Liver regeneration after partial hepatectomy is impaired in steatotic livers of leptin-deficient ob/ob mice. Previous studies have shown that thrombopoietin (TPO) promotes liver regeneration and improves liver cirrhosis by an increase of platelet counts and the expansion of hepatic progenitor cells. Herein we studied whether TPO exerts pro-proliferative and hepatoprotective effects and thereby improves the regenerative capacity of steatotic livers. For this purpose, we studied hepatic regeneration at day 2, 3, 7 and 10 in a model of 55% hepatectomy in obese (ob/ob) and non-obese (C57BL/6J) mice. Liver function and injury, platelet counts, weight of the regenerated liver, proliferating liver cells as well as the number of hepatic (CK19-positive) oval cells were quantified by biochemical and immunohistochemical analysis. As expected, obese mice had a markedly decreased regenerative capacity of livers compared with lean animals. Pretreatment of mice with recombinant TPO (12.5 μg/kg) had no evident effect on regeneration of fatty livers, but ameliorated acute liver damage in obese mice, as indicated by decreased liver enzyme release early after resection. TPO was unable to enhance hepatocyte proliferation, but increased proliferation of non-parenchymal cells, including CK19-positive oval cells, at later observation time points after resection. Interestingly, TPO completely inhibited the resection-induced increase of plasma triglycerides immediately after resection in non-obese mice. In conclusion, TPO slightly prevents acute liver damage after resection in obese mice, but fails to significantly enhance regeneration of fatty livers.

AIMS:Darbepoetin-α (DPO), a long-acting erythropoietin analog, has been shown to protect the liver against cholestatic injury, to exert an antifibrotic effect, and to increase the survival time in a model of common bile duct ligation. Here we evaluate whether these tissue-protective effects are caused by DPO induced regulation of hepatobiliary transporters.MAIN METHODS:C57BL/6J mice underwent common bile duct ligation and were treated with either DPO or physiological saline. Time dependent (2, 5, 14, 28 days after bile duct ligation) protein expression of different hepatobiliary transporters which have been established to play an important role in hepatocellular (i) bile acid uptake, (ii) bile acid excretion, and (iii) retrograde bile acid efflux were assessed. mRNA and protein expression of Lhx2, an important negative regulator of hepatic stellate cell activation, was determined.KEY FINDINGS:Saline treated cholestatic mice impress with increased mRNA expression of Lhx2 as a defense mechanism, while there is less need for such an upregulation in mice treated with DPO. Whereas Ntcp (slc10a1) protein expression is suppressed as early as 2 days after bile duct ligation to 40% in untreated animals, DPO treated mice exhibit decreased protein level not before day 5. Similarly, the steady decline of Mrp4 (abcc4) protein level during extrahepatic cholestasis in control treated animals does not occur upon DPO application.SIGNIFICANCE:The collected data show that DPO affects expression of hepatobilliary transporters during obstructive cholestasis but do not provide sufficient evidence to demonstrate a direct correlation between this regulation and hepatoprotection by DPO.

The induction of angiogenesis is essential for successful engraftment of freely transplanted cells or cellular composites. How to augment angiogenesis to ensure an appropriate viability of the grafts is still under investigation. This study evaluated the proangiogenic capability of different syngeneic free liver transplants and elucidated the origin of the newly formed vascular network via use of an eGFP+/eGFP- (enhanced green fluorescent protein) cross-over design. Using intravital fluorescence microscopy, we found that neonatal and resected murine liver transplants implanted into dorsal skinfold chambers display a significantly enhanced vascularization compared to regular adult transplants. Immunohistochemically, less tissue hypoxia, apoptosis and macrophage infiltration was observed in the neonatal and resected transplants, which is in line with improved vascularization of those grafts. Additionally, electron microscopy revealed morphological hallmarks of liver cells. eGFP+ liver transplants implanted on eGFP- recipients displayed vascular sprouting from the grafts themselves and connection to the recipients` microvasculature, which also undergoes transient proangiogenic response. This process is described as external inosculation, with microvessels exhibiting a chimeric nature of the endothelial lining. These data collectively show that proliferative stimulation is taking effect on angiogenic properties of free transplants and might provide a novel tool for modulating the revascularization of free grafts.

