C. Surrenti

Prolonged n-3 polyunsaturated fatty acid supplementation ameliorates hepatic steatosis in patients with non-alcoholic fatty liver disease: a pilot study

Background  Recent studies suggest a role of n‐3 long‐chain polyunsaturated fatty acids (n‐3 PUFA) as peroxisome proliferator‐activated receptor‐α ligands in improving non‐alcoholic fatty liver disease (NAFLD) in rodents. However, data in humans are still lacking.

Regulation of extracellular matrix synthesis by transforming growth factor β1 in human fat-storing cells

BACKGROUND Fat storing cells (FSC) are nonparenchymal liver cells generally considered the major source of the hepatic extracellular matrix (ECM). Transforming growth factor beta 1 (TGF-beta 1) is a potent regulator of ECM synthesis in various cell types. In this study, the effect of TGF-beta 1 on procollagen types I, III, IV, laminin (Lam), and fibronectin (FN) synthesis in cultured human FSCs was analyzed. METHODS FSCs were isolated from wedge sections of normal human livers. Morphological studies were performed by immunofluorescence and electron microscopy. ECM components in human FSC cultures were measured by an enzyme-linked immunosorbent assay. The expression of messenger RNA (mRNA) was evaluated by Northern blot and in situ hybridization. RESULTS Cultured human FSCs displayed numerous fat droplets in the perinuclear zone, and immunoreactivity for vimentin and alpha-smooth muscle actin. A weak nonfibrillar staining was observed by using a polyclonal antidesmin antibody. TGF-beta 1 induced a dose-dependent increase of procollagen I, III, and FN accumulation in human FSC cultures, whereas procollagen IV and Lam production was not affected. Furthermore, TGF-beta 1 increased the expression of alpha 1 (I), alpha 1 (III) procollagen, FN and TGF-beta 1 mRNA in human FSC cultures. CONCLUSIONS These data indicate that TGF-beta 1 is able to increase the synthesis of procollagen I, III, and FN in cultured human FSCs. Moreover, TGF-beta 1 can induce its own mRNA in the same cells.

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.)

Substance P (Neurokinin-1) and Neurokinin A (Neurokinin-2) Receptor Gene and Protein Expression in the Healthy and Inflamed Human Intestine

Increasing evidence suggests that tachykinins are involved in the control of pathophysiological states, such as inflammation. The precise localization of tachykinin receptors is of paramount importance in the search for their possible physiological and pathological role; in this study, therefore, we attempted to define cellular sites of substance P (NK-1R) and neurokinin A (NK-2R) receptor expression in the healthy and the inflamed human intestine by in situ hybridization and immunohistochemistry. In the normal ileum and colon, NK-1R and NK-2R were localized to smooth muscle cells of the muscularis mucosae and propria and a few inflammatory cells of the lamina propria; NK-1R expression was also found in the muscular wall of submucosal blood vessels, enteric neurons and, to a lesser degree, in surface epithelial cells. Patients with Crohns disease and ulcerative colitis showed a dramatic increase in NK-1R density relative to controls, in both the inflamed and the uninvolved mucosa. Up-regulation of NK-1R was particularly evident on epithelial cells lining the mucosal surface and crypts, as well as on endothelial cells of capillaries and venules. Also, a marked increase in NK-2R expression was found in both groups of patients on inflammatory cells of the lamina propria, especially eosinophils. Our findings demonstrate that in the normal human intestine NK-1R and NK-2R are expressed in multiple cell types, which are endowed with different physiological functions; in addition, they demonstrate that both NK-1R and NK-2R are up-regulated in patients with Crohns disease and ulcerative colitis. Taken together, these observations may have important physiological and pathophysiological implications, and provide the rationale for the use of NK-1R and NK-2R antagonists in the treatment of inflammatory bowel disease.

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

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.

Leisure time but not occupational physical activity significantly affects cardiovascular risk factors in an adult population

Background  A large number of studies have demonstrated that regular physical activity during leisure time (LTPA) accounts for a significant protection against cardiovascular diseases (CVD). On the other hand, conflicting findings on the beneficial effects of occupational physical activity (OPA) have been reported. The aim of this study is to evaluate the possible influence of different amounts of LTPA and OPA on circulating levels of several parameters associated with an increased risk of CVD.

Eradication of helicobacter pylori reduces the rate of duodenal ulcer rebleeding: a long-term follow-up study

Objectives:The long-term efficacy of Helicobacter pylori eradication to reduce the rate of recurrence of peptic ulcer bleeding is still uncertain. We evaluated the rate of duodenal ulcer rebleeding for 48 months after H. pylori eradication.Methods:Thirty-two male patients with H. pylori infection and duodenal ulcer bleeding were treated with omeprazole (40 mg/day for 4 wk), colloidal bismuth (480 mg/day for 2 wk), amoxicillin (2 g/day for 1 wk), and metronidazole (750 mg/day for 1 wk), and followed up for 48 months. Endoscopy and tests for H. pylori infection were repeated every year.Results:Ulcer healed in all patients, but H. pylori infection persisted or recurred in 11 patients. Within 48 months, rebleeding occurred in nine (81.8%) of these patients, whereas the 21 patients who were persistently negative for H. pylori infection remained asymptomatic without rebleeding (0/21 = 0%, p < 0.002) during the whole follow-up.Conclusions:Eradication of H. pylori can reduce the rate of duodenal ulcer rebleeding for at least 4 yr, thus potentially modifying the natural history of the disease.

Effects of calcitonin gene-related peptide (CGRP), neurokinin A and neurokinin A (4-10) on the mitogenic response of human peripheral blood mononuclear cells

Summary(1)We have studied the ability of some regulatory peptides to induce a mitogenic (incorporation of tritiated thymidine) response in human peripheral blood mononuclear cells (PBMC) and to modify the response produced by phytohaemagglutinin (PHA), a well known PBMC mitogen. (2) Human calcitonin gene-related peptide (hCGRP), human or salmon calcitonin (hCT, sCT), neurokinin A (NKA) and neurokinin (4–10) (up to 1 μM for each peptide) did not produce per se any significant PBMC stimulation. (3) hCGRP (0.1 nM −1 μM) produced a concentration dependent enhancement of the response to a submaximal concentration of PHA (1 μg/ml). On the other hand, hCGRP decreased the mitogenic response to a maximal concentration of PHA (25 μg/ml). (4) Neither hCT nor sCT (0.1 nM−1 μM) had a significant influence on the response to PHA (1–25 μg/ml). (5) Both NKA and NKA (4–10) produced a concentration-dependent (1 fM −10 pM) enhancement of the response to 1 μg/ml PHA, while these compounds had no effect on the response to 25 μg/ml PHA. (6) These findings suggest a potent modulatory action of CGRP and NKA, two peptides present in sensory and other nerves, on immune function which is possibly mediated via C2 receptors for CGRP and NK-2 tachykinin receptors, respectively.