Andrea Galli

Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis

A genome-wide association screen for primary biliary cirrhosis risk alleles was performed in an Italian cohort. The results from the Italian cohort replicated IL12A and IL12RB associations, and a combined meta-analysis using a Canadian dataset identified newly associated loci at SPIB (P = 7.9 × 10−11, odds ratio (OR) = 1.46), IRF5-TNPO3 (P = 2.8 × 10−10, OR = 1.63) and 17q12-21 (P = 1.7 × 10−10, OR = 1.38).

Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)‐2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP‐2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide‐producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP‐2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose‐dependent manner. The addition of antioxidants as well as MMP‐2–specific inhibitors impaired these phenomena. X/XO treatment increased MMP‐2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane‐type 1 MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO‐induced MMP‐2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal‐regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3‐kinase (PI3K) abrogated X/XO‐elicited MMP‐2 upregulation and completely prevented X/XO‐induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP‐2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS‐mediated MMP‐2 expression. (HEPATOLOGY 2005;41:1074–1084.)

The Transcriptional and DNA Binding Activity of Peroxisome Proliferator-activated Receptor α Is Inhibited by Ethanol Metabolism A NOVEL MECHANISM FOR THE DEVELOPMENT OF ETHANOL-INDUCED FATTY LIVER

Fatty acids are ligands for the peroxisome proliferator-activated receptor α (PPARα). Fatty acid levels are increased in liver during the metabolism of ethanol and might be expected to activate PPARα. However, ethanol inhibited PPARα activation of a reporter gene in H4IIEC3 hepatoma cells expressing alcohol-metabolizing enzymes but not in CV-1 cells, which lack these enzymes. Ethanol also reduced the ability of the PPARα ligand WY14,643 to activate reporter constructs in the hepatoma cells or cultured rat hepatocytes. This effect of ethanol was abolished by the alcohol dehydrogenase inhibitor 4-methylpyrazole and augmented by the aldehyde dehydrogenase inhibitor cyanamide, indicating that acetaldehyde was responsible for the action of ethanol. PPARα/retinoid X receptor extracted from hepatoma cells exposed to ethanol or acetaldehyde bound poorly to an oligonucleotide containing peroxisome proliferator response elements. This effect was also blocked by 4-methylpyrazole and augmented by cyanamide. Furthermore, in vitro translated PPARα exposed to acetaldehyde failed to bind DNA. Thus, ethanol metabolism blocks transcriptional activation by PPARα, in part due to impairment of its ability to bind DNA. This effect of ethanol may promote the development of alcoholic fatty liver and other hepatic consequences of alcohol abuse.

Clinical ResearchHeart Rhythm DisorderShort- and Long-Term Prognosis of Syncope, Risk Factors, and Role of Hospital Admission: Results From the STePS (Short-Term Prognosis of Syncope) Study

OBJECTIVE We sought to assess short- and long-term prognosis of syncope and associated risk factors. BACKGROUND Syncope is a common clinical event, but our knowledge of its short-term outcome is largely incomplete. Further, it is unknown whether hospital admission might positively affect a patients syncope prognosis. METHODS We screened 2,775 consecutive subjects who presented for syncope at 4 emergency departments between January and July 2004. Short- and long-term severe outcomes (i.e., death and major therapeutic procedures) and related risk factors were compared in all enrolled patients arrayed according to hospital admission or discharge. RESULTS A total of 676 subjects were included in the study. Forty-one subjects (6.1%) experienced severe outcomes (5 deaths, 0.7%; 36 major therapeutic procedures, 5.4%) in the 10 days after presentation. An abnormal electrocardiogram, concomitant trauma, absence of symptoms of impending syncope, and male gender were associated with short-term unfavorable outcomes. Long-term severe outcomes were 9.3% (40 deaths, 6.0%; 22 major therapeutic procedures, 3.3%), and their occurrence was correlated with an age >65 years, history of neoplasms, cerebrovascular diseases, structural heart diseases, and ventricular arrhythmias. Short-term major therapeutic procedures were more common (p < 0.05) in subjects who had been admitted to hospital (13.3%) than in discharged (1.6%), whereas mortality was similar. One-year mortality was greater (p < 0.05) in admitted (14.7%) than in discharged (1.8%) patients. CONCLUSIONS Risk factors for short- and long-term adverse outcomes after syncope differed. Hospital admission favorably influenced syncope short term prognosis. Instead, 1-year mortality was unaffected by hospital admission and related to comorbidity.

