Dual proteotoxic stress accelerates liver injury via activation of p62-Nrf2.

Protein accumulation is the hallmark of various neuronal, muscular, and other human disorders. It is also often seen in the liver as a major protein-secretory organ. For example, aggregation of mutated alpha1-antitrypsin (AAT), referred to as PiZ, is a characteristic feature of AAT deficiency, whereas retention of hepatitis B surface protein (HBs) is found in chronic hepatitis B (CHB) infection. We investigated the interaction of both proteotoxic stresses in humans and mice. Animals overexpressing both PiZ and HBs (HBs-PiZ mice) had greater liver injury, steatosis, and fibrosis. Later they exhibited higher hepatocellular carcinoma load and a more aggressive tumor subtype. Although PiZ and HBs displayed differing solubility properties and distinct distribution patterns, HBs-PiZ animals manifested retention of AAT/HBs in the degradatory pathway and a marked accumulation of the autophagy adaptor p62. Isolation of p62-containing particles revealed retained HBs/AAT and the lipophagy adapter perilipin-2. p62 build-up led to activation of the p62-Nrf2 axis and emergence of reactive oxygen species. Our results demonstrate that the simultaneous presence of two prevalent proteotoxic stresses promotes the development of liver injury due to protein retention and activation of the p62-Nrf2 axis. In humans, the PiZ variant was over-represented in CHB patients with advanced liver fibrosis (unadjusted odds ratio = 9.92 [1.15-85.39]). Current siRNA approaches targeting HBs/AAT should be considered for these individuals. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Quantification of Liver Fibrosis at T1 and T2 Mapping with Extracellular Volume Fraction MRI: Preclinical Results.

Purpose To evaluate MRI T1 and T2 mapping with calculation of extracellular volume (ECV) for diagnosis and grading of liver fibrosis. Materials and Methods Different grades of fibrosis were induced in 60 male Sprague-Dawley rats by bile duct ligation (BDL) and carbon-tetrachloride (CCl4) intoxication. Portal pressure was measured invasively, whereas hepatic fibrosis was quantified by hydroxyproline content, Sirius red staining, and α smooth muscle actin staining. T1 values, T2 values, and ECV were assessed by using quantitative MRI mapping techniques. Results T1 values in animals 4 weeks after BDL were greater than in control animals (718 msec ± 74 vs 578 msec ± 33, respectively; P < .001). T2 values at 4 weeks were also greater in animals that underwent BDL than in control animals (46 msec ± 6 vs 29 msec ± 2, respectively; P < .001). Similar T1 and T2 findings were observed after CCl4 intoxication. ECV was greater in animals 4 weeks after BDL compared with control animals (31.3% ± 1.3 vs 18.2% ± 3.5, respectively; P < .001), with similar results after CCl4 intoxication. High correlations were found between ECV and hepatic hydroxyproline content (BDL: r = 0.68, P < .001; CCl4: r = 0.65, P < .001), Sirius red staining (BDL: r = 0.88, P < .001; CCl4: r = 0.82, P < .001), α smooth muscle actin staining (BDL: r = 0.70, P < .001; CCl4: r = 0.73, P < .001), and portal pressure (BDL: r = 0.54, P = .003; CCl4: r = 0.39, P = .043). Conclusion Elevation of T1 and T2 values and ECV was associated with severity of liver fibrosis and portal hypertension in an experimental animal model.

Meningioma assessment: Kinetic parameters in dynamic contrast-enhanced MRI appear independent from microvascular anatomy and VEGF expression.

BACKGROUND AND PURPOSE: Kinetic parameters of T1-weighted dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) are considered to be influenced by microvessel environment. This study was performed to explore the extent of this association for meningiomas. MATERIALS AND METHODS: DCE-MRI kinetic parameters (contrast agent transfer constants Ktrans and kep, volume fractions vp and ve) were determined in pre-operative 3T MRI of meningioma patients for later biopsy sites (19 patients; 15 WHO Io, no previous radiation, and 4 WHO IIIo pre-radiated recurrent tumors). Sixty-three navigated biopsies were consecutively retrieved. Biopsies were immunohistochemically investigated with endothelial marker CD34 and VEGF antibodies, stratified in a total of 4383 analysis units and computationally assessed for VEGF expression and vascular parameters (vessel density, vessel quantity, vascular fraction within tissue [vascular area ratio], vessel wall thickness). Derivability of kinetic parameters from VEGF expression or microvascularization was determined by mixed linear regression analysis. Tissue kinetic and microvascular parameters were tested for their capacity to identify the radiation status in a subanalysis. RESULTS: Kinetic parameters were neither significantly related to the corresponding microvascular parameters nor to tissue VEGF expression. There was no significant association between microvessel density and its presumed correlate vp (P=0.07). The subgroup analysis of high-grade radiated meningiomas showed a significantly reduced microvascular density (AUC 0.91; P<0.0001) and smaller total vascular fraction (AUC 0.73; P=0.01). CONCLUSIONS: In meningioma, DCE-MRI kinetic parameters neither allow for a reliable prediction of tumor microvascularization, nor for a prediction of VEGF expression. Kinetic parameters seem to be determined from different independent factors.

Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis.

OBJECTIVE: Angiotensin II (AngII) activates via angiotensin-II-type-I receptor (AT1R) Janus-kinase-2 (JAK2)/Arhgef1 pathway and subsequently RHOA/Rho-kinase (ROCK), which induces experimental and probably human liver fibrosis. This study investigated the relationship of JAK2 to experimental and human portal hypertension. DESIGN: The mRNA and protein levels of JAK2/ARHGEF1 signalling components were analysed in 49 human liver samples and correlated with clinical parameters of portal hypertension in these patients. Correspondingly, liver fibrosis (bile duct ligation (BDL), carbon tetrachloride (CCl4)) was induced in floxed-Jak2 knock-out mice with SM22-promotor (SM22Cre+-Jak2f/f). Transcription and contraction of primary myofibroblasts from healthy and fibrotic mice and rats were analysed. In two different cirrhosis models (BDL, CCl4) in rats, the acute haemodynamic effect of the JAK2 inhibitor AG490 was assessed using microsphere technique and isolated liver perfusion experiments. RESULTS: Hepatic transcription of JAK2/ARHGEF1 pathway components was upregulated in liver cirrhosis dependent on aetiology, severity and complications of human liver cirrhosis (Model for End-stage Liver disease (MELD) score, Child score as well as ascites, high-risk varices, spontaneous bacterial peritonitis). SM22Cre+- Jak2f/f mice lacking Jak2 developed less fibrosis and lower portal pressure (PP) than SM22Cre--Jak2f/f upon fibrosis induction. Myofibroblasts from SM22Cre+-Jak2f/f mice expressed less collagen and profibrotic markers upon activation. AG490 relaxed activated hepatic stellate cells in vitro. In cirrhotic rats, AG490 decreased hepatic vascular resistance and consequently the PP in vivo and in situ. CONCLUSIONS: Hepatic JAK2/ARHGEF1/ROCK expression is associated with portal hypertension and decompensation in human cirrhosis. The deletion of Jak2 in myofibroblasts attenuated experimental fibrosis and acute inhibition of JAK2 decreased PP. Thus, JAK2 inhibitors, already in clinical use for other indications, might be a new approach to treat cirrhosis with portal hypertension.

Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease.

UNLABELLED: Pathogen- and injury-related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver-derived plasma protein, histidine-rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver-derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild-type (WT) and Hrg(-/-) mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti-inflammatory M2 subtype in Hrg(-/-) mice. Upon chronic liver damage induced by CCl4 or methionine-choline-deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg(-/-) , compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg(-/-) mice. Moreover, HRG-deficient mice showed significantly enhanced hepatic vascularization by micro-computed tomography and histology, corroborating proangiogenic activities of M2-polarized liver macrophages. Purified HRG protein induced, but HRG-deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg(-/-) mice transplanted with Hrg(+/+) bone marrow, but not Hrg(-/-) -transplanted Hrg(+/+) mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up-regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. CONCLUSIONS: Liver-derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases.

Statins improve NASH via inhibition of RhoA and Ras.

