Objective-We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis. Methods and Results-We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet ϩ10 4 IU/d Vit.D 2 for 12 weeks. Superoxide and H 2 O 2 microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H 2 O 2 signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol (PՅ0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA. Conclusions-Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation. Key Words: calcification Ⅲ atherosclerosis Ⅲ antioxidants Ⅲ valves Ⅲ free radicals D egenerative aortic valve (AV) stenosis, the third most prevalent cardiovascular disease in the elderly, 1 shares common risk factors and pathophysiological features with atherosclerosis. [2][3][4][5][6] Although the role of oxidative stress in atherosclerosis is well explored, 7,8 it is unclear whether redox processes contribute to progression of AV calcification. 2,3,9 -11,15,16 Scarce observations provide indirect support for this hypothesis. 10 In vitro studies showed that exogenous superoxide, hydrogen peroxide, or other oxidants increase the number and activity of calcifying vascular cells (CVCs), 11 referred to as a specific subpopulation of cells, derived from (de)differentiation of vascular smooth muscle cells, 12 pericytes, or mesenchymal cells 13 that can produce hydroxyapathite in the vascular wall. 14 In addition, reactive oxygen species (ROS) mediate increase in BMP2 expression and signaling, favoring osteogenesis. 2 On the other hand, calcium resorption by osteoclasts is dependent on ROS derived from its own NAD(P)H oxidase, 15 whereas nitric oxide induces osteoclast detachment and inhibits calcium resorption. 16 Recent data from an experimental mouse model of aortic stenosis suggested locally increased superoxide generation. 3 Observational clinical studies with statins indicated possible decrease in calcification progression in hypercholesterolemic patients, 4 but ...