DESCRIPTION (provided by applicant): The broad long-term objective is to improve methods of disease assessment and treatment in the liver. Approximately 5.5 million American adults (i.e., 2-3% of the adult US population) suffer from chronic liver disease. Alcoholic liver disease (ALD) accounts for the largest proportion of chronic liver disease in the West and exacerbates liver injury due to non-alcoholic causes. Alcoholic cirrhosis ranks as the 8th most common cause of mortality in the US and the second leading cause of GI-related deaths. There is an unmet need to improve our arsenal of diagnostic options by using modern molecular approaches to analyze biological specimens that can be collected in a minimally-invasive or non-invasive manner, and a new lead has emerged with the discovery of "exosomes". These are nanovesicles that are secreted by many cell types and which contain a complex mixture of microRNAs, mRNAs and proteins that reflect the transcriptional and/or translational activity of their producer cells. We showed that (i) miRs in circulating exosomes undergo dynamic changes during experimental fibrosis in mice; (ii) expression of fibrosis-related miRs or mRNA in exosomes secreted by hepatic stellate cells (HSC, the principal pro-fibrogenic cell type in the liver) reflects the induction status of fibrogenic pathways within the cells; (iii) expression in HC of molecules regulating exosome biogenesis is ethanol-dependent; (iv) fibrosis-related miRs are exosomally shuttled between HSC and are functional after being taken up by recipient cells; and (v) exosomes from HSC are preferentially taken up by HSC both in vivo and in vitro, highlighting the existence of a novel homing mechanism for HSC-derived exosomes. Thus our overall objective is to establish the role of exosomes in liver disease and their potential role in disease diagnosis or therapy. Our central hypothesis is that exosomes have diagnostic and therapeutic utility in liver disease, including that caused by alcohol. The Aims to test our hypothesis are: Specific Aim 1: Establish ethanol-mediated changes in exosome production and molecular payload. We will determine the effect of ethanol on pathways of exosome biogenesis and secretion in HSC and establish miR signatures in exosomes from the serum of ALD patients Specific Aim 2. Establish mechanisms and therapeutic applications of exosome homing to HSC. We will characterize integrin-mediated interactions between exosomes and HSC and, in an in vivo model of experimental liver fibrosis, establish their role in mediating HSC-specific localization and therapeutic efficacy of exosomes loaded with anti-fibrotic siCTGF. The expected outcome of these exploratory R21 studies will be innovative breakthroughs in the diagnosis and therapy of chronic liver disease by improved understanding and exploitation of exosome function. The rationale underlying the application is that identification of exosomal molecular payloads and intercellular shuttling mechanisms will have direct relevance to disease diagnosis and therapy. The positive impact of these studies is that they will improve the health of millions of people world-wide with ALD and other liver diseases.

PUBLIC HEALTH RELEVANCE: Alcoholic liver disease (ALD) ranks as the 8th most common cause of mortality in the US and is the second leading cause of GI-related deaths, but methods for disease assessment and patient treatment require improvement. This grant application seeks to understand the effect of alcohol on the production by liver cells of tiny secreted vesicles called "exosomes' which have an important function in delivering molecular messages between cells and which can be carried to distant sites in body fluids such as the blood. These studies will establish whether the complex molecular cargo in exosomes can be "read" to help assess the presence and severity of ALD, and whether scarring (fibrosis) in livers can be treated by delivery of exosomes that have been modified to carry therapeutic (anti-fibrotic) agents.