Profibrogenic Gene Expression Research Articles

Protective effect of xanthohumol on toxin-induced liver inflammation and fibrosis

Xanthohumol, the major prenylated chalcone found in hops, is known for its anti-inflammatory properties. We have recently shown that xanthohumol inhibits hepatic inflammation and fibrosis in a murine model of nonalcoholic steatohepatitis. The aim of this study was to investigate the effect of xanthohumol in an acute model of liver injury. Carbon tetrachloride (CCl(4)), an industrial solvent, is a hepatotoxic agent and its administration is widely used as an animal model of toxin-induced liver injury. Xanthohumol was applied orally at a dose of 1 mg/g body weight 2 days prior as well as during and after exposure to CCl(4). 72 h after a single CCl(4) application histomorphology and serum levels of transaminases revealed considerable hepatocellular necrosis, which was accompanied by significantly enhanced hepatic expression of pro-inflammatory cytokines. Furthermore, elevated hepatic alpha-smooth muscle actin expression indicated activation of hepatic stellate cells, and in accordance, we detected enhanced hepatic expression levels of TGF-β and collagen type I reflecting a marked fibrogenic response to CCl(4) exposure. While the degree of hepatocellular damage in response to CCl(4) was similar in mice which received xanthohumol and the control group, pro-inflammatory and profibrogenic hepatic gene expression were almost completely blunted in xanthohumol fed mice. Furthermore, xanthohumol fed mice revealed decreased hepatic NFκB activity. These results suggest that the protective effects of xanthohumol in this toxic liver injury model involves direct mechanisms related to its ability to block both hepatic inflammation and the activation of hepatic stellate cells, presumable at least in part via decreasing NFκB activity. Thus, this study further indicates the potential of xanthohumol application to prevent or ameliorate the development and progression of liver fibrosis in response to hepatic injury.

The effects of sorafenib on the portal hypertensive syndrome in patients with liver cirrhosis and hepatocellular carcinoma – a pilot study

Increased intrahepatic vascular resistance and hyperperfusion in the splanchnic circulation are the principal mechanisms leading to portal hypertension in cirrhosis. Several preclinical studies have demonstrated a beneficial effect of the multikinase inhibitor sorafenib on the portal hypertensive syndrome. To investigate the effect of sorafenib on hepatic venous pressure gradient (HVPG), systemic hemodynamics and intrahepatic mRNA expression of proangiogenic, profibrogenic and proinflammatory genes. Patients with liver fibrosis/cirrhosis and hepatocellular carcinoma were treated with sorafenib 400 mg b.d. HVPG measurement and transjugular liver biopsy were performed at baseline and at week 2. Changes in HVPG and intrahepatic mRNA expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), RhoA, tumour necrosis factor-alpha (TNF-α) and placental growth factor (PlGF) were evaluated. Thirteen patients (m/f = 12/1; Child-Pugh class A/B = 10/3) were included. The most common aetiology of liver disease was alcohol consumption (n = 7). Eleven patients had an elevated portal pressure, including eight patients with clinically significant portal hypertension. A significant decrease of HVPG (≥ 20% from baseline) was observed in four subjects. In HVPG responders, we observed mRNA downregulation of VEGF, PDGF, PlGF, RhoA kinase and TNF-α, while no substantial mRNA decrease was found in nonresponders in any of the five genes. In two of the four HVPG responders we observed a dramatic (43-85%) mRNA decrease of all five investigated genes. Larger controlled clinical trials are needed to demonstrate any potential beneficial effect of sorafenib on portal hypertension in patients with cirrhosis.

Accelerated CCl4-Induced Liver Fibrosis in Hjv-/- Mice, Associated with an Oxidative Burst and Precocious Profibrogenic Gene Expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl4. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.

The Renin–Angiotensin System and Reactive Oxygen Species: Implications in Pancreatitis

The renin-angiotensin system (RAS) is a circulating hormonal system involved in the regulation of blood pressure and circulating fluid electrolytes. Recent findings have revealed that locally generated angiotensin (Ang) II plays a pivotal role in normal physiology as well as pathophysiology in various tissues and organs, including the pancreas. This review article summarizes current progress that has been made in elucidating the putative roles of Ang II in both acute and chronic pancreatitis. A convergence of evidence suggests that the underlying mechanism may involve reactive oxygen species (ROS)-generating systems, such as nicotinamide adenine dinucleotide phosphate oxidase, and subsequent elevation of proinflammatory and profibrogenic gene expression as well as protein activity. More importantly, Ang II-induced ROS interacts with other ROS-generating systems to positively feed-forward the ROS-induced signaling. Advances in basic research indicate that RAS blockers may provide potential therapeutic role for the management of pancreatic inflammation and, more importantly, pancreatitis-associated complications. Genetic alterations resulting from a malfunction in the epigenetic control of pancreatic RAS could be a causative factor in the development of pancreatitis.

Subepithelial collagen deposition, profibrogenic cytokine gene expression, and changes after prolonged fluticasone propionate treatment in adult eosinophilic esophagitis: A prospective study

Recent research shows that both pediatric and adult patients with eosinophilic esophagitis (EoE) experience esophageal remodeling marked by increased collagen deposition in which TGF-β plays an important role. However, limited data are available on the intensity and reversibility of fibrous remodeling in adults with EoE. We sought to analyze differences in collagen deposition in the lamina propria (LP) and profibrogenic cytokine gene expression along with other changes induced by prolonged treatment with fluticasone propionate in adults with EoE. Ten adults given consecutive diagnoses of EoE were studied prospectively. Deep esophageal biopsy specimens were obtained before and after 1 year of treatment with fluticasone propionate. Collagen deposition in the LP was assessed in tissue sections with the aid of the Masson trichrome technique. IL5, TGFB1, fibroblast growth factor 9 (FGF9), and CCL18 gene expression was quantified through real-time PCR. EoE results were compared among samples from 10 adult patients with gastroesophageal reflux disease and 10 control subjects with healthy esophagi. Patients with EoE showed a significant increase in subepithelial collagen deposition; this correlated positively with eosinophil density in the LP and the patient's age. Prolonged steroid treatment induced a nonsignificant reduction in subepithelial fibrosis, which remained significantly higher than in control subjects. Profibrogenic cytokine gene expression also increased in patients with EoE, with IL5 (P < .001), FGF9 (P = .005), and CCL18 (P = .008) all significantly upregulated. After 1 year of treatment, a reduction was observed in gene expression; for CCL18 expression, this decrease was statistically significant (P < .001). Esophageal remodeling is associated with upregulated gene expression of profibrogenic cytokines in adults with EoE. Prolonged treatment with fluticasone propionate leads to a nonsignificant reduction in subepithelial collagen deposition accompanied by downregulation of profibrogenic cytokine gene expression, with that of CCL18 being especially significant.

S05 * ALCOHOL AND INFLAMMATION * S05.1 * THE ROLE OF CYTOKINES IN ALCOHOLIC STEATOHEPATITIS

S05 * ALCOHOL AND INFLAMMATION * S05.1 * THE ROLE OF CYTOKINES IN ALCOHOLIC STEATOHEPATITIS

Gut Microbiota Dysbiosis Is Associated with Inflammation and Bacterial Translocation in Mice with CCl4-Induced Fibrosis

BackgroundGut is the major source of endogenous bacteria causing infections in advanced cirrhosis. Intestinal barrier dysfunction has been described in cirrhosis and account for an increased bacterial translocation rate.Hypothesis and AimsWe hypothesize that microbiota composition may be affected and change along with the induction of experimental cirrhosis, affecting the inflammatory response.Animals and MethodsProgressive liver damage was induced in Balb/c mice by weight-controlled oral administration of carbon tetrachloride. Laparotomies were performed at weeks 6, 10, 13 and 16 in a subgroup of treated mice (n = 6/week) and control animals (n = 4/week). Liver tissue specimens, mesenteric lymph nodes, intestinal content and blood were collected at laparotomies. Fibrosis grade, pro-fibrogenic genes expression, gut bacterial composition, bacterial translocation, host's specific butyrate-receptor GPR-43 and serum cytokine levels were measured.ResultsExpression of pro-fibrogenic markers was significantly increased compared with control animals and correlated with the accumulated dose of carbon tetrachloride. Bacterial translocation episodes were less frequent in control mice than in treated animals. Gram-positive anaerobic Clostridia spp count was decreased in treated mice compared with control animals and with other gut common bacterial species, altering the aerobic/anaerobic ratio. This fact was associated with a decreased gene expression of GPR43 in neutrophils of treated mice and inversely correlated with TNF-alpha and IL-6 up-regulation in serum of treated mice along the study protocol. This pro-inflammatory scenario favoured blood bacterial translocation in treated animals, showing the highest bacterial translocation rate and aerobic/anaerobic ratio at the same weeks.ConclusionsGut microbiota alterations are associated with the development of an inflammatory environment, fibrosis progression and bacterial translocation in carbon tetrachloride-treated mice.

