Zhang Danfeng, Liu Lingjuan, Yuan Yuxing, Lv Tiewei, Huang Xupei, Tian Jie
Department of Cardiology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, Chongqing, China.
Chongqing Key Laboratory of Pediatrics, Chongqing, China.
Front Pediatr. 2021 Feb 22;9:618267. doi: 10.3389/fped.2021.618267. eCollection 2021.
E-selectin is a cell adhesion molecule of the vascular endothelium that mediates leukocyte rolling in the early inflammatory responses in many diseases including Kawasaki disease (KD). Previous studies have demonstrated that the expression levels of E-selectin was significantly increased in the sera of KD patients and in endothelial cells of KD patient's autopsy. In this study, we aimed to examine E-selectin levels in endothelial cells treated with sera from KD patients and explore the underlying mechanisms. Human coronary artery endothelial cells (HCAECs) were randomly incubated with sera from either healthy children [healthy control (HC group)] or pediatric KD patients [assigned as KD with coronary artery lesion (KD-CAL+ group) and KD without coronary artery lesion (KD-CAL- group)]. E-selectin levels were determined by RT-qPCR, Western blotting, and immunofluorescence. Cell adhesion assay was performed to quantify the role of E-selectin in intercellular adhesion. High-throughput cell RNA sequencing followed by functional validation was performed to explore the underlying mechanism. E-selectin levels were significantly increased in KD-CAL+ group vs. HC group and KD-CAL- group. Compared with the KD-CAL- group, endothelia-monocyte adhesion was increased in the KD-CAL+ group, while E-selectin-specific siRNA could significantly rescue it. High-throughput cell RNA sequencing analysis also found a significant difference in oxidative phosphorylation (OXPHOS) levels between KD-CAL+ group and KD-CAL- group. Functional validation results further confirmed that the OXPHOS was upregulated in the KD-CAL+ group and KD-CAL- group compared to that in the HC group, while the KD-CAL+ group exhibited a higher OXPHOS than the KD-CAL- group. We also found that the E-selectin levels and endothelia-monocyte adhesion were significantly decreased by OXPHOS inhibitor oligomycin in the KD-CAL+ group and KD-CAL- group, respectively. Sera from KD patients stimulate OXPHOS levels and enhance E-selectin expression in HCAECs, which may contribute to the development of CAL in KD patients.
E选择素是血管内皮细胞的一种细胞粘附分子，在包括川崎病（KD）在内的许多疾病的早期炎症反应中介导白细胞滚动。先前的研究表明，KD患者血清以及KD患者尸检的内皮细胞中，E选择素的表达水平显著升高。在本研究中，我们旨在检测用KD患者血清处理的内皮细胞中的E选择素水平，并探索其潜在机制。人冠状动脉内皮细胞（HCAECs）被随机与健康儿童血清[健康对照（HC组）]或儿科KD患者血清[分为有冠状动脉病变的KD（KD-CAL+组）和无冠状动脉病变的KD（KD-CAL-组）]一起孵育。通过逆转录定量聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法和免疫荧光法测定E选择素水平。进行细胞粘附试验以量化E选择素在细胞间粘附中的作用。进行高通量细胞RNA测序并随后进行功能验证以探索潜在机制。与HC组和KD-CAL-组相比，KD-CAL+组的E选择素水平显著升高。与KD-CAL-组相比，KD-CAL+组的内皮细胞-单核细胞粘附增加，而E选择素特异性小干扰RNA（siRNA）可显著挽救这种情况。高通量细胞RNA测序分析还发现KD-CAL+组和KD-CAL-组之间氧化磷酸化（OXPHOS）水平存在显著差异。功能验证结果进一步证实，与HC组相比，KD-CAL+组和KD-CAL-组的OXPHOS上调，而KD-CAL+组的OXPHOS高于KD-CAL-组。我们还发现，OXPHOS抑制剂寡霉素分别使KD-CAL+组和KD-CAL-组的E选择素水平和内皮细胞-单核细胞粘附显著降低。KD患者的血清刺激OXPHOS水平并增强HCAECs中E选择素的表达，这可能有助于KD患者冠状动脉病变（CAL）的发展。
