: Aims: During calcific aortic valve stenosis (CAVS) progression, oxidative stress and endothelial dysfunction mark the initial pathogenic steps with a parallel dysregulation of the antioxidant systems. Here, we tested whether oxidation-induced protein S-glutathionylation (P-SSG) accounts for a phenotypic switch in human aortic valvular tissue, eventually leading to calcium deposition. Next, we tested whether countering this reactive oxygen species (ROS) surge would prevent these perturbations. Results: We employed state-of-the-art technologies, such as electron paramagnetic resonance (EPR), liquid chromatography-tandem mass spectrometry, imaging flow-cytometry, and live-cell imaging on human excised aortic valves and primary valve endothelial cells (VECs). We observed that a net rise in EPR-detected ROS emission marked the transition from fibrotic to calcific in human CAVS specimens, coupled to a progressive increment in P-SSG deposition. In human VECs (hVECs), treatment with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid triggered highly oxidizing conditions prompting P-SSG accumulation, damaging mitochondria, and inducing endothelial nitric oxide synthase uncoupling. All the events conjured up in morphing these cells from their native endothelial phenotype into a damaged calcification-inducing one. As proof of principle, the use of the antioxidant N-acetyl-L-cysteine prevented these alterations. Innovation: Borne as a compensatory system to face excessive oxidative burden, with time, P-SSG contributes to the morphing of hVECs from their innate phenotype into a damaged one, paving the way to calcium deposition. Conclusion: Our data suggest that, in the human aortic valve, unremitted ROS emission along with a P-SSG build-up occurs and accounts, at least in part, for the morphological/functional changes leading to CAVS. Antioxid. Redox Signal. 37, 1051-1071.
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