Delayed liver regeneration in C3H/HeJ mice: possible involvement of haemodynamic and structural changes in the hepatic microcirculation.

NEW FINDINGS: What is the central question of this study? The liver regenerative process is complex and involves a sequence of signalling events, but the possible involvement of structural and haemodynamic changes in vivo during this process has never been explored. What is the main finding and its importance? Normal sinusoidal blood flow and velocity are crucial for a normal regenerative response, and delays in these haemodynamic events resulted in impaired liver regeneration in lipopolysaccharide-insensitive, C3H/HeJ mice. Toll-like receptor 4 signalling is required for restoration of normal liver architecture during the liver regenerative process. Liver regeneration is delayed in mice with a defective Toll-like receptor 4 (TLR4; C3H/HeJ mice) but is normal in TLR4 knockouts (TLR4-/- ). Here, we investigated the possible involvement of structural and haemodynamic changes in vivo in the underlying mechanism. In lipopolysaccharide-sensitive (C3H/HeN and C57BL/6) and lipopolysaccharide-insensitive (C3H/HeJ and TLR4-/- ) mice, a 70% partial hepatectomy (PH) was performed under inhalational anaesthesia. At days 3 and 7 after PH, the hepatic microcirculation was interrogated using intravital microscopy. Delayed liver regeneration was confirmed in C3H/HeJ, but not in C3H/HeN, C57BL/6 (WT) or TLR4-/- mice by liver weight-to-body-weight ratio, the percentage of proliferating cell nuclear antigen (PCNA)-positive cells and mitotic index data. At day 3 after PH, sinusoidal red blood cell velocity increased by 100% in C3H/HeN mice, but by only 40% in C3H/HeJ mice. Estimated sinusoidal blood flow was significantly higher at day 7 after PH in C3H/HeN than in C3H/HeJ mice. The hepatic cord width was significantly larger in C3H/HeN than in C3H/HeJ mice at day 3 and it was significantly larger in TLR4-/- than in C57BL/6 WT mice at day 7 after PH. Hepatocyte nucleus density and functional sinusoidal density was significantly reduced at days 3 and 7 after PH in all mouse strains compared with their zero-time controls. Functional sinusoidal density was significantly lower in C3H/HeJ compared with C3H/HeN mice at day 7 after PH. The present study indicates that altered sinusoidal blood flow and velocity in C3H/HeJ mice may contribute to the observed delay in the regenerative response in these mice. In addition, restoration of normal liver architecture may be delayed in TLR4-/- mice.

Apoptotic signaling in endothelial cells with neutrophil activation.

As is the case for tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), degranulated substances (DS) released from polymorphonuclear leukocytes (PMN) and H(2)O(2) cause endothelial cell apoptosis through the phosphorylation of members of the mitogen-activated protein kinase (MAPK) family. Stimulation of human umbilical vein endothelial cells (HUVEC) with IL-1ß or TNF-α/cycloheximide (CHX) was found to enhance the phosphorylation of p38 and Jun-N-terminal kinase (JNK) in a time-dependent fashion, but did not affect the time-dependent phosphorylation of extracellular signal-regulated kinase. In addition, IL-1ß and TNF-α/CHX induced the phosphorylation of activating transcription factor-2 (ATF-2), but not c-Jun. Moreover, the p38 in HUVEC was phosphorylated by DS released from PMN and also by H(2)O(2), but not by •O(2) (-) induced by myeloperoxidase (MPO) of PMN. On the other hand, caspase 8 in HUVEC was activated by DS, but not by H(2)O(2) and/or •O(2) (-). In addition, caspases 3 and 7 were cleaved by the treatment of DS and turned into active forms. DS was concentrated, analyzed by electrophoresis, and revealed to contain precursor and subunits of MPO (90, 60, and 14 kDa) and another peptide with a molecular weight of about 28 kDa. Because SB203580 that was an inhibitor of p38 MAPK did not repress phosphorylation of ATF-2 in HUVEC, it was suggested that JNK was more important than p38 in a series of signaling courses. These results suggest the possibility that not only TNF-α/CHX and IL-1ß but also DS released from PMN and the cell-permeable reactive oxygen species H(2)O(2) induce blood vessel injury through endothelial apoptosis.

Protection by glycyrrhizin against warm ischemia-reperfusion-induced cellular injury and derangement of the microcirculatory blood flow in the rat liver.

