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Identification of Robust and
Disease-Specific Stromal Alterations in Spondyloarthritis
Synovitis
Nataliya Yeremenko
1, Gemma M. M. Rigter1,
Iris Simon2, Juan D. Cañete3, 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
Presentation Number:
1591
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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识别强直性脊柱炎高效和疾病特定的基质改变
Nataliya Yeremenko
, et al. ACR 2011. Present
No:1591
背景/目的:强直性脊柱炎（AS）产生滑膜炎症和基质重塑的细胞和分子通路在很大程度上仍未知。SpA和类风湿性关节炎(RA)在结构重塑的方式完全不同，而且重塑过程中的基质通路可能有组织特异性，因此系统比较两者的滑膜组织可能有助于鉴别疾病特异的致病机理。本研究旨在应用无偏倚的微基因筛选法发现SpA滑膜炎特异的细胞和分子通路。
方法:通过关节镜从未经治疗的SpA(n = 55),RA(n = 45)和痛风(10例)患者获得滑膜组织样本。对其中12例SpA和8例RA患者标本提取RNA并用基因芯片表达基因谱(Agilent 44 K)。在3个独立的SpA和对照队列中，通过qPCR方法对差异最显著的基因进行验证，并经免疫组化在蛋白水平再次再次确认。qPCR法还对TNF阻滞剂治疗前后的配对SpA滑膜组织进行检测。
结果:使用非常严格的分析和统计标准，微阵列实验确定了SpA和RA间差异显著增高的359个基因（界定值> 1.5倍的改变,p < 0.001）。通过qPCR对同一样本进行技术验证，确认基因芯片与qPCR结构强相关 (r = 0.93,p = 1.95e-009),其中25个基因差异表达最显著(p < 0.05)。在另一独立的早期未经治疗的SpA和RA患者队列中对这25个基因再分析，再次确认其差异性表达并与最初的基因芯片结果高度一致 (r = 0.97，p =
0.00004)。基因的表达特征结果可重复，而且通路研究中几乎所有差异表达明显的SpA中上调转录基因都与单核细胞／肌成纤维细胞生理相关。其中的数个基因如a平滑肌型肌动蛋白，在SpA滑膜炎中比RA高100倍，证明这些基因在SpA中特异性高表达而在RA中表达下调。a平滑肌肌动蛋白和平滑肌肌球蛋白重链的免疫组化确定这些蛋白的表达,不仅出现在血管壁上，而且表达在滑膜下和滑膜内膜内层的成纤维样细胞。后两个表达区域, SpA就比RA显著增高，而血管内表达无差别。 双重免疫荧光法和FACS分析显示α-平滑肌肌动蛋白和成纤维细胞标记CD90的共定位。最后,SpA患者TNF阻滞剂治疗12周前后样本的配对分析显示这些基因的表达并未随治疗而改变。
结论:本研究发现SpA滑膜中肌纤维母细胞表达显著增高并有疾病特异性。这种增高的原因以及这些细胞在炎症中的作用, 更重要的是它们在SpA结构重构中的作用还在研究中。
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