title: An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture.
abstract: Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.
authors.affiliation: Third Department of Internal Medicine, Hirosaki University School of Medicine, Japan. tmsw@cc.hirosaki-u.ac.jp
authors.forename: N
authors.initials: N
authors.lastname: Tamasawa
authors.forename: H
authors.initials: H
authors.lastname: Murakami
authors.forename: J
authors.initials: J
authors.lastname: Matsui
authors.forename: K
authors.initials: K
authors.lastname: Yamato
authors.forename: G
authors.initials: G
authors.lastname: JingZhi
authors.forename: T
authors.initials: T
authors.lastname: Imaizumi
authors.forename: K
authors.initials: K
authors.lastname: Fujimoto
authors.forename: H
authors.initials: H
authors.lastname: Yoshida
authors.forename: K
authors.initials: K
authors.lastname: Satoh
authors.forename: T
authors.initials: T
authors.lastname: Suda
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004730:Endothelium, Vascular / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007653:Ketocholesterols / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha; D014471:Umbilical Veins; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D006888:Hydroxycholesterols; D007653:Ketocholesterols; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D019716:Tissue Inhibitor of Metalloproteinase-2; C007997:25-hydroxycholesterol; C003001:7-ketocholesterol