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. Author manuscript; available in PMC: 2009 Sep 22.
Published in final edited form as: J Am Coll Cardiol. 2008 Sep 2;52(10):843–850. doi: 10.1016/j.jacc.2008.05.043

Dysregulation of Antioxidant Mechanisms Contributes to Increased Oxidative Stress in Calcific Aortic Valvular Stenosis in Humans

Jordan D Miller *, Yi Chu *,§, Robert M Brooks *, Wayne E Richenbacher ‡, Ricardo Peña-Silva *,†, Donald D Heistad *,†,§
PMCID: PMC2748760  NIHMSID: NIHMS144731  PMID: 18755348

Abstract

Objectives

The aim of this study was to determine whether oxidative stress is increased in calcified, stenotic aortic valves and to examine mechanisms that might contribute to increased oxidative stress.

Background

Oxidative stress is increased in atherosclerotic lesions and might play an important role in plaque progression and calcification. The role of oxidative stress in valve disease is not clear.

Methods

Superoxide (dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence), hydrogen peroxide (H2O2) (dichlorofluorescein fluorescence), and expression and activity of pro- and anti-oxidant enzymes were measured in normal valves from hearts not suitable for transplantation and stenotic aortic valves that were removed during surgical replacement of the valve.

Results

In normal valves, superoxide levels were relatively low and distributed homogeneously throughout the valve. In stenotic valves, superoxide levels were increased 2-fold near the calcified regions of the valve (p < 0.05); noncalcified regions did not differ significantly from normal valves. Hydrogen peroxide levels were also markedly elevated in calcified regions of stenotic valves. Nicotinamide adenine dinucleotide phosphate oxidase activity was not increased in calcified regions of stenotic valves. Superoxide levels in stenotic valves were significantly reduced by inhibition of nitric oxide synthases (NOS), which suggests uncoupling of the enzyme. Antioxidant mechanisms were reduced in calcified regions of the aortic valve, because total superoxide dismutase (SOD) activity and expression of all 3 SOD isoforms was significantly decreased. Catalase expression also was reduced in pericalcific regions.

Conclusions

This study provides the first evidence that oxidative stress is increased in calcified regions of stenotic aortic valves from humans. Increased oxidative stress is due at least in part to reduction in expression and activity of antioxidant enzymes and perhaps to uncoupled NOS activity. Thus, mechanisms of oxidative stress differ greatly between stenotic aortic valves and atherosclerotic arteries.

Keywords: aortic valve, calcification, oxidative stress, stenosis

Replacement of the aortic valve in patients with symptomatic, calcific aortic valve stenosis is the most common valvular surgical procedure performed in the U.S. (1). Risk factors associated with the development of aortic valve stenosis are similar to those of atherosclerosis and include older age (2,3), male gender, hypertension, smoking, hypercholesterolemia (3), and diabetes (1,3).

Calcified lesions in stenotic aortic valves resemble atherosclerotic lesions and contain calcium (4), oxidized low-density lipoproteins, areas of neovascularization (5,6), high levels of matrix-remodeling enzymes (7–9), and apoptotic cells within the valvular plaque (10–12). A population of cells in calcified aortic valves is indistinguishable from osteoblast and osteoclast cells (13,14), which strongly suggests that the deposition of calcium and progression of aortic valve stenosis is an active process. Signaling cascades related to osteoblast differentiation have been examined in calcified aortic valves (5), but upstream activators of these pathways have been elusive.

Increases in oxidative stress might play a critical role in the initiation and progression of atherosclerotic plaques as well as differentiation of cultured vascular smooth muscle cells into a more osteoblast-like phenotype (15). In atherosclerotic plaques, increased oxidative stress seems to be due primarily to increases in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, with no change or an increase in activity of antioxidant enzyme activities (16).

We obtained preliminary evidence for increased oxidative stress in an experimental mouse model of aortic valve stenosis (17). It is not known whether oxidative stress is increased in calcified aortic valves in humans or whether mechanisms that increase or protect against oxidative stress are altered. The purpose of this study was to test the hypothesis that oxidative stress is increased in stenotic aortic valves in humans and to examine mechanisms that contribute to increases in oxidative stress in calcific aortic valve disease.

Methods

Subject characteristics and methods for tissue acquisition, histological analysis (Von Kossa, Alizarin Red), detection of reactive oxygen species (dihydroethidium and dichlorofluorescein fluorescence, lucigenin chemiluminescence), measurements of gene expression (quantitative real-time reverse-transcriptase polymerase chain reaction), spatial distribution of proteins (fluorescent immunohistochemistry), and enzyme activity (NADPH oxidase and superoxide dismutase [SOD] activity) are detailed in the Online Appendix.

Statistics

Group data are expressed as mean ± SEM. Comparisons between groups were made with unpaired t tests assuming unequal variances (Welch t test). Bonferroni corrections were used to control for multiple comparisons. Significance was defined as α = 0.05.

Results

Oxidative stress in aortic valves

Superoxide

In nonstenotic human aortic valve tissue, dihydroethidine (DHE) fluorescence was relatively low and evenly distributed throughout the valve (Fig. 1A). In contrast, valves from patients with aortic stenosis had intense oxyethidium fluorescence near the calcified regions of the valve that progressively declined as the distance from the calcified regions increased (Figs. 1B to 1D) and was markedly reduced by polyethylene glycol SOD (PEG-SOD) (data not shown). Lucigenin-enhanced chemiluminescence confirmed that superoxide levels were much higher in calcified regions of the stenotic valve than in both normal and noncalcified valve regions (Fig. 1E) (p < 0.05); superoxide levels were similar in noncalcified regions of the stenotic valves and in normal tissue (Fig. 1E) (p = NS).

Figure 1. Superoxide Levels Detected With DHE Fluorescence and Lucigenin-Enhanced Chemiluminescence.

Figure 1

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Superoxide (red fluorescence) in a normal (A) and stenotic (B) aortic valve detected with dihydroethidine (DHE) fluorescence. Superoxide levels were markedly elevated near the calcified (calc) region of the valve and were markedly reduced by the addition of polyethylene glycol superoxide dismutase. Magnified images of noncalcified (non-calc) and calcified regions of a stenotic valve with DHE staining are shown in C and D. (E) Superoxide levels measured with lucigenin-enhanced chemiluminescence in corresponding regions of normal and stenotic valves (n = 14 control valves, n = 20 stenotic valves; *p < 0.05 vs. noncalcified stenotic tissue; #p < 0.05 vs. base and tip of normal valves).

H2O2

In nonstenotic human aortic valve tissue, H2O2 levels were very low and evenly distributed throughout the valve, as estimated by the PEG-catalase inhibitable fluorescence of dichlorofluorescein (Figs. 2A and 2B). In calcified valves, however, H2O2 levels were significantly increased in the calcified and peri-calcific regions of the valve versus regions further away from the calcified mass (Figs. 2C to 2E).

Figure 2. H2O2 Detected With DCF Fluorescence.

Figure 2

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Hydrogen peroxide (H2O2) in a normal (A) and stenotic (C) aortic valve detected with dichlorofluorescein (DCF) fluorescence. Levels of H2O2 were markedly elevated near the calcified regions of the valve, and most of the DCF fluorescence was eliminated by pre-incubation of the slide with polyethylene glycol (PEG)-catalase (CAT) (B and D). (E) The PEG-CAT–inhibitable fraction of DCF fluorescence in normal (base and tip regions) and stenotic (calcified and noncalcified regions) aortic valves (n = 4 normal valves, n = 7 stenotic valves; *p < 0.05 vs. noncalcified stenotic tissue, #p < 0.05 vs. base region of normal valves). Abbreviations as in Figure 1.

