Siegfried Matern

Intrahepatic cholestasis of pregnancy: molecular pathogenesis, diagnosis and management

Frank Lammert, Hanns-Ulrich Marschall1, Anna Glantz2 and Siegfried Matern Department of Internal Medicine III, Aachen University of Technology – RWTH, Aachen, Germany, 1Karolinska Institutet, Department of Medicine, Division of Gastroenterology and Hepatology, Huddinge University Hospital, Stockholm, and 2Department of Obstetrics and Gynecology, Sahlgren’s University Hospital, East, Göteborg, Sweden

Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.

Mutation of β- glucosidase 2 causes glycolipid storage disease and impaired male fertility

beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the beta-glucosidase acid 1 (GBA1) defective in subjects with the Gauchers form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease.

Spontaneous cholecysto‐ and hepatolithiasis in Mdr2−/− mice: A model for low phospholipid‐associated cholelithiasis

Previously, we identified needle‐like and filamentous, putatively “anhydrous” cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4) knockout mice that are characterized by phospholipid‐deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non‐fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2−/− mice precipitate needle‐like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2−/− mice develop gallbladder stones, with female mice displaying a markedly higher gallstone‐susceptibility. Although gallbladder biles of Mdr2−/− mice contain only traces (≤ 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2‐phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2−/− mice show intra‐ and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2−/− phenotype. The Mdr2−/− mouse is therefore a model for low phospholipid‐associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (HEPATOLOGY 2004;39:117–128.)

Verification of the intraluminal multiple electrical impedance measurement for the recording of gastrointestinal motility

A new intraluminal electrical impedance procedure for high‐resolution measurements and the quantitative assessment of gastrointestinal motility is examined in healthy volunteers by cineradiography and manometry. The peristalsis in the oesophagus, stomach and small bowel is recorded with a combined impedance‐pressure catheter. Additionally, investigations with a flexible 16‐channel impedance catheter with a closed surface and a diameter of 3 mm are carried out in the oesophagus and small intestine. The 16 measuring segments with a length of 2 cm each record the contractile patterns from a 32‐cm‐long organ section without a gap. The correctness of the physical approach is validated by concurrent impedance and cineradiography recordings of the oesophageal peristalsis. The comparative studies confirm a close relation between the pressure and the impedance changes in the oesophagus and small intestine. The time analysis of the impedance tracings offers information about the bolus transit and the change of the wall compliance along the organ. Regions of high or low compliance of the muscular wall can be recognized. From the impedance tracings the direction of the contraction waves as well as their velocities, lengths, beginnings and ends can be determined. Moreover, the beginning and end of the bolus, and from them the momentary bolus length, can be characterized for any instant during the bolus transit.

Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice

Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) plays a crucial role in the pathogenesis of hepatic fibrosis and thus may represent an important therapeutic target in the design of anti‐fibrotic strategies for chronic liver disease. We present an innovative therapy based on the assignment of inactivated enzymes acting as scavengers for TIMP‐1. Hepatic fibrosis was induced in BALB/c mice by repetitive intraperitoneal CCl4 injection. The animals were treated with proteolytic inactive matrix metalloproteinase‐9 mutants (E402Q, H401A, E402H/H411E) using adenovirus‐mediated gene transfer. Application of these MMP‐9 mutants inhibited fibrogenesis, which was indicated by decreasing portal and periportal accumulation of collagen. Total hydroxyproline of liver tissue, the morphometric stage of fibrosis as well as mRNA expression of marker proteins for hepatic fibrosis in livers of E402Qand H401A‐treated mice were significantly reduced. MMP‐9 mutants suppressed transdifferentiation of hepatic stellate cells to the myofibroblast like phenotype in vitro and in vivo. Moreover, adenoviral application of the mutants MMP‐9‐H401A and ‐E402Q led to increased apoptosis of activated hepatic stellate cells, thought to be the main promoters of hepatic fibrosis. Application of MMP‐9 mutants as TIMP‐1 scavengers may provide a new therapeutic strategy for hepatic fibrosis.—Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M.‐L., Henkel, C., Grötzinger, J., Gressner, A. M., Matern, S., Roeb, E. Inhibition of hepatic fibrogenesis by matrix metalloproteinase‐9 mutants in mice. FASEB J. 20, 444–454 (2006)

Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver

BACKGROUND & AIMS Liver regeneration in response to various forms of injury or surgical resection is a complex process resulting in restoration of the original liver mass and maintenance of liver-specific functions such as bile formation. However, liver regeneration is frequently associated with cholestasis, whose molecular pathogenesis remains unknown. METHODS To study the molecular mechanisms leading to cholestasis, expression of all major hepatic organic anion transporters contributing to bile formation was determined for up to 2 weeks in rats after 70% partial hepatectomy. RESULTS Inversely related to serum bile acid levels, basolateral transporters including the sodium-taurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides Oatp1 and Oatp2 were markedly down-regulated at both protein and steady-state mRNA levels by 50%-60% of controls (P < 0.05) during early replicative stages of regeneration (12 hours to 2 days) with a slightly delayed time course for Oatp2. Expression of all basolateral transporters returned to control values between 4 and 4 days after partial hepatectomy. In contrast, protein and mRNA expression of both the canalicular ATP-dependent bile salt export pump (Bsep) and the multiorganic anion transporter Mrp2 remained unchanged or were slightly increased during liver regeneration, but also returned to control values 7-14 days after partial hepatectomy. CONCLUSIONS The data suggest a differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver. Unaltered expression of Bsep and Mrp2 provides a potential molecular mechanism for regenerating liver cells to maintain or even increase bile secretion expressed per weight of remaining liver. However, down-regulation of basolateral organic anion transporters might protect replicating liver cells by diminishing uptake of potentially hepatotoxic bile salts, because the remaining liver initially cannot cope with the original bile acid pool size.

Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride

BACKGROUND/AIMS Hepatobiliary transporters are down-regulated in cholestasis, but their expression in acute, non-cholestatic, cytokine-mediated liver injury is unknown. Thus we studied the molecular mechanisms, by which sodium taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, multidrug-resistance protein 2 (Mrp2) and bile salt export pump (Bsep) are regulated in liver injury induced by carbon tetrachloride (CCl(4)). METHODS mRNA and protein levels were determined in rats 24 and 72h after CCl(4) injection. Transporter gene transcription and binding activities of Ntcp and Mrp2 transactivators were assessed by nuclear runoff and electrophoretic mobility shift assays. RESULTS mRNA levels significantly declined to 41+/-44% for Ntcp, 65+/-41% for Oatp1 and 64+/-28% for Oatp2, but remained unchanged for Oatp4, canalicular Mrp2 and Bsep. Protein levels declined only for Oatp4 (-50+/-17%) and Ntcp (-23+/-13%) at 24h. Reduced mRNA levels (Ntcp, Oatp1, Oatp2) were associated with decreased transcriptional activities. Binding activity of Ntcp transactivators (hepatocyte nuclear factor 1 alpha (HNF1alpha) and CAAT enhancer binding protein alpha (C/EBPalpha) were reduced by 24h, whereas retinoid X receptor alpha (RXRalpha):retinoid acid receptor alpha (RARalpha) as transactivator of both Ntcp and Mrp2 remained unaltered. Recovery of acute hepatitis and changes in gene expression occurred after 72h. CONCLUSIONS Acute liver injury results in down-regulation of basolateral organic anion transporters similar to liver regeneration after partial hepatectomy, but in contrast to endotoxin-induced cholestasis. Maintained binding activity of RXRalpha:RARalpha may explain differences in Mrp2 expression.

TIMP expression in toxic and cholestatic liver injury in rat

BACKGROUND/AIMS Hepatic fibrosis is a dynamic pathological process with a net accumulation of extracellular matrix proteins. Recent evidence suggests that besides their increased synthesis, inhibition of matrix degradation plays a significant role. ECM degradation occurs via metalloproteinases which are inhibited in situ by specific tissue inhibitors of metalloproteinases (TIMPs). The aim of our studies was to determine the expression of TIMPs during toxic liver injury and cholestatic liver injury leading to fibrosis. METHODS We examined the expression of TIMP-1, -2 and -3 in two different rat models for liver injury (intraperitoneal CCl4 injection and bile duct ligation) by Northern blot analysis and in situ hybridization. For comparison, the mRNA expression of the acute phase protein haptoglobin was measured. RESULTS TIMP-1 was increased during the early phase of toxic liver injury and in cholestasis. Its expression occurred predominantly in areas of inflammation, in hepatocytes, and in mesenchymal and endothelial cells. There was a slight upregulation of TIMP-2 expression during cholestasis. TIMP-3 was not detected at all. CONCLUSIONS Our results emphasize an involvement of TIMP-1 in matrix homeostasis, indicating its possible participation in liver fibrosis.

Regulation of basolateral organic anion transporters in ethinylestradiol-induced cholestasis in the rat

BACKGROUND/AIMS Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na(+)/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes. METHODS Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays. RESULTS All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30-40% of controls, but less pronounced than Ntcp (minus 70-80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40-90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein alpha (C/EBPalpha) and pregnane X receptor (PXR) were also diminished during the time of cholestasis. CONCLUSIONS Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens.