Vitamin D counteracts fibrogenic TGF-ß signalling in human hepatic stellate cells both receptor-dependently and independently.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity and constitutes part of the metabolic syndrome, which have been associated with low serum vitamin D (VD). Due to known crosstalk between VD and transforming growth factor (TGF)-ß signalling, VD has been proposed as an antifibrotic treatment. DESIGN: We evaluated the association between VD, the vitamin D receptor (VDR) and liver fibrosis in primary human hepatic stellate cells (phHSC) and 106 morbidly obese patients with NAFLD. RESULTS: Treating phHSC with VD ameliorated TGF-ß-induced fibrogenesis via both VDR-dependent and VDR-independent mechanisms. Reduction of fibrogenic response was abolished in cells homozygous for GG at the A1012G single nucleotide polymorphisms within the VDR gene. Compared with healthy livers, NAFLD livers expressed higher levels of VDR mRNA and VDR fragments. VDR mRNA was lower in patients homozygous for GG at A1012G and expression of pro-fibrogenic genes was higher in patients carrying the G allele. CONCLUSIONS: VD may be an antifibrotic treatment option early in the onset of fibrosis in specific genotypes for VDR. Known polymorphisms of the VDR may influence the response to VD treatment.

Mycophenolic acid induces apoptosis of hepatic stellate cells in an in vitro model of HCV.

INTRODUCTION: Hepatitis C virus (HCV) infection is an important risk factor for the development of liver fibrosis and progression to cirrhosis. Liver transplantation as terminal treatment option for liver disease requires life-long immunosuppression. However, immunomodulatory therapy may promote reinfection and renewed fibrogenesis. Immunosupressive agents may also affect the life cycle of hepatic stellate cells (HSC), the main source of extracellular matrix. We thus aimed to characterize the effects of three common immunosuppressive agents on HSC apoptosis with or without engulfment of HCV infected apoptotic bodies. MATERIAL AND METHODS: LX2 cells were incubated with three different immunosuppressants (rapamycine, mycophenolic acid or cyclosporine A) and co-incubated for 24 and 48 h with apoptotic bodies (AB), produced from Huh7 cells or from Con1 cells (Huh7-cells containing a subgenomic HCV replicon). The engulfment of AB was confirmed by immunofluorescence staining. HSC viability, apoptosis rate and expression of profibrogenic and proapoptotic genes were quantified. RESULTS: In LX2 cells that engulfed Con1 AB, the treatment with mycophenolic acid induced HSC apoptosis and reduced collagen 1alpha 1 expression compared to cylosporine A or rapamycine treatment. In conclusion mycophenolic acid is a potent inducer of HSC apoptosis and attenuates HSC activation and consecutively fibrogenesis in HCV infection. Translational studies will need to confirm these mono-culture results in vivo.

Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts bile acid-induced liver injury in superobese patients with nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and may proceed to steatohepatitis (NASH). Apoptosis and free fatty acid (FFA)-induced lipotoxicity are important features of NASH pathogenesis. We have shown a hepatoprotective effect of adiponectin in steatotic livers of hepatitis C virus (HCV) patients and recent data links bile acid (BA) metabolism to the pathogenesis of NAFLD. The aim of this study was to identify potential interactions between BA and FFA metabolism in NAFLD. Liver biopsies and serum samples from 113 morbidly obese patients receiving bariatric surgery, healthy individuals, and moderately obese NAFLD patients were studied. Serum FFA, BA, and M30 were increased in NASH versus simple steatosis, while adiponectin was significantly decreased. The NAFLD activity score (NAS) score correlated with BA levels and reversely with adiponectin. Adiponectin reversely correlated with CD95/Fas messenger RNA (mRNA) and hepatocellular apoptosis. The BA transporter high-affinity Na+ /taurocholate cotransporter (NTCP) and the BA synthesizing enzyme cholesterol 7 alpha-hydroxylase (CYP7A1) were significantly up-regulated in obese patients and hepatoma cells exposed to FFA. Up-regulation of NTCP and CYP7A1 indicate failure to activate small heterodimer partner (SHP) upon farnesoid X receptor (FXR) stimulation by increasing BA concentrations. In line with the NAS score, adiponectin levels were reversely correlated with BA levels. Adiponectin correlated with NTCP and affects Cyp7A1 expression both in vivo and in vitro. CONCLUSION: BA synthesis and serum BA levels correlated with disease severity in NAFLD, while adiponectin is reversely correlated. FFA exposure prevented SHP-mediated repression of NTCP and Cyp7A1 expression, which lead to increased BA synthesis and uptake. In NASH, BA accumulation induced hepatocyte cell death and late FXR activation failed to prevent hepatocyte injury due to decreased adiponectin levels. Early treatment with FXR ligands and/or adiponectin-receptor agonists might prevent NASH.