Antioxidant enzymes

SOD

Expression (messenger ribonucleic acid [mRNA] levels) of copper-zinc superoxide dimutase (SOD1), manganese SOD (SOD2), or extracellular SOD (SOD3) did not differ significantly between normal tissue and noncalcified regions of stenotic valves. However, in calcified regions of the stenotic valves, mRNA levels for CuZnSOD, MnSOD, and ecSOD were significantly reduced compared with normal tissue and were decreased by 75 ± 8%, 66 ± 13%, and 81 ± 8%, respectively, when compared with noncalcified regions of the same valve (Fig. 3A). Total SOD activity was also significantly reduced by 47 ± 12% (Fig. 3B).

Figure 3. SOD Expression and Activity in Normal and Stenotic Aortic Valves.

Figure 3

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(A) Expression of the 3 superoxide dismutase (SOD) isoforms in normal and noncalcified and calcified regions of stenotic aortic valves (n = 16 normal valves, n = 15 stenotic valves). (B) Regional total SOD activity in stenotic aortic valves (n = 10 normal valves, n = 11 stenotic valves; *p < 0.05 vs. normal valves; #p < 0.05 vs. noncalcified stenotic tissue). mRNA = messenger ribonucleic acid; other abbreviations as in Figure 1.

Catalase

Catalase mRNA levels were reduced by 63 ± 9% in noncalcified stenotic tissue compared with normal tissue (p < 0.05). Further reductions in catalase expression were observed in the calcified regions of stenotic valves, where mRNA levels for catalase were reduced by 89 ± 3% compared with normal valves (p < 0.05) (Fig. 4A).

Figure 4. Catalase Expression in Normal Valves and Noncalcified and Calcified Regions of Stenotic Aortic Valves.

Figure 4

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n = 10 normal valves, n = 11 stenotic valves; *p < 0.05 versus normal valves; #p < 0.05 versus noncalcified stenotic tissue. Abbreviations as in Figures 1 and 3.

Sources of reactive oxygen species

In all aortic valves, Nox1 mRNA expression was below detectable limits. Expression of Nox2 and Nox4 mRNA did not differ significantly between normal valves and noncalcified regions of stenotic valves. However, Nox2 and Nox4 mRNA levels in calcified regions of stenotic valves were significantly decreased compared with normal valve tissue (Fig. 5A).

Figure 5. Pro-Oxidative Enzymes in Normal and Stenotic Aortic Valves.

Figure 5

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(A) Expression of subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in normal (n = 16) and stenotic aortic valves (n = 15 valves, with 15 noncalcified and 15 calcifed regions). (B) The NADPH oxidase activity in normal (n = 8 base and tip regions) and stenotic aortic valves (n = 14 calcified and noncalcified regions). Effects of inhibitors of enzymatic sources of superoxide in noncalcified (C) and calcified (D) regions of stenotic aortic valves measured with lucigenin-enhanced chemiluminescence (n = 5 to 11/group). APO = apocynin; DPI = diphenylidonium; RLU = relative light units; other abbreviations as in Figures 1 and 3.

Addition of exogenous NADPH produced similar increases in superoxide in tissue homogenates of calcified and noncalcified valves (Fig. 5B). Thus, NADPH oxidase activity was similar in calcified and noncalcified regions of the valve. Addition of the flavin-containing enzyme inhibitor diphenyliodonium decreased superoxide levels in calcified valve homogenates, but the more specific NADPH oxidase inhibitor apocynin did not reduce superoxide levels (Fig. 5D).

Inhibition of the flavin-containing nitric oxide synthases (NOS) with nitro-L-arginine methyl ester (L-NAME) significantly reduced superoxide levels in both noncalcified and calcified regions of stenotic valves (Figs. 5C and 5D), which suggests a possible role for uncoupling of NOS. Neither indomethacin nor allopurinol reduced superoxide levels in noncalcified or calcified aortic valve tissue (data not shown).

Characterization of tissue with increased oxidative stress

We did not observe consistent immunostaining for macrophages in regions with elevated oxidative stress (Fig. 6). However, tissue in calcified and peri-calcified regions of stenotic valves consistently expressed high levels of α-smooth muscle actin (Fig. 6), which is consistent with an activated myofibroblast phenotype.

Figure 6. Macrophage and Activated Myofibroblast Markers in a Stenotic Human Valve.