Background Many aspects of the signaling mechanisms involved in the initiation of hepatic regeneration are under current investigation. Nevertheless, the actual mechanisms switching liver regeneration on and off are still unknown. Hemodynamic changes in the liver following partial hepatectomy have been suggested to be a primary stimulus in triggering liver regeneration. Most of the new knowledge about the impact of hemodynamic changes on liver regeneration is both conceptually important and directly relevant to clinical problems. Purpose The purpose of this review is therefore to exclusively address the hemodynamic signal driving the liver regeneration process.

The characteristics of the hepatic macrocirculation, i.e., the parallel portal-venous and arterial blood supply, is of utmost relevance for liver surgery. With extended hepatectomy or transplantation of a reduced-size liver the remaining or transplanted liver tissue is overperfused because the liver fails to regulate the portal-venous inflow. This portal hyperperfusion is responsible for the initiation of liver cell proliferation but represents at the same time one of the substantial events in the pathogenesis of the small-for-size syndrome. Portal-venous hyperperfusion, the so-called hepatic arterial buffer response, which describes the semi-reciprocal relationship between the portal-venous and hepatic arterial blood flows, leads to an arterial hypoperfusion of the small-for-size liver. In this article experimental and clinical data are discussed which underline the high but so far overseen relevance of this arterial underperfusion in the development of a small-for-size syndrome.

Die Besonderheit der hepatischen Makrozirkulation, d. h. die parallele portalvenöse und arterielle Blutversorgung, ist von erheblicher Relevanz für die Leberchirurgie. Nach ausgedehnter Hepatektomie oder Transplantation einer größenreduzierten Leber wird das verbleibende oder transplantierte Lebergewebe stärker perfundiert, da die Leber den portalvenösen Einstrom kaum regulieren kann. Diese portale Hyperperfusion ist zum einen für die Induktion der Leberzellproliferation verantwortlich, wird auf der anderen Seite aber als einer der wesentlichen Momente in der Pathogenese des „Small-for-size“-Syndroms gesehen. Die sog. „hepatic arterial buffer response“, womit die semireziproke Beziehung zwischen der Durchblutung der Pfortader und der Durchblutung der Leberarterie beschrieben wird, führt gleichzeitig zu einer arteriellen Minderversorgung der „Small-for-size“-Leber. In diesem Beitrag werden experimentelle und klinische Daten diskutiert, die die hohe aber bislang wenig berücksichtigte Relevanz dieser arteriellen Minderversorgung bei der Entwicklung eines „Small-for-size“-Syndroms unterstreichen.

Obstructive cholestasis is caused by mechanical constriction or occlusion leading to reduced bile flow. Serious complications such as jaundice and even death may follow. Little is known about the initial phase of cholestasis and its consequences for the hepatic microarchitecture. This in vivo study aimed to characterize the nature and kinetics of developing obstructive cholestasis and focused on areas with biliary stasis and infarction by visualizing the autofluorescence of bile acids using intravital microscopy of the liver over a period of 30 h after bile duct ligation in rats. The innovation resided in performing fluorescence microscopy without applying fluorescent dyes. In animals subjected to obstructive cholestasis, the most significant changes observed in vivo were the concomitant appearance of (1) areas with bile accumulation increasing in size (6 h: 0.163 ± 0.043, 18 h: 0.180 ± 0.086, 30 h: 0.483 ± 0.176 mm(2)/field) and (2) areas with biliary infarction (6 h: 0.011 ± 0.006, 18 h: 0.010 ± 0.004, 30 h: 0.010 ± 0.050 mm(2)/field) as well as (3) a relation between the formation of hepatic lesions and enzyme activity in serum. The sequential in vivo analysis presented herein is a new method for the in vivo visualization of the very early changes in the hepatic parenchyma caused by obstructive cholestasis.

Transplantation of hepatocytes is a therapeutic approach for diverse acute and chronic liver diseases. As the availability of primary cells is limited, there exists an increasing demand for hepatocyte-like cells that can be obtained by stem cell technology. Among which adipose-derived mesenchymal stem cells (Ad-MSCs) represent a promising source, as they are easily assessable and can be obtained in sufficient amounts.