Fetuin-A mRNA expression is elevated in NASH compared with NAFL patients.

Fetuin-A is a pro-inflammatory protein expressed by hepatocytes. Its course in morbidly obese patients with NAFLD (non-alcoholic fatty liver disease) following weight loss by BAS (bariatric surgery) has not been fully elucidated yet. In the present study, we prospectively examined the effects of weight loss on various metabolic factors at 4 weeks and 6 months after surgery. Blood and liver tissues were retrieved from 108 morbidly obese NAFLD patients before/during BAS, and 50 of these individuals met the criteria for NASH (non-alcoholic steatohepatitis). Fetuin-A expression was measured by qPCR (quantitative real-time PCR), Western blotting and immunohistochemistry. Hepatocyte apoptosis was quantified via M30 (caspase-cleaved cytokeratin-18 fragments). Plasma concentrations of adiponectin and fetuin-A were determined by ELISA. Serum-derived parameters were additionally taken at 4 weeks and 6 months post-operatively. In addition, primary human hepatocytes were treated with NEFA (non-esterified fatty acid) to investigate changes in fetuin-A. BMI (body mass index) decreased significantly from 53.0±1.1 to 36.4±1.9 kg/m2 in the NAFL group and from 53.3±1.1 to 37.6±1.2 kg/m2 in the NASH group (P<0.0001) at 6 months post-surgery. This was associated with diminishing M30 and M65 (total cytokeratin-18) levels over 6 months after surgery. Adiponectin levels increased continuously in NASH patients, whereas NAFL patients plateaued at 4 weeks post-operatively. Hepatic fetuin-A mRNA and protein expression was elevated before surgery-induced weight loss. However, plasma concentrations of fetuin-A increased signficantly in NASH patients 4 weeks post-operatively. Treatment of hepatocytes with NEFA led to up-regulation of fetuin-A expression. BAS probably has a beneficial effect on NAFLD, as indicated by reduced hepatocyte apoptosis and improved adipokine profiles. In addition, fetuin-A expression is more prominent in NASH.

Expression of apoptosis- and vitamin D pathway-related genes in hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and therapeutic options are scarce. As they might represent future targets for cancer therapy, the expression of apoptosis-related genes in HCC is of particular interest. In this pilot study, we further examined apoptosis-related genes in human HCC and also focused on vitamin D signaling as this might be a regulator of HCC cell apoptosis. METHODS: We employed tumor tissue and serum samples from 62 HCC patients as well as 62 healthy controls for these studies. Tissue and serum specimens were analyzed by quantitative RT-PCR, immunohistochemistry and ELISA. RESULTS: In HCC patients the apoptosis marker M30 was found to be elevated and several pro-apoptotic (TRAIL, FasL and FasR) as well as anti-apoptotic genes (Mcl-1 and Bcl-2) were simultaneously upregulated in tumor tissue and especially tumor-surrounding tissue as compared to healthy control livers. Moreover, vitamin D serum levels were decreased in HCC patients whereas vitamin D receptor mRNA expression was increased in tumor tissue and tumor-surrounding tissue as compared to healthy livers. CONCLUSIONS: In human HCC, M30 serum levels are elevated indicating an increased cell turnover. Modulation of the vitamin D pathway might be a supportive, pro-apoptotic HCC therapy.

Differential effects of factor IIa inhibitors on the endogenous thrombin potential.

