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Cancers 13(22), 5680 (2021). doi:10.3390/cancers13225680 special issue: "Special issue "Cell cycle proteins as promising targets in cancer therapy" / special issue editor: Arun K. Rishi, guest editor", Published by MDPI, Basel

Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol‐associated and metabolic‐associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol‐associated liver disease plus metabolic‐associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA‐sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets., DUAL model perfectly mimics all histological, metabolic and transcriptomic gene signatures of human advanced steatohepatitis, and thus serve as a preclinical tool for the development of therapeutic targets.

The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2&minus, /&minus, mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2&alpha, (eIF2&alpha, ), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1&alpha, /TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2&minus, mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2&minus, mice. Lcn2&minus, mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2&minus, and Chop null (Chop&minus, ) primary hepatocytes and found Chop&minus, hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2&alpha, /GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop&minus, hepatocytes resumed activation of IRE1&alpha, /JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop&minus, mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.

International journal of molecular sciences 21(9), 3267 (2020). doi:10.3390/ijms21093267 special issue: "Special Issue "Pathophysiology of Liver Fibrosis and Its Therapies" / Special Issue Editor: Dr. Pavel Strnad, Guest Editor", Published by Molecular Diversity Preservation International, Basel

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45+ cells after single injection. Acute CCl4 -mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45+ leukocytes. Pretreatment with CcnE1-siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl4 for 4-6 weeks. Co-treatment with CcnE1-siRNA once a week was sufficient to continuously block CcnE1 expression and cell-cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1-siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. Conclusion Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257).

The endoplasmic reticulum (ER) is primarily recognized as the site of synthesis and folding of secreted membrane-bound and certain organelle-targeted proteins. Optimum protein folding requires several factors, including ATP, Ca2+ and an oxidizing environment to allow disulphide-bond formation. ER is highly sensitive to stress that perturb cellular energy levels, the redox state or the Ca2+ concentration. Such stresses reduce the protein folding capacity of the ER, resulting in the accumulation and aggregation of unfolded proteins, a condition referred to as unfolded protein response (UPR). Matricellular proteins of the CCN (CYR61, CTGF, NOV) family play essential roles in extracellular matrix signaling and turnover. They exhibit a similar type of organization and share a closely related primary structure, including 38 conserved cysteine residues. Since CCN1/CYR61 overexpression in hepatic stellate cells (HSC) induces ER stress-related apoptosis, we endeavored to investigate whether the adenovirus mediated gene transfer of other members of CCN proteins incurs ER stress in primary HSC and hepatocytes. We found Ad5-CMV-CCN2, Ad5-CMV-CCN3 and Ad5-CMV-CCN4 to induce ER stress and UPR comparable to Ad5-CMV-CCN1. UPR is a pro-survival response to reduce accumulation of unfolded proteins and restore normal ER functioning. If, however protein aggregation is persistent and the stress cannot be resolved, signaling switches from pro-survival to pro-apoptosis. The observed CCN-induced UPR is relevant in wound healing responses and essential for hepatic tissue repair following liver injury. Adenoviral gene transfer induced massive amounts of matricellular proteins proving to effectively mitigate liver fibrosis if targeted cell specific in HSC and myofibroblasts.

Unfolded protein response (UPR) is an adaptive mechanism allowing the endoplasmic reticulum (ER) to react to an accumulation of unfolded proteins in its lumen, also known as ER stress. The UPR is interconnected with inflammation through several pathways such as reactive oxygen species (ROS) production resulting from the protein folding or alternatively, activation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) via IRE1, or induction of acute phase response (APR). Lipocalin 2 (LCN2) is one of the APR proteins induced under inflammatory conditions and up-regulated during ER stress. Upon incubation of Lcn2−/− and wild type (wt) primary hepatocytes with tunicamycin (TM) or thapsigargin (TG) we found the Lcn2−/− hepatocytes to react with strong UPR to the ER stress, as evidenced by significantly increased levels of Grp94, Bip and Chop mRNA and protein compared to the wt. TM and TG-treated hepatocytes activated p65 NF-κB and JNK, the pathways that respond to stress stimuli and playing a central role in inflammation and apoptosis, respectively. ER stress further activated and cleaved full-length CREBH/CREB3L3, the hepatocyte specific transcription factor to induce systemic inflammatory responses. Upregulation of the C/EBP homologous protein (CHOP) was very prominent in Lcn2−/− hepatocytes and sustained until 48 h, resulting in hepatocyte apoptosis as evidenced by increased cleaved caspase 3. We also explored the UPR of the Lcn2 null mouse livers in acute intoxication and inflammation stages with a single application of lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). The Lcn2 null mice clearly developed stronger UPR in LPS- and CCl4-induced ER stress compared to the wt. Our findings indicate that the upregulation of LCN2 during ER stress-induced inflammatory responses protects hepatocytes from being overwhelmed by UPR upon liver injury.

