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 ...

Vascular calcification decreases compliance and increases morbidity. Mechanisms of this process are unclear. The role of oxidative stress and effects of antioxidants have been poorly explored. We investigated effects of the antioxidants lipoic acid (LA) and tempol in a model of atherosclerosis associated with elastocalcinosis. Male New Zealand white rabbits (2.5-3.0 kg) were fed regular chow (controls) or a 0.5% cholesterol (chol) diet+104 IU/day vitamin D2 (vitD) for 12 weeks, and assigned to treatment with water (vehicle, n=20), 0.12 mmol·kg-1·day-1 LA (n=11) or 0.1 mmol·kg-1·day-1 tempol (n=15). Chol+vitD-fed rabbits developed atherosclerotic plaques associated with expansive remodeling, elastic fiber disruption, medial calcification, and increased aortic stiffness. Histologically, LA prevented medial calcification by ∼60% and aortic stiffening by ∼60%. LA also preserved responsiveness to constrictor agents, while intima-media thickening was increased. In contrast to LA, tempol was associated with increased plaque collagen content, medial calcification and aortic stiffness, and produced differential changes in vasoactive responses in the chol+vitD group. Both LA and tempol prevented superoxide signals with chol+vitD. However, only LA prevented hydrogen peroxide-related signals with chol+vitD, while tempol enhanced them. These data suggest that LA, opposite to tempol, can minimize calcification and compliance loss in elastocalcionosis by inhibition of hydrogen peroxide generation.