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Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

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Abstract

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.

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Acknowledgements

The authors thank Mr. R. Yoshii for assistance with the electron microscope, Ms. Y. Saitoh and Ms. Y. Ueno for excellent technical assistance, and Dr. H. Kubo (Molecular & Cancer Research Unit, HMRO, Kyoto University, Kyoto, Japan) for generously donating antibodies against podoplanin.

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Authors and Affiliations

  1. Department of Anatomy, Faculty of Medicine, University of Toyama, Toyama, Japan

    Mitsuhiro Matsuo & Osamu Ohtani

  2. Department of Pathogenic Biochemistry,Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan

    Keiichi Koizumi, Sanae Yamada & Ikuo Saiki

  3. Department of Pharmaceutical Science, Faculty of Pharmaceutical Science, University of Toyama, Toyama, Japan

    Masatoshi Tomi & Ken-ichi Hosoya

  4. 21st Century COE Program, University of Toyama, Toyama, Japan

    Ikuo Saiki

  5. The YS Institute, Utsunomiya, Tochigi, Japan

    Ri-ichi Takahashi & Masatsugu Ueda

  6. Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Sendai, Japan

    Tetsuya Terasaki

  7. Department of Cell Biology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan

    Masuo Obinata

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  1. Mitsuhiro Matsuo
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  2. Keiichi Koizumi
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  3. Sanae Yamada
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  4. Masatoshi Tomi
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  6. Masatsugu Ueda
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  7. Tetsuya Terasaki
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  8. Masuo Obinata
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  9. Ken-ichi Hosoya
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  10. Osamu Ohtani
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  11. Ikuo Saiki
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Corresponding author

Correspondence to Keiichi Koizumi.

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M. Matsuo and K. Koizumi contributed equally to this work.

This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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Matsuo, M., Koizumi, K., Yamada, S. et al. Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats. Cell Tissue Res 326, 749–758 (2006). https://doi.org/10.1007/s00441-006-0229-x

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  • DOI: https://doi.org/10.1007/s00441-006-0229-x

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