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Anti-Angiogenic Action of Plasma Hyaluronan Binding Protein in Human Umbilical Vein Endothelial Cells

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Anti-Angiogenic Action of Plasma Hyaluronan Binding Protein in Human Umbilical Vein Endothelial Cells 209-215 1/6/06 17:37 Page 209 INTERNATIONAL JOURNAL OF ONCOLOGY 29: 209-215, 2006 209 Anti-angiogenic action of plasma hyaluronan binding protein in human umbilical vein endothelial cells JI WON JEON1,2*, HYUN SEOK SONG2*, EUN-JOUNG MOON1, SHI-YOUNG PARK1, MYUNG JIN SON2, SEUNG YOUN JUNG1, JI TAE KIM1, DO-HYUN NAM2,5, NAM-HO CHOI-MIURA3, KYU-WON KIM4 and YUNG-JIN KIM1 1Department of Molecular Biology, Pusan National University, Busan 609-735; 2Department of Neurosurgery, Samsung Medical Center and Samsung Biomedical Research Institute, Sung Kyun Kwan University School of Medicine, Seoul 135-710, South Korea; 3Department of Physiological Chemistry, School of Pharmaceutical Science, Showa University, Tokyo 142-8555, Japan; 4Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742; 5Xenotransplantation Research Center, Seoul, South Korea Received December 27, 2005; Accepted February 21, 2006 Abstract. The kringle domain is a triple loop structure present findings unravel a new function of PHBP as an inhibitor of the in angiostatin and endostatin. The disulfide bond-linked proangiogenic phenotype of vascular endothelial cells and kringle architectures have been known to be essential for demonstrate that the kringle domain of PHBP might be a potent anti-angiogenic activity. Plasma hyaluronan binding protein novel inhibitor of activated endothelial cells in vitro and in vivo. (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, Introduction and a serine protease domain. PHBP can be cleaved auto- catalytically to generate activity and is highly expressed in Angiogenesis, the sprouting of new capillaries from pre- the human blood and liver. To determine the anti-angiogenic existing vasculature, is an essential physiological process in activities of PHBP, we purified recombinant mouse PHBP embryonic development, wound healing, and reproductive from stable cell line overexpressing PHBP and used protein cycles in adult females (1). It is also recognized as a in vivo and in vitro angiogenesis assays. We found that characteristic of pathological conditions such as psoriasis, recombinant PHBP inhibits not only angiogenesis in vivo in proliferative retinopathies, and cancer growth and metastasis chorioallantoic membrane (CAM) assay but also the basic (2). During angiogenesis, endothelial cells need to divide, fibroblast growth factor (bFGF)-induced proliferation, invasion migrate, invade the extracellular matrix, and form capillary and tube formation of human umbilical vein endothelial cells structures from pre-existing blood vessels (3). These complex (HUVECs) in a dose-dependant manner. Moreover, we found processes imply the presence of multiple controls, which can that the kringle domain of PHBP was essential for the anti- be temporarily turned on and off within a short period (4,5). angiogenic action of PHBP by the deletion mutants. These A switch of the angiogenic phenotype by an up-regulation of angiogenesis activators and down-regulation of angiogenesis _________________________________________ inhibitors often leads to the progression of many diseases (1,6,7). An endogenous angiogenesis inhibitor, angiostatin, is an Correspondence to: Dr Yung-Jin Kim, Department of Molecular internal fragment of plasminogen and contains the first three Biology, Pusan National University, Busan 609-735, South Korea or four triple loop structures, known as kringle domains (8). E-mail: [email protected] The primary amino acid sequence of each kringle domain is Dr Kyu-Won Kim, Research Institute of Pharmaceutical Sciences, composed of approximately 80 amino acids and the structure College of Pharmacy, Seoul National University, Seoul 151-742, exists in many proteins. The disulfide bond-linked kringle South Korea architectures are essential for the anti-angiogenic activity of E-mail: [email protected] angiostatin (9). A previous study shows that kringle fragments * of several other proteins also inhibit angiogenesis (8). Most Contributed equally kringles only inhibit angiogenesis when cleaved as fragments from their parental proteins that lack anti-angiogenic activity. Abbreviations: CAM, chorioallantoic membrane; PHBP, plasma PHBP, isolated by adsorption to immobilized hyaluronan, hyaluronan binding protein; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor is a novel glycoprotein found in human plasma. The domain structure of PHBP is composed of one secretion signal Key words: angiogenesis inhibition, plasma hyaluronan binding peptide, three epidermal growth factor (EGF) domains, one protein, factor VII activating protease kringle domain and