Nonalcoholic steatohepatitis (NASH), especially as part of the metabolic syndrome (MS), is an increasing burden in Western countries. Statins are already used in MS and seem to be beneficial in liver diseases. The aim of this study was to investigate the molecular mechanisms underlying pleiotropic effects on small GTPases of statins in NASH. NASH within MS was induced in 12-wk-old apoE-/- mice after 7 wk of Western diet (NASH mice). Small GTPases were inhibited by activated simvastatin (SMV), NSC23766 (NSC), or Clostridium sordellii lethal toxin (LT) by using subcutaneous osmotic minipumps. Hepatic steatosis, inflammation, and fibrosis were assessed by histology, Western blot, and RT-PCR measurements of cholesterol and hydroxyproline content. SMV treatment significantly decreased hepatic inflammation and fibrosis, but had no significant effect on steatosis and hepatic cholesterol content in NASH. SMV blunted fibrosis due to inhibition of both RhoA/Rho kinase and Ras/ERK pathways. Interestingly, inhibition of RAC1 and Ras (by LT) failed to decrease fibrosis to the same extent. Inhibition of RAC1 (by NSC) showed no significant effect at all. Inhibition of RhoA and Ras downstream signaling by statins is responsible for the beneficial hepatic effects in NASH.

Pharmacological inhibition of the chemokine C-C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly-6C(+) macrophage infiltration in mice.

UNLABELLED: Macrophages constitute a major proinflammatory component during chronic liver diseases and are considered a key factor in promoting hepatic fibrosis. However, there is increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified "restorative" macrophages as Ly-6C (Ly6C, Gr1) low-expressing, monocyte-derived cells. We investigated molecular pathways balancing proinflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augmenting beneficial macrophage functionality in fibrosis resolution. Therefore, we employed a Spiegelmer-based inhibitor of the chemokine, C-C motif chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1), termed mNOX-E36, in the regression phase of two murine models of toxic (CCl4 ) and metabolic (methionine-choline-deficient diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, we observed a transient influx of Ly-6C(+) infiltrating monocytes (iMΦ), which are characterized by typical macrophage morphology, up-regulated expression of CCR2, and the pro-inflammatory cytokine, tumor necrosis factor (TNF), in injured liver. By inhibiting the early influx of Ly-6C(+) iMΦ by the CCL2 inhibitor, mNOX-E36, the intrahepatic macrophage equilibration shifted toward the "restorative" Ly-6C(-) subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX-E36 administration in both models. Blocking transient recruitment of infiltrating Ly-6C(+) monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced intrahepatic levels of proinflammatory cytokines. CONCLUSION: Transient CCL2-dependent recruitment of infiltrating Ly-6C(+) monocytes during fibrosis regression counteracts scar resolution by perpetuating inflammatory reactions through release of proinflammatory cytokines such as TNF. Pharmacological inhibition of Ly-6C(+) monocyte recruitment using the CCL2-inhibitor, mNOX-E36, accelerates regression from toxic and metabolic liver fibrosis in two independent experimental models.

Expression of vasoactive proteins in gastric antral mucosa reflects vascular dysfunction in patients with cirrhosis and portal hypertension.

BACKGROUND & AIMS: Patients with cirrhosis display hypocontractility of splanchnic vessels because of dysregulation of vasoactive proteins, such as decreased effect of RhoA/ROCK and increased activity of ß-Arrestin-2 and eNOS. However, it is unknown whether the dysregulation of vasoactive proteins is displayed in other vessels. We investigated whether expression of vasoactive proteins can be evaluated in gastric mucosa vessels. METHODS: Biopsies from the gastric mucosa of 111 patients with cirrhosis were collected at three different centres and from 13 controls. Forty-nine patients had received TIPS. Portal pressure gradient was measured in 49 patients with TIPS and in 16 patients without TIPS. Biopsies from the antrum were conserved in formaldehyde for immunohistochemistry or shock-frozen for PCR and Western blot. RESULTS: The mucosal transcription of vascular markers (αSMA, CD31) was higher in cirrhotic patients than controls, which was confirmed by immunohistochemistry. On average, relative mucosal levels of RhoA and ROCK were lower, while ß-Arrestin-2 levels were higher in cirrhotic patients compared to controls. Transcriptional levels of eNOS increased with presence of ascites and grade of oesophageal varices. Patients with TIPS showed less pronounced markers of vascular dysfunction in gastric mucosa. CONCLUSION: This is the first evidence that the expression of vasoactive proteins in mucosa from the gastric antrum of patients with cirrhosis reflects their vascular dysfunction and possibly changes after therapeutic interventions.

Comparison between modified Dixon MRI techniques, MR spectroscopic relaxometry, and different histologic quantification methods in the assessment of hepatic steatosis.