Figure 6

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Antibodies against CD68 and α-SMA were used to detect macrophages and activated myofibroblasts, respectively, in noncalcified and calcified regions. Representative images from n = 5. Inset images are negative control images from adjacent sections.

Expression of genes related to calcification

Tissue in calcified regions of stenotic valves had intense immunofluorescence for the pro-osteoblast transcription factor Msx2 (Figs. 7C and 7D), and modest immunofluorescence of Msx1 (Figs. 7A and 7B). Interestingly, immunofluorescence for the osteogenic transcription factor CBFA1/Runx2 was significantly increased in noncalcified regions of stenotic valves compared with normal tissue. In contrast, staining for CBFA1 was not prominent in calcified regions of stenotic valves (Figs. 7E and 7F), which is consistent with mRNA levels (Fig. 7H). Expression of osteopontin mRNA in valve tissue was similar between normal valves and noncalcified regions of stenotic valves but was significantly increased in the calcified regions of stenotic valves (Fig. 7G).

Figure 7. Immunofluorescent Staining in Noncalcified and Calcified Regions of Stenotic Aortic Valves.

Figure 7

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Immunofluorescent staining for Msx1 (A and B), Msx2 (C and D), and CBFA1 (E and F) in noncalcified and calcified regions of stenotic aortic valves (inset images are negative control images from adjacent sections; representative images from n = 5/stain). Panels G and H show messenger ribonucleic acid (mRNA) levels of osteopontin and CBFA1/Runx2 mRNA in normal valves and in noncalcified and calcified regions of stenotic aortic valves (*p < 0.05 vs. normal tissue, #p < 0.05 vs. noncalcified stenotic tissue, n = 8/group). Abbreviations as in Figure 1.

Discussion

The main findings of this study are: 1) levels of superoxide and H2O2 are markedly increased in calcified regions of stenotic aortic valves from humans; 2) expression and activity of antioxidant enzymes are reduced in calcified regions of stenotic aortic valves; and 3) superoxide production in calcified aortic valves seems to be related at least in part to uncoupling of NOS but not to increases in NADPH oxidase activity. Thus, oxidative stress is increased in stenotic aortic valves, but mechanisms that account for oxidative stress differ greatly from those observed in atherosclerotic arteries (Fig. 8).

Figure 8. Different Mechanisms Generating Oxidative Stress in Vascular Lesions and Calcifed Stenotic Aortic Valves.

Figure 8

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↔ = no change; +/− = increases or decreases have been reported; NADPH = nicotinamide adenine dinucleotide phosphate; NOS = nitric oxide synthase.

Reactive oxygen species are increased in calcified regions of stenotic valves

We found increases in superoxide and H2O2 levels near calcified regions of stenotic aortic valves. This finding is similar to changes in atherosclerotic plaques, where superoxide seems to play an important role in the local inflammatory response (18,19), migration and proliferation of vascular smooth muscle (20,21), and endothelial dysfunction (22–24). Several of these effects might be mediated by increased oxidative stress, reducing the bioavailability of nitric oxide and the promotion of uncoupled NOS activity (25,26).

The close association between calcification and oxidative stress in the present investigation broaches the question: do increases in oxidative stress cause valvular calcification or are increases in oxidative stress an epiphenomenon associated with aortic valve stenosis? This study does not address this question, but many genes related to soft tissue calcification (15), inflammation (19), and matrix remodeling (27) are regulated by oxidative stress. Increased oxidative stress might play a role in the early stages of valve disease by driving myofibroblast activation (28). These “activated” cells are capable of subsequent transdifferentiation to cells with an osteoblast phenotype. Evidence for a role of oxidative stress in transdifferentiation to an osteoblast-like cell differentiation has been provided in vitro, where generation of either superoxide or H2O2 increases the formation of calcified nodules in vascular muscle (15). Thus, it is reasonable to speculate that oxidative stress might be upstream in several signaling cascades related to changes in the aortic valve. Whether increases in oxidative stress play a key role in initiation and progression of aortic valve disease in vivo has not yet been demonstrated.

