The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

Exploring the impact of severity in hepatic fibrosis disease on the intrahepatic distribution of novel biodegradable nanoparticles targeted towards different disease biomarkers.

Nanoparticle-based drug delivery systems (DDS) have shown promising results in reversing hepatic fibrosis, a common pathological basis of chronic liver diseases (CLDs), in preclinical animal models. However, none of these nanoparticle formulations has transitioned to clinical usage and there are currently no FDA-approved drugs available for liver fibrosis. This highlights the need for a better understanding of the challenges faced by nanoparticles in this complex disease setting. Here, we have systematically studied the impact of targeting strategy, the degree of macrophage infiltration during fibrosis, and the severity of fibrosis, on the liver uptake and intrahepatic distribution of nanocarriers. When tested in mice with advanced liver fibrosis, we demonstrated that the targeting ligand density plays a significant role in determining the uptake and retention of the nanoparticles in the fibrotic liver whilst the type of targeting ligand modulates the trafficking of these nanoparticles into the cell population of interest - activated hepatic stellate cells (aHSCs). Engineering the targeting strategy indeed reduced the uptake of nanoparticles in typical mononuclear phagocyte (MPS) cell populations, but not the infiltrated macrophages. Meanwhile, additional functionalization may be required to enhance the efficacy of DDS in end-stage fibrosis/cirrhosis compared to early stages.

Therapeutic potential of macrophage colony-stimulating factor in chronic liver disease.

Resident and recruited macrophages control the development and proliferation of the liver. We have previously shown in multiple species that treatment with a macrophage colony stimulating factor (CSF1)-Fc fusion protein initiated hepatocyte proliferation and promoted repair in models of acute hepatic injury in mice. Here, we investigated the impact of CSF1-Fc on resolution of advanced fibrosis and liver regeneration, using a non-resolving toxin-induced model of chronic liver injury and fibrosis in C57BL/6J mice. Co-administration of CSF1-Fc with exposure to thioacetamide (TAA) exacerbated inflammation consistent with monocyte contributions to initiation of pathology. After removal of TAA, either acute or chronic CSF1-Fc treatment promoted liver growth, prevented progression and promoted resolution of fibrosis. Acute CSF1-Fc treatment was also anti-fibrotic and pro-regenerative in a model of partial hepatectomy in mice with established fibrosis. The beneficial impacts of CSF1-Fc treatment were associated with monocyte-macrophage recruitment and increased expression of remodelling enzymes and growth factors. These studies indicate that CSF1-dependent macrophages contribute to both initiation and resolution of fibrotic injury and that CSF1-Fc has therapeutic potential in human liver disease.

Overexpression of miRNA-25-3p inhibits Notch1 signaling and TGF-ß-induced collagen expression in hepatic stellate cells.

During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-ß and its type 1 receptor (TGFBR1) mRNA expression, TGF-ß-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-ß-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-ß signaling.

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

Serum MicroRNAs as Biomarkers in Hepatitis C: Preliminary Evidence of a MicroRNA Panel for the Diagnosis of Hepatocellular Carcinoma.