Calibrated automated thrombin generation measurement in clotting plasma (endogenous thrombin potential, ETP) is being used increasingly to monitor the effects of anticoagulant drugs. Calibrated automated thrombography measures the concentration of thrombin in clotting plasma by monitoring the cleavage of a fluorogenic substrate (Z-Gly-Gly-Arg-7-amino-4-methylcoumarin) and comparing it with a constant known thrombin activity in a parallel nonclotting sample. This study compared the concentration-dependent effects of different factor IIa inhibitors on the ETP. In accordance with a theoretical prediction, the monovalent factor IIa inhibitors melagatran and argatroban reduced peak thrombin concentrations without a marked effect on the lag time of thrombin generation. However, both bivalent factor IIa inhibitors lepirudin and bivalirudin markedly prolonged the lag time of thrombin generation and thus behaved like "super" factor Xa inhibitors according to the theoretical model. These findings have important consequences for therapeutic drug monitoring using thrombin generation assays. For monitoring of hirudins, the lag time of thrombin generation seems to be a very sensitive parameter. In contrast, for monitoring of argatroban, the evaluation of the ETP seems to be useful.

Inhibition of Na+/H+-exchanger with sabiporide attenuates the downregulation and uncoupling of the myocardial beta-adrenoceptor system in failing rabbit hearts.

Chronic heart failure (HF) is characterized by left ventricular (LV) structural remodeling, impaired function, increased circulating noradrenaline (NA) levels and impaired responsiveness of the myocardial beta-adrenoceptor (betaAR)-adenylyl cyclase (AC) system. In failing hearts, inhibition of the sodium/proton-exchanger (NHE)-1 attenuates LV remodeling and improves LV function. The mechanism(s) involved in these cardioprotective effects remain(s) unclear, but might involve effects on the impaired betaAR-AC system. Therefore, we investigated whether NHE-1 inhibition with sabiporide (SABI; 30 mg kg(-1) day(-1) p.o.) might affect myocardial betaAR density and AC activity in relation to changes in LV end-diastolic diameter (LVEDD) and LV systolic fractional shortening (LVS-FS) after 3 weeks of rapid LV pacing in rabbits. After 3 weeks of rapid LV pacing LVEDD was significantly increased (Shams 17+/-0.2 mm, n=9 vs 3 wksHF 20+/-0.5 mm, n=8; P<0.05) and LVS-FS decreased (Shams 31+/-1%, n=9 vs 3 wksHF 10+/-1%, n=8; P<0.05). SABI treatment significantly improved LV function independent of whether rabbits were treated after 1 week of pacing (3 wksHF+2 wksSABI (n=7): LVEDD 18+/-1 mm; LVS-FS 16+/-4%) or before pacing (3 wksHF+3wksSABI (n=9): LVEDD 18+/-1 mm; LVS-FS 18+/-6%). After 3 weeks of rapid LV pacing, SABI treatment significantly attenuated increases in serum NA content (Shams 0.83+/-0.19, 3 wksHF 2.68+/-0.38, 3 wksHF+2 wksSABI 1.22+/-0.32, 3 wksHF+3wksSABI 1.38+/-0.33 ng ml(-1)). Moreover, betaAR density (Shams 64+/-5, 3 wksHF 38+/-3, 3 wksHF+2 wksSABI 48+/-4, 3 wksHF+3 wksSABI 55+/-3 fmol mg(-1) protein) and responsiveness (isoprenaline-stimulated AC activity. (Shams 57.6+/-4.9, 3 wksHF 36.3+/-6.0, 3 wksHF+2 wksSABI 56.9+/-6.0, 3 wksHF+3 wksSABI 54.5+/-4.8 pmol cyclic AMP mg(-1) protein(-1) min(-1)) were significantly improved in SABI-treated rabbits. From the present data we cannot address whether the improved betaAR-AC system permitted improved LV function and/or whether the improved LV function resulted in less activation of the sympathetic nervous system and by this in a reduced stimulation of the betaAR-AC system. Accordingly, additional studies are needed to fully establish the cause-and-effect relationship between NHE-1 inhibition and the restoration of the myocardial betaAR system.

Ventricular hypertrophy plus neurohumoral activation is necessary to alter the cardiac beta-adrenoceptor system in experimental heart failure.

