Session Type: Abstract Submissions (ACR)
The histone methyltransferase Enhancer of Zeste 2 (EZH2) is overexpressed in solid tumors and is associated with invasion, malignancy and tumor angiogenesis. EZH2 expression is upregulated in rheumatoid arthritis (RA) synovial fibroblasts (SF) whose activated phenotype drives them to invade and destroy articular cartilage. Another hallmark of the inflamed rheumatoid joint is angiogenesis and RASF are important regulators of this pathway secreting proangiogenic molecules and providing a molecular scaffold for blood vessel formation. Here, we studied the role of EZH2 in the interplay of RASF and endothelial cells in RA.
Fresh biopsies of synovial tissue (ST) from RA patients were stimulated with tumor necrosis factor alpha (TNFα, 10ng/ml) for 24h (explant culture) and RNA was isolated (n=3). Human microvascular endothelial cells (HMVEC) were stimulated with proinflammatory stimuli, Toll-like receptor (TLR) ligands or synovial fluid (2%, 5% and 10%) for different periods of time (n=3). RASF (n=4) were transfected with siRNA targeting EZH2. After 72h and 96h, cells were analyzed for EZH2, interleukin (IL)-6, IL8 and vascular endothelial growth factor (VEGF) expression at mRNA (by quantitative real-time PCR) and protein levels (by ELISA).
Ex vivo, TNFα induced the expression of EZH2 in RA ST (1.8±0.3-fold) which was accompanied by an increase in the expression and copious secretion of proinflammatory and proangiogenic mediators, namely IL6 (from 77±88 ng/ml to 197±163 ng/ml) and IL8 (from 105±53 ng/ml to 220±30 ng/ml). Since RA ST explant cultures maintain the synovial architecture with cell-cell and cell-matrix contacts thus mimicking the in vivo situation, these data indicate that chronic inflammation within the RA synovium upregulates EZH2 expression in vivo. Our previous work demonstrated that TNFα upregulates EZH2 in RASF. Silencing of EZH2 in RASF changed the expression and secretion of IL6 and VEGF into cell culture supernatants whereas no significant changes were observed for IL8. Specifically, IL6 and VEGF were downregulated by EZH2 knockdown both under basal (by 39±17% and 29±8%) and TNFα-stimulated (by 30±18% and 38±6%) conditions which was confirmed at the protein level by ELISA. To elucidate whether other cell types show the same EZH2 expression pattern, HMVEC were studied for their response to pathogenetically relevant stimuli. No significant changes in EZH2 expression, however, were observed following proinflammatory stimuli (TNFα, IL-1β, IL-17), TLR stimulation (TLR2, 3 and 4) or treatment with synovial fluid, ruling out a direct effect of inflammation-induced EZH2 on endothelial cell behaviour.
Here we show that TNFα induces the expression of the epigenetic regulator EZH2 in RA ST explants ex vivo. In vitro, TNFα regulates EZH2 expression in RASF but not in endothelial cells. Silencing of EZH2 in RASF decreased the expression and secretion of IL6 and VEGF. These data imply that induction of EZH2 by TNFα in synovial tissue and RASF drives proinflammatory and proangiogenic mechanisms in RASF.
D. J. Veale,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-proangiogenic-function-of-the-epigenetic-regulator-ezh2-in-synovial-tissue-is-mediated-by-fibroblasts-in-rheumatoid-arthritis/