Decreased antioxidant enzyme function contributes to increases in oxidative stress

We found that expression and activity of CuZnSOD, MnSOD, and ecSOD were decreased in calcified regions of stenotic valves and thus might contribute to increases in superoxide in these regions. These changes are directionally opposite from those observed in atherosclerotic plaques, where expression of CuZnSOD and MnSOD is unchanged and ecSOD expression is increased (16). We speculate that this finding could be explained by: 1) downregulation or repression of SOD expression (29); 2) impairment of mechanisms that upregulate SOD expression (e.g., peroxisome proliferator-activated receptor gamma [30,31]); or 3) paradoxical reductions in pro-inflammatory stimuli that might occur in end-stage aortic valve stenosis (32).

Hydrogen peroxide was increased in stenotic aortic valves despite decreases in SOD activity. In contrast to atherosclerotic plaques, where increased dismutation of superoxide by elevated SOD levels contributes to increases in H2O2 (33), increases in peri-calcific H2O2 in the valve cannot be explained by increases in SOD activity. Instead, reductions in antioxidant enzymes related to H2O2 catabolism might contribute to increases in levels of H2O2. Support for this hypothesis is provided by our observation that expression of catalase was reduced in the calcified regions of the valve. Thus, reduced expression of proteins that catabolize peroxide in calcified valve tissue resembles atherosclerotic lesions, where expression of catalase and glutathione peroxidase-1 are also reduced (34,35).

Determining the role of H2O2 in the pathophysiology of cardiovascular diseases has proven to be difficult. One reason is that altering the expression of enzymes related to the catabolism of H2O2 is likely to alter the expression of other genes (e.g., through epidermal growth factor receptor transactivation). Overexpression of CuZnSOD paradoxically accelerates the development of atherosclerosis in apolipoprotein E−/− mice and can be “rescued” by overexpression of catalase (33). This finding suggests that H2O2 might play an important role in the development of atherosclerosis.

Sources of superoxide in calcified regions of stenotic valves

A surprising finding in the present study was that NADPH oxidase activity was not increased in the calcified regions of stenotic valves, and expression of the catalytic subunits of the oxidase was unchanged or decreased. These findings contrast with observations in atherosclerotic plaques, where upregulation of the catalytic subunits of NADPH oxidase plays a key role in increasing oxidative stress (16,36).

We used several inhibitors to examine potential sources of superoxide production in the calcified regions of stenotic valves. Diphenyliodonium (a nonspecific inhibitor of flavin-containing enzymes, including NADPH oxidase) reduced superoxide levels. We found, however, that specific inhibition of the NOS (which are also flavin-containing enzymes) with L-NAME produced the most consistent reductions in superoxide levels. This finding is surprising, because nitric oxide generated by NOS quenches superoxide, and L-NAME would therefore be expected to increase superoxide levels.

The finding that superoxide is reduced by L-NAME could be explained by “uncoupling” of enzymatic activity of endothelial, inducible, or neuronal NOS, which might occur during a deficiency in 1 or more cofactors required for the generation of nitric oxide (25,26). Depletion of the NOS cofactor tetrahydrobiopterin (BH4) can result in generation of superoxide radicals by NOS (37). Local biopterin levels seem to be a critical determinant of NOS function (38), are modulated by levels of peroxynitrite (which oxidize BH4 to BH3), and can be increased by antioxidants (37).

Expression of genes related to calcification

We found that, similar to previous studies (14), expression of tissue osteopontin was markedly increased in the calcified region of stenotic aortic valves. Although increased plasma osteopontin levels are thought to confer protection against soft tissue calcification, elevated tissue levels of osteopontin might actually augment soft tissue calcification by increasing local inflammation and activity of matrix metalloproteinases (39).