Significant advantage of targeted antitumoral treatment consists in the possibility to restrict maximum therapeutic efficacy to the malignant cell population by reducing toxicity in healthy tissues. Using different clinical models for aggressive medullary thyroid carcinoma (MTC), we have recently identified peptide ligands that bind highly selective to tumor cells. By linking the most convincing SRESPHP peptide to an adenoviral (Ad) vector expressing the MTC-related oncogene inhibitor RETΔTK, gene transfer was specifically directed to neoplastic tissue after systemic virus administration. We show that peptide-mediated delivery of RETΔTK significantly enhanced apoptosis, resulting in a strong inhibition of orthotopic and xenograft tumor growth. Conversely, tumors treated with controls expanded their initial size without notable cell death. According to the therapeutic effect, strong virus accumulation was found exclusively in thyroid carcinomas. Strikingly, application of native tropism depleted viral vector linked to tumor-selective peptide was accompanied by a substantial reduction of Ad binding to the liver. Of note, single systemic injection of a low dose (10e8 pfu/mouse) of MTC-specific Ad.RETΔTK induced regression of multiple tumors at different sites in all treated animals. In sum, our results open up the possibility for an efficient cancer cell-specific therapy of primary MTC, their migrating populations and potentially metastases.

AIMS:Mitochondria not only generate and modulate bioenergy but also serve as biosensors for oxidative stress, and eventually become effector organelles for cell viability. Therefore, the implications of mitochondrial (dys)function in the development of multiple organ failure are profound. We investigated whether a mutation in the ATPase subunit-8 gene affects the course of endotoxemic acute liver failure.MAIN METHODS:C57BL/6J (ATP8 wild type) and C57BL/6J-mt(FVB/N) (ATP8 mutant) mice were challenged with d-galactosamine (GalN) and Escherichia coli lipopolysaccharide (LPS) for induction of acute liver failure, and studied 6 h thereafter. Control mice received physiological saline only. Analysis included in vivo fluorescence microscopy of hepatic microcirculation and levels of hepatocellular apoptosis, hepatic adenosine nucleotides and oxidative stress. Additionally, survival rates were assessed.KEY FINDINGS:Induction of endotoxemic liver failure provoked marked liver damage, which was coexistent with a drop of total adenosine nucleotide levels and increased oxidative stress. Of interest, oxidative stress was higher in the GalN/LPS challenged ATP8 mutants compared to wild types. Concomitantly, adenosine triphosphate (ATP) levels in livers of mice carrying the ATP8 mutation remained higher than those in wild type mice. As net result, ATP8 mutants showed lower transaminase release and a tendency to better survival rate upon GalN/LPS exposure compared to wild types.SIGNIFICANCE:Our findings demonstrate that mutation in the ATPase subunit-8 partially protects mice against endotoxemic stress, most probably due to better hepatic energy status despite elevated oxidative stress. Thus, modulating mitochondrial function to preserve bioenergetic status may be an effective strategy to protect against sepsis-induced multiorgan dysfunction.

Partial ligation of portal branches leads to atrophy of the deprived lobes and hypertrophy of the intact lobes. In this study we investigated the microcirculatory response and their consequences on tissue regeneration after left-sided portal branch ligation (PBL) in Sprague-Dawley rats. At day 1 and 3 after PBL the hepatic microcirculation was assessed by intravital microscopy (IVM). In addition histological, immunohistochemical and biochemical techniques were used to determine alterations of hepatic microarchitecture. IVM analysis of the microcirculation of the ligated hepatic lobes revealed significant alterations with a reduction in sinusoidal perfusion rate, a decrease of red blood cell velocity, an increase of sinusoidal diameter and a marked reduction in shear stress at days 1 and 3 after PBL. On the contrary, the non-ligated lobes presented with higher blood flow velocities, marked sinusoidal vasoconstriction and thus, shear stress elevation. In consequence, ligated liver lobes exhibited marked cell apoptosis and necrosis, being accompanied by massive intrahepatic leukocyte accumulation and a ~30% weight loss. The non-ligated liver tissue showed marked PCNA expression and thereby completely compensated weight loss. Beside full restoration of liver mass, sinusoidal blood flow was comparable in ligated and non-ligated lobes as well as in sham-treated controls. This study shows that the liver aims at constant tissue mass and blood flow, most probably for maintenance of adequate clearance function. In addition, it supports the hypothesis that shear stress plays a pivotal role in triggering liver hypertrophy in the non-ligated lobes.

