Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.

Coupling of mutated Met variants to DNA repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration

BACKGROUND & AIMS: Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. METHODS: Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. RESULTS: After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. CONCLUSIONS: Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation during liver regeneration: evidence for synergism between growth factors and extracellular nucleotides

Crosstalk between elements of the sinusoidal vasculature, platelets and hepatic parenchymal cells influences regenerative responses to liver injury and/or resection. Such paracrine interactions include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6 and small molecules such as serotonin and nucleotides. CD39 (nucleoside triphosphate diphosphohydrolase-1) is the dominant vascular ectonucleotidase expressed on the luminal surface of endothelial cells and modulates extracellular nucleotide signaling. We have previously shown that integrity of P2-receptors, as maintained by CD39, is required for angiogenesis in Matrigel plugs in vivo and that there is synergism between nucleotide P2-receptor- and growth factor-mediated cell proliferation in vitro. We have now explored effects of CD39 on liver regeneration and vascular endothelial growth factor responses in a standard small animal model of partial hepatectomy. The expression of CD39 on liver sinusoidal endothelial cells (LSEC) is substantially boosted during liver regeneration. This transcriptional upregulation precedes maximal sinusoidal endothelial cell proliferation, noted at day 5-8 in C57BL6 wild type mice. In matched mutant mice null for CD39 (n=14), overall survival is decreased to 71% by day 10. Increased lethality occurs as a consequence of extensive LSEC apoptosis, decreased endothelial proliferation and failure of angiogenesis leading to hepatic infarcts and regenerative failure in mutant mice. This aberrant vascular remodeling is associated with biochemical liver injury, elevated serum levels of VEGF (113.9 vs. 65.5pg/ml, p=0.013), and decreased circulating HGF (0.89 vs. 1.43 ng/ml, p=0.001) in mice null for CD39. In agreement with these observations, wild type LSEC but not CD39 null cultures upregulate HGF expression and secretion in response to exogenous VEGF in vitro. CD39 null LSEC cultures show poor proliferation responses and heightened levels of apoptosis when contrasted to wild type LSEC where agonists of P2Y receptors augment cell proliferation in the presence of growth factors. These observations are associated with features of P2Y-desensitization, normal levels of the receptor tyrosine kinase VEGFR-1 (Flt-1) and decreased expression of VEGFR-2 (FLK/KDR) in CD39 null LSEC cultures. We provide evidence that CD39 and extracellular nucleotides impact upon growth factor responses and tyrosine receptor kinases during LSEC proliferation. We propose that CD39 expression by LSEC might co-ordinate angiogenesis-independent liver protection by facilitating VEGF-induced paracrine release of HGF to promote vascular remodeling in liver regeneration.

Granulocyte colony-stimulating factor increases hepatic sinusoidal perfusion during liver regeneration in mice

Conditioning with granulocyte colony-stimulating factor (G-CSF) promotes liver regeneration in an experimental small-for-size liver remnant mouse model. The mechanisms involved in this extraordinary G-CSF effect are unknown. The aim of this study was to investigate the influence of G-CSF on the hepatic microvasculature in the regenerating liver. The hepatic sinusoidal microvasculature and microarchitecture of the regenerating liver were evaluated by intravital microscopy in mice. Three experimental groups were compared: (1) unoperated unconditioned animals (control; n = 5), (2) animals conditioned with G-CSF 48 h after 60% partial hepatectomy (G-CSF-PH; n = 6), and (3) animals sham conditioned 48 h after 60% PH (sham-PH; n = 6). PH led to hepatocyte hypertrophy and increased hepatic sinusoidal velocity in the sham-PH and G-CSF-PH groups. Increased sinusoidal diameter and increased hepatic blood flow were observed in the G-CSF-PH group compared to the sham-PH and control groups. Furthermore, there was a strong positive correlation between spleen weight and hepatic sinusoidal diameter in the G-CSF-PH group. The increased hepatic blood flow could explain the observed benefit of G-CSF conditioning during liver regeneration. These results elucidate an unexplored aspect of pharmacological modulation of liver regeneration and motivate further experiments.

TRAIL-induced apoptosis: between tumor therapy and immunopathology

The death ligand members of the tumor necrosis factor (TNF) family are potent inducers of apoptosis in a variety of cell types. In particular, TNF-related apoptosis-inducing ligand (TRAIL) has recently received much scientific and commercial attention because of its potent tumor cell-killing activity while leaving normal untransformed cells mostly unaffected. Furthermore, TRAIL strongly synergizes with conventional chemotherapeutic drugs in inducing tumor cell apoptosis, making it a most promising candidate for future cancer therapy. Increasing evidence indicates, however, that TRAIL may also induce or modulate apoptosis in primary cells. A particular concern is the potential side effect of TRAIL-based tumor therapies in the liver. In this review we summarize some of the recent findings on the role of TRAIL in tumor cell and hepatocyte apoptosis.

Immune cell-mediated liver injury

Liver diseases represent an important cause of morbidity and mortality in the world. Death of hepatocytes and other hepatic cell types is a characteristic feature of several forms of liver injury such as cholestasis, viral hepatitis, drug- or toxin-induced injury, and alcohol-induced liver damage. Moreover, irrespectively of the reason, liver injury seems to be facilitated by similar immune effector mechanisms common to these various liver diseases. Indeed, common immune effector mechanisms may explain the high prevalence of cirrhosis and cancer development in most forms of liver disease. Improved understanding of the immune cell-mediated mechanisms involved in hepatocyte cell death could be beneficial for the development of common therapeutic strategies against different forms of liver diseases. In this review, we will discuss novel findings on the role of different immune cells in liver disease and immune cell-induced death executioner mechanisms involved in hepatocyte cell death.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response - Biological and Clinical Aspects

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

Analysis of liver stage development in and merozoite release from hepatocytes

Exoerythrocytic Plasmodium parasites infect hepatocytes and develop to huge multinucleated schizonts inside a parasitophorous vacuole. Finally, thousands of merozoites are formed and released into the host cell cytoplasm by complete disintegration of the parasitophorous vacuole membrane. This, in turn, results in death and detachment of the infected hepatocyte, followed by the formation of merosomes. The fast growth of the parasite and host cell detachment are hallmarks of liver stage development and can easily be monitored. Here, we describe how to translate these observations into assays for characterizing parasite development. Additionally, other recently introduced techniques and tools to analyze and manipulate liver stage parasites are also discussed.

HGF Expressing Stem Cells in Usual Interstitial Pneumonia Originate from the Bone Marrow and Are Antifibrotic

BACKGROUND Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia. METHODS Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later. RESULTS In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis. CONCLUSIONS HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.

In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95

Testosterone (TES) 6-β-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-β-hydroxytestosterone showed the largest peak. Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-β-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies.