Abstract 701: Increased NAD(P)H Oxidase Expression and Oxidative Stress in Calcifying Vascular Cells do not Enhance Progression of Aortic Valve and Vascular Calcification
Pathophysiology of degenerative aortic valve (AV) stenosis shares common aspects with atherosclerosis, in which oxidative stress can be important. To investigate whether oxidative stress contributes to progression of AV and vascular calcification, we assessed the occurrence and topography of ROS generation in a rabbit model of AV calcification and studied effects of the antioxidant Tempol. Rabbits were fed 0.5% cholesterol diet + Vitamin D2 10000IU/day for 12 weeks in the absence (HCD, n = 21) or presence of Tempol (0.1mmol/kg/day, n = 9); 0.5% cholesterol only (HC, n = 5); or regular chow (C, n = 22). HC and HCD rabbits showed macrophage infiltration, but only HCD had AV/vascular calcification. Superoxide or H2O2, detected by dihydroethidium (DHE) or dichlorofluorescein fluorescence microtopography plus HPLC analysis (for DHE), were increased not only in macrophage-rich areas, but mainly around calcification foci, in a subpopulation of cells strongly stained for nitrotyrosine (nitroxidative stress marker) and co-expressing NAD(P)H oxidase subunits p22phox, Nox2 and protein disulfide isomerase, a newly-identified oxidase regulator. RT-PCR showed decreased Nox1 and markedly increased Nox4 mRNA. Immunostaining for osteopontin and Cbfa-1 suggest that such are osteoblast-like calcifying vascular cells. Tempol administration decreased ROS production, but was linked to similar (or even greater) increase in histological calcium or valve echogenicity as HCD, vs HC and C rabbits (1.8 ± 0.6 and 1.6 ± 0.5 vs. 0.2 ± 0.4 and 0.4 ± 0.5 echo units, respectively, p ≤ 0,05). Moreover, Tempol decreased expression of osteopontin (calcification inhibitor) in vascular smooth muscle cell calcification model in vitro.The massive vascular expansive remodeling found in HCD rabbits, akin to human elastocalcinosis, was prevented by Tempol. Specimens from AV stenosis patients undergoing surgery (n = 5) or authopsied subjects with AV sclerosis (n = 4) showed analogous increase in ROS generation and protein expression around calcifying foci. Thus, oxidative stress occurs mainly around calcifying foci, due to Nox2 and Nox4 expression in calcifying vascular cells. However, at least Tempol-simile antioxidants do not inhibit and may even increase AV/vascular calcification.