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Portal fibroblasts are mesenchyme-derived fibroblasts surrounding the bile ducts, and activated into portal myofibroblasts (pMF) during cholestatic liver injury. pMF express α-smooth muscle actin (α-SMA) and produce the fibrogenic extracellular matrix (ECM) collagen type I and fibronectin, playing important roles in portal fibrosis. A cholestatic bile duct-ligated (BDL) model is characterized by impaired hepatobiliary excretion of bile, leading to increased bile acid accumulation. Accumulation of bile acids is known to induce endoplasmic reticulum (ER) stress leading to liver damage and cell death. Additionally, a BDL fibrotic model is also associated with upregulation of CCN (CYR61, CTGF and NOV) matricellular proteins and reported to induce ER stress both in vitro and in vivo. To explore the effects of CCN proteins, we used adenovirus-mediated CCN1-4 (Ad-CCN1-4) gene transfers into cultured pMF. Overexpression of CCN proteins leads to protein accumulation in the ER lumen, causing ER stress and unfolded protein response (UPR). We further found ER stress and UPR to mitigate fibrogenesis in pMF by decreased cellular production of fibronectin, collagen type 1 and α-SMA. In this scenario, Tauroursodeoxycholic acid, a pharmaceutical chaperone and ER stress inhibitor, attenuated Ad-CCN1-4 induced pMF apoptosis and restored collagen and fibronectin levels. Since hepatic fibrogenesis is accompanied by ER stress and upregulation of CCN proteins in a BDL, we further evaluated ER stress responses after Ad-CCN1 gene transfer in such a model and found overexpressed CCN1 to enhance the ER stress-associated proteins BiP and CHOP with positive cleaved caspase 3 and 9 staining in periportal nonparenchymal cells. This indicates that these nonparenchymal cells, most likely pMF, have the tendency to undergo apoptosis during later stages of BDL. Ad-CCN1 transduction furthermore sensitized pMF for ER stress and apoptosis. We suggest that CCN proteins are key factors in the fibrotic microenvironment impacting pMF survival during fibrogenesis and pMF apoptosis during fibrosis resolution.

Platelet-derived growth factor-D (PDGF-D) is one member of PDGF growth factors and known to signal by binding to and activating its cognate receptor type β (PDGFR-β). Beside PDGF-B, PDGF-D is a potent growth factor for stellate cell growth and proliferation and therefore potentiates the extracellular matrix deposition in liver fibrogenesis. We aimed to explore the signaling and molecular mechanisms of PDGF-D in liver fibrogenesis using the primary liver portal myofibroblasts and hepatic stellate cells. Unexpectedly we found PDGF-D to bind to PDGFR-α, thus inducing receptor endocytosis and decreasing the amount of PDGFR-α significantly. PDGF-D activates PDGFR-α specific tyrosine 754 and -1018 phosphorylation and CrkII, the adaptor protein that is specifically recruited by activated PDGFR-α. As a novel finding we could also demonstrate that recombinant PDGFR-α-Fc chimera homodimer is able to bind PDGF-D and thus prevent PDGF-D signaling. PDGF-D does induce individual PDGFR-β specific tyrosine phosphorylation similar to the PDGF-B. Additionally, PDGF-D enhances extracellular matrix accumulation comparable to the PDGF-B isoform. Conclusion PDGF-D signaling in pMF and HSC is identical to that of PDGF-B by binding to both PDGFR-α and -β.

