Geir I. Nedredal

Liver sinusoidal endothelial cells represents an important blood clearance system in pigs

BackgroundNumerous studies in rats and a few other mammalian species, including man, have shown that the sinusoidal cells constitute an important part of liver function. In the pig, however, which is frequently used in studies on liver transplantation and liver failure models, our knowledge about the function of hepatic sinusoidal cells is scarce. We have explored the scavenger function of pig liver sinusoidal endothelial cells (LSEC), a cell type that in other mammals performs vital elimination of an array of waste macromolecules from the circulation.Results125I-macromolecules known to be cleared in the rat via the scavenger and mannose receptors were rapidly removed from the pig circulation, 50% of the injected dose being removed within the first 2–5 min following injection. Fluorescently labeled microbeads (2 μm in diameter) used to probe phagocytosis accumulated in Kupffer cells only, whereas fluorescently labeled soluble macromolecular ligands for the mannose and scavenger receptors were sequestered only by LSEC. Desmin-positive stellate cells accumulated no probes. Isolation of liver cells using collagenase perfusion through the portal vein, followed by various centrifugation protocols to separate the different liver cell populations yielded 280 × 107 (range 50–890 × 107) sinusoidal cells per liver (weight of liver 237.1 g (sd 43.6)). Use of specific anti-Kupffer cell- and anti-desmin antibodies, combined with endocytosis of fluorescently labeled macromolecular soluble ligands indicated that the LSEC fraction contained 62 × 107 (sd 12 × 107) purified LSEC. Cultured LSEC avidly endocytosed ligands for the mannose and scavenger receptors.ConclusionsWe show here for the first time that pig LSEC, similar to what has been found earlier in rat LSEC, represent an effective scavenger system for removal of macromolecular waste products from the circulation.

EFFECT OF ALBUMIN DIALYSIS ON INTRACRANIAL PRESSURE INCREASE IN PIGS WITH ACUTE LIVER FAILURE: A RANDOMIZED STUDY

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). Methods:Pigs were randomized into three groups: sham, ALF, and ALF + M-MARS. ALF was induced by hepatic devascularization (time = 0). M-MARS began at time = 2 and ended with the experiment at time = 6. ICP, arterial ammonia, brain water, cerebral blood flow (CBF), and plasma inflammatory markers were measured. Results:ICP and arterial ammonia increased significantly over 6 hrs in the ALF group, in comparison with the sham group. M-MARS attenuated (did not normalize) the increased ICP in the ALF group, whereas arterial ammonia was unaltered by M-MARS. Brain water in the frontal cortex (grey matter) and in the subcortical white matter at 6 hrs was significantly higher in the ALF group than in the sham group. M-MARS prevented a rise in water content, but only in white matter. CBF and inflammatory mediators remained unchanged in all groups. Conclusion:The initial development of cerebral edema and increased ICP occurs independently of CBF changes in this noninflammatory model of ALF. Factor(s) other than or in addition to hyperammonemia are important, however, and may be more amenable to alteration by albumin dialysis.

The mannose receptor on murine liver sinusoidal endothelial cells is the main denatured collagen clearance receptor

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.)

Scavenger properties of cultivated pig liver endothelial cells

BackgroundThe liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC.ResultsEndocytosis of five 125I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells.ConclusionPig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC.

N-acetylcysteine increases cerebral perfusion pressure in pigs with fulminant hepatic failure.

ObjectiveIntravenous administration of N-acetylcysteine beyond 15 hrs reduces mortality rates in patients suffering from paracetamol-induced fulminant hepatic failure, although the mechanism of the therapeutic benefit remains unclear. We hypothesized increased survival to be caused by improved hemodynamic performance. The main objective for the study was to explore the effect of N-acetylcysteine on hemodynamics, oxygen transport, and regional blood flow in pigs with fulminant hepatic failure. DesignProspective, randomized, controlled trial. SettingSurgical research laboratory in a university hospital. SubjectsFemale Norwegian Landrace pigs. InterventionsFulminant hepatic failure was induced by a total liver devascularization procedure. Five hours later, the pigs were allocated to N-acetylcysteine treatment (150 mg·kg−1 in 100 mL of 0.9% saline over 15 mins, followed by 50 mg·kg−1 in 500 mL of 0.9% saline over a period of 4 hrs) or placebo. Measurements and Main ResultsMean arterial pressure stabilized in the N-acetylcysteine group and increased slightly during the last 2 hrs (pGT = .009). Thus, mean arterial pressure was significantly higher compared with placebo after 3 hrs (p = .01). Cerebral perfusion pressure was significantly higher during the last 2 hrs in the N-acetylcysteine group (pGT = .033). Common carotid artery flow also increased and was maintained at a higher level compared with placebo (pG = .027). Systemic vascular resistance index initially decreased but then gradually increased (pGT < .001). Cardiac index increased after 15 mins of N-acetylcysteine infusion, causing a significant interaction (pGT = .038), but did not differ after 3 hrs. No significant differences in hindleg and mesentery hemodynamics were found. A short-lived increase in oxygen delivery caused by a temporary increase in cardiac index was observed but without any corresponding increase in oxygen consumption. ConclusionsIntravenous N-acetylcysteine infusion increases cerebral perfusion pressure in pigs with fulminant hepatic failure. Earlier reported effects on oxygen transport and uptake could not be confirmed.

