Response of sinusoidal mouse liver cells to choline-deficient ethionine-supplemented diet

Abstract

BackgroundProliferation of oval cells, the bipotent precursor cells of the liver, requires impeded proliferation and loss of hepatocytes as well as a specific micro-environment, provided by adjacent sinusoidal cells of liver. Despite their immense importance for triggering the oval cell response, cells of hepatic sinusoids are rarely investigated. To elucidate the response of sinusoidal liver cells we have employed a choline-deficient, ethionine-supplemented (CDE) diet, a common method for inducing an oval cell response in rodent liver. We have utilised selected expression markers commonly used in the past for phenotypic discrimination of oval cells and sinusoidal cells: cytokeratin, E-cadherin and M2-pyruvate kinase for oval cells; and glial fibrillary acidic protein (GFAP) was used for hepatic stellate cells (HSCs).ResultsCDE diet leads to an activation of all cells of the hepatic sinusoid in the mouse liver. Beside oval cells, also HSCs and Kupffer cells proliferate. The entire fraction of proliferating cells in mouse liver as well as endothelial cells and cholangiocytes express M2-pyruvate kinase. Concomitantly, GFAP, long considered a unique marker of quiescent HSCs was upregulated in activated HSCs and expressed also in cholangiocytes and oval cells.ConclusionsOur results point to an important role of all types of sinusoidal cells in regeneration from CDE induced liver damage and call for utmost caution in using traditional marker for identifying specific cell types. Thus, M2-pyruvate kinase should no longer be used for estimating the oval cell response in mouse liver. CDE diet leads to activation of GFAP positive HSCs in the pericentral zone of liver lobulus. In the periportal zone the detection of GFAP in biliary cells and oval cells, calls other cell types as progenitors of hepatocytes into question under CDE diet conditions.

Pathological implications of cadherin zonation in mouse liver

“…1a, d). There was evidence from the literature that N-cadherin was solely expressed in perivenous hepatocytes [39][40][41]. However, staining for N-cadherin demonstrated a gradient of expression with high expression in perivenous and much lower expression in periportal hepatocytes, where it colocalized with E-cadherin (Fig.…”

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…The enrichment of periportal and pericentral hepatocytes in the isolated subpopulations is usually estimated by measuring the activities of several highly zonated enzymes such as glutamine synthetase, alanine aminotransferase and pyruvate kinase (Gebhardt and Mecke 1983; Burger et al 1989). Since E-cadherin in the liver is present only in the periportal zone (~50 % of hepatocytes) (Ueberham et al 2010), it can be used as a suitable marker for revealing the enrichment of periportal cells by immunocytochemical staining.…”

Global Increase of p16INK4a in APC-Deficient Mouse Liver Drives Clonal Growth of p16INK4a-Negative Tumors

“…Immunohistochemistry was performed as described (19,20). Briefly, 5 mm paraffin sections were dewaxed with xylol and hydrated through downward alcohol series.…”

“…Primary hepatocytes were isolated and cultivated as described (20) by collagenase perfusion (21). Pericentral and periportal hepatocytes were isolated by a modified digitonin/collagenase perfusion technique (22,23) and transfected with TOP-Gau reporter plasmids (24) using Effectene (Qiagen).…”

“…Concentration was adjusted to 0.5 mg/mL. qRT-PCR was performed as described (20) using primers listed in Supplementary Table S3. RNA load was normalized with the housekeeping gene cyclophilin (Ppia). Standard curves of serial dilutions from total RNA were used for transforming C t values to concentration values depicted as arbitrary units.…”