BACKGROUND/AIM: The mechanism by which ischemia-reperfusion (I/R)-induced derangement of the hepatic microcirculation leads to tissue injury is not fully understood. We postulated that alterations to the hepatic microcirculation, including hemodynamic derangement and increased leukocyte-endothelium interaction, play a role, and that glycyrrhizin exerts its hepatoprotective effects, in part, by reducing these microcirculatory changes. MATERIALS AND METHODS: Wistar rats were subjected to 30-60 minutes segmental hepatic ischemia, followed by 120 minutes of reperfusion. Glycyrrhizin was administered prior to ischemia. Using intravital fluorescence microscopy, the administration of fluorescein isothiocyanate-conjugated erythrocytes allowed the measurement of erythrocyte-velocity (RBC(vel)), lobular, and sinusoidal perfusion. Bleb formation was observed by electron microscopy. Blood and tissue were taken for the assessment of liver injury. RESULTS: Glycyrrhizin reduced I/R-induced liver injury (histology, liver enzymes) and reduced hepatocyte apoptosis (TUNEL, caspase-3 activity). Glycyrrhizin inhibited hepatocyte bleb formation and reversed the I/R-induced reductions in lobular perfusion and RBC(vel). Leukocyte rolling and adherence in postsinusoidal venules and neutrophil infiltration were reduced by glycyrrhizin. I/R-induced elevation in HMGB1 was prevented by glycyrrhizin. CONCLUSIONS: Early bleb formation with deranged microcirculatory flow and leukocyte-endothelium interaction would appear to contribute to I/R-induced hepatocellular injury. Glycyrrhizin exerts its hepatoprotective effect by preventing these changes, in addition to a direct cellular effect.

Role of F4/80Mac-1 adherent non-parenchymal liver cells in concanavalin A-induced hepatic injury in mice.

AIM: Non-parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80(+)Mac-1(high+) cells were involved in the regulative feedback-modulated regulation of inflammatory responses during concanavalin A (Con A)-induced hepatitis. METHODS: Hepatitis was induced in BALB/c mice by the intravenous injection of Con A. Liver injury was assessed using serum aminotransferase and pathology. The function of NPLC was assessed by FACS analysis. Accessory cell function of adherent Con A NPLC was performed with an ovalbumin specific T-helper 1 (Th1) clone proliferation assay. The culture supernatant nitric oxide (NO) content was quantified by the Griess reaction. Inducible NO synthase (iNOS) expression was demonstrated by immunohistochemistry and Western blot analysis. RESULTS: The number of hepatic F4/80(+)Mac-1(high+)cells increased in a time-dependent manner after Con A administration, which consequently suppressed Th1 cell proliferation by a mechanism likely to involve NO. The iNOS expression of NPLC was elevated at 24 h post-Con A injection. In nude mice, F4/80(+)Mac-1(high+)cells did not increase in the Con A-treated liver; the NPLC did not suppress Th1 clone proliferation. CONCLUSION: These findings suggest that the in vivo activation of F4/80(+)Mac-1(high+)cells by Con A administration suppresses Th1 cell proliferation by increasing NO, and subsequently reducing liver injury.

Vitamin A storage in hepatic stellate cells in the regenerating rat liver: with special reference to zonal heterogeneity.

Under physiological conditions, hepatic stellate cells (HSCs) within liver lobules store about 80% of the total body vitamin A in lipid droplets in their cytoplasm, and these cells show zonal heterogeneity in terms of vitamin A-storing capacity. Vitamin A is essential for the growth and differentiation of cells, and it is well known that liver cells including HSCs show a remarkable growth capacity after partial hepatectomy (PHx). However, the status of vitamin A storage in HSCs in the liver regeneration is not yet known. Therefore, we conducted the present study to examine vitamin A storage in these cells during liver regeneration. Morphometry at the electron microscopic level, fluorescence microscopy for vitamin A autofluorescence, and immunofluorescence microscopy for desmin and alpha-smooth muscle actin (alpha-SMA) were performed on sections of liver from male Wistar strain rats at various times after the animal had been subjected to 70% PHx. The mean area of vitamin A-storing lipid droplets per HSC gradually decreased toward 3 days after PHx, and then returned to normal within 14 days after it. However, the heterogeneity of vitamin A-storing lipid droplet area per HSC within the hepatic lobule disappeared after PHx and did not return to normal by 14 days thereafter, even though the liver volume had returned to normal. These results suggest that HSCs alter their vitamin A-storing capacity during liver regeneration and that the recovery of vitamin A homeostasis requires a much longer time than that for liver volume.

Effects of immunosuppressant FTY720 on renal and hepatic hemodynamics in the rat.

