Published online before print December 27, 2007, doi: 10.1161/ATVBAHA.107.156745
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Oxidant Generation Predominates Around Calcifying Foci and Enhances Progression of Aortic Valve Calcification
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
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Abstract |
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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 (P
0.05). 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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Introduction |
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Degenerative aortic valve (AV) stenosis, the third most prevalent cardiovascular disease in the elderly,1 shares common risk factors and pathophysiological features with atherosclerosis.2–6 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 cells13 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 a prospective clinical trial in normocholesterolemic subjects showed lack of effect.5 In the present study, we investigated the occurrence and microtopography of ROS generation in an experimental rabbit model of AV calcification and sclerosis and in specimens from human with AV sclerosis or stenosis. In addition, the role of redox processes in the progression of AV calcification was tested by assessing the effects of 2 antioxidants, tempol and lipoic acid (LA), both in a rabbit model17 and in an in vitro model of VSMC calcification.18
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Methods |
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An extended Methods section is available as supplemental material (available online at http://atvb.ahajournals.org).
Human Aortic Valves
AV from patients with stenosis (n=5) or individuals with sclerosis (n=4), collected respectively at surgery or autopsy (<6hs postmortem), were analyzed for ROS microtopography, histology, and immunohistochemistry for NAD(P)H oxidase subunits and compared with young autopsied controls (n=6).
Rabbit Model
The rabbit AV calcification model modified from Drolet et al17 involved administration for 3 months of 0.5% cholesterol-enriched diet+vitamin D2 104 IU/d (HC+vitD rabbits, n=34). Some HC+vitD rabbits concomitantly received tempol 100 µmol/kg/d (n=15) or lipoic acid (LA) 120 µmol/kg/d (n=11) in drinking water. Controls received 0.5% cholesterol alone (n=9) or normal chow (n=32). Plasma cholesterol, calcium, phosphorus, and creatinine levels were analyzed.
After the 12th week, AV were collected and processed for morphological analysis and collagen histomorphometry, vonKossa staining for calcium detection, immunohistochemistry for NAD(P)H oxidase subunits, protein disulfide isomerase (PDI), macrophage marker RAM11, nitrotyrosine, Ki67 and osteoblast markers, immunofluorescence for NAD(P)H oxidase subunits, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and TRAP histochemistry for osteoclasts. Quantitative morphometric analysis was performed by Leica Quantimet software.
In Situ ROS Generation
In situ microfluorotopography of dihydroethidium (DHE) oxidation products was performed as described,8 with 3 µmol/L final DHE concentration. Slides were analyzed by confocal microscopy (Zeiss LSM510) with laser excitation at 488 nm and emission at 610 nm, detected by 560 nm longpass filter. We also performed a novel analogous in situ detection of hydrogen peroxide by 2',3'-dichlorofluorescein diacetate (DCF-DA), using a similar protocol, with final DCF-DA concentration of 3 µmol/L. Controls, performed by incubating slides for 30 minutes with Peg-SOD (500U/mL) or Peg-catalase (400U/mL), indicated preferential detection of superoxide with DHE and hydrogen peroxide with DCF-DA. Quantitative analysis of fluorescent images was performed with Image J (NIH) software.
Real-Time Polymerase Chain Reaction
Quantitative expression of Nox1 or Nox4 mRNA was performed in aortic tissue as detailed in supplement, with primers designed according to rabbit sequences.
Echocardiography
Valve area, left ventricular mass and dimensions, peak and mean aortic valve flow velocity were assessed by ultrasound/doppler as detailed elsewhere3 and in supplement, with 12-MHZ phased-array probe and Philips Sonos 5500 system. Exams were performed at beginning of protocol and after 3 months of intervention.
Calcification Analysis
A semi-quantitative index (range 0 to 3) based on opacity and valve motility was used for estimating AV echogenicity, an index of valve thickening and calcification, as follows: 0-absent, 1-mild; 2-moderate, with preserved motility/opening; 3-severe, with decreased motility/opening. Data were analyzed on a blinded fashion. Histomorphometry of calcium conglomerates was performed with Leica Quantimet software in von Kossa and hematoxylin-eosin stained sections. Ex vivo 64-row computed tomography of aortic specimens was performed as detailed in supplement.
