Loss of Nrf2 in mice evokes a congenital intrahepatic shunt that alters hepatic oxygen and protein expression gradients and toxicity.

The transcription factor Nrf2 (Nfe2l2 nuclear factor, erythroid 2-like 2) regulates gene expression directly, controlling pharmacological and toxicological responses. These processes may also be influenced by the structure of the hepatic vasculature, which distributes blood flow through compartmentalized microenvironments to maintain organismal stability. Castings of the hepatic portal vasculature of albino C57BL/6J but not ICR Nrf2(-/-) mice revealed a congenital intrahepatic shunt that was present in two thirds of Nrf2-disrupted mice. This shunt directly connected the portal vein to the inferior vena cava and displayed characteristics of a patent ductus venosus. Immunohistochemistry revealed that Nrf2(-/-) mice with an intrahepatic shunt manifest changes to hepatic oxygen and protein expression gradients when compared with wild-type (WT) and non-shunted Nrf2(-/-) mice. Centrilobular hypoxia found in WT and Nrf2(-/-) mice without shunts was reduced in Nrf2(-/-) livers with a shunt. Hepatic protein expression of phosphoenolpyruvate carboxykinase (Pepck), normally confined to the periportal zone, exhibited both periportal and centrilobular zonal expression in livers from Nrf2(-/-) mice with an intrahepatic shunt. Centrilobular expression of Cytochrome P450 2E1 (Cyp2e1) was diminished in shunted Nrf2(-/-) livers compared with WT and Nrf2(-/-) livers without shunts. The intrahepatic shunt in Nrf2(-/-) mice was further found to diminish acetaminophen hepatoxicity compared with WT and Nrf2(-/-) non-shunted mice following a 6 h challenge with 250 mg/kg acetaminophen. The presence of an intrahepatic shunt influences several physiological and pathophysiological properties of Nrf2(-/-) mice through changes in blood flow, hepatic oxygenation, and protein expression that extent beyond loss of canonical transactivation of Nrf2 target genes.

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia.

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.

Alterations in expression of genes coding for proteins of the neurovascular unit in ischemic liver failure.

There is evidence to suggest that integrity of the neurovascular unit may be compromised in acute liver failure (ALF). In order to address this issue from a molecular standpoint, expression of an array of genes coding for key cerebrovascular endothelial cell and tight junction proteins were measured by reverse transcription-polymerase chain reaction in cerebral cortex of rats with ischemic liver failure resulting from hepatic devascularization (portacaval anastomosis followed 24h later by hepatic artery ligation) compared to appropriate sham-operated controls. Expression of P-glycoprotein, endothelin-1, von Willebrand factor, caveolin-1, occludin, and the endothelial nitric oxide synthase isoform (eNOS) were measured in brain extracts from rats with ALF at coma/edema stages of encephalopathy. The effects of mild hypothermia (35 degrees C) sufficient to prevent cerebral edema in ALF animals on the expression of these genes were also studied. Brain edema and hepatic coma in normothermic ALF rats was accompanied by selective increases in expression of eNOS. Expression of occludin and von Willebrand factor mRNAs were decreased at coma/edema stages of encephalopathy in ALF rats whereas, expression of other cerebrovascular endothelial cell markers endothelin-1, P-glycoprotein, and caveolin-1 were unaffected. Mild hypothermia led to normalization of brain water content and of eNOS mRNA. However, the correlation between increased eNOS expression and encephalopathy/edema grade was poor suggesting the existence of additional mechanisms. These findings underscore the multifactorial nature of brain edema/encephalopathy mechanisms in ALF and question the role of BBB breakdown as a major pathogenetic factor.

Effect of pyrrolidine dithiocarbamate on hepatic vascular stress gene expression during ischemia and reperfusion.

