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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:463.)
© 2008 American Heart Association, Inc.

Integrative Physiology/Experimental Medicine

Oxidant Generation Predominates Around Calcifying Foci and Enhances Progression of Aortic Valve Calcification

Marcel Liberman; Estêvão Bassi; Marina Kamla Martinatti; Fábio Cerqueira Lario; João Wosniak, Jr; Pablo M.A. Pomerantzeff; Francisco R.M. Laurindo

From the Vascular Biology Laboratory, Heart Institute(InCor), University of São Paulo School of Medicine, São Paulo, Brazil.

Correspondence to Francisco R.M. Laurindo, MD, PhD, Vascular Biology Laboratory, Heart Institute (INCOR), University of São Paulo School of Medicine, Av. Eneas Carvalho Aguiar, 44, Annex II, 9th floor, CEP 05403-000, São Paulo, Brazil. E-mail expfrancisco{at}incor.usp.br

Abstract

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 +10⁴ IU/d Vit.D₂ for 12 weeks. Superoxide and H₂O₂ 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 H₂O₂ signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol (P0.05). 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.

We investigated whether oxidants contribute to aortic valve (AV) calcification/stenosis progression. In rabbit model and human AV stenosis, superoxide and NADPH oxidase subunits strongly increased around AV-calcifying foci. Nox4mRNA was increased. Lipoic acid decreased, whereas tempol increased calcification progression in vivo and in vitro. Hydrogen peroxide was implicated in calcification progression.

Key Words: calcification • atherosclerosis • antioxidants • valves • free radicals

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