ROLE OF OSTEOPONTIN IN ACUTE AND CHRONIC MOUSE MODELS of ALCOHOLIC LIVER DISEASE

Supervisors

1. Dr Devanshi Seth 2. A/Prof Paul Haber

Drug Health Services Drug Health Services

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Missenden Rd. Camperdown 2050. Missenden Rd. Camperdown 2050.

Ph: 9515 7201. Fax: 95158970 Ph: 95157838. Fax: 95158970

Email: Devanshi.seth@email.cs.nsw.gov.au Email: phaber@mail.usyd.edu.au

Techniques to be used in this project

· Animal handling: alcohol administration, blood and tissue collection

· Cell culture

· RNA and protein isolation, RT-PCR

· Western blot

· Flow cytometry

· Immunohistochemistry, ELISA

Brief Overview

Role of inflammation in alcoholic liver disease (ALD):

Inflammation is a hallmark of alcoholic liver injury and is characterized by intrahepatic infiltration of neutrophils or mononuclear cells and by altered local and systemic expression of inflammatory cytokines. It is not clear that inflammation initiates liver injury, but there is evidence it contributes to the progression of this disease. Both the innate immune response, induced by the actions of endotoxin, and the adaptive immune response, stimulated by tissue injury or by neo-antigens derived from alcohol metabolism, contribute to inflammation and tissue injury. A number of cytokines are prominent in ALD and these represent potential therapeutic targets.

Osteopontin (OPN) is now recognised as a Th1 cytokine that plays an important role in diverse benign and neoplastic disease processes. OPN binds to many receptors, including several integrins and CD44. In the liver, OPN expression is increased in models of chronic liver injury, where it is localised to Kupffer cells, macrophages and stellate cells. OPN directly mediates hepatic stellate cell activation in vitro and OPN upregulation was immediate and preceded evidence of tissue injury in vivo. In the OPN -/- mouse, liver injury was markedly attenuated with reduced ALT levels, lobular and portal inflammation, and collagen gene expression. Recently, three splice variants of OPN have been cloned. To date, the differential effects of these three splice variants have not been defined in liver disease. We have shown increased OPN in patients with ALD. Initial experiments in our laboratory have shown increased OPN in mouse liver as early as 4 hours after alcohol administration. Interestingly, our preliminary in vitro data (Honours project 2006) in hepatocyte and stellate cell lines indicated functional differences between splice variants in cell migration assays.

Hypothesis:

Osteopontin mediates the progression of alcoholic liver injury. Osteopontin promotes hepatic inflammatory cellular infiltration in response to ethanol.

Plan: We will utilize an acute alcohol C57BL6 mouse model recently developed by us. The Lieber-DeCarli model of ethanol administration will be used for the chronic alcohol C57BL6 mouse model. Animals will be match-fed in groups of 4 (wt control, wt ethanol-acute, wt ethanol-chronic). At the end of the feeding period (8 weeks for chronic; 4h, 8h, 24h for acute), the animals will be sacrificed, weighed, and plasma retained for measurement of LFTs (liver enzymes) and OPN. Plasma OPN would be determined by ELISA. The liver will be removed, weighed and divided for histology, biochemistry, immunohistochemistry, frozen samples and for RNA isolation. Histology will be assessed on formalin fixed, paraffin embedded sections (steatosis, hepatocyte injury and cellular infiltration). mRNA (RT-PCR) and protein (Western, ELISA, immunohistochemistry) levels will be determined for OPN and its receptors (v3, v5, 91 and41 integrins & CD44). For analysis of cellular infiltration, CD3 and CD4 markers for Th1 will be examined by immunohistochemistry and/or flow cytometery analysis to measure the infiltrating cells as described. Antibodies for these studies are commercially available (Pharmingen, USA).

Skills: It is envisaged that the student who undertakes this project will become proficient in all of these methods whilst being exposed to a number of other general laboratory techniques.

Conclusion and Significance: This project has great significance in determining the nature of the response to ethanol in liver injury. This project embodies a number of techniques and builds on established knowledge and expertise with our laboratory.