Nilotinib Attenuates Renal Injury and Prolongs Survival in Chronic Kidney Disease

The tyrosine kinase inhibitor imatinib is beneficial in experimental renal diseases, but the effect of the new tyrosine kinase inhibitor nilotinib on the progression of renal failure is unknown. We administered either nilotinib or vehicle to Sprague-Dawley rats beginning 2 weeks after 5/6 nephrectomy (Nx) or laparotomy and continuing for 8 weeks. Serum creatinine levels were significantly lower in the nilotinib group after 6 and 8 weeks of treatment. Furthermore, nilotinib-treated rats had less proteinuria, attenuated glomerulosclerosis and tubulointerstitial damage, and reduced macrophage infiltration into the tubulointerstitium. Treatment with nilotinib also significantly decreased renal cortical expression of profibrogenic genes, such as IL-1β and monocyte chemotactic protein-1, which correlated closely with the tubulointerstitial damage score and ED1-positive macrophages score. In addition, nilotinib treatment significantly prolonged survival. Taken together, these results suggest that nilotinib may limit the progression of chronic kidney disease.

AT1R-CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II

The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB(1)R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB(1)R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB(1)R is upregulated. We found a significant upregulation of AT1R-CB(1)R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB(1)R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.

Chronic exposure to nicotine induces biliary growth and fibrosis

Nicotine stimulates fibrogeneis that is mediated through α7nAChR. Our Aim was to determine the effects of nicotine on cholangiocyte (BEC) growth and profibrogenic gene expression. Methods α7nAChR expression was assessed in liver sections from normal and BDL rats and NRIC (cell line). Normal and BDL rats were treated with nicotine (24 mg/kg/BW per day) or vehicle for 2 wks by osmotic minipumps. In vivo, proliferation was determined by intrahepatic bile duct mass (IBDM) in liver sections. Collagen deposition was evaluated by Masson's trichrome staining. The effect of nicotine (1 μM, 24–48 hrs) on NRIC growth was determined by MTS assay with/without α-bungarotoxin (ABT, α7nAChR inhibitor). Nicotine-induced CTGF, TGFβ1, and Col1A1 gene expression was evaluated by qPCR. Results BEC expressed α7nAChR. Nicotine induced an increase in IBDM in normal and BDL rats, and increased collagen deposition surrounding bile ducts in normal and BDL rats. Nicotine-stimulated proliferation was blocked by ABT. Nicotine stimulated CTGF, TGFβ1, and Col1A1 gene expression. Conclusion Nicotine stimulates the growth of BEC and induces profibrogenic gene expression.

Leptin and Acetaldehyde Synergistically Promotes αSMA Expression in Hepatic Stellate Cells by an Interleukin 6-Dependent Mechanism