In Vitro VSMC Calcification Model
The model was described by Shioi et al.18 Rabbit aortic vascular smooth muscle cells cultured in DMEM with FBS10%, at 70% confluence and 4th passage, were supplemented during 14 days with 6 mmol/L calcium chloride, alone or in combination with 10 mmol/L β-glycerophosphate, or both in combination with 0.1 µmol/L vitamin-D3. The latter combination was supplied in the absence or presence of 100 µmol/L tempol or 100 µmol/L LA for 14 days, changing medium every 3 days. Exogenous hydrogen peroxide (50 µmol/L), catalase (500U/mL), or the NO donor NOC-18 (30 µmol/L) were added in specific experiments, changing medium every 3 days. We used either primary cells or cells from an immortalized line.19
CVC layer calcium content was assessed by colorimetric assay, as detailed in supplement. Superoxide production was assessed through high-performance liquid chromatography (HPLC) analysis of DHE oxidation products as described20 and membrane fraction NAD(P)H oxidase activity through lucigenin (5 µmol/L) chemiluminescence.
Statistics
Data are mean±SE. Comparisons were tested through 1-way ANOVA plus Student–Newman-Keuls or Dunnet post-hoc tests, at a 0.05 significance level.
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Results |
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Human Aortic Valves
Human stenotic and to a lesser degree sclerotic AV exhibited thickening, fibrosis, elastolysis, and calcification (not shown). Microtopography of superoxide production showed increased fluorescence signals in sclerotic, and specially in stenotic AV, observed mainly around calcifying foci. Signals decreased on specimen incubation with Peg-SOD (Figure 1A through 1D). Expression of p22phox and Nox2 NAD(P)H oxidase subunits was increased around such foci (Figure 1E and 1F; supplemental Figure I).
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Rabbit Model
Preliminary experiments with serial echocardiograms showed progressive increases in AV calcification in HC+vitD rabbits, with accelerated progression after 8 weeks.
Histological Characterization
AV calcification accompanied the increased plasma levels of cholesterol and calcium-phosphorus product, mainly because of phosphate increase, and minor increase in serum creatinine levels (supplemental Table I). AV from HC+vitD rabbits showed early stages of cusp mineralization (supplemental Figures II and III): thickening caused by collagen infiltration and increased cellularity, massive macrophage infiltration in the aortic face, and several conglomerate foci of subendothelial calcification. In 3 intact rabbits fed regular chow, tempol was given at 100 µmol/kg/d for 3 months. There were no changes in blood biochemical parameters and AV calcification, showing that tempol alone had no effects in the study variables (not shown).
Echocardiographic Data
Significant increase in echogenicity occurred in HC+vitD rabbits (supplemental Figure III). Within the time frame of study, the absolute AV area and aortic jet/left ventricular outflow tract velocity ratio did not differ among groups (supplemental Figure IV). Final AV area decreased versus baseline in all HC+vitD rabbits, but such decrease reached statistical significance only in HC+vitD rabbits given tempol. Concomitantly, there was increase in posterior wall thickness (PWT), revealing concentric ventricular remodeling in all HC+vitD rabbits (supplemental Figure IV). Thus, such animal model shows changes consistent with AV sclerosis.
Reactive Oxygen Species Generation
DHE fluorescence microtopography (Figure 2) showed increased superoxide signals corresponding to macrophage infiltrate and an even more pronounced increase, 
6-fold above normal, in cells surrounding calcifying foci, which, as shown below, did not stain for macrophage markers (Figure 2A). Signals were importantly reduced after specimen incubation with Peg-SOD. Superoxide signals were decreased in LA- and specially tempol-treated rabbits (Figure 2G and 2H). Overall cellularity counts using DAPI nuclei staining showed important decrease in cell number around calcifying foci with tempol. Thus, although tempol clearly decreased superoxide signals in most cells, cell loss appears to have contributed to fluorescence decrease around calcifying foci (Figure 2G).
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In situ DCF-DA fluorescence microtopography showed increased signal in AV cusps in HC+vitD rabbits (Figure 3), which was decreased after specimen incubation with Peg-Catalase, thus indicating that signals reflect mainly hydrogen peroxide. Importantly, hydrogen peroxide signals were increased in AV from tempol-treated rabbits (Figure 3E), and markedly decreased in LA-treated rabbits (Figure 3F).