Early diagnosis of cirrhosis and hepatocellular carcinoma (HCC) due to chronic Hepatitis C (CHC) remain clinical priorities. In this pilot study, we assessed serum microRNA (miRNA) expression to distinguish cirrhosis and HCC, alone and in combination with the aminotransferase-to-platelet ratio (APRI), Fibrosis 4 (FIB-4), and alpha-fetoprotein (AFP). Sixty CHC patients were subdivided into 3 cohorts: Mild disease (fibrosis stage F0-2; n = 20); cirrhosis (n = 20); and cirrhosis with HCC (n = 20). Circulating miRNA signatures were determined using a liver-specific real-time quantitative reverse transcription PCR (qRT-PCR) microarray assessing 372 miRNAs simultaneously. Differentially-expressed miRNA candidates were independently validated using qRT-PCR. Serum miRNA-409-3p was increased in cirrhosis versus mild disease. In HCC versus cirrhosis, miRNA-486-5p was increased, whereas miRNA-122-5p and miRNA-151a-5p were decreased. A logistic regression model-generated panel, consisting of miRNA-122-5p + miRNA-409-3p, distinguished cirrhosis from mild disease (area under the curve, AUC = 0.80; sensitivity = 85%, specificity = 70%; p < 0.001). When combined with FIB-4 or APRI, performance was improved with AUC = 0.89 (p < 0.001) and 0.87 (p < 0.001), respectively. A panel consisting of miRNA-122-5p + miRNA-486-5p + miRNA-142-3p distinguished HCC from cirrhosis (AUC = 0.94; sensitivity = 80%, specificity = 95%; p < 0.001), outperforming AFP (AUC = 0.64, p = 0.065). Serum miRNAs are differentially expressed across the spectrum of disease severity in CHC. MicroRNAs have great potential as diagnostic biomarkers in CHC, particularly in HCC where they outperform the only currently-used biomarker, AFP.

Repair of cartilage defects with devitalized osteochondral tissue: A pilot animal study.

Devitalization using high hydrostatic pressure (HHP) treatment inactivates cells while matrix structure and biomechanical properties are maintained. Because of strong chondroinductive potential of HHP-devitalized cartilage matrix, it may be used as scaffold for reconstruction of (osteo-)chondral lesions. In this pilot study, we evaluated the feasibility of HHP-devitalized osteochondral tissue to repair osteochondral defects in a rabbit model. Removal and reimplantation of osteochondral plugs were performed in 12 female New Zealand White rabbits. From the knee joint of each animal, osteochondral plugs (diameter = 4 mm; depth = 2.5 mm) were harvested and devitalized by HHP (452 MPa for 10 min). Afterward, the plugs were reimplanted into the respective cavity, from where they were taken. Animals were sacrificed 12 weeks postoperatively and the integration of osteochondral plugs was examined using µ-CT, MRI, and histological staining. Furthermore, revitalization of HHP-treated osteochondral plugs was characterized by gene expression analyses. Macroscopic evaluation of tissue repair at implantation sites of HHP-treated osteochondral plugs showed an adequate defect filling 12 weeks after implantation. Plug margins were hardly detectable indicating successful tissue integration. Additionally, gene expression analyses demonstrated initial revitalization of the HHP-treated tissue 12 weeks postoperatively. Our preliminary data revealed that HHP-treated osteochondral plugs could be used to refill osteochondral defects in the knee joint and promote cell migration into defect site. Data indicated that HHP-treated tissue has the potential to act as functional scaffolds for reconstruction of cartilage defects. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2354-2364, 2019.

Liver-specific Repin1 deficiency impairs transient hepatic steatosis in liver regeneration.

Transient hepatic steatosis upon liver resection supposes functional relationships between lipid metabolism and liver regeneration. Repin1 has been suggested as candidate gene for obesity and dyslipidemia by regulating key genes of lipid metabolism and lipid storage. Herein, we characterized the regenerative potential of mice with a hepatic deletion of Repin1 (LRep1-/-) after partial hepatectomy (PH) in order to determine the functional significance of Repin1 in liver regeneration. Lipid dynamics and the regenerative response were analyzed at various time points after PH. Hepatic Repin1 deficiency causes a significantly decreased transient hepatic lipid accumulation. Defects in lipid uptake, as analyzed by decreased expression of the fatty acid transporter Cd36 and Fatp5, may contribute to attenuated and shifted lipid accumulation, accompanied by altered extent and chronological sequence of liver cell proliferation in LRep1-/- mice. In vitro steatosis experiments with primary hepatocytes also revealed attenuated lipid accumulation and occurrence of smaller lipid droplets in Repin1-deficient cells, while no direct effect on proliferation in HepG2 cells was observed. Based on these results, we propose that hepatocellular Repin1 might be of functional significance for early accumulation of lipids in hepatocytes after PH, facilitating efficient progression of liver regeneration.

Microcirculatory disturbances and cellular changes during progression of hepatic steatosis to liver tumors.

