Biochimica et Biophysica Acta 1760 (2006) 1465–1474 www.elsevier.com/locate/bbagen The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells ⁎ Toshiyuki Kaji a,b, , Chika Yamamoto a,b, Mami Oh-i a, Yasuyuki Fujiwara a, Yasuo Yamazaki c, Takashi Morita c, Anna H. Plaas d, Thomas N. Wight e a Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan b Organization of Frontier Research in Preventive Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan c Department of Biochemistry, Meiji Pharmaceutical University, Tokyo, 204-8588, Japan d Department of Internal Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA e Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101-2795, USA Received 3 March 2006; received in revised form 29 May 2006; accepted 20 June 2006 Available online 7 July 2006 Abstract A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer. © 2006 Elsevier B.V. All rights reserved. Keywords: Vascular endothelial growth factor; Extracellular matrix; Perlecan; Proteoglycan; Heparan sulfate; Endothelial cell 1. Introduction heparin-binding growth factors such as fibroblast growth factor-2 (FGF-2) via their heparan sulfate chains and promote Proteoglycans (PGs) are macromolecules that consist of a the binding of FGF-2 to the FGF-2 receptor (FGFR) [1] by the core protein and one or more glycosaminoglycan side chains. formation of an FGF-2/HSPG/FGFR ternary complex [2]. PGs not only contribute to the formation of the extracellular During the ternary complex formation, HSPG induces FGF-2 matrix but also influence cellular proliferation and migration by oligomerization that augments FGFR dimerization and signal interacting with growth factors and other ligands. For example, transduction [3]. In addition, FGF-2 is protected from it has been shown that heparan sulfate PGs (HSPGs) bind proteolytic degradation when complexed with the heparan sulfate chains, [4] and it retains its capacity for the long-term stimulation of endothelial cell functions such as proliferation, ⁎ Corresponding author. Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kana- migration, and plasminogen activator activity [5]. The HSPG zawa 920-1181, Japan. Tel./fax: +81 76 229 6208. that regulates FGF-2 activity by the above mechanisms is the E-mail address: [email protected] (T. Kaji). large extracellular matrix HSPG perlecan [6], which is one of 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.06.010 1466 T. Kaji et al. / Biochimica et Biophysica Acta 1760 (2006) 1465–1474 the major HSPG produced in cultured human brain micro- amounts of endotoxin were purchased from Kurabo (Osaka, Japan) and vascular endothelial cells [7]. Serologicals (Kankakee, IL, USA), respectively. Dulbecco's modified Eagle's medium (DMEM) and tissue culture dishes and plates were purchased from Vascular endothelial growth factors (VEGFs) are highly Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan) and Iwaki (Chiba, Japan), specific mitogens for vascular endothelial cells [8,9]. There are respectively. Fetal bovine serum (FBS) was purchased from Equitech-Bio 35 35 four alternative splicing isoforms of the single VEGF gene; (Kerrville, TX, USA). [ S]Na2SO4 (carrier-free) and Tran S-Label Metabolic 35 35 these are designated as VEGF121, VEGF165, VEGF189, and Labeling Reagent, which consists of ∼70% L-[ S]methionine, ∼15% L-[ S] 35 VEGF206, and they have 121, 165, 189, and 206 amino acids, cysteine, and other S-labeled compounds, were obtained from MP Biomedicals (Irvine, CA, USA). Recombinant human VEGF165 was purchased respectively. VEGF189 and VEGF206 are cell associated and from Strathman Biotech GmbH (Hannover, Germany). The VEGF-neutralizing act as vascular permeability factors, whereas VEGF121 and antibody was obtained from Immuno-Biological Laboratories (Fujioka, Japan). VEGF165 are secreted and induce mitogenesis [10,11]. It has Human recombinant PlGF, DEAE-Sephacel, benzamidine, Tris base, dextran been shown that VEGF165, in particular, plays