Virchows Archiv

, Volume 443, Issue 1, pp 78–86 | Cite as

Vinculin: a novel marker for quiescent and activated hepatic stellate cells in human and rat livers

  • Shuji Kawai
  • Hideaki Enzan
  • Yoshihiro Hayashi
  • Yu-Lan Jin
  • Li-Mei Guo
  • Eriko Miyazaki
  • Makoto Toi
  • Naoto Kuroda
  • Makoto Hiroi
  • Toshiji Saibara
  • Hirofumi Nakayama
Original Article


In liver injuries, the quiescent hepatic stellate cells (HSCs) promptly change to activated HSCs, which are easily identified by the intense immunoreactivity for α-smooth muscle actin. However, reproducible markers for quiescent HSCs in formalin-fixed, paraffin-embedded liver tissue sections have not yet been reported. We immunohistochemically examined the expression of vinculin, one major protein of dense plaques, on cultured LI90 cells and on HSCs in normal and diseased human and rat livers. In cultured LI90 cells, vinculin appeared as small linear patches. Although vinculin was consistently negative in the routine liver tissue sections, an antigen retrieval technique using microwave oven heating yielded excellent effects. Using this technique, the formalin-fixed, paraffin-embedded human and rat normal liver tissue sections showed the vinculin immunoreactivity along the sinusoidal wall. Immunoelectron microscopic observation of hepatic parenchyma demonstrated that the vinculin was exclusively expressed in quiescent HSCs. In fetal rat livers, vinculin-positive quiescent HSCs gradually increased in number with gestation. In diseased livers the activated HSCs showed more intense immunoreaction for vinculin. These results indicate that, using microwave pretreatment, vinculin is expressed in quiescent and activated HSCs in routinely processed liver tissue sections. It could allow us to evaluate the development and distribution of quiescent HSCs and to examine the relationship between quiescent and activated HSCs.


Hepatic stellate cell Cell marker Vinculin Immunohistochemistry Antigen retrieval 



We would like to thank Mr. T. Tokaji, Ms. H. Yamasaki, and Ms. T. Kadota, First Department of Pathology, Mr. M. Shirota, Medical Research Laboratory, Kochi Medical School, for their excellent technical assistance. We are also grateful to Mr. D. Ribble, Department of English, Kochi Medical School, for English correction of our manuscript. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, grant C 13670176.