Collagen type I synthesized by pancreatic periacinar stellate cells (PSC) co-localizes with lipid peroxidation-derived aldehydes in chronic alcoholic pancreatitis

Chronic alcoholic pancreatitis (CAP) is characterized by progressive pancreatic fibrosis and loss of the acinar cell mass, but the pathogenesis of pancreatic fibrosis in the human is poorly understood. It has been recently suggested that lipid peroxidation‐derived aldehydes such as 4‐hydroxynonenal (HNE) are involved in tissue damage and fibrosis in other organs. The aim of this study was to evaluate the role of oxidative stress in the development of alcohol‐induced pancreatic fibrosis in humans, and to assess the contribution of pancreatic periacinar stellate cells (PSC) in the in vivo synthesis of extracellular matrix components during CAP. Lipid peroxidation was evaluated in tissue specimens obtained from patients with CAP who underwent surgical procedures, by immunohistochemistry using a monoclonal antibody directed against HNE–protein adducts. Immunohistochemical determination of collagen type I, α‐smooth muscle actin (α‐SMA), and the β subunit of human platelet‐derived growth factor (PDGF‐Rβ) was also performed. In addition, the tissue mRNA expression of procollagen I, PDGF‐Rβ, and transforming growth factor‐β1 (TGF‐β1) was evaluated by in situ hybridization. In CAP, increased formation of HNE–protein adducts was evident in acinar cells adjacent to the interlobular connective tissue that stained positively for collagen type I. HNE staining was absent in normal pancreas. Several non‐parenchymal periacinar cells (PSC) underlay the HNE‐stained acinar cells. Those PSC stained positively for α‐SMA and PDGF‐Rβ and showed active synthesis of procollagen type I by in situ expression of the specific mRNAs. The pattern of expression of PDGF‐Rβ mRNA reflected that observed in immunostaining, showing increased amounts of transcripts in PSC. TGF‐β1 mRNA expression was increased in CAP, but transcripts were found in several cell types including PSC, acinar, and ductal cells. These results indicate that significant lipid peroxidation phenomena occur in CAP and that they are associated with active synthesis of collagen by PSC. Copyright

Pleiotropic effects of thiazolidinediones: Taking a look beyond antidiabetic activity

Thiazolidinediones (TZD) [Troglitazone (TRO), Pioglitazone (PGZ), Rosiglitazone, (RGZ)] are a novel class of antidiabetic drugs for patients with Type-2 diabetes mellitus (T2DM) able to decrease blood glucose, working through a reduction of insulin resistance. The family of TZD exerts its effect specifically bound to peroxisome proliferator- activated receptor ψ (PPARψ). This is a member of the nuclear hormone receptor superfamily of ligand-dependent transcription factors, together with PPARa and dgB. Although PPARψ is essentially expressed in adipose tissue, it has also been found in endothelial cells, macrophages, vascular smooth muscle cells, glomerular mesangial cells, hepatic stellate cells and in several cancer cell lines. In these cells, the PPARψ activation by TZD determines modulatory effects on growth factor release, production of cytokine, cell proliferation and migration, extracellular matrix remodeling and control on cell cycle progression and differentiation. In addition, TZD have been shown to have a potent antioxidant effect. This review, taking a quick look beyond the antidiabetic activity of PPARψ, shows the dramatic ranging of medical implications that the use of TZD could have modulating the PPARψ activity in several diseases with a strong social impact, such as insulin resistance syndrome, chronic inflammation, atherosclerosis and cancer.

Pathogenesis of alcoholic liver disease: Role of oxidative metabolism

Alcohol consumption is a predominant etiological factor in the pathogenesis of chronic liver diseases, resulting in fatty liver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (HCC). Although the pathogenesis of alcoholic liver disease (ALD) involves complex and still unclear biological processes, the oxidative metabolites of ethanol such as acetaldehyde and reactive oxygen species (ROS) play a preeminent role in the clinical and pathological spectrum of ALD. Ethanol oxidative metabolism influences intracellular signaling pathways and deranges the transcriptional control of several genes, leading to fat accumulation, fibrogenesis and activation of innate and adaptive immunity. Acetaldehyde is known to be toxic to the liver and alters lipid homeostasis, decreasing peroxisome proliferator-activated receptors and increasing sterol regulatory element binding protein activity via an AMP-activated protein kinase (AMPK)-dependent mechanism. AMPK activation by ROS modulates autophagy, which has an important role in removing lipid droplets. Acetaldehyde and aldehydes generated from lipid peroxidation induce collagen synthesis by their ability to form protein adducts that activate transforming-growth-factor-β-dependent and independent profibrogenic pathways in activated hepatic stellate cells (HSCs). Furthermore, activation of innate and adaptive immunity in response to ethanol metabolism plays a key role in the development and progression of ALD. Acetaldehyde alters the intestinal barrier and promote lipopolysaccharide (LPS) translocation by disrupting tight and adherent junctions in human colonic mucosa. Acetaldehyde and LPS induce Kupffer cells to release ROS and proinflammatory cytokines and chemokines that contribute to neutrophils infiltration. In addition, alcohol consumption inhibits natural killer cells that are cytotoxic to HSCs and thus have an important antifibrotic function in the liver. Ethanol metabolism may also interfere with cell-mediated adaptive immunity by impairing proteasome function in macrophages and dendritic cells, and consequently alters allogenic antigen presentation. Finally, acetaldehyde and ROS have a role in alcohol-related carcinogenesis because they can form DNA adducts that are prone to mutagenesis, and they interfere with methylation, synthesis and repair of DNA, thereby increasing HCC susceptibility.

