An endogenous aryl hydrocarbon receptor ligand dysregulates endothelial functions, transcriptome, and phosphoproteome

We have reported that an endogenous aryl hydrocarbon receptor (AhR) ligand, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), inhibits functions of human umbilical vein endothelial cells (HUVECs) and induces preeclampsia (PE)-like symptoms in rats. Herein, we tested the hypothesis that ITE impairs endothelial functions via disturbing transcriptome and phosphoproteome in HUVECs. We measured AhR activity in human maternal and umbilical vein sera from PE and normotensive (NT) pregnancies. The serum-induced changes in CYP1A1/B1 mRNA (indexes of AhR activation) in HUVECs were quantified using quantitative reverse transcription polymerase chain reaction (RT-qPCR). ITE's effects on endothelial proliferation and monolayer integrity in female and male HUVECs were determined. We profiled ITE-induced changes in transcriptome and phosphoproteome in HUVECs using RNA-seq and bottom-up phosphoproteomics, respectively. After 12 h of treatment, umbilical vein sera from PE increased CYP1A1 mRNA (1.7-fold of NT) in HUVECs, which was blocked by CH223191, an AhR antagonist. ITE dose-dependently inhibited endothelial proliferation (76%-87% of control) and time-dependently reduced endothelial integrity with a maximum inhibition (∼10%) at 40 h. ITE induced 140 and 80 differentially expressed genes in female and male HUVECs, respectively. ITE altered phosphorylation of 92 and 105 proteins at 4 and 24 h, respectively, in HUVECs. These ITE-dysregulated genes and phosphoproteins were enriched in biological functions and pathways that are relevant to heart, liver, and kidney diseases, vascular functions, and inflammatory responses. Thus, endogenous AhR ligands may impair endothelial functions by disturbing transcriptome and phosphoproteome. These AhR ligand-dysregulated genes and phosphoproteins may be therapeutic and cell sex-specific targets for PE-induced endothelial dysfunction.<b>NEW & NOTEWORTHY</b> Preeclampsia elevates AhR agonistic activities in fetal circulation and alters immune cell gene signatures of human umbilical vein endothelial cells (HUVECs). An endogenous AhR ligand (ITE) decreases cell proliferation and monolayer integrity in HUVECs in vitro. ITE dysregulates transcriptome in HUVECs in a fetal sex-specific manner. ITE also disrupts phosphoproteome in HUVECs. These ITE-dysregulated genes and phosphoproteins are highly relevant to diseases of the heart, vascular function, and inflammatory responses.