BASIC STUDIES Lecithin: retinol acyltransferase protein is distributed in both hepatic stellate cells and endothelial cells of normal rodent and human liver Keisuke Nagatsuma 1,2 , Yoshihiro Hayashi 3 , Hiroshi Hano 2 , Hiroshi Sagara 4 , Kazuhiro Murakami 5 , Masaya Saito 1 , Takahiro Masaki 6 , Tomoe Lu 2 , Mitsugu Tanaka 2 , Hideaki Enzan 7 , Yoshio Aizawa 1 , Hisao Tajiri 1 and Tomokazu Matsuura 1,8 1 Department of Internal Medicine, Division of Gastroenterology and Hepatology, The Jikei University School of Medicine, Tokyo, Japan 2 Department of Pathology, The Jikei University School of Medicine, Tokyo, Japan 3 Department of Pathology, Kochi Medical School, Kochi University, Kochi, Japan 4 Department of Basic Medical Science, Division of Fine Morphology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 5 Division of Clinical Pathology, Tohoku Welfare Pension Hospital, Sendai, Japan 6 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan 7 Department of Diagnostic Pathology, Chikamori Hospital, Kochi, Japan 8 Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan Keywords endothelial cell – hepatic stellate (Ito) cell – human – lecithin:retinol acyltransferase (LRAT) – rodent – tissue marker Correspondence Tomokazu Matsuura, MD, PhD, Department of Laboratory Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato- ku, Tokyo 105-8461, Japan Tel: 181 3 3433 1111 ext. 3210 Fax: 181 3 3437 5560 e-mail: matsuurat@jikei.ac.jp Received 28 December 2007 Accepted 16 March 2008 DOI:10.1111/j.1478-3231.2008.01773.x Abstract Background: To determine the extent to which hepatic stellate cell (HSC) activation contributes to liver fibrosis, it was found necessary to develop an alternative structural and functional stellate cell marker for in situ studies. Although several HSC markers have been reported, none of those are associated with particular HSC functions. Aim: The present study was undertaken to examine whether lecithin:retinol acyltrans- ferase (LRAT), the physiological retinol esterification enzyme of the liver, is a potential and relevant tissue marker for HSC. Methods: An antibody specific to mouse and human LRAT was prepared based on the amino acid sequences. Antibodies to LRAT were used for immunohistochemical studies to assess the distribution of LRAT-positive cells in the liver with the aid of fluorescence and immunogold electron microscopy. Results: LRAT-positive cells were found to be confined in the space of Disse, correspond- ing with the location of desmin-positive HSC in rodent liver, also in human liver. Interestingly, LRAT-positive staining was also observed along the liver sinusoidal endothelial lining. Furthermore, immune electron microscopic studies revealed that LRAT was mainly distributed in HSC within the rough-endoplasmic reticulum (RER) and multivesicular bodies, whereas LRAT staining within the endothelial cells was largely confined to the perinuclear area and to some extent to the RER. Conclusion: Evidence has been accumulated that LRAT might serve as an excellent alternative HSC marker for future structural and functional studies. Furthermore, the presence of LRAT in endothelial cells might suggest a currently unknown function of this enzyme in liver endothelial biology. Hepatic stellate cells (HSCs), also referred to as Ito cells, fat-storing cells or pericytes, play two major physiological roles during the quiescent phase. Firstly, they serve as fat-storing cells (1). In playing this role, retinoid taken up by hepatocytes after being absorbed from the digestive tract in the form of chylomicrons is released into space of Disse in the form of retinol bound to retinol- binding protein (RBP), and HSC then store retinol in the form of retinyl esters, i.e. in vitamin A-containing fat droplets. Secondly, HSCs serve as pericytes with which liver endothelial cells in sinusoids are lined. In playing this role, long subendothelial processes of HSC are wound around multiple sinusoids, and these processes expand and contract to adjust sinusoidal blood flow (2). When activated in the presence of liver damage, HSCs release cytokines, primarily transforming growth factor-b (TGF-b), and transform into myofibroblasts lacking fat droplets, which produce collagen and promote liver fibrosis. Candidates for specifically expressed proteins, possibly serving as markers of quiescent HSC, which store retinoids in the space of Disse, are cellular RBP type 1 (CRBP1) and lecithin:retinol acyltransferase (LRAT). In 1984, Kato et al. (3) demonstrated immunohistochemically that CRBP1 is highly expressed in HSC. However, CRBP1 is also abundant in hepatocytes. On the other hand, LRAT activity is strongly expressed in the liver, retinal pigment epithelial (RPE) cells, intestinal mucosa, basal keratinocytes, testis, lung, etc. LRAT plays the following roles: storing systemic retinoid in the liver, incorporating retinol into the retina and adjusting its concen- tration in the retinal pigment epithelium to maintain visual function, adjusting the regional concentration of retinoid to differentiate the epithelium in the skin and lung and regulating the concentration of retinoid to maintain a level optimal for maturation of spermatozoa in the testis (4–10). The physiolo- gical retinol esterification enzyme that stores retinoid in the liver is LRAT. Retinyl esters are biosynthesized by removing the fatty acid at position sn-1 of lecithin, using CRBP1-bound retinol as a substrate. The expression of LRAT in the liver is regulated by vitamin A status. Whereas hepatic LRAT activity is low in the liver of vitamin A-deficient rats, the decreased LRAT activity is rapidly elevated by repletion with retinol, retinoic acid or RAR agonists (11–13). Furthermore, LRAT activity is Liver International (2009) c  2009 The Authors. Journal compilation c  2009 Blackwell Publishing Ltd 47 Liver International ISSN 1478-3223