  1. 1.
    Asijee GM, Sturk A, Bruin T, Wilkinson JM, Ten Cate JW (1990) Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organizing cytoskeleton upon platelet activation. Eur J Biochem 189:131–136PubMedGoogle Scholar
  2. 2.
    Ballardini G, Fallani M, Biagini G, Bianchi FB, Pisi E (1988) Desmin and actin in the identification of Ito cells and in monitoring their evolution to myofibroblasts in experimental liver fibrosis. Virchows Arch 56:45–49Google Scholar
  3. 3.
    Buniatian G, Gebhardt R, Schrenk D, Hamprecht B (1996) Colocalization of three types of intermediate filament proteins in perisinusoidal stellate cells: glial fibrillary acidic protein as a new cellular marker. Eur J Cell Biol 70:23–32PubMedGoogle Scholar
  4. 4.
    Buniatian G, Hamprecht B, Gebhardt R (1996) Glial fibrillary acidic protein as a marker of perisinusoidal stellate cells that can distinguish between the normal and myofibroblast-like phenotypes. Biol Cell 87:65–73CrossRefPubMedGoogle Scholar
  5. 5.
    Burridge K, Feramisco JR (1980) Microinjection and localization of a 130 K protein in living fibroblasts: a relationship to actin and fibronectin. Cell 19:587–595Google Scholar
  6. 6.
    Burridge K, Fath K, Kelly T, Nuckolls G, Turner C (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Ann Rev Cell Biol 4:487–525CrossRefPubMedGoogle Scholar
  7. 7.
    Burt AD, Robertson JL, Heir J, MacSween RNM (1986) Desmin-containing stellate cells in rat liver; distribution in normal animals and response to experimental acute liver injury. J Pathol 150:29–35PubMedGoogle Scholar
  8. 8.
    Cassiman D, Van Pelt J, De Vos R, Van Lommel F, Desmet V, Yap S-H, Roskams T (1999) Short communication. Synaptophysin: a novel marker for human and rat hepatic stellate cells. Am J Pathol 155:1831–1839PubMedGoogle Scholar
  9. 9.
    Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, Bifulco C, Key G, D'amato L, Sabattini E, Feudale E, Reynolds F, Gerdes J, Rilke F (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:83–98PubMedGoogle Scholar
  10. 10.
    Di Sario A, Bendia E, Svegliati-Baroni G, Marzioni M, Ridolfi F, Trozzi L, Ugili L, Saccomanno S, Jeqequel AM, Benedetti A (2002) Rearrangement of the cytoskeletal network induced by platelet-derived growth factor in rat hepatic stellate cell: role of different intracellular signaling pathways. J Hepatol 36:179–190PubMedGoogle Scholar
  11. 11.
    Duband J-L, Thiery JP (1990) Spatio-temporal distribution of the adherens junction-associated molecules vinculin and talin in the early avian embryo. Cell Differ Dev 30:55–76CrossRefPubMedGoogle Scholar
  12. 12.
    Enzan H, Himeno H, Iwamura S, Saibara T, Onishi S, Yamamoto Y, Hara H (1994) Immunohistochemical identification of Ito cells and their myofibroblastic transformation in adult human liver. Virchows Arch 424:249–256PubMedGoogle Scholar
  13. 13.
    Enzan H, Himeno H, Hiroi M, Kiyoku H, Saibara T, Onishi S (1997) Development of hepatic sinusoidal structure with special reference to the Ito cells. Microsc Res Tech 39:336–349CrossRefPubMedGoogle Scholar
  14. 14.
    Eyden BP (1993) Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis. Ultrastruct Pathol 17:611–622PubMedGoogle Scholar
  15. 15.
    Gard AL, White FP, Dutton GR (1985) Extra-neural glial fibrillary acidic protein (GFAP) immunoreactivity in perisinusoidal stellate cells of rat liver. J Neuroimmunol 8:359–375PubMedGoogle Scholar
  16. 16.
    Geiger B, Tokuyasu KT, Dutton AH, Singer SJ (1980) Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl Acad Sci U S A 77:4127–4131PubMedGoogle Scholar
  17. 17.
    Grove BD, Pfeiffer DC, Allen S, Vogl AW (1990) Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells. Am J Anat 188:44–56PubMedGoogle Scholar
  18. 18.
    Hemmings L, Barry ST, Critchley DR (1995) Cell-matrix adhesion: structure and regulation. Biochem Soc Trans 23:619–626PubMedGoogle Scholar
  19. 19.
    Ikeda K, Kawada N, Wang YQ, Kadoya H, Nakatani K, Sato M, Kaneda K (1998) Short communication. Expression of cellular prion protein in activated hepatic stellate cells. Am J Pathol 153:1695–1700PubMedGoogle Scholar
  20. 20.
    Imai K, Senoo H (1998) Morphology of sites of adhesion between hepatic stellate cells (vitamin A-storing cells) and a three-dimensional extracellular matrix. Anat Rec 250:430–437CrossRefPubMedGoogle Scholar
  21. 21.
    Jin Y-L, Enzan H, Kuroda N, Hayashi Y, Nakayama H, Zhang Y-H, Toi M, Miyazaki E, Hiroi M, Guo L-M, Saibara T (2003) Tissue remodelling following submassive hemorrhagic necrosis in rat livers induced by an intraperitoneal injection of dimethylnitrosamine. Virchows Arch 442:39–47PubMedGoogle Scholar
  22. 22.
    Kitada T, Seki S, Ikeda K, Nakatani K, Sakaguchi H, Kawada N, Kadoya H, Kaneda K (2000) Clinicopathological characterization of prion: a novel marker of activated human hepatic stellate cells. J Hepatol 33:751–757PubMedGoogle Scholar
