Laminar shear stress inhibits inflammation by activating autophagy in human aortic endothelial cells through HMGB1 nuclear translocation. Laminar shear stress inhibits inflammation by activating autophagy in human aortic endothelial cells through HMGB1 nuclear translocation

Laminar shear stress (LSS) plays a variety of biological roles, one of which is regulation of gene expression in vascular endothelial cells. Prevention and treatment of atherosclerosis by targeting the inflammatory response in vascular endothelial cells has attracted much attention in recent years. In this study, we found that LSS could specifically inhibit the increased expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and matrix metallopeptidase-9 (MMP-9) caused by tumor necrosis factor-α (TNF-α) stimulation in an autophagy-dependent pathway in human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs). Whole-transcriptome sequencing analysis revealed that erythropoietin-producing hepatocyte receptor B2 (EPHB2) was a key gene in response to LSS. Moreover, co-immunoprecipitation (co-IP) assay indicated that LSS could activate autophagy flux by enhancing the EPHB2-mediated nuclear translocation of high mobility group box-1 (HMGB1), which interacts with Beclin-1 (BECN1) and finally leads to autophagy in HAECs. We also found that overexpression of EPHB2 can activate autophagy and exert the same anti-inflammatory effect as LSS. Simultaneously, we identified a novel LSS-sensitive long non-coding RNA (lncRNA), LOC10798635, and constructed an LSS-related LOC107986345/miR-128-3p/EPHB2 regulatory axis. Further research revealed the inhibition of endothelial inflammatory by LSS depends on autophagy activation resulting from the nuclear translocation of HMGB1 via the LOC107986345/miR-128-3p/EPHB2 axis. In conclusion, our study demonstrates for the first time that LSS regulates the expression of EPHB2 in HAECs, and the LOC107986345/miR-128-3p/EPHB2 axis plays a vital role in atherosclerosis development. Overall design: This study included 3 samples, each with 3 replicates, where HAEC_0dy_0h was the control group.

层流剪切应力（Laminar shear stress, LSS）具有多种生物学功能，其中之一是调控血管内皮细胞的基因表达。近年来，以靶向血管内皮细胞炎症反应防治动脉粥样硬化的研究方向受到广泛关注。本研究发现，在人主动脉内皮细胞（human aortic endothelial cells, HAECs）与人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）中，层流剪切应力可通过自噬依赖通路，特异性抑制肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）刺激所诱导的细胞间黏附分子-1（intercellular adhesion molecule-1, ICAM-1）、血管细胞黏附分子-1（vascular cell adhesion molecule-1, VCAM-1）、环氧合酶-2（cyclooxygenase-2, COX-2）及基质金属蛋白酶-9（matrix metallopeptidase-9, MMP-9）的表达升高。全转录组测序分析显示，促红细胞生成素产生肝细胞受体B2（erythropoietin-producing hepatocyte receptor B2, EPHB2）是响应层流剪切应力的关键基因。此外，免疫共沉淀（co-immunoprecipitation, co-IP）实验表明，层流剪切应力可通过增强EPHB2介导的高迁移率族蛋白B1（high mobility group box-1, HMGB1）核转位激活自噬流；HMGB1可与Beclin-1（BECN1）相互作用，最终使人主动脉内皮细胞发生自噬。本研究还发现，过表达EPHB2可激活自噬并发挥与层流剪切应力一致的抗炎效应。同时，本研究鉴定出一种新型层流剪切应力敏感性长链非编码RNA（long non-coding RNA, lncRNA）LOC10798635，并构建了LSS相关的LOC107986345/miR-128-3p/EPHB2调控轴。进一步研究揭示，层流剪切应力对内皮炎症的抑制作用依赖于通过LOC107986345/miR-128-3p/EPHB2轴介导HMGB1核转位所激活的自噬过程。综上，本研究首次证实，层流剪切应力可调控人主动脉内皮细胞中EPHB2的表达，且LOC107986345/miR-128-3p/EPHB2调控轴在动脉粥样硬化发生发展中发挥关键作用。整体实验设计：本研究共纳入3组样本，每组设置3次生物学重复，其中HAEC_0dy_0h为对照组。