Non-alcoholic fatty liver disease is closely associated with metabolic syndrome and comprises a pathological spectrum of liver disease ranging from steatosis to steatohepatitis and can progress to fibrosis/cirrhosis and hepatocellular carcinoma. In 2013, a mouse model was described that mimics non-alcoholic fatty liver disease progression from steatohepatitis to tumors in a short time span and with high incidence. As microcirculatory disturbances play a crucial role in liver disease, the suitability of the steatosis-inflammation-tumor model for microcirculatory studies was assessed. Herein, we present a comprehensive view on morphological, microvascular, cellular, and functional aspects of non-alcoholic fatty liver disease progression in the steatosis-inflammation-tumor model using intravital microscopy, biochemical, and histological techniques. Mice develop steatohepatitis, mild fibrosis, and liver tumors at ages of 6, 12, and 20 weeks, respectively. Non-alcoholic fatty liver disease progression was accompanied by several general aspects of disease severity like increasing liver/body weight index, non-alcoholic fatty liver disease activity score, and hepatocellular apoptosis. Intravital microscopic analysis revealed significant changes in hepatic microcirculation with increasing structural alterations, elevated leukocyte adherence, and impaired nutritive perfusion. Non-alcoholic fatty liver disease was further characterized by a lower sinusoidal density with a striking rise at 20 weeks. The characteristic microcirculatory changes make the model a convenient tool for analysis of microcirculation during progression from steatosis to liver tumor. Impact statement Significant alterations of microcirculation contribute to progression of NAFLD, a chronic liver disease with increasing medical and socio-economic impact. Characterization of microcirculation in a NAFLD model reflecting all relevant stages of disease progression was still missing. Thus, we evaluated microcirculatory and cellular changes in a steatosis-inflammation-tumor model using in vivo microscopy. Analyses revealed increasing structural alterations, elevated leukocyte-endothelial interaction, and impaired nutritive perfusion. Thus, this model is suitable for further studies investigating therapeutic approaches targeting these progressive microcirculatory disturbances.

No significant impact of Foxf1 siRNA treatment in acute and chronic CCl4 liver injury.

Chronic liver injury of any etiology is the main trigger of fibrogenic responses and thought to be mediated by hepatic stellate cells. Herein, activating transcription factors like forkhead box f1 are described to stimulate pro-fibrogenic genes in hepatic stellate cells. By using a liver-specific siRNA delivery system (DBTC), we evaluated whether forkhead box f1 siRNA treatment exhibit beneficial effects in murine models of acute and chronic CCl4-induced liver injury. Systemic administration of DBTC-forkhead box f1 siRNA in mice was only sufficient to silence forkhead box f1 in acute CCl4 model, but was not able to attenuate liver injury as measured by liver enzymes and necrotic liver cell area. Therapeutic treatment of mice with DBTC-forkhead box f1 siRNA upon chronic CCl4 exposition failed to inhibit forkhead box f1 expression and hence lacked to diminish hepatic stellate cells activation or fibrosis development. As a conclusion, DBTC-forkhead box f1 siRNA reduced forkhead box f1 expression in a model of acute but not chronic toxic liver injury and showed no positive effects in either of these mice models. Impact statement As liver fibrosis is a worldwide health problem, antifibrotic therapeutic strategies are urgently needed. Therefore, further developments of new technologies including validation in different experimental models of liver disease are essential. Since activation of hepatic stellate cells is a key event upon liver injury, the activating transcription factor forkhead box f1 (Foxf1) represents a potential target gene. Previously, we evaluated Foxf1 silencing by a liver-specific siRNA delivery system (DBTC), exerting beneficial effects in cholestasis. The present study was designed to confirm the therapeutic potential of Foxf1 siRNA in models of acute and chronic CCl4-induced liver injury. DBTC-Foxf1 siRNA was only sufficient to silence Foxf1 in acute CCl4 model and did not ameliorate liver injury or fibrogenesis. This underlines the significance of the experimental model used. Each model displays specific characteristics in the pathogenic nature, time course and severity of fibrosis and the optimal time point for starting a therapy.

