Hk Seitz

Hypoxia-inducible factor 1α under rapid enzymatic hypoxia: Cells sense decrements of oxygen but not hypoxia per se

HIF1 (hypoxia-inducible factor 1 alpha) is considered a central oxygen-threshold sensor in mammalian cells. In the presence of oxygen, HIF1 is marked by prolyl hydroxylases (PHDs) at the oxygen-dependent degradation (ODD) domain for ubiquitination followed by rapid proteasomal degradation. However, the actual mechanisms of oxygen sensing by HIF1 are still controversial. Thus, HIF1 expression correlates poorly with tissue oxygen levels, and PHDs are themselves target genes of HIF1 considered to readjust to new oxygen thresholds. In contrast to hypoxia chambers, we here establish an enzymatic model that allows both the rapid induction of stable hypoxia and independent control of H(2)O(2). Rapid enzymatic hypoxia only transiently induced HIF1 in various cell types and the HIF1 was completely degraded within 8-12 h despite sustained hypoxia. HIF1 degradation under sustained hypoxia could be blocked by a competitive ODD-GFP construct and PHD siRNA, but also by cobalt chloride and micromolar H(2)O(2) levels. Concomitant induction of PHDs further confirmed their role in degrading HIF1 under enzymatic hypoxia. The rapid and complete degradation of HIF1 under enzymatic hypoxia suggests that, in addition to hypoxia sensing, the HIF1/PHD loop may also compensate for fluctuations of tissue oxygen staying tuned to other, e.g., metabolic, signals. In addition to hypoxia chambers, enzymatic hypoxia provides a valuable tool for independently studying the regulatory functions of hypoxia and oxidative stress in vitro.

In vitro–targeted gene identification in patients with hepatitis C using a genome‐wide microarray technology

Iron in association with reactive oxygen species (ROS) is highly toxic, aggravating oxidative stress reactions. Increased iron not only plays an important role in the progression of hereditary hemochromatosis (HH) but also in common liver diseases such as chronic hepatitis C. The underlying mechanisms of hepatitis C virus (HCV)‐mediated iron accumulation, however, are poorly understood. We introduce an in vitro–targeted approach to identify ROS/iron‐regulated genes in patients with HCV using a genome‐wide DNA microarray. The sensitivity of the 32,231 complementary DNA clone‐carrying microarray was approximately 20% as estimated by detecting target genes of the genome‐wide transcription factor hypoxia inducible factor 1α. Upon in vitro challenge to iron and oxidative stress, 265 iron‐related and 1326 ROS‐related genes could be identified in HepG2 cells; 233 significantly regulated genes were found in patients with mild (HCV) or severe (HH) iron deposition. Notably, 17 of the in vitro–selected genes corresponded to the genes identified in patients with HCV or HH. Among them, natriuretic peptide precursor B (NPPB) was the only iron‐regulated gene identified in vitro that was differentially regulated between HCV and HH. Reverse‐transcription polymerase chain reaction confirmed most of the microarray‐identified genes in an even larger group of patients (n = 12). In patients with HCV, these included genes that are associated with RNA processing (MED9/NFAT, NSUN2), proliferation, differentiation, hypoxia, or iron metabolism (ISG20, MIG6, HIG2, CA9, NDRG1), whereas none of the nine known iron‐related genes showed significant differences between HCV and HH. Conclusion: Although high‐density microarray technology is less suitable for routine liver diagnosis, its use in combination with prior in vitro selection is a powerful approach to identify candidate genes relevant for liver disease. (HEPATOLOGY 2009;49:378–386.)

Primary liver injury and delayed resolution of liver stiffness after alcohol detoxification in heavy drinkers with the PNPLA3 variant I148M