Pyrrolidine dithiocarbamate, an antioxidant and a potent inhibitor of nuclear factor-kappa B (NF-kappaB), is known to have protective effect against ischemia and reperfusion injury. This study examined the cytoprotective mechanism of pyrrolidine dithiocarbamate against the microcirculatory failure caused by hepatic ischemia and reperfusion. Rats were subjected to 60 min of hepatic ischemia followed by 5 h of reperfusion. Pyrrolidine dithiocarbamate (100 mg/kg) or the vehicle was administered intraperitoneally 24 h before ischemia. The level of serum aminotransferases and hepatic lipid peroxides significantly increased, and the glutathione contents fell in the ischemia/reperfusion group. Pyrrolidine dithiocarbamate prevented the increase in the level of serum enzymes and hepatic lipid peroxides, and the decrease in the glutathione contents. The NF-kappaB DNA-binding activity was inhibited by a pre-treatment with pyrrolidine dithiocarbamate. Ischemia and reperfusion significantly increased the mRNA expression of the endothelin-1 and endothelin ET(B) receptor, which was prevented by pyrrolidine dithiocarbamate. There were significant increases in the mRNA expressions of inducible nitric oxide synthase, tumor necrosis factor-alpha, and cyclooxygenase-2, in the livers after ischemia and reperfusion. These increases were attenuated by the pyrrolidine dithiocarbamate treatment. In a rat model of hepatic ischemia and reperfusion, our results suggest that the hepatoprotective actions of pyrrolidine dithiocarbamate may be mediated in part through the modulation of imbalanced expression of vascular stress genes.

Detection of a significant association between mutations in the ACVRL1 gene and hepatic involvement in German patients with hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is a heterogeneous multisystemic dysplasia of the vascular tissue. This autosomal dominant inherited disorder shows a wide variation in its phenotypic expression. Between 8 and 78% of the HHT patients show arteriovenous malformations of the liver. The molecular basis for hepatic manifestation is still unknown. Two genes are known to play a major role in the development of HHT: activin A receptor type II-like 1 gene (ACVRL1) and ENG. Previously, we and others showed that hepatic involvement is associated with mutations in the ACVRL1 gene, but rarely caused by ENG mutations. Here, we report about the sequencing analysis of a new cohort of 18 adult HHT patients. In these patients, we identified eight novel (four in ACVRL1 and four in ENG) and eight already known mutations. Statistical analysis of our entire data revealed significant differences in the distribution of ACVRL1 and ENG mutations among HHT patients with and without liver involvement (p = 0.0016). The positive predictive value for type 2 HHT (ACVRL1 positive) patients to develop liver disease until the age of 52 years is 68.4%. We conclude that molecular genetic testing of HHT patients is important for prognosis with respect to liver disease.

Liver deformation in Ahr-null mice: evidence for aberrant hepatic perfusion in early development.

Mice harboring mutations in the Ahr locus display a patent ductus venosus and smaller livers throughout life. We tested the hypothesis that these hepatic aberrations are secondary to a developmental defect in hepatovascular blood flow by performing a detailed analysis of hepatic development in wild-type and Ahr-/- mice. This study revealed necrotic lesions in the peripheries of Ahr-/- fetal livers as early as embryonic day 15.5, with an increasing incidence up to postnatal day 1 and resolution by 2 weeks post partum. To visualize perfusion of fetal livers, we injected fluorescein isothiocyanate-labeled dextran into the cranial artery and monitored hepatic fluorescence by microscopy. The peripheries of the median and left lobes displayed decreased perfusion in regions corresponding to those regions that displayed necrosis at later developmental times. An examination of adult Ahr-/- animals revealed that smaller livers are predominantly due to decreased sizes of the left and right lobes, corresponding to regions of decreased perfusion and hepatic necrosis observed in fetal livers. Histological aberrations in the portal vein also support a model in which perfusion is compromised in the Ahr-/- liver. Taken in sum, these results indicate that the Ahr locus is required for normal perfusion of the developing liver and that disruption of the AHR signaling pathway gives rise to fetal hepatic necrosis and consequent liver deformation which persists through adult-hood.

From molecules to mammals: what's NOS got to do with it?

Nitric oxide synthases (NOSs) generate nitric oxide (NO) and the by-product l-citrulline, via the catalytic combination of l-arginine and molecular oxygen. In mammals, there are three NOS genes: nNOS (NOS1), iNOS (NOS2) and eNOS (NOS3). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important processes. The studies of NOS enzymes using knockout and transgenic mouse models have provided an invaluable contribution, highlighting critical roles in neuronal, renal, pulmonary, gastro-intestinal, skeletal muscle, reproductive and cardiovascular biology. This review will outline the data gleaned from complementary knockout and transgenic over-expression models in mice, and focus on the interactions between NOS enzymes and pathophysiology of the vascular system. These studies are a paradigm for the near future, which will involve the translation of an enormous amount of genomic data into physiological insights that penetrate the realms of both health care and biology.