Differential Effects of Axin2 Deficiency on the Fibrogenic and Regenerative Response in Livers of Bile Duct-Ligated Mice.

BACKGROUND: Wnt signaling is involved in the pathogenesis of liver fibrosis. Axin2 is a negative regulator of the canonical Wnt pathway by promoting ß-catenin degradation. ß-Catenin-activating and loss-of-function mutations of Axin2 are thought to be functionally relevant for liver diseases and cancer. Thus, we hypothesized that Axin2 deficiency promotes fibrogenesis. METHODS: As the functions and mechanisms of how Axin2/ß-catenin signaling participates in the progression of liver fibrosis are unclear, we investigated the progression of liver fibrosis in Axin2-deficient mice using Axin2-LacZ reporter mice (Axin2+/-, Axin2-/-, and Axin2+/+) which underwent bile duct ligation (BDL). RESULTS: Here, we show that the expression of Axin2 is downregulated during fibrogenesis in wild-type mice, which is consistent with a decreased expression of the reporter gene LacZ in Axin2+/- and Axin2-/- mice. Surprisingly, no alteration in active ß-catenin/Wnt signaling occurs in Axin2-deficient mice upon BDL. Despite a less pronounced liver injury, Axin2 deficiency had only minor and no significant effects on the fibrogenic response upon BDL, i.e. slightly reduced hepatic stellate cell activity and collagen mRNA expression. However, livers of Axin2-/- mice shared a stronger cell proliferation both already at baseline as well as immediately after BDL. CONCLUSION: Our results strongly suggest, contrary to expectation, that a deficiency in Axin2 is not equivalent to an increase in active ß-catenin and target genes, indicating no functional relevance of Axin2-dependent regulation of the canonical Wnt/ß-catenin pathway in the progression of cholestatic liver injury. This also suggests that the negligible effects of Axin2 deficiency during fibrogenesis may be related to an alternative pathway.

Foxf1 siRNA delivery to hepatic stellate cells by DBTC lipoplex formulations ameliorates fibrosis in livers of bile duct ligated mice.

Activation of hepatic stellate cells (HSCs) is a key event in pathogenesis of liver fibrosis and represents an orchestral interplay of inhibiting and activating transcription factors like forkhead box f1 (Foxf1), being described to stimulate pro-fibrogenic genes in HSCs. Here, we evaluated a lipidbased liver-specific delivery system (DBTC) suitable to transfer Foxf1 siRNA specifically to HSCs and examined its antifibrotic potential on primary HSCs and LX-2 cells as well as in a murine model of bile duct ligation (BDL)-induced secondary cholestasis. Foxf1 silencing reduced proliferation capacity and attenuated contractility of HSCs. Systemic administration of DBTC-lipoplexes in mice was sufficient to specifically silence genes expressed in different liver cell types. Using intravital and immunofluorescence microscopy we confirmed the specific delivery of Cy3-labeled DBTC to the liver, and particularly to HSCs. Repeated treatment with DBTC-lipoplexes resulted in siRNA-mediated silencing of Foxf1 early after BDL and finally attenuated progression of the fibrotic process. Decreased HSC activation in-effect ameliorated liver injury as shown by substantial reduction of necrotic area and deposition of extracellular matrix. Our findings suggest that Foxf1 may serve as a target gene to disrupt progression of liver fibrosis and DBTC might provide a potentially feasible and effective tool for HSC-specific delivery of therapeutic RNA.

Protein z exerts pro-angiogenic effects and upregulates CXCR4.

