Reactivation of eNOS in the endothelium rescues eNOS KO mice from endothelial dysfunction and hypertension

Abstract

Production of nitric oxide (NO) by endothelial NO synthase (eNOS; NOS3) plays a key role in regulation of vascular tone and blood pressure. The aim of this study was to establish “loss” and “gain” of function models for eNOS by using tissue specific Cre‐induced gene inactivation or reactivation. To accomplish this, two independent genotypes were created: 1) a conventional floxed eNOS (eNOSflox/flox) against exon 2 or 2) a Cre‐inducible eNOS knock‐in (KI) gene construct (eNOSinv/inv) allowing targeted removal or reactivation of eNOS expression. Both the eNOSflox/flox and eNOSinv/inv were crossed with endothelial cell (EC)‐specific tamoxifen (TAM)‐inducible Cre mice to create EC‐specific knock‐out (EC eNOS KO) and EC eNOS KI mice. We found that Cre‐specific DNA recombination occurred specifically in endothelium of both EC eNOS KO and EC eNOS KI, as assessed by quantitative polymerase chain reaction (qPCR). TAM treatment fully abolished mRNA and protein expression of eNOS in the aorta of EC eNOS KO (eNOSflox/flox Crepos) mice, but not in Cre negative or untreated mice, as determined by retro transcription qPCR and western blot analysis. Functionally, the EC eNOS KO demonstrated inhibition of Ach‐induced vasodilation of aortic rings ex vivo, as well as flow‐mediated dilation (FMD) of the arteria iliaca in vivo (assessed by ultrasound). Moreover, absence of eNOS in the endothelium of EC eNOS KO was correlated with spontaneous hypertension, assessed by Millar catheter (MAP was 80.37 ± 2.135 mmHg in EC eNOS KO against 67.88 ± 2.723 mmHg in control mice). Next, we tested blood pressure of eNOSinv/inv which fully lacked eNOS expression in all tissues and was hypertensive. In EC eNOS KI (eNOSinv/inv Crepos treated with TAM) the expression of eNOS was completely restored in endothelium, leading to a significant decrease of the blood pressure as compared to eNOSinv/inv Creneg mice. In conclusion, we report the first inducible “loss” and “gain” of function models of eNOS in the vascular endothelium demonstrating that reactivation of eNOS rescues from endothelial dysfunction and hypertension. These mice models can further be applied to non endothelial tissues to further investigate the role of eNOS in different tissues in modulating blood pressure and systemic hemodynamics.Support or Funding InformationThis work was supported by the German Research Council (DFG CO 1305/2‐1 to M.M.C.‐K.; F.L. is a scholar of the IRTG1902 to M.M.C.‐K. and B.E.I. and by a research grant of the Forschungskommission to M.M.C.‐K., Medical Faculty, Heinrich Heine University of Düsseldorf.