Objective During abdominal sepsis, the activation of hepatic Kupffer cells (KC) and its consequences are of central interest. This study evaluates the impact of selective KC depletion on hepatic microcirculation, cytokine release, and systemic alterations in the colon ascendens stent peritonitis (CASP), a model of polymicrobial abdominal sepsis.Methods For KC depletion clodronate liposomes were injected 24 h before CASP surgery in female C57BL/6N mice. Three and 12 h after CASP, in-vivo fluorescence microscopy of the liver was performed. Analysis of hepatocellular apoptosis was conducted by immunohistochemistry. In addition, levels of tumor necrosis factor (TNF), IL-6, and IL-10 in the liver, lungs, spleen, and plasma were determined, and bacteriology and survival analysis were performed.Results CASP led to significant sinusoidal perfusion failure, increased leukocyte recruitment, hepatocellular apoptosis and increased levels of TNF, IL-6, and IL-10 in the liver and plasma. KC depletion before CASP significantly reduced leukocyte recruitment to the liver and hepatocellular apoptosis. IL-10 secretion decreased dramatically in the liver and plasma of KC-depleted septic mice. In contrast, TNF levels were clearly elevated after clodronate treatment. In the lung and spleen, a compensatory upregulation of IL-10 could be detected after KC depletion. Clodronate treatment resulted in a significant reduction in survival.Conclusion The results indicate that KC depletion is locally protective in polymicrobial abdominal sepsis, as it reduces hepatic inflammation and apoptosis. These effects could be observed in the presence of clearly elevated TNF levels. However, the lack of IL-10 in KC-depleted mice resulted in a detrimental systemic proinflammation. Eur J Gastroenterol Hepatol 22: 1039-1049 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

The interest in the liver dates back to ancient times when it was considered to be the seat of life processes. The liver is indeed essential to life,not only due to its complex functions in biosynthesis,metabolism and clearance,but also its dramatic role as the blood volume reservoir. Among parenchymal organs,blood flow to the liver is unique due to the dual supply from the portal vein and the hepatic artery. Knowledge of the mutual communication of both the hepatic artery and the portal vein is essential to understand hepatic physiology and pathophysiology. To distinguish the individual importance of each of these inflows in normal and abnormal states is still a challenging task and the subject of on-going research. A central mechanism that controls and allows constancy of hepatic blood flow is the hepatic arterial buffer response. The current paper reviews the relevance of this intimate hepatic blood flow regulatory system in health and disease. We exclusively focus on the endogenous interrelationship between the hepatic arterial and portal venous inflow circuits in liver resection and transplantation,as well as inflammatory and chronic liver diseases. We do not consider the hepatic microvascular anatomy,as this has been the subject of another recent review.

Background. Uncoupling protein-2 (UCP2) might play an important role in mediating ischemia/reperfusion (I/R) injury due to its function in uncoupling of oxidative phosphorylation and in the proton leak-associated increase of reactive oxygen species (ROS) production. The aim of this study was to elucidate the role of UCP2 in hepatic I/R injury.Materials and Methods. UCP2 wild type and UCP2 deficient mice were subjected to I/R of the left liver lobe. Sham-operated animals without I/R served as controls. Intravital fluorescence microscopy was used for assessing postischemic microcirculatory dysfunction. Indicators of hepatic inflammatory response, oxidative stress, and bioenergetic status as well as histomorphology were investigated.Results. Under sham conditions UCP2 -/- mice presented slightly but not significantly higher levels of hepatic ATP and energy charge than wild type mice. In addition, they exhibited higher systemic IL-6 levels and intrahepatic leukocyte adherence. After exposure to I/R, the extent of reperfusion injury did not differ between UCP2 +/+ and UCP2 -/- mice, as indicated by a comparable loss of sinusoidal perfusion, hepatic ATP, and energy charge levels, as well as rise of transaminases and disintegration of liver structures. Intrahepatic leukocyte adherence and plasma IL-6 levels of postischemic UCP2 -/- mice still exceeded those of UCP2 +/+ mice.Conclusions. UCP2 appears to be of minor relevance for the manifestation and extent of postischemic reperfusion injury in nondiseased livers with the increased ATP availability being counteracted by the higher pro-inflammatory IL-6 levels in UCP2 deficient mice. (C) 2011 Elsevier Inc. All rights reserved.