AIM To investigate the influence of PNPLA3 genotype in heavy drinkers on serum markers and liver stiffness (LS) during alcohol withdrawal and its association with histology. METHODS Caucasian heavy drinkers (n = 521) with a mean alcohol consumption of 192.1 g/d (median alcohol consumption: 169.0 g/d; 95%CI: 179.0-203.3) were enrolled at the Salem Medical Center, University of Heidelberg. LS was measured by transient elastography (Fibroscan, Echosens SA, Paris, France). LS and serum markers were prospectively studied in these patients with all stages of alcoholic liver disease (steatosis, steatohepatitis, fibrosis) prior and after alcohol detoxification with a mean observation interval of 6.2 ± 3.2 d. A liver biopsy with histological analysis including the Kleiner score was obtained in 80 patients. RESULTS The PNPLA3 rs738409 genotype distribution for CC, CG and GG was 39.2%, 52.6% and 8.2%. GG genotype primarily correlated with histological steatohepatitis (r = 0.404, P < 0.005), ballooning (r = 0.319, P < 0.005) and less with steatosis (r = 0.264, P < 0.05). Mean LS was lowest in CC carriers (13.1 kPa) as compared to CG and GG carriers (17.6 and 17.2 kPa). Notably, LS primarily correlated with fibrosis stage (r = 0.828, P < 0.005), ballooning (r = 0.516, P < 0.005), steatohepatitis (r = 0.319, P < 0.005) but not with steatosis. After alcohol withdrawal, LS did not change in CC carriers, significantly decreased in CG-carriers from 17.6 to 12.7 kPa but to a lesser extent in GG carriers from 17.6 to 14.5 kPa. This was due to prolonged resolution of inflammation with significantly elevated aspartate transaminase levels after alcohol withdrawal in GG carriers. Non-invasive fibrosis assessment by LS in all patients showed a significantly higher F0 rate as compared to the biopsy cohort (47% vs 6%) with 3.8% more CC carriers while 3.7% less were seen in the F4 cirrhosis group. Thus, about 20% of patients with alcoholic liver cirrhosis would be attributable to PNPLA3 G variants. The OR to develop cirrhosis corrected for age, gender and body mass index was 1.295 (95%CI: 0.787-2.131) for CG + GG carriers. CONCLUSION In heavy drinkers, PNPLA3 GG primarily correlates with ballooning/steatohepatitis but not steatosis resulting in a delayed inflammation-associated resolution of LS. Consequently, sustained ballooning-associated LS elevation seems to be a potential risk factor for fibrosis progression in PNPLA3 GG carriers.

Arterial pressure suffices to increase liver stiffness

Noninvasive measurement of liver stiffness (LS) has been established to screen for liver fibrosis. Since LS is also elevated in response to pressure-related conditions such as liver congestion, this study was undertaken to learn more about the role of arterial pressure on LS. LS was measured by transient elastography (μFibroscan platform, Echosens, Paris, France) during single intravenous injections of catecholamines in anesthetized rats with and without thioacetamide (TAA)-induced fibrosis. The effect of vasodilating glycerol trinitrate (GTN) on LS was also studied. Pressures in the abdominal aorta and caval and portal veins were measured in real time with the PowerLab device (AD Instruments, Dunedin, New Zealand). Baseline LS values in all rats (3.8 ± 0.5 kPa, n = 25) did not significantly differ from those in humans. Epinephrine and norepinephrine drastically increased mean arterial pressure (MAP) from 82 to 173 and 156 mmHg. Concomitantly, LS almost doubled from 4 to 8 kPa, while central venous pressure remained unchanged. Likewise, portal pressure only showed a slight and delayed increase. In the TAA-induced fibrosis model, LS increased from 9.5 ± 1.0 to 25.6 ± 14.7 kPa upon epinephrine injection and could efficiently be decreased by GTN. We finally show a direct association in humans in a physiological setting of elevated cardiac output and MAP. During continuous spinning at 200 W, MAP increased from 84 ± 8 to 99 ± 11 mmHg while LS significantly increased from 4.4 ± 1.8 to 6.7 ± 2.1 kPa. In conclusion, our data show that arterial pressure suffices to increase LS. Moreover, lowering MAP efficiently decreases LS in fibrotic livers that are predominantly supplied by arterial blood.

Gastrointestinal Stromal Tumor in a Patient with Undiagnosed Neurofibromatosis Type I: an Uncommon Cause of Extrahepatic Cholestasis

In this case report we present a 61-year-old patient with obstructive jaundice. Bile duct obstruction was caused by a tumor at the duodenal papilla and bile flow was restored by a plastic stent. Using endoscopic ultrasound and computed tomography imaging two additional tumors of the same morphology were found in the stomach wall and the pelvic region suggesting a multilocular gastrointestinal stroma tumor (GIST). Diagnosis of GIST was confirmed cytologically from the gastric lesion. Based on typical cutaneous manifestations (café-au-lait spots, several tiny dermal neurofibromata and Lisch nodules in the iris), a thus far unidentified neurofibromatosis type I was diagnosed which is known to promote multilocular GIST formation. Tumor resection failed because of cardiac decompensation due to a Takotsubo cardiomyopathy during induction of anesthesia. The patient has been started on imatinib instead and shows so far a stable disease over 6 months.