OBJECTIVE: Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human peripheral arterial disease. However, the role of PZ in ischemia-driven angiogenesis and vascular healing processes has not been elucidated so far. APPROACH: Angiogenic potency of PZ was assessed in established in vitro assays using endothelial cells. PZ-deficient (PZ(-/-)) mice and their wild-type littermates (PZ(+/+)) were subjected to hindlimb ischemia. Furthermore, PZ(-/-) mice were exposed to PZ expressing adenovirus (AdV-PZ) or control adenovirus (AdV-GFP). In an additional set of animals, PZ(-/-) mice were exposed to AdV-PZ and AdV-GFP, each in combination with the CXCR4 antagonist AMD3100. RESULTS: In vitro, PZ stimulated migratory activity and capillary-like tube formation of endothelial cells comparable to SDF-1. PZ(-/-) mice exhibited diminished hypoxia-driven neovascularization and reperfusion in post-ischemic hindlimbs, which was restored by adenoviral gene transfer up to levels seen in PZ(+/+) mice. The stimulatory impact of PZ on endothelial cells in vitro was abolished by siRNA targeting against PZ and PZ was not able to restore reduced migration after knock-down of CXCR4. The increased surface expression of CXCR4 on PZ-stimulated endothelial cells and the abrogated restoration of PZ(-/-) mice via AdV-PZ after concomitant treatment with the CXCR4 antagonist AMD3100 supports the idea that PZ mediates angiogenesis via a G-protein coupled pathway and involves the SDF-1/CXCR4 axis. This is underlined by the fact that addition of the G-protein inhibitor PTX to PZ-stimulated endothelial cells abolished the effect of PZ on capillary-like tube formation. CONCLUSIONS: The results of the current study reveal a role of PZ in ischemia-induced angiogenesis, which involves a G-protein coupled pathway and a raised surface expression of CXCR4. Our findings thereby extend the involvement of PZ from the coagulation cascade to a beneficial modulation of vascular homeostasis.

Protein Z-deficiency is associated with enhanced neointima formation and inflammatory response after vascular injury in mice.

BACKGROUND: Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human atherosclerotic vascular diseases. The aim of this study was to investigate the role of PZ in vascular arterial disease. MATERIAL AND METHODS: PZ-deficient (PZ(-/-)) mice and their wild-type littermates (PZ(+/+)) were subjected to unilateral carotid artery injury by using ferric chloride and dissected 21 days thereafter for histological analysis. Human aortic smooth muscle cells (SMC) were used for in vitro wound healing assay to assess the influence of PZ on SMC migration and for cell proliferation studies. RESULTS: Morphometric analysis of neointima formation revealed a significantly increased area and thickness of the neointima and subsequently increased luminal stenosis in carotid arteries of PZ(-/-) mice compared to PZ(+/+) mice (p < 0.05, n = 9). Immunohistochemical analysis of neointima lesion composition revealed significantly higher numbers of PCNA-positive and α-SMA-positive cells in the neointima of PZ(-/-) mice. Furthermore, PZ showed an anti-migratory potency in in vitro wound healing assay with SMCs, while no effect of PZ on SMC proliferation was detectable. Conclusion: PZ contributes to a reduced neointima formation after vascular injury, underlining the modulatory role of the coagulation cascade in vascular homeostasis.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC.

Diabetes increases pancreatic fibrosis during chronic inflammation.

Diabetes and fibrosis can be concurrent processes in several diseases such as cystic fibrosis or chronic pancreatitis. To evaluate whether diabetes can influence fibrosis and thus aggravate the pathological process, the progression of chronic pancreatitis was assessed in diabetic and non diabetic mice. For this purpose, insulin producing beta-cells in C57Bl/6J mice were selectively impaired by administration of streptozotocin. Chronic pancreatitis was then induced by repetitive administration of cerulein in normoglycaemic and hyperglycaemic mice. Diabetes caused enhanced collagen I deposition within three weeks of the onset of chronic pancreatitis and increased the proliferation of interstitial cells. This was accompanied by an increased number of interlobular fibroblasts, which expressed S100A4 (fibroblast-specific protein-1) and stimulation of α-smooth muscle actin expression of pancreatic stellate cells. In addition, the observed aggravation of chronic pancreatitis by diabetes also led to a significantly enhanced atrophy of the pancreas, increased infiltration of inflammatory chloracetate esterase positive cells and enhanced acinar cell death. We conclude that diabetes has a detrimental influence on the progression of chronic pancreatitis by aggravating fibrosis, inflammation and pancreatic atrophy.

