Jordi Colmenero

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.

BACKGROUND & AIMS The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II. METHODS Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1. CONCLUSIONS After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.

Ascites and hepatorenal syndrome in cirrhosis: pathophysiological basis of therapy and current management

Ascites is the most common complication of cirrhosis [1,2]. It develops late during the course of the disease, when there is severe portal hypertension and hepatic insufficiency. Not surprisingly, it is associated with a poor survival [3] (50% mortality rate within 3 years, Fig. 1). The development of ascites is, therefore, a clear indication for liver transplantation. There are several studies indicating that parameters estimating systemic hemodynamics and renal function are better predictors of survival than those estimating hepatic function [4–6]. The prognosis of patients with dilutional hyponatremia, refractory ascites and hepatorenal syndrome (HRS) is extremely poor and liver transplantation should be indicated prior to the development of these complications [3,7]. Other parameters with prognostic value in cirrhotic patients with ascites are mean arterial pressure, plasma renin activity, plasma norepinephrine concentration, urinary sodium excretion, the renal ability to excrete free water, liver size, serum bilirubin, serum albumin concentration, and prothrombin time [8,9]. The current article is focused on the treatment of ascites and HRS in cirrhosis. To provide the reader with the rationale of the therapeutic measures used in patients with cirrhosis and ascites or HRS, the pathophysiology of these complications is briefly reviewed. The role of liver transplantation in the management of decompensated cirrhotic patients with ascites is not included in this review. 2. The pathophysiological basis of therapy of ascites and HRS

A New Scoring System for Prognostic Stratification of Patients With Alcoholic Hepatitis

OBJECTIVES: Prognostic stratification of patients with alcoholic hepatitis (AH) may improve the clinical management and facilitate clinical trials. We aimed at developing a scoring system capable of providing prognostic stratification of patients with AH.METHODS: Patients with biopsy-proven AH were prospectively included between 2000 and 2006. The biochemical, clinical, portal hemodynamic and histological parameters were evaluated. A Cox regression model was used for univariate and multivariate analyses. A predictive score was built using variables obtained at admission identified in the multivariate analysis. The resulting score was validated in an independent prospective cohort.RESULTS: In total, 103 patients with biopsy-proven AH were included in the study cohort. Age, serum bilirubin, serum creatinine, and international normalized ratio (INR) independently predicted 90-day mortality. We generated the Age, serum Bilirubin, INR, and serum Creatinine (ABIC) score: (age × 0.1) + (serum bilirubin × 0.08) + (serum creatinine × 0.3) + (INR × 0.8). The area under the curve (AUC) was 0.82. Using the Kaplan-Meier analysis with the cutoff values of 6.71 and 9.0, we identified patients with low, intermediate, and high risk of death at 90 days (100%, 70%, and 25% of survival rate, respectively). Using the same cutoff values, the ABIC score also stratified patients according to their risk of death at 1 yr. These results were validated by a confirmatory cohort (N = 80).CONCLUSIONS: The ABIC score is a new tool that allows the stratification of risk of death in patients with AH at 90 days and 1 yr. This score can help improve the management of these patients and also help to perform clinical trials.

Modulation of hepatic fibrosis by c-Jun-N-terminal kinase inhibition

BACKGROUND & AIMS c-Jun N-terminal kinase (JNK) is activated by multiple profibrogenic mediators; JNK activation occurs during toxic, metabolic, and autoimmune liver injury. However, its role in hepatic fibrogenesis is unknown. METHODS JNK phosphorylation was detected by immunoblot analysis and confocal immunofluorescent microscopy in fibrotic livers from mice after bile duct ligation (BDL) or CCl(4) administration and in liver samples from patients with chronic hepatitis C and non-alcoholic steatohepatitis. Fibrogenesis was investigated in mice given the JNK inhibitor SP600125 and in JNK1- and JNK2-deficient mice following BDL or CCl(4) administration. Hepatic stellate cell (HSC) activation was determined in primary mouse HSCs incubated with pan-JNK inhibitors SP600125 and VIII. RESULTS JNK phosphorylation was strongly increased in livers of mice following BDL or CCl(4) administration as well as in human fibrotic livers, occurring predominantly in myofibroblasts. In vitro, pan-JNK inhibitors prevented transforming growth factor (TGF) beta-, platelet-derived growth factor-, and angiotensin II-induced murine HSC activation and decreased platelet-derived growth factor and TGF-beta signaling in human HSCs. In vivo, pan-JNK inhibition did not affect liver injury but significantly reduced fibrosis after BDL or CCl(4). JNK1-deficient mice had decreased fibrosis after BDL or CCl(4), whereas JNK2-deficient mice displayed increased fibrosis after BDL but fibrosis was not changed after CCl(4). Moreover, patients with chronic hepatitis C who displayed decreased fibrosis in response to the angiotensin receptor type 1 blocker losartan showed decreased JNK phosphorylation. CONCLUSIONS JNK is involved in HSC activation and fibrogenesis and represents a potential target for antifibrotic treatment approaches.

