Cellular retinol-binding protein-1 expression in normal and fibrotic/cirrhotic human liver: different patterns of expression in hepatic stellate cells and (myo)fibroblast subpopulations

https://doi.org/10.1016/j.jhep.2004.01.008Get rights and content

Abstract

Background/Aims

Cellular retinol-binding protein-1 (CRBP-1) which is involved in vitamin A metabolism is highly expressed in liver cells, particularly in hepatic stellate cells (HSCs). In this work, the CRBP-1 expression was studied by immunohistochemistry in the different liver cell populations, including HSCs and portal fibroblasts, of normal liver and of fibrotic and cirrhotic liver.

Methods

Normal liver, fibrotic liver in different stages and cirrhotic liver sections were studied. Immunohistochemistry was performed using antibodies against CRBP-1, α-smooth muscle actin (SMA), CD 68 and CD 34.

Results

In normal liver, quiescent HSCs expressed CRBP-1, while portal fibroblasts did not. In fibrotic or cirrhotic liver, activated HSCs co-expressed CRBP-1 and α-SMA; a variable proportion of portal and septal (myo)fibroblasts, more important in cirrhosis, neo-expressed both CRBP-1 and α-SMA. Biliary epithelial cells both in normal and pathological situations expressed CRBP-1. Neither Kupffer cells, nor endothelial cells showed CRBP-1 expression.

Conclusions

Our study demonstrates that CRBP-1 is a good marker to identify HSC in normal human liver. Furthermore, in fibrotic or cirrhotic liver, the different patterns of expression for CRBP-1 and α-SMA allow the distinction of different subsets of fibroblastic cells involved in fibrogenesis and septa formation.

Introduction

In normal liver, quiescent hepatic stellate cells (HSC) are the main storage site for retinoids, whereas hepatocytes play a major role in the primary uptake and processing of retinoids [1]. Numerous intracellular or plasmatic carriers are involved in retinoid metabolism. Among them, cellular retinol-binding protein-1 (CRBP-1) mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid [2]. CRBP-1 is highly expressed in the liver: hepatocytes account for more than 90% of hepatic CRBP-1, while the concentration of the protein (per protein unit) in HSC is 22 times greater than in hepatocytes [3].

It has been shown that during wound healing of a full-thickness rat skin wound, CRBP-1 is transiently expressed by a significant proportion of fibroblastic cells, including α-smooth muscle actin (SMA)-expressing myofibroblasts [4], suggesting that CRBP-1 plays a role in the evolution of granulation tissue. In normal rat liver, contrary to HSC, portal fibroblasts do not express CRBP-1 [5]. This finding adds to the concept of heterogeneity of liver fibroblastic cells that we and others have recently proposed [5], [6], [7], [8], [9]. After carbon tetrachloride injury, CRBP-1 expression is maintained in myofibroblastic α-SMA-positive HSC; after bile duct ligation, portal fibroblasts (which proliferate around ductular structures) acquire expression of both α-SMA [10] and CRBP-1 [5]. These studies show that, during myofibroblastic differentiation, HSC which lose their stores of retinol, maintain a high level of CRBP-1 expression, while portal fibroblasts acquire CRBP1 expression. Together, these data suggest again a correlation between CRBP-1 expression and myofibroblastic differentiation, and highlight the role of the different fibroblastic populations during liver fibrogenesis, including HSC and portal fibroblasts.

α-SMA is a good marker of activated HSC which acquire a myofibroblastic phenotype [11], [12]. However, a specific marker of quiescent HSC in normal human liver has not been described up to now.

The aims of our work were 1/ to investigate whether CRBP-1 represents a reliable marker of HSC in normal human liver, and 2/ to study the expression of CRBP-1 in activated HSC during fibrotic/cirrhotic liver disease. Furthermore, the expression of CRBP-1 in other (myo)fibroblastic subpopulations of the liver, i.e. portal fibroblasts and fibroblasts of the Glisson's capsule, was analyzed in normal liver and in pathological conditions.

Section snippets

Human liver specimens

Forty-four liver samples were studied, obtained from 44 patients. They corresponded either to percutaneous liver biopsies (n=22), large surgical specimens (n=8), or explanted livers (n=14). Eight corresponded to histologically normal livers and 36 to pathological specimens with various fibrotic stages whatever the degree of activity. The stage of fibrosis and the grade of inflammatory activity were classified according to the Metavir score [13].

Tissue sampling and processing

A part of fresh tissue samples was routinely

Normal liver

Eight histologically non-pathological liver tissues were studied. They came from four women and four men aged 33–70 years (average 50 years). Six specimens were obtained from macroscopically normal parts of hepatectomy, taken at a distance from a benign or malignant tumoral lesion: inflammatory pseudotumor around biliary cyst (n=1), focal nodular hyperplasia (n=2), or colorectal adenocarcinoma metastasis (n=3). Two specimens were obtained from total hepatectomy: one was a donor liver, not used

Discussion

In normal rat liver, several markers of quiescent HSC have been described, such as vimentin [21], desmin [22], and neural/neuroendocrine markers including glial fibrillary acidic protein [23], neural cell adhesion molecule [24] and nestin [25]. However, Ballardini et al. [26] have underlined the existence of desmin-negative HSC, mainly located in pericentral areas; they suggested that desmin cannot be viewed as a phenotypical marker but rather as a differentiation marker of HSC, possibly

Acknowledgements

We thank Pascal Correia Gomez and Albano Meli for excellent technical assistance. We are very grateful to Maria Rosa Winnock (Institut de Santé Publique, d'Epidémiologie et de Développement, Université Bordeaux 2, France) for helpful discussion concerning statistical analysis. This work was supported in part by the Région Aquitaine.

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    Copyright © 2004 European Association for the Study of the Liver. Published by Elsevier Ireland Ltd. All rights reserved.