Novel Treatment of Hypertension by Specifically Targeting E2F for Restoration of Endothelial Dihydrofolate Reductase and eNOS Function Under Oxidative Stress

Abstract

We have shown that hydrogen peroxide (H2O2) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H2O2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression, and effects on blood pressure regulation of targeting these novel mechanisms. H2O2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F‐binding sites, but not those of Sp1, abolished H2O2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H2O2 on DHFR promoter activity. H2O2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H2O2. E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)‐infused mice, adenovirus‐mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H2O2 transcriptionally via an E2F‐dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.Support or Funding InformationThis study was supported by National Institute of Health National Heart, Lung and Blood Institute (NHLBI) Grants HL077440 (HC), HL088975 (HC), HL108701 (HC, DGH), HL119968 (HC), an American Heart Association Established Investigator Award (EIA) 12EIA8990025 (HC), and an AHA Postdoctoral Fellowship Award 14POST20380966 (QL), an AHA Postdoctoral Fellowship Award 14POST20380995 (YXZ) and a Guangdong Elite Program for Visiting Graduate Student JY201222 (HL). We thank Dr. Gustavo Leone (The Ohio State University, USA) for kindly providing uspBabe‐Hygro‐E2F1 (pBH‐E2F1), pBH‐E2F2, pBHE2F3a, pBH‐E2F7 and pBH‐E2F8 plasmids. We thank Dr. Xiangming Ding (UCLA, USA) and Dr. Yibin Wang (UCLA, USA) for generous gifts of pGL3 basic luciferase reporter plasmid and pRLrenilla luciferase reporter plasmid, respectively. We also thank Dr. Alan Pollack (University of Miami, USA) for the generous gift of adenoviral vector constructs incorporating full‐length E2F1 or firefly luciferase.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.