Novel Treatment of Hypertension by Specifically Targeting E2F for Restoration of Endothelial Dihydrofolate Reductase and eNOS Function Under Oxidative Stress
We have shown that hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H 2 O 2 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 (specificity protein 1), abolished H 2 O 2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H 2 O 2 on DHFR promoter activity. H 2 O 2 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 H 2 O 2 . 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 H 2 O 2 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.