Aging vasculature exhibits a self‑reinforcing cycle where endothelial dysfunction promotes arterial stiffness, which then worsens endothelial function via impaired mechanotransduction [1]. While oxidative stress from NADPH oxidases and mitochondrial dysfunction is recognized as a contributor to NO degradation and senescence [1], the specific role of mitochondrially derived reactive oxygen species (mtROS) as a direct mechanosensor that triggers eNOS uncoupling remains unexplored. We hypothesize that cyclic pulsatile stretch in aged arteries increases mitochondrial superoxide production in endothelial cells, leading to S‑glutathionylation of eNOS at Cys443. This post‑translational modification shifts eNOS from a NO‑producing to a superoxide‑producing enzyme, exacerbating NO scarcity, increasing peroxynitrite formation, and accelerating senescence‑associated secretory phenotype (SASP) expression. Consequently, heightened arterial stiffness further augments mechanical stretch, creating a feed‑forward loop that drives vascular aging independently of NADPH oxidase activity.

Testable predictions:

1. In old mice, endothelial‑specific overexpression of glutaredoxin‑1 (which reverses S‑glutathionylation) will reduce eNOS uncoupling, lower PWV, and improve endothelium‑dependent vasodilation without altering NADPH oxidase levels.
2. Pharmacological scavenging of mtROS with mitoTEMPO will decrease eNOS S‑glutathionylation, increase NO bioavailability, and attenuate the rise in PWV induced by angiotensin‑II–mediated hypertension, whereas a NADPH oxidase inhibitor (apocynin) will have a lesser effect on PWV under the same conditions.
3. Human endothelial cells cultured on tunable substrates mimicking young (≈5 kPa) vs. aged (≈15 kPa) stiffness will show increased mitochondrial superoxide, eNOS S‑glutathionylation, and SASP IL‑6 secretion only on the stiff substrate; mitoTEMPO pretreatment will block these changes.

Falsifiable outcomes: If glutaredoxin‑1 overexpression or mitoTEMPO fails to improve PWV or endothelial function in aged animals, or if NADPH oxidase inhibition yields comparable or greater benefits than mtROS scavenging, the hypothesis would be refuted. Conversely, confirming that targeting mitochondrial ROS specifically disrupts the mechanotransductive link between stretch and eNOS uncoupling would provide a novel mechanistic node for combination therapies (e.g., senolytics plus mito‑targeted antioxidants) aimed at breaking the dysfunction‑stiffness cycle [8].

This framework extends the current model by positioning mtROS‑driven eNOS uncoupling as a mechanosensitive amplifier of vascular aging, offering a precise, falsifiable target for intervention beyond conventional antioxidant or senolytic approaches [5,6].