic effect of albumin dialysis in ALF with use of the Molecular Adsorbents Recirculating System without hemofiltration/dialysis (modified, M-MARS).

Background: Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared.

Background Significant morbidity associated with acute liver failure (ALF) is from the systemic inflammatory response syndrome (SIRS). Toll-like receptor 4 (TLR4) has been shown to play an integral role in the modulation of SIRS. However, little is known about the mechanistic role of TLR4 in ALF. Also, no cell type has been identified as the key mediator of the TLR4 pathway in ALF. This study examines the role of TLR4 and Kupffer cells in the development of the SIRS following acetaminophen (APAP)-induced ALF. Materials and Methods Five groups of mice were established: untreated wild-type, E5564-treated (a TLR4 antagonist), gadolinium chloride (GdCl3)-treated (Kupffer cell-depleted), clodronate-treated (Kupffer cell-depleted), and TLR4-mutant. Following APAP administration, 72-hour survival, biochemical and histologic liver injury, extent of lung injury and edema, and pro-inflammatory gene expression were studied. Additionally, TLR4 expression was determined in livers of wild-type and Kupffer cell-depleted mice. Results Following APAP administration, wild-type, TLR4-mutant, E5564-treated, and Kupffer cell-depleted mice had significant liver injury. However, wild-type mice had markedly worse survival compared to the other 4 treatment groups. TLR4-mutant, E5564-treated, and Kupffer cell-depleted mice had less lung inflammation and edema than wild-type mice. Selected pro-inflammatory gene expression (il1b, il6, tnf) in TLR4-mutant, E5564-treated, and Kupffer cell-depleted mice was significantly lower compared to wild-type mice after acute liver injury. Conclusion This study demonstrates that survival in APAP-induced ALF potentially correlates with the level of pro-inflammatory gene expression. This study points to a link between TLR4 and Kupffer cells in the APAP model of ALF, and, more importantly, demonstrates benefits of TLR4 antagonism in ALF.

This study characterizes a reproducible large animal model for fulminant hepatic failure that seems suitable for the assessment of bioartificial liver support systems.

Cultivation of primary hepatocytes as spheroids creates an efficient three-dimensional model system for hepatic studies in vitro and as a cell source for a spheroid reservoir bioartificial liver. The mechanism of spheroid formation is poorly understood, as is an explanation for why normal, anchorage-dependent hepatocytes remain viable and do not undergo detachment-induced apoptosis, known as anoikis, when placed in suspension spheroid culture. The purpose of this study was to investigate the role of E-cadherin, a calciumdependent cell adhesion molecule, in the formation and maintenance of hepatocyte spheroids. Hepatocyte spheroids were formed by a novel rocker technique and cultured in suspension for up to 24 h. The dependence of spheroid formation on E-cadherin and calcium was established using an E-cadherin blocking antibody and a calcium chelator. We found that inhibiting E-cadherin prevented cell-cell attachment and spheroid formation, and, surprisingly, E-cadherin inhibition led to hepatocyte death through a caspase-independent mechanism. In conclusion, E-cadherin is required for hepatocyte spheroid formation and may be responsible for protecting hepatocytes from a novel form of caspase-independent cell death.Key words: Hepatocyte spheroids; E-Cadherin; Anoikis; Caspase-independent cell death INTRODUCTIONcules on adjacent cells, is therefore a likely candidate required for spheroid formation and maintenance. E-Cadherin, one of the most important and well-studWe have previously shown that spheroids of primary (nontransformed) rat hepatocytes can be formed rapidly ied cell adhesion proteins, is a known factor involved in anoikis (5). The loss of E-cadherin adhesions has been and efficiently using a rocked suspension technique (2). Under these conditions, freshly isolated rat hepatocytes implicated in the induction of anoikis in tumorigenic epithelial cells (6) and during the normal shedding of the aggregate spontaneously to form spheroids of 60-120 µm diameter by 24 h and viability exceeding 95%. As intestinal epithelial cells at the villus tip (3). Therefore, we asked whether E-cadherin was required for spheroid previously shown by others (1), differentiated hepatocyte functions were better maintained in spheroid culformation and as a necessary step to protect hepatocytes from anoikis cell death. Our findings suggest that Eture. However, cell loss became problematic over extended culturing periods. While culturing hepatocytes in cadherin was required both for spheroid formation and the protection of hepatocytes from cell death under ananchorage-independent conditions has many advantages, little is known about how this cell loss occurred over chorage-independent rocked spheroid conditions. In contrast to previous studies, our results suggest that cell time. Anchorage-independent spheroid cell cultures, such as our novel rocked culturing technique (13), rely death can occur via a caspase-independent mechanism. upon calcium-dependent cell-cell adhesions for survival and function, which occur in the absence of a...

The results of the study show that MARS can remove both albumin and other protein-bound drugs efficiently from the plasma, and it may have a place for the treatment of patients suffering from intoxication with this class of compounds.