Platelet-derived growth factor-D (PDGF-D) is a more recent recognized growth factor involved in the regulation of several cellular processes, including cell proliferation, transformation, invasion, and angiogenesis by binding to and activating its cognate receptor PDGFR-β. After bile duct ligation or in the carbon tetrachloride-induced hepatic fibrosis model, PDGF-D showed upregulation comparable to PDGF-B. Moreover, adenoviral PDGF-D gene transfer induced hepatic stellate cell proliferation and liver fibrosis. We here investigated the molecular mechanism of PDGF-D involvement in liver fibrogenesis. Therefore, the GRX mouse cell line was stimulated with PDGF-D and evaluated for fibrotic markers and PDGF-D signaling pathways in comparison to the other PDGF isoforms. We found that PDGF-D failed to enhance Col I and α-smooth muscle actin (α-SMA) production but has capacity to upregulate expression of the tissue inhibitor of metalloprotease 1 (TIMP-1) resulting in attenuation of MMP-2 and MMP-9 gelatinase activity as indicated by gelatinase zymography. This phenomenon was restored through application of a PDGF-D neutralizing antibody. Unexpectedly, PDGF-D incubation decreased both PDGFR-α and -β in mRNA and protein levels, and PDGF-D phosphorylated typrosines specific for PDGFR-α and -β. We conclude that PDGF-D intensifies fibrogenesis by interfering with the fibrolytic activity of the TIMP-1/MMP system and that PDGF-D signaling is mediated through both PDGF-α and -β receptors.

Cysteine-rich protein 61 (CCN1/CYR61) is a CCN ( C YR61, C TGF (connective tissue growth factor), and N OV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. Conclusion: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G0/G1-S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. Conclusion: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo. (Hepatology 2014;59:651–660)

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-kappa B essential modulator in hepatocytes (IKK gamma/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKK gamma/Nemo-deleted livers. We crossed hepatocyte-specific IKK gamma/Nemo knockout mice (Nemo(Delta hepa)) with constitutive TNFR1(-/-) and TRAIL(-/-) mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKK gamma/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury-a finding that has direct implications for treating chronic liver disease.

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2−/− mice were exposed to short term application of CCl4, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl4 intoxication showed increased liver damage in LCN2−/− mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2−/− mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4 weeks bile duct ligation or 8 weeks CCl4 application), LCN2−/− mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.

Das Adipositas-Schulungsprogramm M.O.B.I.L.I.S. wird in Kooperation mit lokalen Trainer-Arzte-Teams bundesweit an uber 100 Standorten angeboten. Es richtet sich an stark ubergewichtige Erwachsene (BMI 30–40 kg/m 2 ) und arbeitet interdisziplinar mit qualifizierten Fachkraften aus den Bereichen Bewegung, Psychologie/Padagogik, Ernahrung und Medizin. Die korperliche Aktivitat steht mit 40 praktischen Einheiten zwar im Fokus des einjahrigen Gruppenprogramms, aber Ernahrungsumstellung und die Frage der Verhaltensanderung sind ebenso zentrale Themen der insgesamt 20 theoretischen Sitzungen. Aufgrund einer Rahmenvereinbarung zwischen M.O.B.I.L.I.S. und der BARMER GEK ist eine pauschalisierte Kostenerstattung des Programms moglich. Fur 5025 Teilnehmer liegen Ergebnisse der Prozessevaluation vor; diese belegen Effizienz und Wirksamkeit des Konzepts als kostengunstige Therapieoption zum Einsatz bei adiposen Erwachsenen.

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257).

Liver fibrosis is a chronic wound healing process leading to liver scarring by excessive accumulation of extracellular matrix proteins including collagen. The main producers of liver collagen are myofibroblasts derived from activated hepatic stellate cells (HSC). Additionally, other cell types such as portal fibroblasts and bone marrow derived cells may contribute to extracellular matrix (ECM) production. Liver fibrosis develops on the basis of chronic liver injury induced e.g. by chronic viral hepatitis B or C infection, excessive alcohol abuse or fatty liver disease frequently associated with obesity (1). Although immune cells play an essential role in the modulation of liver fibrosis, its pathogenesis implicitly involves injury and proliferation of hepatic stellate cells, hepatocytes and potentially other cell species. Upon liver damage, dying hepatocytes stimulate remnant hepatocytes to re-enter the cell cycle in order to restore the original liver mass and function (2). Liver injury also stimulates HSC activation via complex mechanisms. This involves the conversion of a resting, vitamin A storing cell into a proliferating HSC without vitamin A droplets but capable of producing pro-inflammatory cytokines and ECM components such as collagen (3). The transition from quiescent (G0) cells into the active phase of the cell cycle is predominantly controlled by E-type cyclins and their associated kinase, Cdk2 (4). In mammals, two E-cyclins are known termed Cyclin E1 (CcnE1) and Cyclin E2 (CcnE2), respectively (5, 6). Despite their anticipated essential function for developmental and regenerative processes, single genetic inactivation of CcnE1, CcnE2 or Cdk2 does not affect viability or development in mice (7–10). However, fibroblasts deficient for both E-cyclins are unable to re-enter the cell cycle from G0 (9). We recently demonstrated that CcnE1 and CcnE2 play antagonistic roles in the regenerating liver following partial hepatectomy (11). Accordingly, CcnE2−/− livers show increased and prolonged CcnE1/Cdk2 activity, resulting in earlier and sustained DNA synthesis, hepatomegaly and excessive endoreplication of hepatocytes, whereas ablation of CcnE1 provoked only a moderate delay of hepatocyte proliferation. Earlier work using rat HSC indicated that HSC activation is associated with increased gene expression of CcnE, Cyclin D and induction of polyploidy (12). However, the precise role of E-type cyclins for activation and proliferation of HSC and subsequent liver fibrogenesis has remained elusive. In the present study, we aimed to investigate the contribution of E-type cyclins for liver fibrosis in vivo using constitutive CcnE1−/− and CcnE2−/− knockout mice and derived primary HSC. Our current work demonstrates that CcnE1, but not CcnE2 is essential for HSC survival, proliferation and liver fibrogenesis.