The mechanisms whereby patients with obesity/overweight are more susceptible to alcohol-associated liver fibrosis are unclear. Leptin, a peptide hormone secreted by white adipose tissue is increased in association with overweight/obesity and is recognized as mediator of liver fibrosis. We sought to assess whether leptin contributes to alcoholic liver fibrosis by in vitro studies in hepatic stellate cells (HSC). Rat HSCs in second or third passage were utilised. Leptin, Acetaldehyde or combination with leptin and acetaldehyde were incubated for specific periods in cultured HSCs. Profibrogenic gene and protein expression were determined and associated-signalling pathways were assessed. Interleukin 6 (IL-6) antibody neutralization was used to evaluate the role of IL-6. Leptin did not promote acetaldehyde-induced gene expression of collagen I, transforming growth factor β1 (TGFβ1) and tissue inhibitor of metalloproteinase 1 (TIMP1) in vitro. However, combined treatment of leptin with acetaldehyde synergistically enhanced the protein expression of smooth muscle actin (αSMA), an activation marker of HSCs, and of Interleukin-6 (IL-6). The combination of leptin and acetaldehyde also augmented MAPK/p38 and MAPK/ERK1/2 phosphoprotein expression. IL-6 neutralization down-regulated protein expression of pp38, pERK1/2 and αSMA, while exogenous rat recombinant IL-6 administration up-regulated αSMA. Similarly, MAPK/p38 and MAPK/ERK1/2 inhibition attenuated αSMA expression. H(2)O(2) induction by acetaldehyde was not potentiated by co-treatment with leptin nor did IL-6 neutralization reduce acetaldehyde-induced H(2)O(2) production. We conclude that leptin potentiates acetaldehyde-induced HSC activation and αSMA expression by an IL-6-dependent mechanism.

Deficiency of phosphatidylinositol 3-kinase (PI3K) aggravates hepatic inflammation and fibrosis in experimental NASH but protects against bile duct ligation induced liver fibrosis

Aims: Non alcoholic steatohepatitis (NASH) leads to liver fibrosis characterized by activation of hepatic stellate cells (HSC) and enhanced collagen expression. We were able to show that PI3K, an ubiquitary heterodimeric lipid kinase, participates in HSC proliferation and collagen expression. Therefore, the aim of this study was to evaluate the role of PI3K in experimental NASH and biliary fibrosis. Methods: PI3K knockout mice (PI3K-/-, C57Bl/6 background) and C57Bl/6 wild-type control mice (WT) were allocated to 4 experimental groups (n=6) receiving either standard chow (SC) or a high fat (HF) diet according. In addition, bile duct ligation (BDL) and sham operations were performed. Results: Histological analysis revealed hepatic steatosis, inflammation and fibrosis in WT mice receiving a HF fat diet. However, these histological features of NASH were more pronounced PI3K-/- mice. Serum analysis showed elevated liver enzymes in the HF WT group, which were significantly higher in PI3K-/- mice. Hepatic mRNA expression of proinflammatory and profibrogenic genes was significantly induced in PI3K-/- HF mice compared to WT HF mice, as was expression of TLR4 and -9. Accordingly, elevated collagen I protein and alpha-smooth muscle actin (SMA) expression was evident. Furthermore, gene expression indicating oxidative stress was increased in PI3K-/- mice. In contrast, PI3K-/- mice were protected from BDL-induced liver injury indicated by lower serum transaminases, as well as decreased proinflammtory and profibrogenic gene expression, and less fibrosis. Interestingly, increased hepatic expression of PI3K p110 gamma was deteced in WT mice following BDL but not in mice with NASH. Conclusion: PI3K deficiency differently affects hepatic inflammation and fibrosis, depending on the type of injury and the predominant cell-type involved. These differences may have implications with regards to diagnosis and therapy of (fibrosis progression in) non-alcoholic liver disease.