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In HC+vitD rabbits, nitro/oxidative stress preferentially occurred around calcifying foci, and to a lesser extent in macrophage staining area, as shown from increased 3-nitrotyrosine immunostaining (Figure 4), which was decreased with LA and particularly with tempol (supplemental Figure V).
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Expression of NAD(P)H Oxidase Subunits
NAD(P)H oxidase subunits p22phox, Nox2, and particularly its newly-described regulator protein disulfide isomerase19 were strongly expressed around calcification, coincident with the site of RAM11-negative cells and highest ROS production (Figure 4). Both Nox2 and protein disulfide isomerase colocalized with p22phox in such cells (supplemental Figure VI). Analysis of Nox1 and Nox4 mRNA expression by real-time PCR showed increase in Nox4 expression in HC+vitD rabbits, reflected by increased Nox4/Nox1 ratio, which was especially increased with tempol (Figure 5).
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P<0.05 vs all other groups.
Phenotypic Markers of CVC/Osteoclasts
Several markers were analyzed for assessing the phenotype of cells surrounding calcifying foci. Importantly, staining for macrophage marker RAM11 was absent around calcifying foci (Figure 2A; supplemental Figure II). There was increased immunoreactivity for the osteoblast differentiation markers osteopontin and Cbfa-1 in HC+vitD rabbits (supplemental Figure VIIE and VIIF). Osteopontin mRNA was also increased and was unchanged by antioxidants (supplemental Figure VIIC). In addition, osteoclasts were identified by positive TRAP histochemistry (supplemental Figure VIID). This implicates ROS generation in cellular events related to calcification process.
Effects of Antioxidants on AV Calcification and Cell Infiltration
Effects of tempol or LA in extent of AV calcification were assessed by 3 methods (Figure 5). Echogenicity, a semiquantitative AV thickness/calcification index, was increased in HC+vitD rabbits and unchanged by tempol. Importantly, LA-treated rabbits showed significant 70% decrease in echogenicity, not statistically different vs Normal. Histomorphometric analysis, able to detect larger mineral conglomerates, showed increased calcification in HC+vitD rabbits, which was further enhanced by tempol, and prevented by 43% with LA (Figure 5B). Ex vivo 64-row multidetector CT-scan, a method able to identify only large well-organized calcium deposits, showed increased opacity signals only in aortas from tempol-treated HC+vitD rabbits. These findings were independent of calcium-phosphorus product or creatinine levels (supplemental Table I), which were similar among all groups.
Of note, TUNEL-positive cells increased in HC+vitD rabbits and were further enhanced with tempol, especially around calcifying foci, whereas overall cell proliferation, which was increased in HC+vitD rabbits, was decreased with tempol (supplemental FigureVIIIA and VIIIB). Macrophage infiltration was increased in HC+vitD rabbits and unchanged by tempol or LA, being 2-fold greater in rabbits given cholesterol only (supplemental FigureVIIID). Collagen deposition showed minor nonstatistically significant changes among groups (supplemental Figure VIIIC).
In Vitro Model
In cultured CVCs, calcium deposition increased in proportion with strength of calcifying stimuli during 14 days. Such deposition was further enhanced with tempol, but not with LA (Figure 6). CVC incubation with exogenous 50 µmol/L hydrogen peroxide amplified mineralization response irrespective of antioxidant coincubation. Incubation with catalase did not change basal calcification, but completely prevented increased calcification induced by tempol. Incubation with NO donor NOC-18 (30 µmol/L) for 14 days did not change CVC calcium deposition (not shown). Thus, while exogenous oxidants increased cell calcification, the mechanism of calcification appears to differ in vitro (versus in vivo) enough that baseline CVC calcification is redox-insensitive. Superoxide production, assessed by HPLC analysis of 2-hidroxyethidium (Figure 6), as well as membrane fraction NAD(P)H oxidase activity (supplemental Figure IX) was increased with calcifying stimuli, partially reduced with tempol and strongly decreased with LA.