OBJECTIVES: To compare systematically quantitative MRI, MR spectroscopy (MRS), and different histological methods for liver fat quantification in order to identify possible incongruities. METHODS: Fifty-nine consecutive patients with liver disorders were examined on a 3 T MRI system. Quantitative MRI was performed using a dual- and a six-echo variant of the modified Dixon (mDixon) sequence, calculating proton density fat fraction (PDFF) maps, in addition to single-voxel MRS. Histological fat quantification included estimation of the percentage of hepatocytes containing fat vesicles as well as semi-automatic quantification (qHisto) using tissue quantification software. RESULTS: In 33 of 59 patients, the hepatic fat fraction was >5% as determined by MRS (maximum 45%, mean 17%). Dual-echo mDixon yielded systematically lower PDFF values than six-echo mDixon (mean difference 1.0%; P < 0.001). Six-echo mDixon correlated excellently with MRS, qHisto, and the estimated percentage of hepatocytes containing fat vesicles (R = 0.984, 0.967, 0.941, respectively, all P < 0.001). Mean values obtained by the estimated percentage of hepatocytes containing fat were higher by a factor of 2.5 in comparison to qHisto. Six-echo mDixon and MRS showed the best agreement with values obtained by qHisto. CONCLUSIONS: Six-echo mDixon, MRS, and qHisto provide the most robust and congruent results and are therefore most appropriate for reliable quantification of liver fat. KEY POINTS: • Six-echo mDixon correlates excellently with MRS, qHisto, and the estimated percentage of fat-containing hepatocytes. • Six-echo mDixon, MRS, and qHisto provide the most robust and congruent results. • Dual-echo mDixon yields systematically lower PDFF values than six-echo mDixon. • The percentage of fat-containing hepatocytes is 2.5-fold higher than fat fraction determined by qHisto. • Performance characteristics and systematic differences of the various methods should be considered.

Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis.

Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6(+/gfp) and Cxcr6(gfp/gfp) mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6(-/-) mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6(-/-) mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.

CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis.

OBJECTIVES: In chronic liver injury, angiogenesis, the formation of new blood vessels from pre-existing ones, may contribute to progressive hepatic fibrosis and to development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesised that inflammation may endorse hepatic angiogenesis already at early stages of fibrosis. DESIGN: Angiogenesis in livers of c57BL/6 mice upon carbon tetrachloride- or bile duct ligation-induced chronic hepatic injury was non-invasively monitored using in vivo contrast-enhanced micro computed tomography (µCT) and ex vivo anatomical µCT after hepatic Microfil perfusion. Functional contributions of monocyte-derived macrophage subsets for angiogenesis were explored by pharmacological inhibition of CCL2 using the Spiegelmer mNOX-E36. RESULTS: Contrast-enhanced in vivo µCT imaging allowed non-invasive monitoring of the close correlation of angiogenesis, reflected by functional hepatic blood vessel expansion, with experimental fibrosis progression. On a cellular level, inflammatory monocyte-derived macrophages massively accumulated in injured livers, colocalised with newly formed vessels in portal tracts and exhibited pro-angiogenic gene profiles including upregulated VEGF and MMP9. Functional in vivo and anatomical ex vivo µCT analyses demonstrated that inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sprouting angiogenesis within the portal vein tract. Portal vein diameter as a measure of portal hypertension depended on fibrosis, but not on angiogenesis. CONCLUSIONS: Inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression. Innovative in vivo µCT methodology can accurately monitor angiogenesis and antiangiogenic therapy effects in experimental liver fibrosis.

A common polymorphism in the NCAN gene is associated with hepatocellular carcinoma in alcoholic liver disease.

BACKGROUND & AIMS: The genetic background of alcoholic liver diseases and their complications are increasingly recognized. A common polymorphism in the neurocan (NCAN) gene, which is known to be expressed in neuronal tissue, has been identified as a risk factor for non-alcoholic fatty liver disease (NAFLD). We investigated if this polymorphism may also be related to alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). METHODS: We analysed the distribution of the NCAN rs2228603 genotypes in 356 patients with alcoholic liver cirrhosis, 126 patients with alcoholic HCC, 382 persons with alcohol abuse without liver damage, 362 healthy controls and in 171 patients with hepatitis C virus (HCV) associated HCC. Furthermore, a validation cohort of 229 patients with alcoholic cirrhosis (83 with HCC) was analysed. The genotypes were determined by LightSNiP assays. The expression of NCAN was studied by RT-PCR and immunofluorescence microscopy. RESULTS: The frequency of the NCAN rs2228603 T allele was significantly increased in patients with HCC due to ALD (15.1%) compared to alcoholic cirrhosis without HCC (9.3%), alcoholic controls (7.2%), healthy controls (7.9%), and HCV associated HCC (9.1%). This finding was confirmed in the validation cohort (15.7% vs. 6.8%, OR=2.53; 95%CI: 1.36-4.68; p=0.0025) and by multivariate analysis (OR=1.840; 95%CI: 1.22-2.78; p=0.004 for carriage of the rs2228603 T allele). In addition, we identified and localised NCAN expression in human liver. CONCLUSIONS: NCAN is not only expressed in neuronal tissue, but also in the liver. Its rs2228603 polymorphism is a risk factor for HCC in ALD, but not in HCV infection.