Das hereditäre Hyperferritinämie-Katarakt-Syndrom -die erste Familie in Deutschland

We report on a 23-year-old woman who presented with elevated serum ferritin values at our department. She had undergone cataract surgery at the age of 14 and her family pedigree showed hereditary autosomal-dominant cataract. The combination of isolated hyperferritinemia with autosomal-dominant hereditary cataract led to the diagnosis of the hereditary hyperferritinemia cataract syndrome (HHCS) which we now describe in a German family for the first time. HHCS was confirmed by detection of a causal mutation at position 32 within the iron responsive element (IRE) of L-ferritin leading to a guanine to adenine exchange and the pathognomonic star-shaped cataract. This mutation interrupts the post-transcriptional control of L-ferritin. It prevents binding of the iron regulatory protein 1 (IRP1) to the 5alpha untranslated region of L-ferritin resulting in uncontrolled L-ferritin synthesis and high serum ferritin levels independent of the body iron stores. Premature cataract is eventually caused by deposition of L-ferritin crystals in the lens of the eye. Our family shows the typical autosomal-dominant inheritance of HHCS over four generations affecting a total of 17 family members. The causal mutation, star-shaped cataract and typical laboratory configuration were confirmed in five patients. Thus, in gastroenterological practice, HHCS should be added as a differential diagnosis of hyperferritinemia in Germany. Importantly, patients with HHCS can be spared from invasive diagnostics such as liver biopsy.

413 INCREASED LIVER STIFFNESS IN ALCOHOLIC LIVER DISEASE: DISSECTING FIBROSIS FROM STEATOHEPATITIS

413 INCREASED LIVER STIFFNESS IN ALCOHOLIC LIVER DISEASE: DISSECTING FIBROSIS FROM STEATOHEPATITIS S. Mueller, G. Millonig, S. Friedrich, F. Stickel, T. Longerich, P. Schirmacher, H.-K. Seitz. Center for Alcohol Research and Salem Medical Center, University of Heidelberg, Heidelberg, Germany; Clinical Pharmacology, University of Bern, Bern, Switzerland; Institute of Pathology, University of Heidelberg, Heidelberg, Germany E-mail: sebastian.mueller@urz.uni-heidelberg.de

Hypoxia enhances H 2 O 2 -mediated upregulation of hepcidin: Evidence for NOX4-mediated iron regulation

The exact regulation of the liver-secreted peptide hepcidin, the key regulator of systemic iron homeostasis, is still poorly understood. It is potently induced by iron, inflammation, cytokines or H2O2 but conflicting results have been reported on hypoxia. In our current study, we first show that pronounced (1%) and mild (5%) hypoxia strongly induces hepcidin in human Huh7 hepatoma and primary liver cells predominantly at the transcriptional level via STAT3 using two hypoxia systems (hypoxia chamber and enzymatic hypoxia by the GOX/CAT system). SiRNA silencing of JAK1, STAT3 and NOX4 diminished the hypoxia-mediated effect while a role of HIF1α could be clearly ruled out by the response to hypoxia-mimetics and competition experiments with a plasmid harboring the oxygen-dependent degradation domain of HIF1α. Specifically, hypoxia drastically enhances the H2O2-mediated induction of hepcidin strongly pointing towards an oxidase as powerful upstream control of hepcidin. We finally provide evidences for an efficient regulation of hepcidin expression by NADPH-dependent oxidase 4 (NOX4) in liver cells. In summary, our data demonstrate that hypoxia strongly potentiates the peroxide-mediated induction of hepcidin via STAT3 signaling pathway. Moreover, oxidases such as NOX4 or artificially overexpressed urate oxidase (UOX) can induce hepcidin. It remains to be studied whether the peroxide-STAT3-hepcidin axis simply acts to continuously compensate for oxygen fluctuations or is directly involved in iron sensing per se.