Genetic dissection of plexin signaling in vivo.

Mammalian plexins constitute a family of transmembrane receptors for semaphorins and represent critical regulators of various processes during development of the nervous, cardiovascular, skeletal, and renal system. In vitro studies have shown that plexins exert their effects via an intracellular R-Ras/M-Ras GTPase-activating protein (GAP) domain or by activation of RhoA through interaction with Rho guanine nucleotide exchange factor proteins. However, which of these signaling pathways are relevant for plexin functions in vivo is largely unknown. Using an allelic series of transgenic mice, we show that the GAP domain of plexins constitutes their key signaling module during development. Mice in which endogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versions harboring mutations in the GAP domain recapitulate the phenotypes of the respective null mutants in the developing nervous, vascular, and skeletal system. We further provide genetic evidence that, unexpectedly, the GAP domain-mediated developmental functions of plexins are not brought about via R-Ras and M-Ras inactivation. In contrast to the GAP domain mutants, Plexin-B2 transgenic mice defective in Rho guanine nucleotide exchange factor binding are viable and fertile but exhibit abnormal development of the liver vasculature. Our genetic analyses uncover the in vivo context-dependence and functional specificity of individual plexin-mediated signaling pathways during development.

Impact of hyperglycemia and acute pancreatitis on the receptor for advanced glycation endproducts.

Since hyperglycemia aggravates acute pancreatitis and also activates the receptor for advanced glycation endproducts (RAGE) in other organs, we explored if RAGE is expressed in the pancreas and if its expression is regulated during acute pancreatitis and hyperglycemia. Acute pancreatitis was induced by cerulein in untreated and streptozotocin treated diabetic mice. Expression of RAGE was analyzed by Western blot and immunohistochemistry. To evaluate signal transduction the phosphorylation of ERK1/ERK2 was assessed by Western blot and the progression of acute pancreatitis was monitored by evaluation of lipase activity and the pancreas wet to dry weight ratio. RAGE is mainly expressed by acinar as well as interstitial cells in the pancreas. During acute pancreatitis infiltrating inflammatory cells also express RAGE. Using two distinct anti-RAGE antibodies six RAGE proteins with diverse molecular weight are detected in the pancreas, whereas just three distinct RAGE proteins are detected in the lung. Hyperglycemia, which aggravates acute pancreatitis, significantly reduces the production of two RAGE proteins in the inflamed pancreas.

Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo.

Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGFp) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGFp to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGFp to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGFp transduce activated HSCs better than Ad.GFP-PEG-NGFp. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGFp.

Chimeric rabies viruses for trans-species comparison of lyssavirus glycoprotein ectodomain functions in virus replication and pathogenesis.

The glycoprotein G of lyssaviruses is the major determinant of virus pathogenicity and serves as a target for immunological responses to virus infections. However, assessment of the exact contribution of lyssavirus G proteins to observed differences in the pathogenicity of lyssavirus species is challenging, since the direct comparison of natural lyssaviruses does not allow specific ascription to individual virus proteins or domains. Here we describe the generation and characterization of recombinant rabies viruses (RABV) that express chimeric G proteins comprising of a RABV cytoplasma domain fused to transmembrane and ectodomain G sequences of a virulent RABV (challenge virus standard; CVS-11) or two European bat lyssaviruses (EBLV- and EBLV-2). These "envelope-switched" recombinant viruses were recovered from cDNAs. Similar growth kinetics and protein expression in neuroblastoma cell cultures and successful targeting of primary neurons showed that the chimeric G proteins were able to replace the authentic G protein in a RABV based virus vector. Inoculation of six week old CD-1 mice by the intracranial (i. c.) route of infection further demonstrated that all recombinant viruses were able to spread in the brain and to induce disease. The "envelope-switched" RABV therefore represent an important tool to further investigate the influence of lyssavirus ectodomains on virus tropism, and pathogenicity.