Platelet-derived growth factor (PDGF)-C neutralization reveals differential roles of PDGF receptors in liver and kidney fibrosis.

Platelet-derived growth factors (PDGF) are key mediators of organ fibrosis. We investigated whether PDGF-C(-/-) mice or mice treated with neutralizing PDGF-C antibodies are protected from bile duct ligation-induced liver fibrosis, and we compared the effects with those of PDGF-C deficiency or neutralization on kidney fibrosis induced by unilateral ureteral obstruction. Unexpectedly, and in contrast to kidney fibrosis, PDGF-C deficiency or antagonism did not protect from liver fibrosis or functional liver impairment. Furthermore, the hepatic infiltration of monocytes/macrophages/dendritic cells and chemokine mRNA expression (CC chemokine ligand [CCL]5, CCL2, and CC chemokine receptor 2 [CCR2]) remained unchanged. Transcript expression of PDGF ligands increased in both liver and kidney fibrosis and was not affected by neutralization of PDGF-C. In kidney fibrosis, PDGF-C deficiency or antagonism led to reduced expression and signaling of PDGF-receptor (R)-α- and PDGFR-ß-chains. In contrast, in liver fibrosis there was either no difference (PDGF-C(-/-) mice) or even an upregulation of PDGFR-ß and signaling (anti-PDGF-C group). Finally, in vitro studies in portal myofibroblasts pointed to a predominant role of PDGF-B and PDGF-D signaling in liver fibrosis. In conclusion, our study revealed significant differences between kidney and liver fibrosis in that PDGF-C mediates kidney fibrosis, whereas antagonism of PDGF-C in liver fibrosis appears to be counteracted by significant upregulation and increased PDGFR-ß signaling. PDGF-C antagonism, therefore, may not be effective to treat liver fibrosis.

DCE-MRI in Glioma, Infiltration Zone and Healthy Brain to Assess Angiogenesis: A Biopsy Study.

PURPOSE: To explore the focal predictability of vascular growth factor expression and neovascularization using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in glioma. METHODS: 120 brain biopsies were taken in vital tumor, infiltration zone and normal brain tissue of 30 glioma patients: 17 IDH(isocitrate dehydrogenase)-wildtype glioblastoma (GBM), 1 IDH-wildtype astrocytoma °III (together prognostic group 1), 3 IDH-mutated GBM (group 2), 3 anaplastic astrocytomas IDH-mutated (group 3), 4 anaplastic oligodendrogliomas and 2 low-grade oligodendrogliomas (together prognostic group 4). A mixed linear model evaluated the predictabilities of microvessel density (MVD), vascular area ratio (VAR), mean vessel size (MVS), vascular endothelial growth factor and receptors (VEGF-A, VEGFR­2) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) expression from Tofts model kinetic and model-free curve parameters. RESULTS: All kinetic parameters were associated with VEGF­A (all p < 0.001) expression. Ktrans, kep and ve were associated with VAR (p = 0.006, 0.004 and 0.01, respectively) and MVS (p = 0.0001, 0.02 and 0.003, respectively) but not MVD (p = 0.84, 0.74 and 0.73, respectively). Prognostic groups differed in Ktrans (p = 0.007) and ve (p = 0.004) values measured in the infiltration zone. Despite significant differences of VAR, MVS, VEGF­A, VEGFR­2, and VE-PTP in vital tumor tissue and the infiltration zone (p = 0.0001 for all), there was no significant difference between kinetic parameters measured in these zones. CONCLUSION: The DCE-MRI kinetic parameters show correlations with microvascular parameters in vital tissue and also reveal blood-brain barrier abnormalities in the infiltration zones adequate to differentiate glioma prognostic groups.