Biliary obstruction and cholestasis result in hepatocellular necro-inflammation and lead to the development of liver fibrosis. Herein, we evaluated the therapeutic efficacy of Darbepoetin-α (DPO), a recombinant longer acting analogue of erythropoietin in a model of extrahepatic cholestasis. C57BL/6J mice underwent common bile duct ligation (BDL) and were treated either with DPO (10 μg/kg i. p.) or physiologic saline every third day, beginning 24 h after BDL. Mice were sacrificed at 2, 5 and 14 days after BDL. BDL provoked cholestatic hepatitis characterized by biliary infarcts with accumulation of macrophages followed by marked collagen deposition and increased gene expression of profibrotic transcripts. DPO treatment significantly diminished the area of focal necrosis, reduced the infiltration of macrophages, lowered collagen deposition, decreased levels of profibrotic transcripts, and inverted the pattern of Smad mRNA expression. Moreover, DPO prevented systemic anemia caused by BDL. Finally, DPO treatment significantly prolonged the survival time after BDL. These findings suggest that DPO, a clinically well established substance, could represent an efficient therapeutic option for patients with cholestatic liver disease.

The (dys)function of mitochondria plays an important role in the development of multiple organ failure. Therefore, the implications of mitochondrial (dys)function in the development of multiple organ failure are profound. We investigated whether a mutation in the ATPase subunit-8 gene exerts effects on the course of endotoxemic acute liver failure in mice. For this purpose, wildtype and ATP8 mutant mice were challenged with D-galactosamine (GalN) and Escherichia coli lipopolysaccharide (LPS) and studied 6 hrs thereafter. Control mice received physiological saline only. Analysis included in vivo fluorescence microscopy of hepatic microcirculation and determination of hepatocellular apoptosis, levels of hepatic adenosine nucleotides as well as concentrations of plasma malondialdehyde as a marker of oxidative stress. Induction of endotoxemic liver failure provoked marked liver damage, characterized by microvascular perfusion failure and transaminase release. Of interest, oxidative stress was significantly higher in the GalN/LPS challenged ATP8 mutants compared to wild types. In contrast, the ATP/ADP ratio in livers of mice carrying the ATP8 mutation remained significantly higher than those in wild type mice. As a net result, ATP8 mutant mice showed lower transaminase release and better survival rate compared to wild types. Our data demonstrate that mutation in the ATPase subunit 8 partially protects mice under endotoxemic stress conditions most probably due to better hepatic energy status despite elevated overall oxidative stress. These findings underline the role of genetic polymorphisms in the pathophysiology of sepsis.

Background. Organ shortage in liver transplantation has justified usage of marginal donor livers to expand the donor organ pool. The particular susceptibility of steatotic livers to I/R injury necessitates optimal preservation conditions in order to minimize preservation-reperfusion injury for successful transplantation.Methods. The effect of erythropoietin (EPO) as additive to HTK preservation solution was studied in a mouse model. Lean and steatotic livers were harvested, stored for 24 h in 4 degrees C HTK solution containing either EPO or saline and reperfused for 2 h with 37 degrees C Krebs-Henseleit buffer. Livers without cold storage served as sham controls.Results. Flushing of livers upon cold storage revealed a transaminase release, which was 2-to 10-fold higher in steatotic versus lean livers. EPOwas effective in reducing the enzyme release to 50% in steatotic but not in lean livers. EPO prevented cold storageinduced denudation of the endothelial lining in steatotic livers, but aggravated it in lean livers. During reperfusion, steatotic livers presented with lower oxygen consumption and higher enzyme release than lean livers. In all livers, parameters of reperfusion injury remained unaffected by EPO. Expression of UCP2 was found markedly higher in steatotic livers. After I/R, steatotic livers revealed a significant drop of UCP2, whereas expression in lean livers was only slightly affected. EPO diminished Erk phosphorylation to almost the same extent in both mouse strains.Conclusion. Fortification of the preservation solution by EPO ameliorates cold ischemic injury of stea-totic livers and may thus be considered for use as an adjunctive agent to increase the success of transplanting steatotic livers. (C) 2012 Elsevier Inc. All rights reserved.