Notch 信号通路介导滑膜血管生成以响应血管内皮生长因子和血管生成素 2。
Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.
机构信息
Department of Rheumatology, Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
出版信息
Ann Rheum Dis. 2013 Jun;72(6):1080-8. doi: 10.1136/annrheumdis-2012-201978. Epub 2012 Nov 17.
OBJECTIVE
Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function.
METHODS
Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, Hrt-2) mRNA and/or protein expression was measured by Real-time PCR and/or western blot. VEGF/Ang2 induced EC function was assessed using transwell invasion chambers, matrigel tube formation assays and wound repair scratch assays±Notch-1 siRNA or an γ-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) in RA synovial explants or human microvascular EC. Interleukin (IL)-6 and IL-8 were measured by ELISA and MMP2 and 9 by gelatine zymography.
RESULTS
Notch-1 IC and Notch-4 IC protein expressions were demonstrated in RA and psoriatic arthritis synovial biopsies, with minimal expression observed in Osteoarthritis (OA). VEGF and Ang2 induced Notch-1 IC/ Notch-4 IC protein expression in synovial explant cultures and human microvascular EC levels were further potentiated by VEGF/Ang2 stimulation in combination. Notch-1, Delta-like-ligand 4, and Hrt-2 mRNA expression were significantly induced by VEGF and Ang2 alone and in combination. Furthermore VEGF/Ang2-induced EC invasion, angiogenesis and migration were inhibited by Notch-1 siRNA or DAPT. Conditioned media from VEGF/Ang2 stimulated RA synovial explants induced EC tube formation, an effect that was inhibited by DAPT. Finally, DAPT significantly decreased VEGF/Ang2 induced IL-6, IL-8, MMP2 and 9 expressions in RA synovial explants.
CONCLUSIONS
Notch-1 mediates VEGF/Ang2-induced angiogenesis and EC invasion in inflammatory arthritis.
目的
Notch 信号通路对血管生成和内皮细胞（EC）命运至关重要；然而，在炎症关节中调节这些过程的机制仍有待阐明。在这里，我们研究了 Notch 信号是否介导血管内皮生长因子（VEGF）和血管生成素 2（Ang2）诱导的血管功能。
方法
通过实时 PCR 和/或 Western blot 测量 Notch-1 细胞内结构域（Notch-1 IC）、Notch-4 IC、Delta-like 配体 4、Hes 相关转录抑制因子-1 和 2（Hrt-1、Hrt-2）mRNA 和/或蛋白表达。使用 Transwell 侵袭室、Matrigel 管形成测定和划痕修复划痕测定评估 VEGF/Ang2 诱导的 EC 功能，在 RA 滑膜外植体或人微血管 EC 中使用 Notch-1 siRNA 或 γ-分泌酶抑制剂 N-（N-（3、5-二氟苯乙酰-L-丙氨酸基））-S-苯甘氨酸-t-丁酯（DAPT）。通过 ELISA 测量白细胞介素（IL）-6 和 IL-8，通过凝胶酶谱法测量 MMP2 和 9。
结果
在 RA 和银屑病关节炎滑膜活检中证实了 Notch-1 IC 和 Notch-4 IC 蛋白表达，在骨关节炎（OA）中观察到最小表达。VEGF 和 Ang2 在滑膜外植体培养物中诱导 Notch-1 IC/Notch-4 IC 蛋白表达，并且 VEGF/Ang2 刺激的组合进一步增强了人类微血管 EC 的水平。VEGF 和 Ang2 单独和组合诱导 Notch-1、Delta-like 配体 4 和 Hrt-2 mRNA 表达显著增加。此外，Notch-1 siRNA 或 DAPT 抑制 VEGF/Ang2 诱导的 EC 侵袭、血管生成和迁移。来自 VEGF/Ang2 刺激的 RA 滑膜外植体的条件培养基诱导 EC 管形成，DAPT 抑制了这种作用。最后，DAPT 显著降低了 RA 滑膜外植体中 VEGF/Ang2 诱导的 IL-6、IL-8、MMP2 和 9 的表达。
结论
Notch-1 介导血管生成和炎症性关节炎中 EC 侵袭的 VEGF/Ang2 诱导。
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