In a randomized, placebo-controlled, double-blind study, we tested a 4-week program in normobaric hypoxia that is commercially offered for the prevention of acute mountain sickness (AMS). Twenty-two male and 18 female healthy subjects [mean age 33 +/- 7 (SD) years] exercised 70 min, 3 x /week for 3 weeks on a bicycle ergometer at workloads of 60% VO2max either in normoxia (normoxia group, NG) or in normobaric hypoxia (hypoxia group, HG), corresponding to altitudes of 2500, 3000, and 3500 m during weeks 1, 2, and 3, respectively. Four passive exposures of 90 min in normoxia (NG) or hypoxia corresponding to 4500 m (HG) followed in week 4. Five days after the last session, subjects ascended within 24 h from sea level to 4559 m (one overnight stay at 3611 m) and stayed there for 24 h. AMS was defined as LL (Lake Louise score) > or =5 and AMS-C > or =0.70. The AMS incidence (70% in NG vs. 60% in HG, p = 0.74), LL scores (7.1 +/- 4.3 vs. 5.9 +/- 3.4, p = 0.34), and AMS-C scores (1.50 +/- 1.22 vs. 0.93 +/- 0.81, p = 0.25) at the study endpoint were not significantly different between the groups. However, the incidence of AMS at 3611 m (6% vs. 47%, p = 0.01) and the functional LL score at 4559 m were lower in HG. SpO2 at 3611 m, heart rate during ascents, and arterial blood gases at 4559 m were not different between groups. We conclude that the tested program does not reduce the incidence of AMS within a rapid ascent to 4559 m, but our data show that it prevents AMS at lower altitudes. Whether such a program would prevent AMS at higher altitudes, but with slower ascent, remains to be tested.

Bowel dysfunction is a frequent and serious side effect of opioid analgetics. In spite of its common occurrence, in the course of clinical routine, it is frequently ignored or underestimated. Authors of the analysed literature widely agree that a prophylactic and routine pharmacotherapy is necessary. For this purpose, laxatives, enemas and suppositories, prokinetic agents and opioid antagonists can be considered. Bulk-forming laxatives did not prove to be effective, since the quantity of fluid intake required for the action usually cannot be provided. Furthermore, the benefit of emollient agents is doubted. As a monotherapy they are not sufficient. By contrast, stimulant and osmotic laxatives proved to be active. Prokinetic drugs are not recommended because of their serious side effects. Effective abatement of opioid-induced obstipation by opioid antagonists has been proven in numerous studies. However, the loss of analgesia and opioid withdrawal symptoms were described as adverse effects. Development of quaternary opioid antagonists such as methylnaltrexone was allowed for mitigating these adverse effects.

