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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識別強直性脊柱炎高效和疾病特定的基質改變

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

背景/目的:強直性脊柱炎（AS）產生滑膜炎癥和基質重塑的細胞和分子通路在很大程度上仍未知。SpA和類風濕性關節炎(RA)在結構重塑的方式完全不同，而且重塑過程中的基質通路可能有組織特異性，因此系統比較兩者的滑膜組織可能有助于鑒別疾病特異的致病機理。本研究旨在應用無偏倚的微基因篩選法發現SpA滑膜炎特異的細胞和分子通路。

方法:通過關節鏡從未經治療的SpA(n = 55),RA(n = 45)和痛風(10例)患者獲得滑膜組織樣本。對其中12例SpA和8例RA患者標本提取RNA并用基因芯片表達基因譜(Agilent 44 K)。在3個獨立的SpA和對照隊列中，通過qPCR方法對差異最顯著的基因進行驗證，并經免疫組化在蛋白水平再次再次確認。qPCR法還對TNF阻滯劑治療前后的配對SpA滑膜組織進行檢測。

結果:使用非常嚴格的分析和統計標準，微陣列實驗確定了SpA和RA間差異顯著增高的359個基因（界定值> 1.5倍的改變,p < 0.001）。通過qPCR對同一樣本進行技術驗證，確認基因芯片與qPCR結構強相關 (r = 0.93,p = 1.95e-009),其中25個基因差異表達最顯著(p < 0.05)。在另一獨立的早期未經治療的SpA和RA患者隊列中對這25個基因再分析，再次確認其差異性表達并與最初的基因芯片結果高度一致 (r = 0.97，p = 0.00004)。基因的表達特征結果可重復，而且通路研究中幾乎所有差異表達明顯的SpA中上調轉錄基因都與單核細胞／肌成纖維細胞生理相關。其中的數個基因如a平滑肌型肌動蛋白，在SpA滑膜炎中比RA高100倍，證明這些基因在SpA中特異性高表達而在RA中表達下調。a平滑肌肌動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,不僅出現在血管壁上，而且表達在滑膜下和滑膜內膜內層的成纖維樣細胞。后兩個表達區域, SpA就比RA顯著增高，而血管內表達無差別。 雙重免疫熒光法和FACS分析顯示α-平滑肌肌動蛋白和成纖維細胞標記CD90的共定位。最后,SpA患者TNF阻滯劑治療12周前后樣本的配對分析顯示這些基因的表達并未隨治療而改變。

結論:本研究發現SpA滑膜中肌纖維母細胞表達顯著增高并有疾病特異性。這種增高的原因以及這些細胞在炎癥中的作用, 更重要的是它們在SpA結構重構中的作用還在研究中。