Kurowska-Stolarska Mariola, Distler Jörg H W, Jüngel Astrid, Rudnicka Weronika, Neumann Elena, Pap Thomas, Wenger Roland H, Michel Beat A, Müller-Ladner Ulf, Gay Renate E, Maslinski Wlodzimierz, Gay Steffen, Distler Oliver
University Hospital Zurich, University of Zurich, Zurich, Switzerland.
Arthritis Rheum. 2009 Dec;60(12):3663-75. doi: 10.1002/art.25001.
To map hypoxic areas in arthritic synovium and to establish the relevance of low oxygen levels to the phenotype of synovial fibroblasts, with special focus on bone degradation.
To analyze the distribution of hypoxia in arthritic joints, the hypoxia marker EF5 was administered to mice with collagen-induced arthritis (CIA). To evaluate the effect of hypoxia on rheumatoid arthritis synovial fibroblasts (RASFs), reverse suppression subtractive hybridization and complementary DNA array were used. Real-time polymerase chain reaction, Western blotting, and immunohistochemistry were used to evaluate the expression of inhibitor of DNA binding/differentiation 2 (ID-2). To investigate the function of ID-2 in RASFs, cells were transfected either with ID-2 vector or with ID-2-specific small interfering RNA.
EF5 staining showed the presence of hypoxia in arthritic joints, particularly at sites of synovial invasion into bone. Differential expression analysis revealed that ID-2 was strongly induced by hypoxia in RASFs. Immunohistochemical analysis of CIA mouse synovium and human RA synovium showed a strong expression of ID-2 by RASFs at sites of synovial invasion into bone. Overexpression of ID-2 in RASFs significantly induced the expression of several factors promoting osteoclastogenesis. The biologic relevance of the potent osteoclastogenesis-promoting effects was shown by coculture assays of ID-2-overexpressing RASFs with bone marrow cells, leading to an increased differentiation of osteoclasts from bone marrow precursors.
The data show that hypoxic conditions are present at sites of inflammation and synovial invasion into bone in arthritic synovium. Hypoxia-induced ID-2 may contribute to joint destruction in RA patients by promoting synovial fibroblast-dependent osteoclastogenesis.
绘制关节炎滑膜中的缺氧区域，并确定低氧水平与滑膜成纤维细胞表型的相关性，特别关注骨降解。
为分析关节炎关节中缺氧的分布情况，将缺氧标志物EF5给予胶原诱导性关节炎（CIA）小鼠。为评估缺氧对类风湿性关节炎滑膜成纤维细胞（RASFs）的影响，采用了反向抑制消减杂交和互补DNA阵列技术。运用实时聚合酶链反应、蛋白质印迹法和免疫组织化学法评估DNA结合/分化抑制因子2（ID-2）的表达。为研究ID-2在RASFs中的功能，用ID-2载体或ID-2特异性小干扰RNA转染细胞。
EF5染色显示关节炎关节中存在缺氧现象，尤其是在滑膜侵入骨的部位。差异表达分析表明，缺氧可强烈诱导RASFs中ID-2的表达。对CIA小鼠滑膜和人类风湿性关节炎滑膜进行免疫组织化学分析显示，在滑膜侵入骨的部位，RASFs强烈表达ID-2。RASFs中ID-2的过表达显著诱导了几种促进破骨细胞生成的因子的表达。ID-2过表达的RASFs与骨髓细胞共培养试验表明了其促进破骨细胞生成作用的生物学相关性，导致骨髓前体细胞向破骨细胞的分化增加。
数据表明，在关节炎滑膜的炎症部位和滑膜侵入骨的部位存在缺氧情况。缺氧诱导的ID-2可能通过促进滑膜成纤维细胞依赖性破骨细胞生成，导致类风湿性关节炎患者的关节破坏。
