The rapid progress in genome sequencing and the development of methodologies for large-scale high-throughput gene expression analysis on microarrays and laser capture microdissection (LCM) has made it feasible to obtain a complete representation of the patterns of gene expression in different cells and tissues with high spatial resolution, providing large amounts of detailed information relevant to specific disease states or other physiological conditions. The overall objective of this IRPG is to carry out these tasks in the context of acute or chronic alcohol exposure, using a well-established animal model. We have created substantial functional genomics and computational capabilities that will enable us to use obtain this information at a scale and throughput that is sufficient to yield very large and coherent quantitative datasets. This proposal will focus on the characterization of gene expression patterns in different cell types in the liver of control and ethanol-treated animals. The underlying hypothesis is that ethanol treatment affects liver function so as to impair its capacity to respond appropriately to stress conditions. We propose that a broad range of cell type-specific gene expression patterns will characterize the response of the normal liver to stress and that these responses are altered in livers of ethanol-fed animals. Analysis of these changes in gene expression patterns with appropriate computational kinetic methods will generate new levels of understanding of the mechanisms underlying the defects caused by ethanol treatment.
In Aim 1, we will isolate parenchymal cells, Kupffer cells, stellate cells and endothelial cells from the livers of ethanol-treated and control rats and analyze the gene expression patterns using microarrays generated in our shared resource or obtained commercially to characterize cell type-specific differences in gene expression, generating """"""""signatures"""""""" of gene expression patterns in different liver cell types and illustrate how these profiles are affected by ethanol intake.
In Aim 2, we will analyze extracts of different liver cell types obtained by LCM from pericentral and periportal zones of the liver acinus.
In Aim 3 animals will be exposed to acute stress, by acute endotoxin treatment or by ischemia-reperfusion treatment. We expect that the defective capacity of ethanol-fed animals to respond to these stress treatments will be reflected in changes in characteristic gene expression profiles for different liver cell types in different acinar zones. The concerted efforts of the different projects in the IRPG will prepare the ground for a fundamentally deeper evaluation of the actions of ethanol than can be pursued in a single project and a large array of new mechanistic hypothesis can be expected to result from these studies.