An experimental large animal model for the assessment of bioartificial liver support systems in fulminant hepatic failure.

Background: Pre-clinical assessment of bioartificial liver support systems requires a highly reproducible large animal model. The main objective of the present study was to develop a valid large animal model for assessing novel bioartificial liver support systems in fulminant hepatic failure. Methods: A complete liver devascularization procedure was performed in 10 female pigs weighing 25-38 kg. Five matched pigs were sham-operated and served as controls. Results: Pigs with fulminant hepatic failure developed a hyperdynamic circulation, with increased cardiac index ( P GT < .0001), decreased systemic vascular resistance index ( P GT < .0001) and mean arterial pressure ( P GT = .001). Furthermore, intracranial hypertension developed ( P GT < .0001), with increased common carotid artery flow ( P GT < .0001) and decreased common carotid resistance ( P G = .003). Femoral artery flow increased ( P = .036), while hindleg resistance ( P < .001) and renal artery resistance decreased ( P = .019). Oxygen consumption ( P GT = .050) and oxygen extraction ratio ( P GT = .001) increased compared to controls. Arterial ammonia, venous aspartate aminotransferase and bilirubin levels increased ( P GT < .0001, respectively). Abnormal haemostasis developed with significant loss of platelets ( P GT = .010), decreasing fibrinogen levels ( P G = .001) and increasing international normalized ratio ( P GT = .012) and activated clotting time ( P GT < .001). Urine became hypo-osmotic ( P < .001, P G = .011), with decreased sodium levels ( P = .08) and increased potassium levels ( P G = .025). Conclusions: This study characterizes a reproducible large animal model for fulminant hepatic failure that seems suitable for the assessment of bioartificial liver support systems.

ASSOCIATION OF REDUCED EXTRACELLULAR BRAIN AMMONIA, LACTATE, AND INTRACRANIAL PRESSURE IN PIGS WITH ACUTE LIVER FAILURE

We previously demonstrated in pigs with acute liver failure (ALF) that albumin dialysis using the molecular adsorbents recirculating system (MARS) attenuated a rise in intracranial pressure (ICP). This was independent of changes in arterial ammonia, cerebral blood flow and inflammation, allowing alternative hypotheses to be tested. The aims of the present study were to determine whether changes in cerebral extracellular ammonia, lactate, glutamine, glutamate, and energy metabolites were associated with the beneficial effects of MARS on ICP. Three randomized groups [sham, ALF (induced by portacaval anastomosis and hepatic artery ligation), and ALF+MARS] were studied over a 6‐hour period with a 4‐hour MARS treatment given beginning 2 hours after devascularization. Using cerebral microdialysis, the ALF‐induced increase in extracellular brain ammonia, lactate, and glutamate was significantly attenuated in the ALF+MARS group as well as the increases in extracellular lactate/pyruvate and lactate/glucose ratios. The percent change in extracellular brain ammonia correlated with the percent change in ICP (r2 = 0.511). Increases in brain lactate dehydrogenase activity and mitochondrial complex activity for complex IV were found in ALF compared with those in the sham, which was unaffected by MARS treatment. Brain oxygen consumption did not differ among the study groups. Conclusion: The observation that brain oxygen consumption and mitochondrial complex enzyme activity changed in parallel in both ALF‐ and MARS‐treated animals indicates that the attenuation of increased extracellular brain ammonia (and extracellular brain glutamate) in the MARS‐treated animals reduces energy demand and increases supply, resulting in attenuation of increased extracellular brain lactate. The mechanism of how MARS reduces extracellular brain ammonia requires further investigation. (HEPATOLOGY 2007.)

Protein S-100ß: A Biochemical Marker for Increased Intracranial Pressure in Pigs with Acute Hepatic Failure