Does Hypoxia Cause Carcinogenic Iron Accumulation in Alcoholic Liver Disease (ALD)

Alcoholic liver disease (ALD) is a leading health risk worldwide. Hepatic iron overload is frequently observed in ALD patients and it is an important and independent factor for disease progression, survival, and the development of primary liver cancer (HCC). At a systemic level, iron homeostasis is controlled by the liver-secreted hormone hepcidin. Hepcidin regulation is complex and still not completely understood. It is modulated by many pathophysiological conditions associated with ALD, such as inflammation, anemia, oxidative stress/H2O2, or hypoxia. Namely, the data on hypoxia-signaling of hepcidin are conflicting, which seems to be mainly due to interpretational limitations of in vivo data and methodological challenges. Hence, it is often overlooked that hepcidin-secreting hepatocytes are physiologically exposed to 2–7% oxygen, and that key oxygen species such as H2O2 act as signaling messengers in such a hypoxic environment. Indeed, with the recently introduced glucose oxidase/catalase (GOX/CAT) system it has been possible to independently study hypoxia and H2O2 signaling. First preliminary data indicate that hypoxia enhances H2O2-mediated induction of hepcidin, pointing towards oxidases such as NADPH oxidase 4 (NOX4). We here review and discuss novel concepts of hypoxia signaling that could help to better understand hepcidin-associated iron overload in ALD.

Sonographisch dokumentierte Rückbildung der Lebersteatose während stationärer Langzeittherapie bei alkoholabhängigen Patienten

Hintergrund/Einleitung: Die potentielle Reversibilitat der Lebersteatose (LS) ist seit langem bekannt. Unklar ist, inwieweit sonographische Verlaufskontrollen diese zuverlassig dokumentieren konnen. Zielsetzung: Es sollte untersucht werden, in welcher Zeit sich die sonographischen Leberveranderungen normalisieren und welche Kofaktoren eine Rolle spielen. Ausserdem sollten Angaben zur Reproduzierbarkeit und Standardisierbarkeit der Lebersonographie gemacht werden. Material und Methoden: Konsekutive Patienten mit Alkoholabhangigkeit wurden zu Beginn und Ende der stationaren Entwohnungstherapie sonographiert. Parallel wurden erfasst: Abstinenzzeit vor Aufnahme, gGT, GPT, GOT, Body Mass Index (BMI, kg/m2). Die Graduierung der LS erfolgte qualitativ: Keine, leichte, mittelschwere, ausgepragte LS. Pro Untersuchung wurden vier Leberscans digital gespeichert. Zwei unabhangige Gutachter erhielten diese zum externen Rating. Zusatzlich wurden die digital gespeicherten Leberscans einer Pixelintensitatsanalyse (MaZda, V 3.20, Inst.Politech., Lodz) unterzogen. Als Mas fur die Pixelintensitat (PI) wurde der Mittelwert des Intensitatshistogrammes analysiert. Die PI der Niere diente jeweils als Referenzwert, um die Gerateverstarkung zu kontrollieren. Ergebnisse: Von 71 mannlichen Patienten zeigten 59 eine LS: 18 ausgepragt, 19 mittelschwer, 22 leicht. Zwischen Abstinenzzeit vor Aufnahme (Median=4 Tage, Range: 0–820 Tage) und Schwere der LS zeigte sich ein deutlicher Zusammenhang (p<0.02) und zwar unabhangig vom initialen BMI (25,9±3,7kg/m2). Insgesamt zeigten 37 Patienten (63%) in einer Behandlungszeit von 79±26 Tagen eine sonographische Besserung des initialen Befundes (p<0,01). Die Fremdratings ergaben eine Befundbesserung in 53 bzw. 60% der Falle (Interrater-Reliabilitat: r=0.719). Die mittlere PI zeigte eine Besserung von 20% (p<0.0001). Die PI der Niere zeigte eine Schwankung von 6,8% (p=0.15). Die Zunahme des Korpergewichtes betrug 2,2±5,4kg. Hohe initiale Leberwerte waren mit einer Besserung der LS assoziiert (AUC=0.785). Schlussfolgerung: Die Lebersonographie ist geeignet, die Ruckbildung der LS zu dokumentieren. Durch die elektronische Texturanalyse lasst sich die Methode wahrscheinlich besser standardisieren. Der Effekt der Abstinenz kann trotz Gewichtszunahme klar dokumentiert werden.