BACKGROUND: FTY720 is a novel immunomodulator that may provide an opportunity for a reduction in calcineurin inhibitor dosage in transplant recipients with renal/hepatic side effects. However, the effects of FTY720 on renal or hepatic hemodynamics are unknown. The aim of this study was to establish the hemodynamic and renal actions of FTY720 at therapeutically relevant dosages. METHODS: The effects of acute and repeat oral administration of FTY720 on systemic, renal, and hepatic hemodynamics were investigated in the anesthetized male Lewis rat. Renal function and renal tubular parameters were examined in animals that received repeat high dosage of FTY720. RESULTS: Seven-day oral administration of FTY720 did not cause any significant changes in markers of hepatocyte injury, nor did it cause any reduction in renal function (elevated urea and creatinine). Histological examination of liver and kidney from animals treated with repeat FTY720 for 1 or 3 weeks did not reveal any sclerosis, tubular changes, infiltrates, or fibrosis. Hepatocyte, vascular, and biliary structures were normal. Compared with the vehicle (saline), oral administration of FTY720 at dosages up to 5 mg/kg/day for 1 week did not have any significant effects on systemic, hepatic, or renal hemodynamics. Five min after intravenous FTY720 administration (1 mg/kg), mean arterial pressure (MAP) rose to 114+/-3.3% of baseline (P <0.01) before returning to the normal range within 30-45 min. Lower doses of FTY720 (0.3 and 0.5 mg/kg, i.v.) did not affect MAP. Renal cortical perfusion, renal artery blood flow, and renal vascular resistance were not altered by FTY720 at i.v. doses up to 1 mg/kg. Animals that received FTY720 (5 mg/kg/day) for 3 weeks showed a significant reduction in body weight (-4.8+/-1% of baseline at 3 weeks, P <0.001); however, weight-adjusted creatinine clearance, 24 h urine production, and urine osmolality were not different from those in control animals (0.71+/-0.1 vs. 0.74+/-0.1 ml/min/100 g, 2.63+/-0.2 vs. 3.12+/-0.2 ml/100 g, and 2003+/-33 vs. 1966+/-56 mOsm/kg, respectively). FTY720 at the same repeat oral dosage was, nevertheless, associated with a significantly lower 24 h sodium excretion and a significantly lower fractional excretion of sodium compared with those in control animals (223.4+/-35 vs. 304.5+/-50 micromol/100 g and 1.75+/-0.3 vs. 2.23+/-0.3%, respectively; P <0.05). CONCLUSIONS: Our data indicate that, at least in the short term, oral FTY720 does not cause any significant adverse effects on renal or hepatic hemodynamics, nor does it cause any reduction in glomerular perfusion and thus may provide reasonable rescue/add-on therapy in calcineurin-inhibitor treated transplant recipients. At high repeat oral dosages, however, FTY720 may alter renal handling of sodium.

Role of Hepatic Stellate Cells in the Early Phase of Liver Regeneration in Rat: Formation of Tight Adhesion to Parenchymal Cells.

We investigated activation mechanisms of hepatic stellate cells (HSCs) that are known to play pivotal roles in the regeneration process after 70% partial hepatectomy (PHx). Parenchymal liver cells (PLCs) and non-parenchymal cells (NPLCs) were isolated and purified from the regenerating livers at 1, 3, 7, 14 days after PHx. Each liver cell fraction was stained by immunocytochemistry using an anti-desmin antibody as a marker for HSCs, anti-alpha-smooth muscle actin (alpha-SMA) as a marker for activated HSCs, and 5-bromo-2'-deoxyuridine (BrdU) for detection of proliferating cells. Tissue sections from regenerating livers were also analyzed by immunohistochemistry and compared with the results obtained for isolated cell fractions. One and 3 days after PHx, PLC-enriched fraction contained HSCs adhered to PLCs. The HSCs adhered to PLCs were double positive for BrdU and alpha-SMA, and formed clusters suggesting that these HSCs were activated. However, HSC-enriched fraction contained HSCs not adhered PLCs showed positive staining for anti-desmin antibody but negative for anti-alpha-SMA antibody. These results suggest that HSCs are activated by adhering to PLCs during the early phase of hepatic regeneration.

Up-regulation of adhesion molecule expression in glomerular endothelial cells by anti-myeloperoxidase antibody.

BACKGROUND: Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) has been implicated in pauci-immune crescentic glomerulonephritis. It stimulates primed neutrophils to adhere to glomerular endothelial cells (GECs), thereby releasing reactive oxygen and other toxic substances and ultimately damaging the GECs. Though, a pathogenic role for MPO-ANCA is not fully understood, we hypothesized that MPO-ANCA modulates GEC functions by the increases in expression of adhesion molecules. METHODS: A polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO IgG) was evaluated in mouse GEC (mGEC) for its effect on adhesion molecule expression. The primary culture of mGEC was incubated with anti-rmMPO IgG or isotype control and the expression of intercellular adhesion molecules-1 (ICAM-1) was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis and ICAM-1 cell ELISA. RESULTS: The real-time RT-PCR analysis showed that a treatment with 100 microg/ml anti-rmMPO IgG increased the expression of mRNAs for ICAM-1, vascular cell adhesion molecule-1 and E-selectin by approximately 12.5, 7.5 and 10.5-fold, respectively. ICAM-1 cell ELISA also substantiated increased expression of ICAM-1. This enhancement of ICAM-1 expression was mediated by the antigen specificity of anti-rmMPO IgG. In addition, there were several proteins in mGEC specifically immunoprecipitated with anti-rmMPO IgG. CONCLUSIONS: These results showed that anti-MPO antibody activates not only neutrophils, but also GEC, indicating that anti-rmMPO IgG-induced direct activation of GEC contributes to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis.

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.