  23. 23.
    Knittel T, Aurisch S, Neubauer K, Eichhorst S, Ramadori G (1996) Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair. Am J Pathol 149:449–462PubMedGoogle Scholar
  24. 24.
    Knook DL, Seffelaar AM, de Leeuw AM (1982) Fat-storing cells of the rat livers, their isolation and purification. Exp Cell Res 139:468–471PubMedGoogle Scholar
  25. 25.
    Kojima N, Sato M, Imai K, Miura M, Matano Y, Senoo H (1998) Hepatic stellate cells (vitamin A-storing cells) change their cytoskeleton structure by extracellular matrix components through a signal transduction system. Histochem Cell Biol 110:121–128CrossRefPubMedGoogle Scholar
  26. 26.
    Levy MT, McCaughan GW, Abbott CA, Park JE, Cunningham AM, Müller E, Rettig WJ, Gorrell MD (1999) Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology 29:1768–1778PubMedGoogle Scholar
  27. 27.
    Levy MT, McCaughan GW, Marinos G, Gorrrell MD (2002) Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection. Liver 22:93–101CrossRefPubMedGoogle Scholar
  28. 28.
    Murakami K, Abe T, Miyazawa M, Yamaguchi M, Masuda T, Matsuura T, Nagamori S, Takeuchi K, Abe K, Kyogoku M (1995) Establishment of a new human cell line, LI90, exhibiting characteristics of hepatic Ito (fat-storing) cells. Lab Invest 72:731–739PubMedGoogle Scholar
  29. 29.
    Nakatani K, Seki S, Kawada N, Kobayashi K, Kaneda K (1996) Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver. Cell Tissue Res 283:159–165PubMedGoogle Scholar
  30. 30.
    Neubauer K, Knittel T, Aurisch S, Fellmer P and Ramadori G (1996) Glial fibrillary acidic protein—a cell type specific marker for Ito cells in vivo and in vitro. J Hepatol 24:719–730CrossRefPubMedGoogle Scholar
  31. 31.
    Niki T, De Bleser PJ, Xu G, Van Den Berg K, Wisse E, Geerts A (1996) Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers. Hepatology 23:1538–1545PubMedGoogle Scholar
  32. 32.
    Niki T, Pekny M, Hellemans K, De Bleser P, Van Den Berg K, Vaeyens F, Quartier E, Schuit F, Geerts A (1999) Class VI intermediate filament protein nestin is induced during activation of rat hepatic stellate cells. Hepatology 29:520–527PubMedGoogle Scholar
  33. 33.
    Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, Santini D, Piccaluga PP, Leone O, Damiani S, Ercolessi C, Sandri F, Pieri F, Leoncini L, Falini B (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116–123CrossRefPubMedGoogle Scholar
  34. 34.
    Ramadori G, Veit T, Schwögler S, Dienes HP, Knittel T, Rieder H, Meyer zum Büschenfelde K-H (1990) Expression of the gene of the α-smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells. Virchows Arch 59:349–357Google Scholar
  35. 35.
    Schmitt-Gräff A, Krüger S, Bochard F, Gabbiani G, Denk H (1991) Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol 138:1233–1242PubMedGoogle Scholar
  36. 36.
    Shi S-R, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748PubMedGoogle Scholar
  37. 37.
    Shi S-R, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem 45:327–343PubMedGoogle Scholar
  38. 38.
    Shi S-R, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49:931–937PubMedGoogle Scholar
  39. 39.
    Takase S, Maria Leo MA, Nouchi T, Lieber CS (1988) Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat. J Hepatol 6:267–276PubMedGoogle Scholar
  40. 40.
    Taylor CR, Shi S-R, Cote RJ (1996) Review. Antigen retrieval for immunohistochemistry. Status and need for greater standardization. Appl Immunohistochem 4:144–166Google Scholar
  41. 41.
    Trim N, Morgan S, Evans M, Issa R, Fine D, Afford S, Wilkins B, Iredale J (2000) Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation. Am J Pathol 156:1235–1243PubMedGoogle Scholar
  42. 42.
    Vassy J, Rigaut JP, Briane D, Kraemer M (1993) Confocal microscopy immunofluorescence localization of desmin and other intermediate filament proteins in fetal rat livers. Hepatology 17:293–300PubMedGoogle Scholar
  43. 43.
    Williamis JH, Mepham BL, Wright DH (1997) Tissue preparation for immunocytochemistry. J Clin Pathol 50:422–428PubMedGoogle Scholar
  44. 44.
    Yokoi Y, Namihisa T, Kuroda H, Komatsu I, Miyazaki A, Watanabe S, Usui K (1984) Immunocytochemical detection of desmin in fat-storing cells (Ito Cells). Hepatology 4:709–714PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Shuji Kawai
    • 1
  • Hideaki Enzan
    • 1
  • Yoshihiro Hayashi
    • 1
  • Yu-Lan Jin
    • 1
  • Li-Mei Guo
    • 1
  • Eriko Miyazaki
    • 1
  • Makoto Toi
    • 1
  • Naoto Kuroda
    • 1
  • Makoto Hiroi
    • 1
  • Toshiji Saibara
    • 2
  • Hirofumi Nakayama
    • 3
  1. 1.First Department of PathologyKochi Medical SchoolKochiJapan
  2. 2.First Department of Internal MedicineKochi Medical SchoolKochiJapan
  3. 3.Department of Molecular PathologyGraduate School of Biomedical Sciences, Hiroshima UniversityHiroshimaJapan

Personalised recommendations