DataSheet_1_The role of FOXO4/NFAT2 signaling pathway in dysfunction of human coronary endothelial cells and inflammatory infiltration of vasculitis in Kawasaki disease.docx

AimsThe Ca+/NFAT (Nuclear factor of activated T cells) signaling pathway activation is implicated in the pathogenesis of Kawasaki disease (KD); however, we lack detailed information regarding the regulatory network involved in the human coronary endothelial cell dysfunction and cardiovascular lesion development. Herein, we aimed to use mouse and endothelial cell models of KD vasculitis in vivo and in vitro to characterize the regulatory network of NFAT pathway in KD.
Methods and ResultsAmong the NFAT gene family, NFAT2 showed the strongest transcriptional activity in peripheral blood mononuclear cells (PBMCs) from patients with KD. Then, NFAT2 overexpression and knockdown experiments in Human coronary artery endothelial cells (HCAECs) indicated that NFAT2 overexpression disrupted endothelial cell homeostasis by regulation of adherens junctions, whereas its knockdown protected HCAECs from such dysfunction. Combined analysis using RNA-sequencing and transcription factor (TF) binding site analysis in the NFAT2 promoter region predicted regulation by Forkhead box O4 (FOXO4). Western blotting, chromatin immunoprecipitation, and luciferase assays validated that FOXO4 binds to the promoter and transcriptionally represses NFAT2. Moreover, Foxo4 knockout increased the extent of inflamed vascular tissues in a mouse model of KD vasculitis. Functional experiments showed that inhibition NFAT2 relieved Foxo4 knockout exaggerated vasculitis in vivo.
ConclusionsOur findings revealed the FOXO4/NFAT2 axis as a vital pathway in the progression of KD that is associated with endothelial cell homeostasis and cardiovascular inflammation development.

研究目的：钙/活化T细胞核因子（Nuclear factor of activated T cells, NFAT）信号通路的激活与川崎病（Kawasaki disease, KD）的发病机制相关，但目前学界对人类冠状动脉内皮细胞功能异常及心血管病变发生过程中的调控网络仍缺乏详细认知。本研究旨在通过体内（in vivo）与体外（in vitro）的川崎病血管炎小鼠模型及内皮细胞模型，解析NFAT通路在川崎病中的调控网络。
方法与结果：在NFAT基因家族中，NFAT2在川崎病患者的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中展现出最强的转录活性。随后，在人冠状动脉内皮细胞（Human coronary artery endothelial cells, HCAECs）中开展的NFAT2过表达与敲低实验表明：NFAT2过表达可通过调控黏附连接破坏内皮细胞稳态，而敲低NFAT2则可保护HCAECs免受此类功能异常的影响。结合RNA测序（RNA-sequencing）与NFAT2启动子区域的转录因子（transcriptional factor, TF）结合位点分析，本研究预测叉头框O4（Forkhead box O4, FOXO4）可对其实施调控。蛋白质免疫印迹（Western blotting）、染色质免疫沉淀（chromatin immunoprecipitation）与荧光素酶实验（luciferase assays）验证了FOXO4可结合NFAT2启动子并对其进行转录抑制。此外，FOXO4基因敲除可加重川崎病血管炎小鼠模型的炎症血管组织损伤程度。功能实验显示，抑制NFAT2可缓解FOXO4敲除所加剧的体内血管炎症。
结论：本研究结果揭示了FOXO4/NFAT2信号轴作为川崎病进展过程中的关键通路，其与内皮细胞稳态及心血管炎症的发生发展密切相关。