The role of PRG-B which is a novel angiogenesis-related gene in rheumatoid arthritis and osteoarthritis
| Project/Area Number |
15591602
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Keio University |
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Principal Investigator |
MORIOKA Hideo Keio University, Department of Orthopaedic Surgery, School of Medicine, Lecturer, 医学部, 講師 (10230096)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUMOTO Hideo Keio University, Department of Orthopaedic Surgery, School of Medicine, Assistant Professor, 医学部, 助教授 (50138038)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Joint disease / Rheumatoid arthritis / Osteoarthritis / Bone and Cartilage metabolism / Angiogenesis / Cartilage / Synovium / Synovitis / 軟骨(Cartilage) / 滑膜(Synovium) / 血管新生(Angiogenesis) / 関節リウマチ / plasminogen related gene B / 血管内皮細胞増殖因子(VEGF) / 分子標的治療 / 変形性関節症 / rheumatoid arthritis / osteoarthritis / angiogenesis / plasminogen / cartilage / synovium / plasminogen related gene / vascular endothelial growth factor / angiogenesis inhibitor / recombinant protein |
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| Research Abstract |
Cartilage is an avascular tissue and this avascularity may be due to the local production of angiogenesis inhibitors. During our previous studies on the expression of plasminogen in human articular cartilage, we isolated a unique cDNA fragment named plasminogen related gene-B (PRG-B) which predicts a 9kDa polypeptide designated as plasminogen-related protein-B (PRP-B). We reported that recombinant PRP-B (rPRP-B) inhibited angiogenesis and that this effect was, at least partly, mediated through the inhibition of basic fibroblast growth factor (bFGF) -induced tyrosine kinase signaling in endothelial cells. In rheumatoid arthritis (RA), activation of angiogenesis through the up-regulation of pro-angiogenic cytokines in synovium has been reported. Therefore, attempts have recently been made to employ angiogenesis-inhibitors for the treatment of RA. We also reported that rPRP-B down-regulates VEGF expression in human fibroblast-like synoviocytes (FLS). The aim of this study is to investigat
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e the expression of PRG-B and the regulation of angiogenesis by PRG-B in RA cartilage.
MATERIALS AND METHODS :
Recombinant protein : The isolation and characterization of rPRP-B has been described previously.
Cell culture : Human Chondrocytes (HC) were purchased from CELL APPLICATIONS, INC. Human chondrosarcoma cell line (CS-1) which has phenotype as chondrocyte was also used in this study.
Clinical samples : Cartilage samples were obtained at arthroplasty surgery from knee joints of patients with RA.
Reverse transcription- polymerase chain reaction (RT-PCR) : PCR was performed using specific primer pairs for PRG-B or GAPDH, and the reaction products were analyzed by agarose gel electrophoresis. The expression levels of PRG-B mRNA in HC and CS-1 with or without bFGF were compared.
Antibodies and immunohistochemistry : Purified rPRP-B was applied to rabbits as antigen and antibody against PRP-B was raised. Serum was collected and subject to the protein-A purification. Antibody against plasminogen (PLG) was obtained from DAKO. Immunohistochemistry for detection of PRP-B and plasminogen was performed using paraffin embedded cartilage samples using the standard technique.
Real-time polymerase chain reaction (Real-time PCR) : We investigated the expression of VEGF mRNA in CS-1 with or without PRP-B treatment by Real-time PCR.
RESULTS :
RT-PCR : In analysis for cell lines, PRG-B expression was induced by bFGF stimulation in HC and CS-1.
Immunohistochemistry : In immunohistochemical staining of cartilage using anti-PRP-B antibody, PRP-B expression was shown to be up-regulated in cartilage from RA patients.
Real-time PCR : rPRP-B reduced the expression level of VEGF mRNA of CS-1.
Discussion :
In this study, we investigated the role of PRG-B as an anti-angiogenesis gene in the arthritic cartilage. The results of RT-PCR showed that bFGF induced up-regulation of PRG-B in vitro. In addition, we detected expression of protein derived from PRG-B in clinical samples of RA cartilages. Our results also showed that rPRP-B down-regulated VEGF expression in vitro. In arthritic joint, articular cartilage is stimulated by pro-angiogenic and inflammatory cytokines such as bFGF and IL-1β, which are produced from synovium. We reported that rPRP-B down-regulates VEGF expression in FLS in vitro at the 51^<st> ORS meeting. Therefore, our results suggest that PRG-B is expressed in articular cartilage and has autocrine and paracrine anti-angiogenesis effects on synoviocyte and chondrocyte in the arthritic joint. Therefore, PRG-B seems to be a candidate for anti-angiogenesis targeted gene in arthritic diseases. Less
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Report
(4 results)
Research Products
(8 results)