Hepatic Expression of CXC Chemokines Predicts Portal Hypertension and Survival in Patients With Alcoholic Hepatitis

BACKGROUND & AIMS Alcoholic hepatitis (AH) is characterized by hepatocellular damage, inflammation, and fibrosis. We performed a prospective study to associate hepatic expression of the CXC subfamily of chemokines with histology findings and prognosis of patients with AH. METHODS Liver biopsy samples from 105 patients with AH and 5 normal liver samples (controls) were evaluated for steatosis, inflammation, fibrosis, and cholestasis. Computer-based morphometric analysis assessed the numbers of infiltrating CD3+ T cells and CD15+ cells (neutrophils); terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining was used to quantify apoptosis. Expression of CXC and CC chemokines and selected signaling components were assessed by quantitative reverse-transcription polymerase chain reaction; protein levels of interleukin (IL)-8 and Gro-alpha also were determined by immunohistochemistry. Serum levels of IL-8 and Gro-alpha were measured by enzyme-linked immunosorbent assay. The Cox regression model identified variables associated with mortality. RESULTS Most patients (75%) had severe AH; their 90-day mortality rate was 21.9%. In AH liver samples, expression of the CXC subfamily members IL-8, Gro-alpha, CXCL5, CXCL6, CXCL10, and platelet factor 4 was up-regulated and compared with controls. The CC chemokine CCL2, but not CCL5, also was up-regulated. Higher expression levels of IL-8, CXCL5, Gro-gamma, and CXCL6 were associated with worse prognosis. Expression of CXC components correlated with neutrophil infiltration and the severity of portal hypertension. In the multivariate analysis, IL-8 protein levels were an independent predictor of 90-day mortality. IL-8 and Gro-alpha serum levels did not correlate with prognosis. CONCLUSIONS Hepatic expression of CXC components correlates with prognosis of patients with AH. Reagents that target CXC chemokines might be developed as therapeutics.

Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans.

There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild‐type and ghrelin‐deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct–ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin‐deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (−994CT and −604GA) influenced the progression of liver fibrosis. Conclusion: Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy. (HEPATOLOGY 2010;51:974–985.)

Angiotensin II Activates IκB Kinase Phosphorylation of RelA at Ser536 to Promote Myofibroblast Survival and Liver Fibrosis

BACKGROUND & AIMS The transcription factor nuclear factor-kappaB (NF)-kappaB promotes survival of hepatic myofibroblasts and fibrogenesis through poorly defined mechanisms. We investigated the activities of angiotensin II and I kappaB kinase (IKK) in regulation of NF-kappaB activity and the role of these proteins in liver fibrosis in rodents and humans. METHODS Phosphorylation of the NF-kappaB subunit RelA at serine 536 (P-Ser(536)-RelA) was detected by immunoblot and immunohistochemical analyses. P-Ser(536)-RelA function was assessed using vectors that expressed mutant forms of RelA, cell-permeable blocking peptides, and assays for RelA nuclear transport and apoptosis. Levels of P-Ser(536)-RelA were compared with degree of fibrosis in liver sections from chronically injured rats and patients with hepatitis C virus-mediated fibrosis who had been treated with the AT1 antagonist losartan. RESULTS Constitutive P-Ser(536)-RelA is a feature of human hepatic myofibroblasts, both in vitro and in situ in diseased livers. Autocrine angiotensin II stimulated IKK-mediated phosphorylation of RelA at Ser(536), which was required for nuclear transport and transcriptional activity of NF-kappaB. Inhibition of angiotensin II, the angiotensin II receptor type 1 (AT1), or IKK blocked Ser(536) phosphorylation and stimulated myofibroblast apoptosis. Treatment of fibrotic rodent liver with the angiotensin converting enzyme (ACE) inhibitor captopril or the IKK inhibitor sulphasalazine resulted in loss of P-Ser(536)-RelA-positive myofibroblasts and fibrosis regression. In human liver samples, increased numbers of P-Ser(536)-RelA-positive cells were associated with fibrosis that regressed following exposure to losartan. CONCLUSIONS An autocrine pathway that includes angiotensin II, IKK, and P-Ser(536)-RelA regulates myofibroblast survival and can be targeted to stimulate therapeutic regression of 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.

Cigarette smoking exacerbates nonalcoholic fatty liver disease in obese rats

The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal‐regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase‐1 and procollagen‐alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). Conclusion: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD. (HEPATOLOGY 2010.)

Atorvastatin attenuates angiotensin II-induced inflammatory actions in the liver.

Statins exert beneficial effects in chronically damaged tissues. Angiotensin II (ANG II) participates in liver fibrogenesis by inducing oxidative stress, inflammation, and transforming growth factor-beta1 (TGF-beta1) expression. We investigate whether atorvastatin modulates ANG II-induced pathogenic effects in the liver. Male Wistar rats were infused with saline or ANG II (100 ng kg(-1) min(-1)) for 4 wk through a subcutaneous osmotic pump. Rats received either vehicle or atorvastatin (5 mg kg(-1) day(-1)) by gavage. ANG II infusion resulted in infiltration of inflammatory cells (CD43 immunostaining), oxidative stress (4-hydroxynonenal), hepatic stellate cells (HSC) activation (smooth muscle alpha-actin), increased intercellular adhesion molecule (ICAM-1), and interleukin-6 hepatic gene expression (quantitative PCR). These effects were markedly blunted in rats receiving atorvastatin. The beneficial effects of atorvastatin were confirmed in an additional model of acute liver injury (carbon tetrachloride administration). We next explored whether the beneficial effects of atorvastatin on ANG II-induced actions are also reproduced at the cellular level. We studied HSC, a cell type with inflammatory and fibrogenic properties. ANG II (10(-8)M) stimulated cell proliferation, proinflammatory actions (NF-kappaB activation, ICAM-1 expression, interleukin-8 secretion) as well as expression of procollagen-alpha(1(I)) and TGF-beta1. All of these effects were reduced in the presence of atorvastatin (10(-7)M). These results indicate that atorvastatin attenuates the pathogenic events induced by ANG II in the liver both in vivo and in vitro. Therefore, statins could have beneficial effects in conditions characterized by hepatic inflammation.