NOX5-induced uncoupling of endothelial NO synthase is a causal mechanism and theragnostic target of an age-related hypertension endotype

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase (Nox) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5—but not NOX1, NOX2, or NOX4—with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human Nox5 in endothelial cells developed—upon aging—severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

Methods
CD144+ microparticles were isolated as described with modifications (see paper). Briefly, Dynabeads G (Invitrogen, Carlsbad, CA) were washed with PBS containing 0.1% BSA and then reconstituted with PBS. Anti-CD144 antibody (Santa Cruz Biotechnology, Dallas, TX), which specifically targets endothelial cells, was mixed with prewashed Dynabeads G for 2 hours and then incubated with plasma samples at 1:200 dilution overnight at 4°C. After precipitation, Dynabeads G were washed with PBS and 1% Tween-20 three times. The purity of CD144+ MPs, determined by FACS analysis, was 70% ± 5.6%. With the use of FITC-conjugated beads as size references, the size of such particles was assessed to be <0.5 μm in diameter. The baseline characteristics of those patients are listed in S1 Table. The values provided for Fig. S1 in the article’s S1 Data file are ng/mL values calculated from optical density (O.D.) measurements.