Liver fibrosis and regeneration in dogs and cats: An immunohistochemical approach

Abstract

In this thesis we focus on liver tissue repair processes in canine and feline hepatitis, on formalin fixed paraffin embedded archival liver specimens. Hepatitis was diagnosed using histological standard criteria, and always includes hepatocellular cell death and an inflammatory infiltrate. Subsequently, the liver may react with regeneration and fibrosis (a detectable deposit of extracellular matrix). Fibrosis can develop into cirrhosis, which has a poor prognosis. Based on a better understanding of the underlying reaction pattern of hepatic tissue repair we hope to facilitate the development of new anti-fibrotic therapies. Moreover, the liver is a very interesting model organ for tissue repair in general. In contrast to most other organs, disease progress and associated tissue repair attempts can be monitored relatively easily. This study contains the first identification of a liver progenitor cell compartment in the dog and cat, comparable to human liver. Several key cell types regarding hepatic fibrosis and regeneration were identified immunohistochemically: hepatocytes, cholangiocytes, fibroblasts including hepatic stellate cells (HSCs) and progenitor cells. Also, immunohistochemical localization of major signaling factors determining the outcome of hepatic tissue repair were investigated: hepatocyte growth factor (HGF), HGF receptor (c-Met) and TGF-beta-receptor1 (TGF-β-R1) as occurring in spontaneous canine hepatitis. These findings were compared to previously determined associated messenger-RNA (mRNA) and protein expression levels. Remarkably, the absolute HGF availability did not seem the limiting factor in hepatic regeneration. In acute hepatitis, hepatic stellate cell upregulation of HGF was restricted to locations adjacent to necrotic areas. All proliferating progenitor cells (reactive ductules) showed mitogenic stimulation, but could be restrained from further differentiation by TGF-beta signaling from encompassing TGF-β-R1 positive cells. Interface locations of chronic hepatitis (CH) showed highest activity of the inflammatory process During fibrosis, the extracellular matrix (ECM) increases in volume. We studied the distribution of the ECM protein tenascin-C (TN-C). TN-C distribution in CH matched the distribution of the active interface phenotype. Possibly, TN-C staining combined with α-SMA could be useful to assess the type or activity of the fibrosing process, and HSC activation, respectively. At sites (as seen in CH) with enhanced ECM turnover, the disease process is active, with generally a poorer prognosis when widely present. However, we expect that this activity also implies potential reversibility of fibrosis, thus an indication of fibrosis activity may predict potential therapeutical accessibility and success rate. To optimize the use of a single liver biopsy and to minimize the number of biopsies per animal we evaluated different fixation- and RNA isolation methods available in our laboratory. Conclusively, we found that at least two biopsies are needed. One for histology (formalin 1-4 hrs) and one for molecular assays: RNA-later. A logical next step will be the further identification of relations between all relevant cells and their microenvironment: the cellular niche consisting of the surrounding cells, matrix and signaling factors. The cellular niche likely plays a decisive role in the fate of cells. Development of markers to assess the potential of scar regression in patients justifies great interest as scar regression potentially means clinical improvement in currently untreatable diseases like liver cirrhosis.