We also found increases in expression of CBFA1/Runx2 in stenotic aortic valves, although these increases were most pronounced in noncalcified regions of the valve. This finding might reflect early stages of myofibroblast transdifferentiation to a more osteoblast-like phenotype, because alterations in Smad tone and increases in CBFA1/Runx2 play a key role in transdifferentiation in myoblastic (40) and vascular smooth muscle cells (41). Interestingly, we observed marked increases in Msx2 immunofluorescence in calcified regions of stenotic valve, which suggest that Msx2 might be promoting further differentiation of activated myofibroblasts to an osteoblast-like phenotype in the calcified regions of the valve, perhaps through canonical Wnt signaling pathways (42).

Study limitations

An inherent limitation of our study of diseased tissue from humans with end-stage disease is that we are unable to examine experimentally whether increases in oxidative stress drive valvular calcification or whether oxidative stress is an epiphenomenon associated with the disease. Nevertheless, these findings provide strong evidence for an association but not necessarily a causal relationship between oxidative stress and aortic stenosis and lay the groundwork for mechanistic studies.

We did not collect clinical information from the patients whose valves were used in these studies. Consequently, we are not able to examine variability within a group by drug treatment or comorbid conditions. Numerous drugs reduce oxidative stress via increases in antioxidant enzyme expression or by reducing NADPH oxidase activity. Consequently, oxidative stress might be considerably higher in animal models of aortic valve stenosis or in stenotic valves from patients who are not receiving optimal pharmacotherapy. In addition, there are compelling data linking acceleration of lesion formation and calcification by diabetes to activation of NADPH oxidase (39,43). Thus, in some patients (perhaps especially diabetic patients), we cannot exclude the possibility that increases in NADPH oxidase activity contribute to oxidative stress in the aortic valve.

An unusual aspect of our tissue acquisition process is that the time from tissue explantation to placing the tissue in ice-cold buffer is <15 min. Additionally, our normal valve group was not composed of tissues gathered at autopsy but instead were “transplant quality” tissues acquired from organ procurement organizations (see Online Appendix). Thus, changes in oxidative stress or enzymatic activity probably cannot be attributed to differences in time to post-explant tissue preservation.

Clinical implications

Treatments that effectively slow the progression of aortic valve stenosis have proven elusive (32,44,45). Retrospective studies suggest that “statins” (46) and angiotensin-converting enyme inhibitors (47) might slow the progression of aortic valve stenosis, although prospective studies have yielded conflicting results (32,48). Both statins and angiotensin-converting enyme inhibitors also reduce oxidative stress (49), and it is possible that interventions specifically targeting oxidative stress during earlier stages of the disease (e.g., when patients have aortic sclerosis) might slow the progression of aortic valve stenosis.

Conclusions

Oxidative stress is increased in calcified regions of stenotic aortic valves. In striking contrast to atherosclerotic lesions, increased oxidative stress seems to be due in part to reductions in antioxidant enzyme expression and activity. Furthermore, NOS uncoupling might play an important role in the generation of superoxide in calcified aortic valves. Additional studies are needed to experimentally test whether oxidative stress is a viable therapeutic target to slow the progression of aortic valve stenosis.

Added note in proofs

After submission of this paper, another article was published by Liberman et al. (50) that also indicated that oxidative stress occurs in stenotic aortic valves.

Supplementary Material

supplementary data

Acknowledgments

Studies were supported by National Institutes of Health grants HL-086160, HL-62984, and NS-24621; funds provided by the Veterans Affairs Medical Service; and a Carver Research Program of Excellence.

The authors thank Kristine M. Serrano and Donald D. Lund, PhD, for assistance with data acquisition and analysis; Kathleen Walters for staining tissue sections; Jeffrey E. Everett, MD, for assisting in the procurement of diseased aortic valve tissue; Teresa Ruggle for assistance with figures and illustrations; and Frank M. Faraci, PhD, Francis J. Miller, MD, and Robert M. Weiss, MD, for insightful discussions regarding the data and study design.

Abbreviations and Acronyms

DHE

dihydroethidine

L-NAME

nitro-L-arginine methyl ester

mRNA

messenger ribonucleic acid

NADPH

nicotinamide adenine dinucleotide phosphate

NOS

nitric oxide synthase

SOD

superoxide dismutase

Footnotes

For supplementary methods, please see the online version of this article.

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