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Isolation and characterization of primary lymphatic endothelial cells from rat thoracic duct
Author(s) -
Ohta Hideki,
Katsube Kenichi,
Ogawa Jyunichi,
Yanagishita Masaki
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a619-c
Subject(s) - lymphangiogenesis , thoracic duct , lymphatic endothelium , lymphatic system , biology , pathology , endothelial stem cell , microbiology and biotechnology , immunohistochemistry , andrology , in vitro , immunology , medicine , metastasis , biochemistry , genetics , cancer
The objective of the present study was to refine the isolation and culture methods of primary rat lymphatic endothelial cells (rLECs) to investigate lymphangiogenic factors. rLECs were isolated from rat thoracic duct using intraluminal digestion method and were cultured in endothelial growth medium‐2 with 10% fetal bovine serum supplement. rLECs were characterized by their morphology and by immunohistochemistry for the expression of typical endothelial marker proteins. They were also examined for the expression of lymphatic specific gene by RT‐PCR. We then used the cultured rLECs to investigate the effects of angiogenic and anti‐angiogenic factors, i.e., hypoxia and endorepellin, especially in reference to primary rat venous endothelial cells (rVECs) and HUVECs. Under hypoxic conditions, rLECs formed non‐overlapping cell junctions in a tile‐like appearance with upregulated VEGFR‐3 mRNA level and downregulated VEGFR‐2 level. In contrast, morphological features and the transcription of VEGFR‐2 mRNA and VEGFR‐3 mRNA of rVECs and HUVECs were less affected by oxygen concentrations. Endorepellin, an antiangiogenic peptide fragment of perlecan, induced disassembly of actin filaments similarly in rLECs, rVECs and HUVECs. In conclusion, we have established isolation and culture methods for rLECs from rat thoracic duct, and they would provide useful tools for investigating lymphangiogenesis in vitro .