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.

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.

The dataset contains repeated values for Fig. S1A because NOX1 concentrations were very close to the limit of detection, i.e., close to the O.D. of the buffer control for which we obtained a value of 0.09. The repeated values in the 0.07-0.16 range were interpreted as essentially negative results, and from these data we concluded that NOX1 was not detectable using this assay for most subjects. Only one normotensive and two hypertensive patients with microalbuminuria yielded O.D. readings above the 0.07-0.16 range.