a critical role in blue, phenylmethanesulfonyl fluoride, papain (1.7U/mg solid), unsaturated proliferation, migration, and tube formation [12]. VEGF165 glucuronic acid-6-O-sulfated N-acetylgalactosamine (GlcA-GalNAc(6S)), and heparan sulfate disaccharide units were obtained from Sigma-Aldrich (St. mediated signal transduction occurs via receptor tyrosine Louis, MO, USA). Protease-free chondroitinase ABC (EC 4.2.2.4, derived from kinases fms-like tyrosine kinase-1 (Flt-1) [13,14] and/or kinase Proteus vulgaris), chondroitinase ACII (EC 4.2.2.5, derived from Arthrobactor insert domain-containing receptor (KDR) [15,16] that are aurescens), heparinase II (derived from Flavobacterium heparinum), and designated as VEGF receptor 1 (VEGFR-1) and VEGF heparinase III (EC 4.2.2.8, derived from F. heparinum) were obtained from receptor-2 (VEGFR-2), respectively. In addition related growth Seikagaku (Tokyo, Japan). Enhanced chemiluminescence (ECL) Western blotting detection reagents, horseradish peroxidase-linked protein A, nitrocel- factors, such as that placenta growth factor (PlGF) or Vammin lulose membranes (Hybond ECL), Hyperfilm ECL Quick Prep Micro mRNA and VR-1, snake venom-derived VEGFs, have been shown to purification kit, Sepharose CL-4B, Sepharose CL-6B, and PD-10 columns selectively activate VEGFR-1 or VEGFR-2, respectively (disposable, Sephadex G-25M) were obtained from Amersham Biosciences [17,18]. (Piscataway, NJ, USA). Proteinase K (fungal) was purchased from Invitrogen It has been reported that VEGF and FGF-2 interact during (Carlsbad, CA, USA); urea, phenol red, and sodium dodecyl sulfate (SDS) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Rabbit angiogenesis and neovascularization. Angiogenesis that is antiserum against the perlecan core protein (EY-9) was kindly provided by induced by either VEGF or FGF-2 is inhibited by VEGFR-2 Dr. John Hassell (Shriners Hospital for Children, Tampa, FL, USA). Microcon antagonists in vivo and in vitro [19]; VEGF-neutralizing YM3 (3000MW cut-off) ultrafiltration devices were obtained from Millipore antibodies markedly reduce FGF-2-induced vascularization in (Billerica, MA, USA); 2-aminoacridone hydrochloride was obtained from the mouse cornea [20], and further, VEGFR-1-blocking Molecular Probes (Eugene, OR, USA). Cetylpyridinium chloride and other reagents were from Nacalai Tesque (Kyoto, Japan). antibodies and dominant-negative VEGFR-1 expression reduce the capillary morphogenesis of endothelial cells [21]. These 2.2. Incorporation of [35S]sulfate into glycosaminoglycans (GAGs) results suggest that FGF-2 induces angiogenesis indirectly by the activation of the VEGF/VEGFR system. In fact, FGF-2 Human brain microvascular endothelial cells were cultured in HuMedia induces an increase in the expression of VEGF [20], VEGFRs, EG-2 in 100-mm dishes in a humid atmosphere of 5% CO2 in air until confluent. They were transferred into 24-well culture plates at a density of and FGFRs [22] in endothelial cells. As a result, VEGF and 4 2 FGF-2 synergistically induce angiogenesis in vivo [23] and in 1×10 cells/cm and cultured until confluent. After washing the cells with DMEM supplemented with 10% FBS, the medium was replaced with fresh vitro [24]. However, the role of VEGF in the activation of the DMEM supplemented with 10% FBS, and the cells were subsequently FGF-2/FGFR system is not completely understood. incubated at 37 °C for 8, 24, or 48 h with VEGF165 (25, 50, 100, 150, Among the predominant forms of VEGF, VEGF121 does not or 200 ng/ml) and/or VEGF-neutralizing antibody in the presence of [35S] bind to heparan sulfate chains [25], while VEGF165 activity can sulfate (1MBq/ml). In another experiment, the cells were treated with PlGF be potentiated by HSPGs [26]. On the other hand, HSPGs may (100 ng/ml) or vammin (5, 10, 25, or 50 ng/ml) for 48 h. After incubation, the conditioned medium was harvested, and solid urea was added up to a bind to VEGFRs and modulate the binding