BMP6 protects against hepatic fibrosis in experimental NASH models

A significant number of patients with non-alcoholic fatty disease (NAFLD) develop significant inflammation (NASH) and fibrosis, which may progress to cirrhosis. Different studies have uncovered bone morphogenetic proteins (BMPs) as important molecules being involved in organ fibrosis, however, studies regarding hepatic fibrosis are sparse and mostly focused on BMP7, of which anti-fibrogenic effects have been described. The aim of this study was to analyze the expression and function of BMP6 in NASH induced fibrosis. Methods and Results: BMP6 deficient (BMP6-/-) and 129Sv/Ev wild type control mice (wt) were subjected to two established dietary NASH-models: (i) a high fat diet (HFD; for 3 months) or (ii) a methionine choline deficient diet (MCD; for 5 weeks). In both models hepatic BMP6 expression was markedly increased compared to the control group fed with standard chow. In contrast, no induction of BMP7 was observed. In both models BMP6-/- mice exhibited increased hepatic expression of pro-inflammatory (MCP-1, TNF-alpha) and pro-fibrogenic (TGF-β, Collagen I) genes compared to wt mice. Additionally, alpha-SMA immunohistochemistry revealed activation of hepatic stellate cells (HSC) and reticulin and sirius red staining evidenced significant histological fibrosis. Noteworthy, hepatic inflammation and fibrosis were significantly higher in BMP6-/- mice in both models. Ex vivo analysis showed that neither freshly isolated nor in vitro activated HSC express BMP6. Also in primary hepatocytes basal BMP6 expression is on the detection limit. However and interestingly, in an in vitro model of hepatocellular lipid accumulation we observed a marked up-regulation of BMP6 expression in primary hepatocytes. Conclusion: Hepatic steatosis induces hepatic BMP6 expression, which inhibits NASH induced inflammation and fibrosis. Thus, (recombinant) BMP6 appears as potential therapy to inhibit the progression of NAFLD.

A novel in vitro model of alcohol-induced hepatic fibrogenesis

Chronic alcohol consumption is one of the main etiological factors for liver disease worldwide, however, only a fraction of drinkers develops significant hepatic inflammation, and even less progresses to significant hepatic fibrosis and cirrhosis. Still, there are no experimental models that lead to relevant hepatic fibrosis, and there is a high need to unravel the mechanisms and factors promoting hepatic injury in alcoholic liver disease (ALD).The aim of this study was to establish an in vitro model to study the hepatocyte mediated effects of alcohol on hepatic stellate cells (HSCs), the central mediators of liver fibrogenesis. Methods and Results: HepG2 hepatoma cells and primary human hepatocytes (PHH) were incubated with different concentrations of alcohol (0.1–1%) for 48h, which did not affect cell viability or mitochondrial activity but led to dose-dependent hepatocellular lipid accumulation as assessed by analysis of triglyceride content and free fatty acid levels, and as visualized by oil red o staining. Subsequently, human HSC were incubated with conditioned media (CM) from alcohol stimulated (Alc) or control hepatocytes. Further controls included control medium with and without alcohol (at concentrations determined in the CM from Alc-treated cells). Noteworthy, incubation of HSC with CM from Alc-treated hepatocytes significantly induced proliferation as well as pro-fibrogenic (TGF-beta) and NFkapaB-mediated pro-inflammatory gene (IL-8, RANTES) expression compared to the control groups. Conclusion: Our in vitro data indicate release of soluble factors as a potential mechanism for development and progression of fibrogenesis in alcoholic liver disease, the identification of which may lead to novel prognostic markers. Thus, this study provides an attractive in vitro model to study molecular mechanisms of alcohol-induced fibrogenesis, and to identify and test novel targets for anti-fibrotic therapies in alcoholic liver disease.

All-trans retinoic acid diminishes collagen production in a hepatic stellate cell line via suppression of active protein-1 and c-Jun N-terminal kinase signal

Following acute and chronic liver injury, hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content, but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood. The influence of retinoids on HSCs and hepatic fibrosis remains controversial. The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation, mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), fibrolytic genes (MMP-3, MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G). Cell proliferation was evaluated by measuring BrdU incorporation. The mRNA expression levels of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and fibrolytic genes (MMP-3, MMP-13) were quantitatively detected by using real-time PCR. The mRNA expression of JNK and AP-1 was quantified by RT-PCR. The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)] and profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1. These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal, then decrease the mRNAs expression of profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly induce the mRNA expression of MMP-3 and MMP-13.

Xanthohumol, a chalcon derived from hops, inhibits hepatic inflammation and fibrosis

Xanthohumol (XN) is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. In this study, we first investigated the effects of XN on hepatocytes and hepatic stellate cells (HSC), the central mediators of liver fibrogenesis. XN inhibited the activation of primary human HSC and induced apoptosis in activated HSC in vitro in a dose dependent manner (0-20 microM). In contrast, XN doses as high as 50 microM did not impair viability of primary human hepatocytes. However, in both cell types XN inhibited activation of the transcription factor NFkappaB and expression of NFkappaB dependent proinflammatory genes. In vivo, feeding of XN reduced hepatic inflammation and expression of profibrogenic genes in a murine model of non-alcoholic steatohepatitis. These data indicate that XN has the potential as functional nutrient for the prevention or treatment of non-alcoholic steatohepatitis or other chronic liver disease.

