Activation of stellate cells, a key issue in the pathogenesis of hepatic fibrosis, is mediated by factors released from damaged hepatocytes and activated Kupffer cells. Understanding the mechanisms by which Kupffer cells modulate the formation and secretion of the components of the scar tissue is of great relevance for potential therapeutic intervention, and this constitutes the major goal of this application. We hypothesize that Kupffer cell-derived factors/reactive species play a critical role in the stellate cell fibrogenic response. In this application we propose: 1) to explore the impact of Kupffer cells on stellate cell collagen I production using an in vitro co-culture model of primary rat Kupffer cells and primary stellate cells. We will analyze whether the effect on collagen I expression involves transcriptional regulation by carrying out nuclear in vitro transcription run-on assays, promoter studies, mRNA stability, and Northern blot analysis. To study if protein synthesis is affected, the rate of collagen I synthesis and degradation will be calculated;2) to determine if Kupffer cell-derived reactive species are the mediators for collagen I up-regulation in stellate cells, concentration of prooxidant and nitrosative species will be measured and the effect of incubating the co-cultures with antioxidants and inhibitors will be studied. Studies to identify the source of reactive species in the co-culture (e.g. NADPH oxidase, xanthine oxidase, mitochondria, cytochrome P450 2E1, and inducible nitric oxide synthase) will involve addition of inhibitors or chemical inducers. The use of conditioned medium from Kupffer cells added to stellate cells, and transfection with siRNA to selectively silence the sources of reactive species in one cell type or the other will help to dissect the contribution of each cell type to the effects observed in the co-culture;3) to characterize the contribution of arachidonic acid, as a representative polyunsaturated fatty acid, on collagen I expression in the co-cultures and to compare it to that of other fatty acids. Mechanistic approaches will include evaluating the contribution of lipid peroxidation reactions and the arachidonic acid metabolic pathways;and 4) to assess the contribution of chronic ethanol feeding to collagen I expression by stellate cells in co-culture with Kupffer cells. The basal expression of collagen I in control stellate cells will be compared to that of stellate cells from ethanol-treated rats, and the contribution of reactive species will be assessed. Mechanistic studies will follow to understand how Kupffer cells modulate the fibrogenic response in stellate cells from ethanol-treated rats. We hope that this co-culture model will help us to define the effects of Kupffer cells on the stellate cell fibrogenic response and to dissect potential therapeutic targets for preventing liver disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK069286-05
Application #
7643259
Study Section
Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
Program Officer
Serrano, Jose
Project Start
2005-09-15
Project End
2011-06-30
Budget Start
2009-07-01
Budget End
2011-06-30
Support Year
5
Fiscal Year
2009
Total Cost
$242,555
Indirect Cost
Name
Icahn School of Medicine at Mount Sinai
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
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Magdaleno, Fernando; Blajszczak, Chuck C; Nieto, Natalia (2017) Key Events Participating in the Pathogenesis of  Alcoholic Liver Disease. Biomolecules 7:
Arriazu, Elena; Ge, Xiaodong; Leung, Tung-Ming et al. (2017) Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury. Gut 66:1123-1137
Magdaleno, Fernando; Arriazu, Elena; Ruiz de Galarreta, Marina et al. (2016) Cartilage oligomeric matrix protein participates in the pathogenesis of liver fibrosis. J Hepatol 65:963-971
Lu, Yongke; Ward, Stephen C; Nieto, Natalia (2014) Ethanol plus the Jo2 Fas agonistic antibody-induced liver injury is attenuated in mice with partial ablation of argininosuccinate synthase. Alcohol Clin Exp Res 38:649-56
Ge, Xiaodong; Leung, Tung-Ming; Arriazu, Elena et al. (2014) Osteopontin binding to lipopolysaccharide lowers tumor necrosis factor-? and prevents early alcohol-induced liver injury in mice. Hepatology 59:1600-16
Arriazu, Elena; Ruiz de Galarreta, Marina; Cubero, Francisco Javier et al. (2014) Extracellular matrix and liver disease. Antioxid Redox Signal 21:1078-97
Wang, Xiaodong; Lopategi, Aritz; Ge, Xiaodong et al. (2014) Osteopontin induces ductular reaction contributing to liver fibrosis. Gut 63:1805-18
Leung, Tung-Ming; Nieto, Natalia (2013) CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease. J Hepatol 58:395-8
Leung, Tung-Ming; Wang, Xiaodong; Kitamura, Naoto et al. (2013) Osteopontin delays resolution of liver fibrosis. Lab Invest 93:1082-9

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