Effects of Liver Fibrosis Progression on Tissue Relaxation Times in Different Mouse Models Assessed by Ultrahigh Field Magnetic Resonance Imaging.

Recently, clinical studies demonstrated that magnetic resonance relaxometry with determination of relaxation times T1 and T2⁎ may aid in staging and management of liver fibrosis in patients suffering from viral hepatitis and steatohepatitis. In the present study we investigated T1 and T2⁎ in different models of liver fibrosis to compare alternate pathophysiologies in their effects on relaxation times and to further develop noninvasive quantification methods of liver fibrosis. MRI was performed with a fast spin echo sequence for measurement of T1 and a multigradient echo sequence for determination of T2⁎. Toxic liver fibrosis was induced by injections of carbon tetrachloride (1.4 mL CCl4 per kg bodyweight and week, for 3 or 6 weeks) in BALB/cJ mice. Chronic sclerosing cholangitis was mimicked using the ATP-binding cassette transporter B4 knockout (Abcb4 -/-) mouse model. Untreated BALB/cJ mice served as controls. To assess hepatic fibrosis, we ascertained collagen contents and fibrosis scores after Sirius red staining. T1 and T2⁎ correlate differently to disease severity and etiology of liver fibrosis. T2⁎ shows significant decrease correlating with fibrosis in CCl4 treated animals, while demonstrating significant increase with disease severity in Abcb4 -/- mice. Measurements of T1 and T2⁎ may therefore facilitate discrimination between different stages and causes of liver fibrosis.

Novel Rat Model of Repetitive Portal Venous Embolization Mimicking Human Non-Cirrhotic Idiopathic Portal Hypertension.

BACKGROUND: Non-cirrhotic idiopathic portal hypertension (NCIPH) is characterized by splenomegaly, anemia and portal hypertension, while liver function is preserved. However, no animal models have been established yet. This study assessed a rat model of NCIPH and characterized the hemodynamics, and compared it to human NCIPH. METHODS: Portal pressure (PP) was measured invasively and coloured microspheres were injected in the ileocecal vein in rats. This procedure was performed weekly for 3 weeks (weekly embolization). Rats without and with single embolization served as controls. After four weeks (one week after last embolization), hemodynamics were investigated, hepatic fibrosis and accumulation of myofibroblasts were analysed. General characteristics, laboratory analyses and liver histology were collected in patients with NCIPH. RESULTS: Weekly embolization induced a hyperdynamic circulation, with increased PP. The mesenteric flow and hepatic hydroxyproline content was significantly higher in weekly embolized compared to single embolized rats (mesenteric flow +54.1%, hydroxyproline +41.7%). Mesenteric blood flow and shunt volumes increased, whereas splanchnic vascular resistance was decreased in the weekly embolization group. Fibrotic markers αSMA and Desmin were upregulated in weekly embolized rats. DISCUSSION: This study establishes a model using repetitive embolization via portal veins, comparable with human NCIPH and may serve to test new therapies.

Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases.

Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities.

Seven weeks of Western diet in apolipoprotein-E-deficient mice induce metabolic syndrome and non-alcoholic steatohepatitis with liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation and fibrosis, which might progress to cirrhosis. Human NASH is associated with metabolic syndrome (MS). Currently, rodent NASH models either lack significant fibrosis or MS. ApoE(-/-) mice are a MS model used in cardiovascular research. The aim of this work was to establish and characterise a novel mouse NASH model with significant fibrosis and MS. ApoE(-/-) and wild-type mice (wt) were fed either a western-diet (WD), methionine-choline-deficient-diet (MCD) or normal chow. Liver histology, RT-PCR, hepatic hydroxyproline content, triglycerides and cholesterol levels, and fasting glucose levels assessed hepatic steatosis, inflammation and fibrosis. Further, portal pressure was measured invasively, and kidney pathology was assessed by histology. ApoE(-/-) mice receiving WD showed abnormal glucose tolerance, hepatomegaly, weight gain and full spectrum of NASH including hepatic steatosis, fibrosis and inflammation, with no sign of renal damage. MCD-animals showed less severe liver fibrosis, but detectable renal pathological changes, besides weight loss and unchanged glucose tolerance. This study describes a murine NASH model with distinct hepatic steatosis, inflammation and fibrosis, without renal pathology. ApoE(-/-) mice receiving WD represent a novel and fast model with all characteristic features of NASH and MS well suitable for NASH research.

Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl4)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.