Abstract FR416: USP8 regulates αEnNaC to induce endothelial stiffening

Excessive activation of endothelial cell (EC) mineralocorticoid receptor (MR) (ECMR) and its downstream epithelial sodium channels in ECs (EnNaC) promotes oxidative stress, inflammation, leading to vascular remodeling and stiffening. Our previous studies have indicated that elevated circulating aldosterone in diet-induced obesity enhances ECMR/αEnNaC signaling, contributing to endothelial stiffness by reducing endothelial nitric oxide synthase. However, the specific role of ECMR/αEnNaC signaling and its underlying molecular mechanisms remain unclear. We hypothesized that EC ubiquitin-specific protease 8 (ECUSP8) mediates ECMR activation-induced increases in endosomal αEnNaC, its trafficking, plasma membrane αEnNaC accumulation and endothelial stiffening. To investigate this, in vitro mouse ECs were treated with 30 nmol aldosterone to active MRs. Six-week-old ECMR -/- and wild-type littermate male mice were fed either standard chow or a Western diet (WD) high in fat (46%) and fructose (17.5%) for 16 weeks. In vitro aldosterone treatment and in vivo WD feeding increased ECMR and ECUSP8 expression, leading to endothelial stiffening, as determined by atomic force microscopy. While ECUSP8 mediated aldosterone-induced increases in colocalization of αEnNaC and Rab7, as well as the accumulation αEnNaC at the membrane, it also mediated aldosterone induced inhibition of αEnNaC localization in lysosomes and the ratio of lysosomal αEnNaC to late endosomal αEnNaC distribution. There abnormalities were blunted by spironolactone treatment. These data indicated that ECUSP8 mediates ECMR activation-induced inhibition of lysosomal αEnNaC localization and degradation, leading to increases in endosomal αEnNaC and its accumulation at the plasma membrane and subsequent endothelial stiffening.