Extra-cellular signal regulated kinase-5 (Erk-5), a transcriptional activator and regulator of endothelial cells (ECs) homeostasis, has been implicated in shear stress-induced endothelial dysfunction (ED), however its role in oxidized low-density lipoprotein (oxLDL)- induced ED during metabolic stress is not known. Herein, regulation and function of Erk-5 in oxLDL-induced EC death, inflammation and dysfunction has been investigated. Primary Human Umbilical Vein Endothelial Cells (pHUVECs) were stimulated with oxLDL. MTT and Trypan blue exclusion assays to assess cell viability, RT-qPCR and Western blotting assays to determine expression of endothelial and inflammatory markers and ED mediators at mRNA and protein levels, respectively were performed. Monocyte adhesion assay was performed to examine monocytes adherence to oxLDL-stimulated pHUVECs. The exposure of oxLDL induced a dose- and time-dependent decrease in pHUVECs viability, which concurred with decreased Erk-5 expression. Further, oxLDL (100 μg/ml) decreased the expression of endothelial markers eNOS and vWF, and increased the expression of ICAM-1, at both mRNA and protein levels. SiRNA-mediated silencing of Erk-5 or its inhibition showed that changes in eNOS, vWF and ICAM-1 expression could be mediated through Erk-5. Furthermore, oxLDL decreased the levels of Erk-5's upstream regulator MEK5 and downstream regulators Mef2c and KLF2, which were similar to their expressions in Erk-5 silenced cells. Fisetin, a phytochemical and bioflavonoid, could reduce the effect of oxLDL in ECs by upregulating the expression of endothelial markers including Erk-5, and downregulating the expression of inflammation markers. These results suggest that Erk-5 could be a critical regulator of oxLDL-induced EC death, inflammation and dysfunction via downregulation of Erk-5/Mef2c-KLF2 signaling pathway, which can be ameliorated by a bioflavonoid, fisetin.

细胞外信号调节激酶5（Erk-5），一种内皮细胞（ECs）稳态的转录激活剂和调节剂，与剪切应力诱导的内皮功能障碍（ED）有关，但是它在氧化型低密度脂蛋白中的作用（oxLDL）诱导的代谢应激中的ED尚不清楚。在本文中，已经研究了Erk-5在oxLDL诱导的EC死亡，炎症和功能障碍中的调节和功能。oxLDL刺激人原代脐静脉内皮细胞（pHUVECs）。分别进行了MTT和台盼蓝排除法以评估细胞活力，RT-qPCR和Western印迹法以分别测定mRNA和蛋白质水平上的内皮和炎性标志物以及ED介体的表达。进行单核细胞粘附测定以检查单核细胞对oxLDL刺激的pHUVEC的粘附。oxLDL的暴露引起pHUVEC生存力的剂量和时间依赖性降低，这与Erk-5表达降低有关。此外，oxLDL（100μg/ ml）在mRNA和蛋白质水平上均降低了内皮标志物eNOS和vWF的表达，并增加了ICAM-1的表达。siRNA介导的Erk-5沉默或它的抑制作用表明eNOS，vWF和ICAM-1表达的变化可以通过Erk-5介导。此外，oxLDL降低了Erk-5上游调节剂MEK5和下游调节剂Mef2c和KLF2的水平，这与它们在Erk-5沉默细胞中的表达相似。Fisetin是一种植物化学物质和生物类黄酮，可通过上调包括Erk-5在内的内皮标志物的表达并下调炎症标志物的表达来降低oxLDL在EC中的作用。