Ergocalciferol significantly inhibits profibrogenic and proinflammatory gene expression by hepatic stellate cells: Ergocalciferol a potential supplement in the therapy of liver fibrosis

Aims: Patients with Fibrosis exhibit Vitamin-D (VD) deficiency. HSC store VD, express the VD-Receptor1 (VDR1) and play a key role in hepatic fibrogenesis. Thus we studied the effect of VD and its derivatives on HSC. Methods: LX2 cells were treated with VD2 (10–6 M), 25-hydroxyvitamin D3 (25VD; 10–7 M), or 1,25-dihydroxyvitamin D3 (1,25VD; 10–8 M) for 24 or 48h. Expression of α-SMA, COL1A, TGF-β, MCP1, PDGFR, VDR and IL-6 were determined in qRT-PCR and Western Blot (TGF-β). Metabolization of VD2 into 25VD was measured in culture supernatants (25VD EIA-Kit). 20 low or high fibrosis patients (LF, HF; identified by NAS) and 4 healthy controls were assayed for VDR1 expression and 25 VD in serum. Results: LX2 cells metabolized VD concentration- and time-dependently. VD2 treatment significantly reduced expression of COL1A (0.77±0.045; 0.81±0.068), TGF-β (0.78±0.038; 0.73±0.045), MCP1 (0.78±0.065; 0.59±0.04), IL-6 (0.72±0.065; 0.52 ±0.055), α-SMA (0.69±0.059; 0.65±0.074) and PDGFR (0.79±0.065; 0.82±0.102) 24 and 48h post treatment. 25VD led to a significant decrease of TGF-β (0.75±0.042), MCP1 (0.81±0.059), and α-SMA (0.78±0.063) mRNA at 24h, while PDGFR was reduced 24h and 48h after treatment (0.74±0.054; 0.73±0.048). 1,25VD significantly inhibited the expression of MCP1 at 48h (0.78±0.035), of TGF-β (0.81 ±0.035; 0.81±0.087) and PDGFR (0.83±0.072; 0.77±0.055) at 24h and 48h. Western blot displayed lowered TGF-β protein concentration 48h after VD2 or 25VD application. Patients with fibrosis exhibited significantly reduced serum 25 VD compared to controls. HF patients exposed increased VDR expression (LF: 1.1±0.2; HF: 1.9±0.41) Conclusions: VD depletion and high VDR1 expression seem indicative for progression from mild to extended fibrosis. Since VD in vitro reduces VDR and profibrotic/proinflammatory expression in HSC, it may be a potential supplement for liver fibrosis.

Effects of losartan on hepatic expression of nonphagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C

Angiotensin II promotes liver fibrogenesis by stimulating nonphagocytic NADPH oxidase (NOX)-induced oxidative stress. Angiotensin II type 1 (AT1) receptor blockers attenuate experimental liver fibrosis, yet their effects in human liver fibrosis are unknown. We investigated the effects of losartan on hepatic expression of fibrogenic, inflammatory, and NOX genes in patients with chronic hepatitis C (CHC). Fourteen patients with CHC and liver fibrosis received oral losartan (50 mg/day) for 18 mo. Liver biopsies were performed at baseline and after treatment. The degree of inflammation and fibrosis was evaluated by histological analysis (METAVIR). Collagen content was measured by morphometric quantification of Sirius red staining. Overall collagen content and fibrosis stage remained stable in the whole series, yet the fibrosis stage decreased in seven patients. Inflammatory activity improved in seven patients. The effect of losartan on hepatic expression of 31 profibrogenic and inflammatory genes and components of the NOX complex was assessed by quantitative PCR. Losartan treatment was associated with a significant decrease in the expression of several profibrogenic and NOX genes including procollagen alpha1(I) and alpha1(IV), urokinase-type plasminogen activator, metalloproteinase type 2, NOX activator 1 (NOXA-1) and organizer 1 (NOXO-1), and Rac-1. Losartan was well tolerated in all patients and was effective in attenuating the activity of the systemic renin-angiotensin system. No effects on serum liver tests or viral load were observed. We conclude that prolonged administration of losartan, an oral AT1 receptor blocker, is associated with downregulation of NOX components and fibrogenic genes in patients with CHC. Controlled studies are warranted to assess the effect of AT1 receptor blockers in chronic liver injury.

