title: Suppression of lymphangiogenesis by soluble vascular endothelial growth factor receptor-2 in a mouse lung cancer model.
abstract: The vascular endothelial growth factor (VEGF) family has a key role in the formation of blood vessels and lymphatics. Among the members of this family, VEGF-C is one of the most important factors involved in lymphangiogenesis via binding with two receptors (vascular endothelial growth factor receptor-2 and -3: VEGFR-2 and VEGFR-3). Soluble VEGFR-2 (sVEGFR-2) has a role in maintaining the alymphatic state of the cornea associated with binding to VEGF-C, and selectively inhibits lymphangiogenesis but not angiogenesis. In this study, we introduced sVEGFR-2 into lung cancer cells and evaluated the influence on tumor progression and on genes regulating lymphatic formation and metastasis in vivo. A retroviral vector was used to introduce the sVEGFR-2 gene into Lewis lung carcinoma cells (LLC), which were designated as LLC-sVEGFR-2 cells. Proteins secreted into the culture supernatant by these cells were detected by western blotting using specific antibodies. To examine lymphangiogenesis by primary lung cancer in vivo, LLC-sVEGFR-2 cells were subcutaneously injected into C57BL/6 mice. At 14days after injection, immunohistochemistry was performed using an antibody directed against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a marker of lymphatics. Expression of mRNA for VEGFR-2, VEGFR-3 and matrix metalloproteinases (MMPs) was also determined by real-time PCR. Furthermore, LLC-sVEGFR-2 cells were directly inoculated into the left lung in C57BL/6 mice and the number of micro-metastases in pulmonary lymph nodes was determined. Introduction of sVEGFR-2 into LLC cells resulted in secretion of sVEGFR-2 protein into the culture supernatant. There were fewer LYVE-1 positive lymphatics after inoculation of LLC-sVEGFR-2 into mice compared with the control group. In addition, VEGFR-2, VEGFR-3, and MMPs gene expression was suppressed in the primary tumors of the LLC-sVEGFR-2 group compared with the control group. Furthermore, there were fewer micro-metastases in the pulmonary lymph nodes of the LLC-sVEGFR-2 group compared with the control group after cells were directly inoculated into the lung. These findings indicate that introduction of sVEGFR-2 suppressed lymphangiogenesis in primary lung cancer and also suppressed lymphogenic metastasis by inhibiting VEGF-C, followed by down-regulation of VEGFR-2, VEGFR-3 and MMPs. Accordingly, sVEGFR-2 might be a promising target for treatment of cancer by regulating lymphangiogenesis and lymphogenic metastasis.
authors.affiliation: Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan. Electronic address: dm14021q@st.kitasato-u.ac.jp.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Maehana
authors.affiliation: Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan; Department of Microbiology, Kitasato University School of Allied Health Sciences, Kanagawa, Japan. Electronic address: m-nakamu@kitasato-u.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Pharmacology, Kitasato University School of Medicine, Kanagawa, Japan. Electronic address: fumihiro@med.kitasato-u.ac.jp.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ogawa
authors.affiliation: Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan. Electronic address: mm14010h@st.kitasato-u.ac.jp.
authors.forename: Rimika
authors.initials: R
authors.lastname: Imai
authors.affiliation: Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan. Electronic address: reireireirei0109reireireirei@yahoo.co.jp.
authors.forename: Rei
authors.initials: R
authors.lastname: Murakami
authors.affiliation: Department of Pharmacology, Kitasato University School of Allied Health Sciences, Kanagawa, Japan. Electronic address: kojimaf@kitasato-u.ac.jp.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Kojima
authors.affiliation: Department of Pharmacology, Kitasato University School of Medicine, Kanagawa, Japan. Electronic address: mmajima@med.kitasato-u.ac.jp.
authors.forename: Masataka
authors.initials: M
authors.lastname: Majima
authors.affiliation: Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Kanagawa, Japan; Department of Microbiology, Kitasato University School of Allied Health Sciences, Kanagawa, Japan. Electronic address: hkita@kitasato-u.ac.jp.
authors.forename: Hidero
authors.initials: H
authors.lastname: Kitasato
mesh_terms: D000818:Animals; D018827:Carcinoma, Lewis Lung / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D042583:Lymphangiogenesis; D008207:Lymphatic Metastasis; D042601:Lymphatic Vessels / Q000378:metabolism* / Q000473:pathology; D008297:Male; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D061206:Neoplasm Micrometastasis; D015398:Signal Transduction; D012995:Solubility; D014162:Transfection; D015854:Up-Regulation; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D020782:Matrix Metalloproteinases; D026901:Membrane Transport Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2; D040321:Vascular Endothelial Growth Factor Receptor-3; C492273:LYVE1 protein, mouse; C000592875:Kdr protein, mouse
keywords: Lymphangiogenesis; Lymphogenic metastasis; Soluble VEGFR-2