Background & Aims Progression of alcoholic liver disease (ALD) can be influenced by genetic factors, which potentially include specific oncogenes and tumor suppressors. In the present study, we tested the hypothesis that aberrant expression of the proto-oncogene c-myc might exert a crucial role in the development of ALD. Methods Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR and immunohistochemistry. Mice with transgenic expression of c-myc in hepatocytes (alb-myc tg ) and wild-type (WT) controls were fed either control or ethanol (EtOH) containing Lieber-DeCarli diet for 4weeks to induce ALD. Results Hepatic c-myc was strongly upregulated in human patients with advanced ALD and in EtOH-fed WT mice. Transcriptome analysis indicated deregulation of pathways involved in ER-stress, p53 signaling, hepatic fibrosis, cell cycle regulation, ribosomal synthesis and glucose homeostasis in EtOH-fed alb-myc tg mice. Transgenic expression of c-myc in hepatocytes with simultaneous EtOH-uptake led to early ballooning degeneration, increased liver collagen deposition and hepatic lipotoxicity, together with excessive CYP2E1-derived reactive oxygen species (ROS) production. Moreover, EtOH-fed alb-myc tg mice exhibited substantial changes in mitochondrial morphology associated with energy dysfunction. Pathway analysis revealed that elevated c-myc expression and ethanol uptake synergistically lead to strong AKT activation, Mdm2 phosphorylation and as a consequence to inhibition of p53. Conclusions Expression of c-myc and EtOH-uptake synergistically accelerate the progression of ALD most likely due to loss of p53-dependent protection. Thus, c-myc is a new potential marker for the early detection of ALD and identification of risk patients.

Cellular signalling 28(1), 34.42 (2015). doi:10.1016/j.cellsig.2015.10.013, Published by Elsevier, Amsterdam [u.a.]

Der Deutsche Pflegerat begrust in seiner Stellungnahme zum Gesetzentwurf eines „Dritten Gesetzes zur Starkung der pflegerischen Versorgung und zur Anderung weiterer Vorschriften“ (PSG III) die Initiative der Bundesregierung, die kommunalen Strukturen zu starken und durch entsprechende Regelungen auszubauen. Weiter sieht der Deutsche Pflegerat e.V. (DPR) jedoch auch enormen Verbesserungsbedarf.

Gegenstand der vorliegenden Arbeit ist die Interrater Reliabilität des «Functional Independence Measure» (FIM). Der FIM ist ein psychometrisches Instrument zur Einschätzung der funktionalen Selbständigkeit bei Patienten mit Funktionseinschränkungen. Er erfasst Aktivitäten des Lebens mit 18 Items. Anhand einer siebenstufigen Ordinalskala zu jedem Item wird der gegenwärtige Grad der Selbständigkeit bewertet. Die Interrater Reliabilität wurde anhand einer Gelegenheitsstichprobe von 128 doppelten Einschätzungen mit dem FIM untersucht. Fünfzehn Pflegende führten diese Einschätzungen bei 30 Patienten mit Schädel-Hirn-Verletzungen in einer Rehabilitationseinrichtung durch. Das Design war korrelationell. Die Übereinstimmung der Einschätzungen wurde anhand der Kappa-Koeffizienten nach Cohen berechnet. Die Kappa-Koeffizienten der Item-Scores lagen zwischen kappa = 0,56 und kappa = 0,78, der Median der Kappa-Koeffizienten bei kappa = 0,65. Das Ergebnis belegt eine mittlere bis hohe Interrater Reliabilität des FIM bei der Einschätzung von Patienten mit Schädel-Hirn-Verletzungen durch Pflegende.

Background and Aims CCN3/NOV, a matricellular protein of the CYR61-CTGF-NOV (CCN) family, comprises six secreted proteins that associate specifically with the extracellular matrix. CCN proteins lack specific high-affinity receptors; instead, they regulate crucial biological processes, such as fibrosis, by signalling via integrins and proteoglycans. Recent studies have linked overexpression of CCN3/NOV to mitigate kidney fibrosis. This study aims to investigate CCN3/NOV overexpression in liver fibrogenesis in vivo. Methods The biological efficacy of adenoviral expressed CCN3/NOV directed under transcriptional control of the constitutively active Cytomegalovirus promoter (Ad-NOV) was analysed in a bile duct ligation model and in cultured primary hepatocytes. Results and Conclusions Even though Ad-NOV gene transfer in a 3-week bile duct ligation mouse model showed the expected high levels of CCN3/NOV in both mRNA and protein, it failed to reduce liver fibrogenesis, but instead enhanced hepatocyte apoptosis. Furthermore, overexpressed CCN3/NOV in cultured primary hepatocytes resulted in decreased levels of CCN2/CTGF, the profibrotic marker protein in liver fibrosis. Both Ad-NOV and Ad-CTGF induced reactive oxygen species production, enhanced p38 and JNK activation. Therefore, we conclude that CCN3/NOV overexpression in vivo is insufficient to mitigate liver fibrogenesis because of the induction of hepatocyte injury and apoptosis.

Journal of inflammation 9(1), 49 (2012). doi:10.1186/1476-9255-9-49, Published by Wiley-Liss, New York, NY [u.a.]