Treatment of rats with monocrotaline (MCT) leads to pulmonary hypertension, right ventricular (RV) hypertrophy, and finally to RV heart failure. This is associated with characteristic changes in right ventricular beta-adrenoceptors (beta-AR), neuronal noradrenaline transporter (NAT) density and activity (uptake1), and G protein-coupled receptor kinase (GRK) activity. This study aimed to find out factors that determine beta-AR, uptake1, and GRK changes. Thus, 6-week-old rats were treated with 50 mg/kg MCT subcutaneous or 0.9% saline. Within 13 to 19 days after MCT application (group A), RV weight (222+/-6 versus 147+/-5 mg) and RV/left ventricular (LV) weight ratio (0.42+/-0.01 versus 0.29+/-0.01) were significantly increased, whereas plasma noradrenaline, RV beta-AR density, RV NAT density and activity, and RV GRK activity were not significantly altered. Twenty-one to twenty-eight days after MCT (group B), however, not only RV weight (316+/-4 versus 148+/-2 mg) and RV/LV weight ratio (0.61+/-0.01 versus 0.3+/-0.01) were markedly increased but also plasma noradrenaline (645+/-63 versus 278+/-18 pg/mL); now, RV beta-AR density (13.4+/-1.3 versus 26.5+/-1.1 fmol/mg protein), RV NAT density (50.9+/-11.3 versus 79.6+/-2.9 fmol/mg protein), and RV NAT activity (65.4+/-7.4 versus 111.8+/-15.9 pmol [3H]-NA/mg tissue slices/15 min) were significantly decreased and RV-membrane GRK activity (100+/-15 versus 67+/-6 [32P]-rhodopsin in cpm) significantly increased. LV parameters of MCT-treated rats were only marginally different from control LV. We conclude that in MCT-treated rats ventricular hypertrophy per se is not sufficient to cause characteristic alterations in the myocardial beta-AR system often seen in heart failure; only if ventricular hypertrophy is associated with neurohumoral activation beta-ARs are downregulated and GRK activity is increased.

G-protein-coupled receptor kinase activity in human heart failure: effects of beta-adrenoceptor blockade.

OBJECTIVES: In human end-stage heart failure as well as in experimental animal models of heart failure, G-protein-coupled receptor kinase activity (GRK) is increased while beta-adrenoceptor responsiveness is diminished. In animal studies, beta-adrenoceptor blockers reverse the GRK-mediated desensitization and down-regulation of myocardial beta-adrenoceptors. The aim of this study was to investigate whether alterations in GRK activity are an early or late accompaniment of human heart failure and whether also in humans beta-adrenoceptor blocker treatment is able to influence myocardial GRK activity. METHODS: We assessed in right atria, obtained from patients at different stages of heart failure, treated with or not treated with beta-adrenoceptor blockers, and in the four chambers of explanted hearts, obtained from patients with end-stage heart failure, beta-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and GRK activity (by an in vitro rhodopsin phosphorylation assay). RESULTS: With increasing severity of heart failure, plasma noradrenaline levels increased while myocardial beta-adrenoceptor density decreased with a maximum in GRK activity in end-stage heart failure. However, in relation to the progression of heart failure, we found that GRK activity transiently increased at an early stage of heart failure (NYHA I and II) but decreased back to control values in patients at NYHA III and IV. beta-Adrenoceptor blockers were able to reduce the early increase in GRK activity at NYHA I and II to control levels, whereas in those patients who did not have increased GRK activity (NYHA III and IV), they had only a marginal effect. CONCLUSION: According to our results, an increase in GRK activity is an early and transient event in the course of heart failure that can be prevented by beta-adrenoceptor blocker treatment.

Unchanged G-protein-coupled receptor kinase activity in the aging human heart.

OBJECTIVES: We sought to find out whether G-protein-coupled receptor kinase (GRK) activity is also increased in the aging human heart. BACKGROUND: In the aging and failing human heart, cardiac beta-adrenoceptors (beta-AR) are desensitized. In heart failure (HF), an increase in cardiac GRK activity considerably contributes to this beta-AR desensitization. METHODS: We assessed GRK activity (by in vitro rhodopsin phosphorylation) in the right atria (RA) from 16 children (mean age 9 +/- 2 years) and 17 elderly patients (mean age 67 +/- 2 years) without apparent HF and in the RA from four patients with end-stage HF.Cytosolic and membranous GRK activities in the RA from children were not significantly different from those in elderly patients; in contrast, cytosolic and membranous GRK activities in the RA from patients with end-stage HF were significantly increased. CONCLUSIONS: In contrast to the failing human heart, in the aging human heart, GRK activity is not increased. Thus, GRK activity appears to not play an important role in beta-AR desensitization in the aging human heart.

