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

软骨是一种无血管组织，这种无血管性可能是由于局部产生血管生成抑制剂。我们在研究纤溶酶原在人关节软骨中的表达过程中，分离到一个独特的cDNA片段，命名为纤溶酶原相关基因-B（plasminogen related gene-B，PRG-B），它预测了一个分子量为9 kDa的多肽，命名为纤溶酶原相关蛋白-B（plasminogen related protein-B，PRP-B）。我们报道了重组PRP-B（rPRP-B）抑制血管生成，并且这种作用至少部分是通过抑制内皮细胞中碱性成纤维细胞生长因子（bFGF）诱导的酪氨酸激酶信号传导介导的。在类风湿性关节炎（RA）中，通过滑膜中促血管生成细胞因子的上调激活血管生成已被报道。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下调人成纤维细胞样滑膜细胞（FLS）中VEGF的表达。本研究的目的是探讨 关于我们 材料和方法：重组蛋白：rPRP-B的分离和表征已经在前面描述。临床标本：取关节置换术后RA患者膝关节软骨标本，采用PRG-B和GAPDH特异性引物进行RT-PCR，琼脂糖凝胶电泳分析反应产物。比较有或无bFGF时HC和CS-1中PRG-B mRNA的表达水平。抗体和免疫组化：将纯化的rPRP-B作为抗原应用于兔，产生针对PRP-B的抗体。收集血清并进行蛋白A纯化。抗纤溶酶原（PLG）的抗体获自DAKO。使用标准技术，使用石蜡包埋的软骨样品进行PRP-B和纤溶酶原的免疫组织化学检测。（Real-time PCR）：我们通过Real-time PCR研究了有或没有PRP-B处理的CS-1中VEGF mRNA的表达。结果：RT-PCR：在细胞系分析中，在HC和CS-1中，PRG-B表达被bFGF刺激诱导。用抗B抗体免疫组化染色显示PRP-B在RA患者软骨中表达上调，实时荧光定量PCR：rPRP-B降低CS-1的VEGF mRNA表达水平。RT-PCR结果显示bFGF在体外可诱导PRG-B表达上调。此外，我们还检测了PRG-B蛋白在RA软骨组织中的表达。我们的研究结果还表明，rPRP-B下调VEGF的表达在体外。在关节炎关节中，关节软骨受到滑膜产生的促血管生成和炎性细胞因子如bFGF和IL-1β的刺激。我们在第51届ORS会议上报道了rPRP-B在体外下调FLS中VEGF的表达<st>。因此，我们的研究结果表明，PRG-B在关节软骨中表达，并对关节炎关节的滑膜细胞和软骨细胞具有自分泌和旁分泌的抗血管生成作用。因此，PRG-B可能是抗血管生成靶基因在关节炎疾病中的候选者。少

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