Adenoviral delivery of dominant‐negative transforming growth factor β type II receptor up‐regulates transcriptional repressor SKI‐like oncogene, decreases matrix metalloproteinase 2 in hepatic stellate cell and prevents liver fibrosis in rats

Dominant-negative transforming growth factor beta type II receptor (TbetaRIIDeltacyt) is a protein that blocks transforming growth factor (TGF-beta) signaling. Because the consequences of blocking TGF-beta have not been completely elucidated in liver fibrosis, we analysed the effects of adenoviral delivery of TbetaRIIDeltacyt on profibrogenic genes and matrix metalloproteinase (MMP) proteins, as well as on TGF-beta signal repressor SKI-like oncogene (SnoN), in cultured hepatic stellate cells (HSCs) and in a rat model of liver fibrosis. To induce liver fibrosis, rats were treated with thioacetamide for 7 weeks and administrated once with Ad-TbetaRIIDeltacyt or Ad-betagal through the iliac vein. Fibrosis was measured by morphometric analysis. We evaluated SnoN by western blot, immunocytochemistry and immunohistochemistry; MMP activity was determined by zymography and profibrogenic gene expression by the real-time reverse transcriptase-polymerase chain reaction in cultured HSCs and liver tissue. Profibrogenic gene expression of collagen alpha1 (I), TGF-beta1, platelet-derived growth factor-B, plasminogen activator inhibitor (PAI)-1, tissue inhibitor of matrix metalloproteinase-1 and MMP-2 was down-regulated; whereas MMP-3 was over-expressed in response to Ad-TbetaRIIDeltacyt in HSCs. Moreover, zymography assays corroborated MMP-2 and MMP-3 changes in activity. Surprisingly, anti-TGF-beta molecular intervention increased nuclear SnoN in HSCs. In vivo, Ad-TbetaRIIDeltacyt reduced liver fibrosis, increased nuclear SnoN in sinusoidal cells, and also produced significant suppression in collagen alpha1 (I), TGF-beta1, PAI-1, MMP-2 and over-expression in MMP-3 in thioacetamide-intoxicated animals. The results obtained in the present study suggest that the molecular mechanism for the blocking effects of Ad-TbetaRIIDeltacyt in TGF-beta signaling acts via up-regulation of the transcriptional repressor SnoN, which antagonizes TGF-beta signaling (TGF-beta/Smad-pathway inhibitor). Consequently, profibrogenic genes are down-regulated.

Role of the receptor for advanced glycation end products inhepatic fbrosis

To study the role of advanced glycation end products (AGE) and their specific receptor (RAGE) in the pathogenesis of liver fibrogenesis. In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells (HSC) were measured after stimulation with the two RAGE ligands, advanced glycation end product-bovine serum albumin (AGE-BSA) and N(epsilon)-(carboxymethyl) lysine (CML)-BSA, or with tumor necrosis factor-alpha (TNF-alpha). In vivo RAGE expression was examined in models of hepatic fibrosis induced by bile duct ligation or thioacetamide. The effects of AGE-BSA and CML-BSA on HSC proliferation, signal transduction and profibrogenic gene expression were studied in vitro. In hepatic fibrosis, RAGE expression was enhanced in activated HSC, and also in endothelial cells, inflammatory cells and activated bile duct epithelia. HSC expressed RAGE which was upregulated after stimulation with AGE-BSA, CML-BSA, and TNF-alpha. RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation, apoptosis, fibrogenic signal transduction and fibrosis- or fibrolysis-related gene expression, except for marginal upregulation of procollagen alpha1(I) mRNA by AGE-BSA. Despite upregulation of RAGE in activated HSC, RAGE stimulation by AGE does not alter their fibrogenic activation. Therefore, RAGE does not contribute directly to hepatic fibrogenesis.