Rainer M. Bohle

The etiology of liver damage imparts cytokines transforming growth factor β1 or interleukin-13 as driving forces in fibrogenesis†

It is unknown whether transforming growth factor β1 (TGF‐β1) signaling uniformly participates in fibrogenic chronic liver diseases, irrespective of the underlying origin, or if other cytokines such as interleukin (IL)‐13 share in fibrogenesis (e.g., due to regulatory effects on type I pro‐collagen expression). TGF‐β1 signaling events were scored in 396 liver tissue samples from patients with diverse chronic liver diseases, including hepatitis B virus (HBV), hepatitis C virus (HCV), Schistosoma japonicum infection, and steatosis/steatohepatitis. Phospho‐Smad2 staining correlated significantly with fibrotic stage in patients with HBV infection (n = 112, P < 0.001) and steatosis/steatohepatitis (n = 120, P < 0.01), but not in patients with HCV infection (n = 77, P > 0.05). In tissue with HBx protein expression, phospho‐Smad2 was detectable, suggesting a functional link between viral protein expression and TGF‐β1 signaling. For IL‐13, immunostaining correlated with fibrotic stage in patients with HCV infection and steatosis/steatohepatitis. IL‐13 protein was more abundant in liver tissue lysates from three HCV patients compared with controls, as were IL‐13 serum levels in 68 patients with chronic HCV infection compared with 20 healthy volunteers (72.87 ± 26.38 versus 45.41 ± 3.73, P < 0.001). Immunohistochemistry results suggest that IL‐13–mediated liver fibrogenesis may take place in the absence of phospho–signal transducer and activator of transcription protein 6 signaling. In a subgroup of patients with advanced liver fibrosis (stage ≥3), neither TGF‐β nor IL‐13 signaling was detectable. Conclusion: Depending on the cause of liver damage, a predominance of TGF‐β or IL‐13 signaling is found. TGF‐β1 predominance is detected in HBV‐related liver fibrogenesis and IL‐13 predominance in chronic HCV infection. In some instances, the underlying fibrogenic mediator remains enigmatic. (HEPATOLOGY 2009.)

Progranulin antibodies in autoimmune diseases

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasus arteritis (4/13), classical panarteritis nodosa (4/10), Behcets disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

IGF2 mRNA binding protein p62/IMP2-2 in hepatocellular carcinoma: antiapoptotic action is independent of IGF2/PI3K signaling

The insulin-like growth factor II (IGF2) mRNA binding protein (IMP) p62/IMP2-2, originally isolated from a hepatocellular carcinoma (HCC) patient, induces a steatotic phenotype when overexpressed in mouse livers. Still, p62 transgenic livers do not show liver cell damage but exhibit a pronounced induction of Igf2 and activation of the downstream survival kinase AKT. The aim of this study was to investigate the relation between p62 and IGF2 expression in the human system and to study potential antiapoptotic actions of p62. p62 and IGF2 mRNA levels were assessed by real-time RT-PCR. For knockdown and overexpression experiments, human hepatoma HepG2 and PLC/PRF/5 cells were transfected with siRNA or plasmid DNA. Phosphorylated AKT and ERK1/2 were analyzed by Western blot. Investigations of 32 human HCC tissues showed a strong correlation between p62 and IGF2 expression. Of note, p62 expression was increased markedly in patients with poor outcome. In hepatoma cells overexpression of p62 lowered levels of doxorubicin-induced caspase-3-like activity. Vice versa, knockdown of p62 resulted in increased doxorubicin-induced apoptosis. However, neither PI3K inhibitors nor a neutralizing IGF2 antibody showed any effects. Western blot analysis revealed increased levels of phosphorylated ERK1/2 in hepatoma cells overexpressing p62 and decreased levels in p62 knockdown experiments. When p62-overexpressing cells were treated with ERK1/2 inhibitors, the apoptosis-protecting effect of p62 was completely abrogated. Our data demonstrate that p62 exerts IGF2-independent antiapoptotic action, which is facilitated via phosphorylation of ERK1/2. Furthermore, p62 might serve as a new prognostic marker in HCC.

OVEREXPRESSION OF THE IGF2-mRNA BINDING PROTEIN p62 IN TRANSGENIC MICE INDUCES A STEATOTIC PHENOTYPE

BACKGROUND & AIMS The insulin-like growth-factor 2 (IGF2) mRNA binding protein p62 is highly expressed in hepatocellular carcinoma tissue. Still, its potential role in liver disease is largely unknown. In this study, we investigated pathophysiological implications of p62 overexpression in mice. METHODS We generated mice overexpressing p62 under a LAP-promotor. mRNA expression levels and stability were examined by real-time RT-PCR. Allele-specific expression of Igf2 and H19 was assessed after crossing mice with SD7 animals. The Igf2 downstream mediators pAKT and PTEN were determined by Western blot. RESULTS Hepatic p62 overexpression neither induced inflammatory processes nor liver damage. However, 2.5week old transgenic animals displayed a steatotic phenotype and improved glucose tolerance. p62 overexpression induced the expression of the imprinted genes Igf2 and H19 and their transcriptional regulator Aire (autoimmune regulator). Neither monoallelic expression nor mRNA stability of Igf2 and H19 was affected. Investigating Igf2 downstream signalling pathways showed increased AKT activation and attenuated PTEN expression. CONCLUSIONS The induction of a steatotic phenotype implies that p62 plays a role in hepatic pathophysiology.

