Cryostat: Preparation of liver
tissue for dissociation

Microscope: Separation of active HSC population

Genomics and Proteomics of Fibrosis

Fibrosis Group: Geraldine Grant, Mike Estep, Linda O'Reilly

Introduction:
Liver fibrosis is a central feature of chronic liver injury due to metabolic, genetic, viral disease. Extensive liver fibrosis, resulting from the deposition of collagen type I within the liver, can ultimately lead to cirrhosis and chronic liver failure, due to an unnatural hardening of the organ, thus inhibiting its function. Currently, there is no known treatment, with the exception of replacing the affected organ via transplant.

Hepatic stellate cells play a central role in the process of fibrosis, as they are the major source of collagen in the liver. Under normal conditions, stellate cells are “inactive”, and are mainly involved in retinol storage. Following liver injury, stellate cells become “activated” fibroblast-like cells. These cells dump their retinol store, mobilize to the site of injury, and promote fibrosis via collagen deposition, forming a scar. Scar formation is a natural defense against injury and under normal circumstances should resolve with the dissolution of the scar, once the injury is removed. The stellate cells should also be signaled to die at this point. However, in fibrosis the scar remains. The exact steps involved in this fibrogenic process and the associated molecular pathways are not entirely clear. We hope to gain insight into the fibrotic processes by determining the differential gene expression of fibrotic liver tissue.

Aims:
To investigate the molecular mechanisms of fibrosis, we will examine the transcriptional profile of the inactive and active stellate cells from patients with end stage cirrhosis, by differential gene expression, utilizing a custom cDNA microarray. The hepatic transcriptome is second only to the brain in size, with 25-40% of human genes expressed in the liver. This could make total genome analysis too complex. By custom building our microarray of 5700 genes, to include genes involved with fibrotic-specific pathways, such as apoptosis, and cell proliferation, we can focus specifically on fibrosis data.

Liver specimens from 20 patients undergoing orthotopic liver transplantation due to idiopathic fibrosis. The tissue is dissociated; the hepatic stellate cells are collected and segregated to their active and inactive populations. These samples are then hybridized to the microarray. The data obtained is then further investigated by RT-PCR, and cell culture experiments

Methodology:
Liver tissue is dissociated by collagenase perfusion for 1 hour. The resulting released cells are harvested by centrifugation. This cell pellet is then separated on a nycodenz density gradient. Due to the additional boyant density of the stored retinol, the inactive stellate cells will be at the top of the gradient, the activated stellate cells present in the pellet fraction. The active stellate cells are then separated from other liver cells, by plating on tissue culture treated plastic culture flasks for 1 hour. The active stellate cells, due to the fibroblast-like nature, bind to the flask surface, and the other cells can be washed away. The RNA will be extracted from both cells populations and investigated using the custom cDNA microarray, and by semi-quantitative RT-PCR.

Microarray: Examination of microarray data