Genotype-dependent time course of lymphocyte beta 2-adrenergic receptor down-regulation.

BACKGROUND: Volunteers homozygous for Glu27 beta(2)-adrenergic receptor (beta(2)AR) polymorphism have delayed onset of agonist-induced desensitization of cardiac beta(2)AR responses. METHODS AND RESULTS: To determine whether this can also be demonstrated for Glu27Glu beta(2)AR natively expressed in circulating lymphocytes, we assessed the effects of 2 weeks of oral treatment with 3 x 5 mg/d terbutaline on lymphocyte beta(2)AR density (determined by [-]-[iodine 125]iodocyanopindolol binding) and responsiveness (assessed as [-]-isoproterenol hydrochloride [INN, isoprenaline] [1 nmol/L to 1 micromol/L]-induced lymphocyte cyclic adenosine monophosphate increases) in 23 healthy volunteers (13 with wild-type beta(2)AR [group A], 5 homozygous for Glu27 with Gly16Gly or Arg16Gly [group B], and 5 homozygous for Gly16 with Gln27Gln or Gln27Glu [group C]). Before terbutaline treatment, lymphocyte beta(2)AR density and isoproterenol-induced lymphocyte cyclic adenosine monophosphate accumulation were not significantly different in the genotype groups; 2 weeks of terbutaline treatment significantly decreased lymphocyte beta(2)AR density and responsiveness in the 3 genotype groups to a nearly identical extent, and no differences were observed. In time-course studies, however, in groups A and C lymphocyte beta(2)AR showed significant (P <.05, repeated-measures ANOVA) down-regulation as early as 24 hours after the first terbutaline intake, whereas in group B significant (P <.05, repeated-measures ANOVA) beta(2)AR decreases were observed only 72 hours after the first terbutaline intake. Thus the time course of lymphocyte beta(2)AR down-regulation in group B was significantly (P <.01, 2-way ANOVA) different from that in groups A and C. CONCLUSION: The extent of lymphocyte beta(2)AR down-regulation after long-term terbutaline treatment in volunteers homozygous for the Gly16 or Glu27 beta(2)AR polymorphism was genotype-independent and was nearly identical to that in wild-type beta(2)AR volunteers. However, the onset of beta(2)AR down-regulation was delayed in volunteers homozygous for the Glu27 beta(2)AR polymorphism.

Vitamin D ameliorates stress ligand expression elicited by free fatty acids in the hepatic stellate cell line LX-2.

BACKGROUND/AIMS: Hepatic stellate cells play an important role as the major source of fibrillar and non-fibrillar matrix proteins in the process of liver fibrosis. Natural killer cells have an anti-fibrotic effect through the killing of activated hepatic stellate cells. Major histocompatibility complex class I-related molecules, MICA and MICB, function as ligands for the NKG2D receptor and play an important role in hepatic stellate cells susceptibility to natural killer cells during hepatic inflammation. The aim of this study was therefore to investigate the effect of vitamin D2 and free fatty acids on stress ligands and pro-fibrotic activity in LX-2 cells and human primary hepatic stellate cells. METHODS: LX-2 cells and primary human hepatic stellate cells were treated with vitamin D2 (10-6 M) and free fatty acids at different concentrations (0.25 mM, 0.5 mM, and 1 mM) for 24 hours, and expressions of the stress ligands MICA/B as well as of transforming growth factor-ß, α-smooth muscle actin and collagen 1α were assessed by quantitative real time-polymerase chain reaction. RESULTS: Treatment of cells with 0.5 mM and 1 mM free fatty acids induced α-smooth muscle actin and transforming growth factor-ß expression in LX-2 cells. Moreover, 1 mM free fatty acids resulted in increased expression of MICA. Surprisingly, collagen 1α expression was reduced after addition of free fatty acids. MICA/B expression in primary hepatic stellate cells was not affected by free fatty acids treatment. Vitamin D2 treatment significantly downregulated the free fatty acids-induced expression of transforming growth factor-ß and α-smooth muscle actin in LX-2 cells. Further, in hepatic stellate cells, a significant decrease in MICA/B mRNA with vitamin D2, independent of free fatty acids treatment, was detectable. CONCLUSIONS: These results indicate that vitamin D2 may reduce inflammatory and pro-fibrogenic activity of stellate cells in vitro.