Clinically driven design of multi-scale cancer models: the ContraCancrum project paradigm

The challenge of modelling cancer presents a major opportunity to improve our ability to reduce mortality from malignant neoplasms, improve treatments and meet the demands associated with the individualization of care needs. This is the central motivation behind the ContraCancrum project. By developing integrated multi-scale cancer models, ContraCancrum is expected to contribute to the advancement of in silico oncology through the optimization of cancer treatment in the patient-individualized context by simulating the response to various therapeutic regimens. The aim of the present paper is to describe a novel paradigm for designing clinically driven multi-scale cancer modelling by bringing together basic science and information technology modules. In addition, the integration of the multi-scale tumour modelling components has led to novel concepts of personalized clinical decision support in the context of predictive oncology, as is also discussed in the paper. Since clinical adaptation is an inelastic prerequisite, a long-term clinical adaptation procedure of the models has been initiated for two tumour types, namely non-small cell lung cancer and glioblastoma multiforme; its current status is briefly summarized.

Sec62 bridges the gap from 3q amplification to molecular cell biology in non-small cell lung cancer.

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Fatty Acid Elongation in Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma

Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.

In Vitro and In Vivo Characterization of Nonbiomedical- and Biomedical-Grade Alginates for Articular Chondrocyte Transplantation

Alginate is a key hydrogel for cartilage tissue engineering. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to support the in vitro culture and in vivo transplantation of articular chondrocytes. Chondrocytes in all ultrapure alginates maintained high cell viability. Spheres composed of biomedical-grade, low-viscosity, high-mannuronic acid content alginate showed the lowest decrease in size over time. Biomedical-grade, low-viscosity, high-guluronic acid content alginate allowed for optimal cell proliferation. Biomedical-grade, medium-viscosity, high-mannuronic acid content alginate promoted the highest production of proteoglycans. When transplanted into osteochondral defects in the knee joint of sheep in vivo, empty spheres were progressively surrounded by a granulation tissue. In marked contrast with these observations, all alginate spheres carrying allogeneic chondrocytes were gradually invaded by a granulation tissue containing multinucleated giant cells, lymphocytes, and fibroblasts, regardless whether they were based on biomedical- or nonbiomedical-grade alginates. After 21 days in vivo, transplanted chondrocytes were either viable or underwent necrosis, and apoptosis played a minor role in their early fate. The individual characteristics of these alginates may be valuable to tailor specific experimental and clinical strategies for cartilage tissue engineering.

3-Deazaadenosine inhibits vasa vasorum neovascularization in aortas of ApoE(-/-)/LDL(-/-) double knockout mice.

BACKGROUND Atherosclerosis and inflammation/angiogenesis are strongly associated including growth of vasa vasorum (VV) and plaque neovascularization, but a causative role for neovascularization has still not been established. Hence, we investigated the effect of 3-deazaadenosine (c(3)Ado), an anti-inflammatory and anti-proliferative drug, on plaque progression and VV neovascularization in apoE(-/-)/LDL(-/-) double knockout mice. METHODS The arterial trees from apoE(-/-)/LDL(-/-) mice with, or without c(3)Ado at the age of 16 weeks (n=10), 18 weeks (n=8) and 20 weeks (n=7) were infused in situ with Microfil, and the aortas harvested and scanned with micro-CT (12mum cubic voxel). We characterized plaque volume and VV luminal volume along the descending aorta using Analyze 6.0 software. Cellular effects of c(3)Ado on human endothelial and vascular smooth muscle cells were investigated in cell cultures and on nylon cDNA expression arrays. RESULTS Lesions spatially connected to VV increased from 16 to 20 weeks significantly (p<0.001). The volume of atherosclerotic lesions was significantly reduced in animals treated with c(3)Ado (p<0.01). This was accompanied by a significant decrease of vasa vasorum neovascularization along the descending aorta (p<0.01). Using nylon cDNA expression arrays, we identified the regulation of anti-proliferative, anti-inflammatory genes in human smooth muscle cells which might be involved in the anti-angiogenic effects of c(3)Ado. Moreover, c(3)Ado dose-dependently prevented the proliferation and migration of human coronary artery endothelial cells in vitro. CONCLUSION The smaller lesion volume in animals treated with c(3)Ado was closely associated with a reduced VV neovascularization, suggesting a direct relationship between lesion growth and VV development.

AP-1 and STAT-1 decoy oligodeoxynucleotides attenuate transplant vasculopathy in rat cardiac allografts

AIMS Cardiac allograft vasculopathy (CAV) continues to be an unsolved clinical problem requiring the development of new therapeutic strategies. We have previously demonstrated that ex vivo donor allograft treatment with decoy oligodeoxynucleotides (ODN) targeting the transcription factors, activator protein-1 (AP-1) or signal transducer and activator of transcription-1 (STAT-1), delays acute rejection and prolongs cardiac allograft survival. Here, we investigated whether this treatment regime also prevents the occurrence of CAV in a fully allogeneic rat heart transplantation model. METHODS AND RESULTS Wistar-Furth rat cardiac allografts were perfused ex vivo with AP-1 decoy ODN, STAT-1 decoy ODN, or buffer solution and transplanted into the abdomen of Lewis rats immunosuppressed with cyclosporine. Treatment with both decoy ODNs but not vehicle significantly attenuated the incidence and severity of CAV. Laser-assisted microdissection/real-time polymerase chain reaction as well as immunohistochemistry analyses revealed a significant increase in CD40 abundance in the coronary endothelial cells and medial smooth muscle cells on day 1 post transplantation which was virtually abolished upon AP-1 or STAT-1 decoy ODN treatment. While the AP-1 decoy ODN primarily attenuated basal CD40 expression, the STAT-1 decoy ODN suppressed tumour necrosis factor-alpha-/interferon-gamma-stimulated expression of CD40 in rat native endothelial cells. CONCLUSION Treating donor hearts with decoy ODNs neutralizing AP-1 or STAT-1 at the time of transplantation prevents upregulation of CD40 expression in the graft coronary arteries and effectively inhibits CAV.