Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2)

Article Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2) Haishan Xu 1,#, Jinhong Duan 1,#, Jun Tao 2, Wen Wang 3, Yunqing Wu 1,^, Shunling Dai 1,*, and Jun Ren 4,5, 1 Faculty of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005 China 2 Department of Cardiovascular Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510000 China 3 Department of Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China 4 Department of Cardiology, Zhongshan Hospital Fudan University, Shanghai 200032, China 5 National Clinical Research Center for Interventional Medicine, Shanghai 200032, China # These two authors contributed equally to this work ^ Deceased * Correspondence: daishunling@aliyun.com (Shunling Dai); corresponding author:jren_aldh2@outlook.com (Jun Ren)     Abstract: It is perceived that oxidized low density lipoprotein (oxLDL) perturbs endothelial function and fosters endothelin-1 (ET-1) secretion although the underlying mechanism remains elusive. This study was designed to decipher potential mechanisms underscoring oxLDL-evoked regulation of ET-1 and signaling pathways involved in endothelial cells. ET-1 mRNA expression, secretion and promoter function were determined using RT-PCR, enzyme immunometric and luciferase assays, respectively. GO and GSEA bioinformatics analyses depicted differentially expressed genes (DEGs) mainly associated with cell proliferation, cell division, cellular structure, energy supply, and apoptosis in oxLDL-challenged endothelial cells. Incubation of oxLDL overtly increased ROS production, apoptosis, mRNA level, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), the effects were mitigated by N-Acetyl Cysteine (NAC). Moreover, oxLDL challenge evoked phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) in HUVECs, the effect was reversed by NAC and MEK inhibitor PD98059. NAC and PD98059 nullified oxLDL- induced rises in mRNA expression, secretion and promoter activity of ET-1. Truncation of 5’-flanking sequence of ET-1 (–566 bpLuc to –250 bpLuc) displayed elevated luciferase activity with 24-h oxLDL incubation. Fusion plasmid from –233 and –185 bp Luc drastically dampened luciferase activity in basal and oxLDL-challenged HUVECs. Transfection of reporter construct –250 bp Luc with a 2 bp mutation at AP-1 locus, removed basal and oxLDL- evoked rises in ET-1 promoter activity. Collectively, our findings support that oxLDL evoked activation of ERK1/2 signaling likely through ROS production, en route to upregulation of endothelial transcriptional factor AP-1, resulting in expression and secretion of ET-1.