BACKGROUND Acute hepatic failure (AHF) may cause encephalopathy. Intracranial pressure (ICP) is frequently monitored to guide therapy, but such monitoring may cause intracerebral haemorrhagic complications. We hypothesize that determination of serum levels of S-100beta, a protein synthesized in astroglial cells, will provide useful clinical information on the presence and extent of intracranial hypertension in AHF. METHODS Continuous intraparenchymatous ICP monitoring and serial S-100beta measurements in serum were performed in 11 Norwegian Landrace pigs with surgically induced AHF and in 4 sham-operated controls. RESULTS ICP increased hour by hour in the devascularized pigs in parallel with increased serum levels of protein S-100beta. In the sham-operated controls S-100beta was not detectable at any time point. CONCLUSIONS Serum levels of S-100beta are increased early in experimental AHF. Determination of protein S-100beta may provide useful information on the presence and extent of intracranial hypertension in AHF.Background: Acute hepatic failure (AHF) may cause encephalopathy. Intracranial pressure (ICP) is frequently monitored to guide therapy, but such monitoring may cause intracerebral haemorrhagic complications. We hypothesize that determination of serum levels of S-100 b, a protein synthesized in astroglial cells, will provide useful clinical information on the presence and extent of intracranial hypertension in AHF.Methods: Continuous intraparenchymatous ICP monitoring and serial S-100 b measurements in serum were performed in 11 Norwegian Landrace pigs with surgically induced AHF and in 4 sham-operated controls. Results: ICP increased hour by hour in the devascularized pigs in parallel with increased serum levels of protein S-100 b. In the sham-operated controls S-100 b was not detectable at any time point. Conclusions: Serum levels of S-100 b are increased early in experimental AHF. Determination of protein S-100 b may provide useful information on the presence and extent of intracranial hypertension in AHF.

Systemic and regional haemodynamics in pigs with acute liver failure and the effect of albumin dialysis

Objective. Acute liver failure (ALF) is haemodynamically characterized by a hyperdynamic circulation. The aims of this study were to investigate the systemic and regional haemodynamics in ALF, to measure changes in nitric oxide metabolites (NOx) and to evaluate whether these haemodynamic disturbances could be attenuated with albumin dialysis. Material and methods. Norwegian Landrace pigs (23–30 kg) were randomly allocated to groups as controls (sham-operation, n=8), ALF (hepatic devascularization, n=8) and ALF + albumin dialysis (n=8). Albumin dialysis was started 2 h after ALF induction and continued for 4 h. Systemic and regional haemodynamics were monitored. Creatinine clearance, nitrite/nitrate and catecholamines were measured. A repeated measures ANOVA was used to analyse the data. Results. In the ALF group, the cardiac index increased (PGT<0.0001), while mean arterial pressure (PG=0.02) and systemic vascular resistance decreased (PGT<0.0001). Renal resistance (PG=0.04) and hind-leg resistance (PGT=0.003) decreased in ALF. There was no difference in jejunal blood flow between the groups. ALF pigs developed renal dysfunction with increased serum creatinine (PGT=0.002) and decreased creatinine clearance (P=0.02). Catecholamines were significantly higher in ALF, but NOx levels were not different. Albumin dialysis did not attenuate these haemodynamic or renal disturbances. Conclusions. The haemodynamic disturbances during the early phase of ALF are characterized by progressive systemic vasodilatation with no associated changes in metabolites of NO. Renal vascular resistance decreased and renal dysfunction developed independently of changes in renal blood flow. After 4 h of albumin dialysis there was no attenuation of the haemodynamic or renal disturbances.

Porcine liver sinusoidal endothelial cells contribute significantly to intrahepatic ammonia metabolism

Ammonia metabolism in the liver has been largely credited to hepatocytes (HCs). We have shown that liver nonparenchymal cells that include liver sinusoidal endothelial cells (LSECs) produce ammonia. To address the limited knowledge regarding a role for LSECs in ammonia metabolism, we investigated the ammonia metabolism of isolated LSECs and HCs under three different conditions: (1) bioreactors containing LSECs (LSEC‐bioreactors), (2) bioreactors containing HCs (HC‐bioreactors), and (3) separate bioreactors containing LSECs and HCs connected in sequence (Seq‐bioreactors). Our results showed that LSEC‐bioreactors released six‐fold more ammonia (22.2 nM/hour/106 cells) into the growth media than HC‐bioreactors (3.3 nM/hour/106 cells) and Seq‐bioreactors (3.8 nM/hour/106 cells). The glutamate released by LSEC‐bioreactors (32.0 nM/hour/106 cells) was over four‐fold larger than that released by HC‐bioreactors and Seq‐bioreactors (<7 nM/hour/106 cells). LSEC‐bioreactors and HC‐bioreactors consumed large amounts of glutamine (>25 nM/hour/106 cells). Glutaminase is known for catalyzing glutamine into glutamate and ammonia. To determine if this mechanism may be responsible for the large levels of glutamate and ammonia found in LSEC‐bioreactors, immunolabeling of glutaminase and messenger RNA expression were tested. Our results demonstrated that glutaminase was present with colocalization of an LSEC‐specific functional probe in lysosomes of LSECs. Furthermore, using a nucleotide sequence specific for kidney‐type glutaminase, reverse‐transcription polymerase chain reaction revealed that this isoform of glutaminase was expressed in porcine LSECs. Conclusion: LSECs released large amounts of ammonia, perhaps due to the presence of glutaminase in lysosomes. The ammonia and glutamate released by LSECs in Seq‐bioreactors were used by hepatocytes, suggesting an intrahepatic collaboration between these two cell types. (HEPATOLOGY 2009.)