PPARγ and Oxidative Stress: Con(β) Catenating NRF2 and FOXO

Peroxisome-proliferator activator receptor γ (PPARγ) is a nuclear receptor of central importance in energy homeostasis and inflammation. Recent experimental pieces of evidence demonstrate that PPARγ is implicated in the oxidative stress response, an imbalance between antithetic prooxidation and antioxidation forces that may lead the cell to apoptotic or necrotic death. In this delicate and intricate game of equilibrium, PPARγ stands out as a central player devoted to the quenching and containment of the damage and to foster cell survival. However, PPARγ does not act alone: indeed the nuclear receptor is at the point of interconnection of various pathways, such as the nuclear factor erythroid 2-related factor 2 (NRF2), Wnt/β-catenin, and forkhead box proteins O (FOXO) pathways. Here we reviewed the role of PPARγ in response to oxidative stress and its interaction with other signaling pathways implicated in this process, an interaction that emerged as a potential new therapeutic target for several oxidative-related diseases.

Alcohol induced hepatic fibrosis: Role of acetaldehyde

Alcohol abuse is one of the major causes of liver fibrosis worldwide. Although the pathogenesis of liver fibrosis is a very complex phenomenon involving different molecular and biological mechanisms, several lines of evidence established that the first ethanol metabolite, acetaldehyde, plays a key role in the onset and maintenance of the fibrogenetic process. This review briefly summarizes the molecular mechanisms underlying acetaldehyde pro-fibrogenic effects. Liver fibrosis represents a general wound-healing response to a variety of insults. Although mortality due to alcohol abuse has been constantly decreasing in the past 20 years in Southern Europe and North America, in several Eastern-European countries and Great Britain Alcoholic Liver Disease (ALD) shows a sharply increasing trend [Bosetti, C., Levi, F., Lucchini, F., Zatonski, W.A., Negri, E., La, V.C., 2007. Worldwide mortality from cirrhosis: an update to 2002. J. Hepatol. 46, 827-839]. ALD has a complex pathogenesis, in which acetaldehyde (AcCHO), the major ethanol metabolite, plays a central role. Ethanol is mainly metabolized in the liver by two oxidative pathways. In the first one ethanol is oxidized to acetaldehyde by the cytoplasmic alcohol dehydrogenase enzyme (ADH), acetaldehyde is then oxidized to acetic acid by the mitochondrial acetaldehyde dehydrogenase (ALDH). The second pathway is inducible and involves the microsomal ethanol-oxidizing system (MEOS), in which the oxidation of ethanol to acetaldehyde and acetic acid also leads to generation of reactive oxygen species (ROS). Chronic ethanol consumption significantly inhibits mitochondrial ALDH activity while the rate of ethanol oxidation to acetaldehyde is even enhanced, resulting in a striking increase of tissue and plasma acetaldehyde levels [Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clin. Chim. Acta 257, 59-84]. This review will focus on the molecular mechanisms by which acetaldehyde promote liver fibrosis.

Antidiabetic thiazolidinediones inhibit invasiveness of pancreatic cancer cells via PPARγ independent mechanisms

Background/Aims: Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of some epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferators activated receptor γ (PPARγ) the mechanism by which TZD exert their anticancer effect is currently unclear. Furthermore, the effect of TZD on local motility and metastatic potential of cancer cells is unknown. The authors analysed the effects of two TZD, rosiglitazone and pioglitazone, on invasiveness of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma. Methods: Expression of PPARγ in human pancreatic adenocarcinomas and pancreatic carcinoma cell lines was measured by reverse transcription polymerase chain reaction and confirmed by western blot analysis. PPARγ activity was evaluated by transient reporter gene assay. Invasion assay was performed in modified Boyden chambers. Gelatinolytic and fibrinolytic activity were evaluated by gel zymography. Results: TZD inhibited pancreatic cancer cells’ invasiveness, affecting gelatinolytic and fibrinolytic activity with a mechanism independent of PPARγ activation and involving MMP-2 and PAI-1 expression. Conclusion: TZD treatment in pancreatic cancer cells has potent inhibitory effects on growth and invasiveness suggesting that these drugs may have application for prevention and treatment of pancreatic cancer in humans.