原文

譯文

Identification of Robust and Disease-Specific Stromal Alterations in Spondyloarthritis Synovitis

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Nataliya Yeremenko ¹, Gemma M. M. Rigter¹, Iris Simon², Juan D. Ca?ete³, Paul P. Tak¹ and Dominique L. Baeten¹, ¹Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ²Agendia BV, Amsterdam, Netherlands, ³Hospital Clínic de Barcelona, IDIBAPS, Barcelona, Spain

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Presentation Number: 1591

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Background/Purpose:?The cellular and molecular pathways driving synovial inflammation and stromal remodeling in spondyloarthritis (SpA) remain largely unknown. As SpA and rheumatoid arthritis (RA) show clearly distinct patterns of structural remodeling and since stromal pathways of remodeling may be specific to the target tissues, systematic comparison of the inflamed synovial tissue in both conditions may help to identify disease-specific pathogenic mechanisms. We conducted this study in order to identify cellular and molecular pathways specific for SpA synovitis by an unbiased microarray screening approach.

Method:?Synovial tissue samples were obtained by arthroscopy from untreated individuals with SpA (n=55), RA (n=45) and gout (n=10). RNA was extracted and gene expression profiling was performed on a test cohort of 12 SpA versus 8 RA samples using microarrays (Agilent 44K). Top differentially expressed genes were validated on three independent cohorts of SpA versus control samples by qPCR and confirmed on the protein level by immunohistochemistry. qPCR was also performed on paired SpA synovial biopsies before and after TNF blockade.

Result:?Using very stringent analysis and statistical criteria, the microarray experiments identified a signature set of 359 genes that discriminated with high certainty (a cut-off >1.5-fold change, p<0.001) between patients with SpA and RA. Technical validation by qPCR on the same samples yielded a strong correlation between the microarray and qPCR data (r=0.93, p= 1.95e-009) with 25 of the top genes being confirmed as differentially expressed (p < 0.05). Reanalysis of the top 25 genes in an independent cohort of early, untreated SpA and RA confirmed the differential expression of the genes with again a very good correlation with the original microarray results (r=0.97. p= 0.00004). The gene signature was not only reproducible but also consistent as pathway analysis revealed that almost all top-ranking upregulated transcripts in SpA were related to myocyte/myofibroblast biology. Several of these genes, including the alpha smooth muscle form of actin, showed up to 100-fold upregulation in SpA versus RA synovitis. Additional analysis of gout versus SpA samples confirmed that these genes were specifically upregulated in SpA synovitis rather than downregulated in RA. Immunohistochemistry for α-smooth muscle actin and smooth muscle myosin heavy chain identified expression of these proteins not only around the vessel walls but also in fibroblast-like cells in the intimal lining layer and synovial sublining. Expression in the latter two regions, but not in the vessels, was significantly increased in SpA versus RA. Double immunofluorescence and FACS analyses revealed a colocalization of α-smooth muscle actin and fibroblasts marker CD90. Finally, paired analysis of SpA samples obtained before and after 12 weeks of TNF blockade showed that the expression of these genes was not altered by this treatment.?

Conclusion:?This study identified a robust and disease-specific increase in myofibroblasts in SpA synovitis. The reason for this increase and the potential role of these cells in inflammation and, more importantly, structural remodeling in SpA are currently under investigation.

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識(shí)別強(qiáng)直性脊柱炎高效和疾病特定的基質(zhì)改變

Nataliya Yeremenko , et al. ACR 2011. Present No:1591

背景/目的:強(qiáng)直性脊柱炎（AS）產(chǎn)生滑膜炎癥和基質(zhì)重塑的細(xì)胞和分子通路在很大程度上仍未知。SpA和類(lèi)風(fēng)濕性關(guān)節(jié)炎(RA)在結(jié)構(gòu)重塑的方式完全不同，而且重塑過(guò)程中的基質(zhì)通路可能有組織特異性，因此系統(tǒng)比較兩者的滑膜組織可能有助于鑒別疾病特異的致病機(jī)理。本研究旨在應(yīng)用無(wú)偏倚的微基因篩選法發(fā)現(xiàn)SpA滑膜炎特異的細(xì)胞和分子通路。

方法:通過(guò)關(guān)節(jié)鏡從未經(jīng)治療的SpA(n = 55),RA(n = 45)和痛風(fēng)(10例)患者獲得滑膜組織樣本。對(duì)其中12例SpA和8例RA患者標(biāo)本提取RNA并用基因芯片表達(dá)基因譜(Agilent 44 K)。在3個(gè)獨(dú)立的SpA和對(duì)照隊(duì)列中，通過(guò)qPCR方法對(duì)差異最顯著的基因進(jìn)行驗(yàn)證，并經(jīng)免疫組化在蛋白水平再次再次確認(rèn)。qPCR法還對(duì)TNF阻滯劑治療前后的配對(duì)SpA滑膜組織進(jìn)行檢測(cè)。

結(jié)果:使用非常嚴(yán)格的分析和統(tǒng)計(jì)標(biāo)準(zhǔn)，微陣列實(shí)驗(yàn)確定了SpA和RA間差異顯著增高的359個(gè)基因（界定值> 1.5倍的改變,p < 0.001）。通過(guò)qPCR對(duì)同一樣本進(jìn)行技術(shù)驗(yàn)證，確認(rèn)基因芯片與qPCR結(jié)構(gòu)強(qiáng)相關(guān) (r = 0.93,p = 1.95e-009),其中25個(gè)基因差異表達(dá)最顯著(p < 0.05)。在另一獨(dú)立的早期未經(jīng)治療的SpA和RA患者隊(duì)列中對(duì)這25個(gè)基因再分析，再次確認(rèn)其差異性表達(dá)并與最初的基因芯片結(jié)果高度一致 (r = 0.97，p = 0.00004)?；虻谋磉_(dá)特征結(jié)果可重復(fù)，而且通路研究中幾乎所有差異表達(dá)明顯的SpA中上調(diào)轉(zhuǎn)錄基因都與單核細(xì)胞／肌成纖維細(xì)胞生理相關(guān)。其中的數(shù)個(gè)基因如a平滑肌型肌動(dòng)蛋白，在SpA滑膜炎中比RA高100倍，證明這些基因在SpA中特異性高表達(dá)而在RA中表達(dá)下調(diào)。a平滑肌肌動(dòng)蛋白和平滑肌肌球蛋白重鏈的免疫組化確定這些蛋白的表達(dá),不僅出現(xiàn)在血管壁上，而且表達(dá)在滑膜下和滑膜內(nèi)膜內(nèi)層的成纖維樣細(xì)胞。后兩個(gè)表達(dá)區(qū)域, SpA就比RA顯著增高，而血管內(nèi)表達(dá)無(wú)差別。 雙重免疫熒光法和FACS分析顯示α-平滑肌肌動(dòng)蛋白和成纖維細(xì)胞標(biāo)記CD90的共定位。最后,SpA患者TNF阻滯劑治療12周前后樣本的配對(duì)分析顯示這些基因的表達(dá)并未隨治療而改變。

結(jié)論:本研究發(fā)現(xiàn)SpA滑膜中肌纖維母細(xì)胞表達(dá)顯著增高并有疾病特異性。這種增高的原因以及這些細(xì)胞在炎癥中的作用, 更重要的是它們?cè)?span lang="en-us">SpA結(jié)構(gòu)重構(gòu)中的作用還在研究中。