one serine protease domain from its amino-terminus (10). PHBP has the ability of the protease to 209-215 1/6/06 17:37 Page 210 210 JEON et al: PHBP IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS activate coagulation factor VII (FVII), named FVII-activating Carlsbad, CA) and selected in DMEM with 10% FBS and protease (FSAP) (11,12), and to activate tissue- or urinary- 400 μg/ml G418. The stable cell line was incubated with plasminogen activators (11,17). DMEM containing 1% FBS, and then the conditioned medium Single-chain PHBP is a 70-kDa zymogen that exists in was harvested and analyzed by sodium dodecyl sulfate- human plasma at a concentration of 12 μg/ml and can be polyacrylamide gel electrophoresis (SDS-PAGE) and immuno- cleaved autocatalytically to generate the active two-chain blotting with anti-V5 antibody (Invitrogen, Carlsbad, CA). form (50 kDa and 27 kDa) linked by a disulfide bond (13,14). Recombinant mouse PHBP from conditioned medium was PHBP exhibits a strong affinity for negatively charged concentrated using Vivaspin concentrator (Sartorius, Hannover, substances such as hyaluronic acid, dextran sulfate, or heparin, Germany), purified using Ni-NTA magnetic agarose beads all of which enhance autoactivation. At a cellular level, PHBP (Qiagen, Valencia, CA) under native conditions, according interacts with glycosaminoglycans (GAGs) and directly to the manufacturer's instructions. cleaves matrix proteins such as fibronectin, and fibrinogen (17,18). In addition, PHBP specifically binds to vascular Chorioallantoic membrane (CAM) assay. To determine the smooth muscle cells and reduces platelet-derived growth anti-angiogenic activity of recombinant mouse PHBP in vivo, factor (PDGF)-dependent smooth muscle cell proliferation. a CAM assay was performed as previously described (21,22). The complex formation between PHBP and PDGF has been Fertilized eggs (Pulmuone, Kyungki-do, Korea) were incubated reported as the major cause (16). at 37˚C and 90% relative humidity. After 3 days, approx- Recently, it has been reported that PHBP inhibits bFGF/ imately 2-3 ml of albumin was removed and a window was EGF-induced proliferation of HUVECs (18). The authors made. At the 4.5-day-old CAM, test samples or retinoic acid present two possible mechanisms involved. First, PHBP (1 μg) loaded on a quarter size of Thermanox coverslip (Nunc cleaves adhesion molecules which are required for attachment International, Naperville, IL) were applied on the CAM of and proliferation. Second, PHBP strongly binds to and partially individual embryos. After 48-h incubation, 10% fat emulsion hydrolyses bFGF. However, even though PHBP affects the (Intralipose; Korea Green Cross, Seoul, Korea) was injected proliferation of endothelial cells, the precise mechanisms of into the CAM for observation of the inhibition zone of angio- PHBP on the process of angiogenesis were not elucidated. genesis under a microscope. Therefore, more detailed studies are required to determine the effects of PHBP on the process of angiogenesis. [3H]-thymidine incorporation assay. To examine the anti- In order to determine whether PHBP containing kringle proliferative effect of PHBP, HUVECs were seeded at a density domain can be a novel angiogenesis inhibitor, we performed of 2x104 cells/well in a 24-well plate. Cells were incubated in in vivo and in vitro angiogenesis assays using recombinant EGM and allowed to attach for 24 h. Cells were washed two mouse PHBP. times with endothelial basal medium (EBM) and incubated for 6 h in EBM containing 1% FBS. Cells were stimulated Materials and methods by the addition of the indicated concentration of PHBP and 25 ng/ml of bFGF (Upstate, Lake Placid, NY) for 24 h, and Cell culture. HUVECs were grown on 0.3% gelatin-coated were subjected for 4 h to the addition of 1 μCi/ml [3H]- dishes and maintained in endothelial cell growth medium thymidine (Amersham Pharmacia Biotechnology, Piscataway, EGM-2 kit (Clonetics, San Diego, CA). HUVECs were used NJ). After fixing cells with methanol, high molecular mass at passage from 2 to 8. Human embryonic kidney (HEK) 293 [3H]-radioactivity was precipitated using 5% trichloroacetic cells were cultured in Dulbecco's modified Eagle's medium acid at 4˚C for 16 h. After two washes with PBS, [3H]- (DMEM) supplemented with heat-inactivated 10% fetal radioactivity was solubilized in 0.2 N NaOH and 0.1% SDS bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml and determined using a liquid scintillation counter (Beckman streptomycin. Cells were grown in a 37˚C incubator with a Instruments, Fullerton, CA). Each experiment was performed humidified atmosphere containing 5% CO2, 95% air. in triplicate. Construction of the expression vector. The E. coli expression Invasion assay. The invasive chemotactic motility of HUVECs vector (pBluescript, Stratagene) of
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