The role of PRG-B which is a novel angiogenesis-related gene in rheumatoid arthritis and osteoarthritis

新型血管生成相关基因PRG-B在类风湿关节炎和骨关节炎中的作用

基本信息

批准号：
15591602
负责人：
MORIOKA Hideo
金额：
$ 2.24万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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 … More 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

软骨是一种血管组织，这种血管性可能是由于血管生成抑制剂的局部产生。在我们先前关于人类关节软骨中纤溶酶原的表达的研究中，我们分离了一种独特的cDNA片段，称为纤溶酶原相关基因-B（PRG-B），该片段预测了指定为纤溶酶原相关蛋白-B（PRP-B）的9KDA多肽。我们报告说，重组PRP-B（RPRP-B）抑制了血管生成，并且至少部分是通过抑制基本成纤维细胞生长因子（BFGF）诱导的内皮细胞中酪氨酸激酶信号传导而介导的。在类风湿关节炎（RA）中，据报道，促促促血管生成细胞因子在滑膜上的激活。因此，最近已经尝试采用血管生成抑制剂来治疗RA。我们还报道了RPRP-B在人成纤维细胞样细胞（FLS）中下调VEGF的表达。 The aim of this study is to investigat … More 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在这项研究中也使用了软骨细胞。临床样品：在膝关节成形术手术中，从膝关节置换术手术中获得了RA. RA.RESSERCTRACTION-聚合酶链反应（RT-PCR）的膝关节手术：PCR使用特异性引物对PRG-B或GAPDH进行特异性引物，并通过Agarose gelorothorsis分析了反应产物。比较了有或没有BFGF的HC和CS-1中PRG-B mRNA的表达水平。抗体和免疫组织化学：将纯化的RPRP-B用于兔子，作为抗原和针对PRP-B的抗体。收集血清并进行蛋白质A纯化。抗纤溶酶原（PLG）的抗体是从Dako获得的。免疫组织化学使用标准技术使用石蜡嵌入的软骨样品对PRP-B和纤溶酶原进行检测。真实时代聚合酶链反应（实时PCR）：我们研究了CS-1中VEGF mRNA在CS-1中的表达，并通过或无需通过PRP-B处理，通过实时pCR iNTS：rt-bf-bf. rt-bf。 CS-1。免疫组织化学：在使用抗PRP-B抗体对软骨的免疫组织化学染色中，PRP-B表达被证明在RA患者的软骨中被上调。真实时间PCR：RPRP-B：RPRP-B降低了CS-1.-Discussion的VEGF mRNA的表达水平：在这项研究中，我们研究了eraniation，我们研究了eraniation，我们研究了eraniation of ernition of ernities or nis the ernitiation of ernition of ers he and heris of ernities nis and of ers of ernities or nisiati软骨。 RT-PCR的结果表明，BFGF在体外诱导了PRG-B的上调。此外，我们在RA软骨的临床样品中检测到了源自PRG-B的蛋白质的表达。我们的结果还表明，RPRP-B在体外下调VEGF表达。在关节炎关节中，关节软骨是由促血管生成和炎性细胞因子（例如BFGF和IL-1β）刺激的，这些因子是由基质素产生的。我们报告说，RPRP-B在51^<ST>会议上的FLS中下调VEGF表达。因此，我们的结果表明，PRG-B在关节软骨中表达，并且对促性关节中滑膜和软骨细胞的自分泌和旁分泌抗血管生成作用。因此，PRG-B似乎是关节疾病中抗血管生成靶向基因的候选者。较少的