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Discussion |
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Convergence between ROS and valve calcification is usually regarded in a chronic inflammation context. Thus, in our rabbit model, the observed topography of ROS production around calcifying foci from cells displaying phenotypic markers of osteoblasts or osteoclasts, but not of macrophages, is an important novel finding, although contribution of other phenotypes cannot be excluded. Analogous patterns occurred in human AV sclerosis or stenosis, despite advanced disease in the latter. The rabbit model—mimicking earlier human disease—displayed massive AV infiltration of macrophage foam cells exhibiting increased superoxide signals versus control, but not as marked as in cells around calcification. This probably reflects the role of ROS as signaling intermediates of cellular metabolic or developmental processes occurring during CVC or osteoclast formation and activity.2,6,11,16 Moreover, these data stress that even with the likely occurrence of substantial lipoprotein oxidation, cellular signaling still seems of prime or at least comparable importance as ROS source.7 Thus, our data show that oxidant stress pattern in valve calcification shares partial resemblance with atherosclerosis, and extends beyond vascular or inflammatory cell activation. Of note, tempol and LA decreased superoxide levels without changes in macrophage density (supplemental Figure VIIID).
Decreased calcification progression with LA in our rabbit model indicates that ROS potentiate AV calcification. The large effect of LA verified at echogenicity analysis contrasts with the smaller effect evident at histomorphometry. Echogenicity reflects AV thickening and microcalcification, suggesting LA effects in halting early onset of calcium conglomerates, because macrophage and collagen densities were unchanged (supplemental FigureVIIIC and VIIID). Contrarily, histology preferentially shows larger mineral conglomerates. This could indicate that once a threshold conglomerate size is reached, further growth occurs rapidly and overrides antioxidant effects, an explanation in line with known exponential growth rate of calcification.6 This could also reflect a limitation of our models, related to abnormally high calcification stimulus attributable to induced increase in calcium-phosphorus product, which can activate CVC differentiation via phosphate cotransporter Pit-1.21 Although such increase is found in some diseases, degenerative AV calcification usually occurs under normal calcium-phosphorus product. In fact, in our in vitro model, LA or catalase showed little effect in basal calcification, whereas exogenous peroxide clearly increased calcium deposits. Another methodological caveat is that total DHE fluorescence analysis, used because calcium deposits and small rabbit AV size limited use of other methods, lacks specificity for superoxide.20 Thus, whereas Peg-SOD controls indicate that superoxide contributes to DHE fluorescence, other oxidants, heme or peroxidases might play additional roles.20 Analogous observations apply to in situ DCF studies regarding hydrogen peroxide detection. On the other hand, 2-hydroxyethidium measurements in CVC in vitro provide more specific evidence for superoxide.20
The NAD(P)H oxidase complex exhibited dynamic expression changes during calcification, with upregulation of Nox4 and Nox2, but not Nox1. ROS topography around calcifying foci coincided with increased expression of subunits p22phox and Nox2 and enhanced 3-nitrotyrosine staining. Protein disulfide isomerase, recently described by us to assist NAD(P)H oxidase activity,19 was strongly upregulated at this location. This pattern is in line with known roles of Nox4 in cell differentiation/apoptosis and of Nox2 in inflammation.22 The precise correlation between each Nox isoform and cell type remains undefined, considering lack of available information about Nox subtypes in CVCs/osteoblats. In osteoclasts, Nox2 is prominent, whereas Nox4 is upregulated on Nox2 knockout, both supporting bone resorption.23
Together, our observations implicate hydrogen peroxide as a species contributing to vascular/AV calcification. Tempol, which increased hydrogen peroxide production, promoted enhanced calcification in vivo or in vitro, contrary to LA, which promoted hydrogen peroxide decrease and halted calcification progression in vivo. Of note, both antioxidants promoted comparable superoxide decrease. Moreover, CVC incubation with exogenous hydrogen peroxide promoted enhanced calcification, whereas analogous incubation with nitric oxide donor was without effect. In addition, catalase prevented tempol-associated increase in CVC calcification. The importance of hydrogen peroxide is in line with observed increase in Nox4, which may preferentially generate hydrogen peroxide and, in turn, is induced by this species.22 Indeed, in our CVC, Nox4/Nox1 ratio increased 1.6-fold versus control VSMCs, whereas 14-day exposure to hydrogen peroxide (see Methods) further enhanced such ratio by 4- to 5-fold in both cases (not shown).
Mechanisms whereby ROS enhance vascular/valve calcification are yet poorly understood. ROS underlie signaling and expression of BMP family proteins such as BMP22 and BMP4,24 as well as Cbfa-1 and alkaline phosphatase.2,11 Oscillatory shear stress (to which AV is exposed) induces BMP4 via p47phox/Nox1-dependent NAD(P)H oxidase, further inducing intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion.24 ROS may also trigger apoptosis, a potential seed for calcification.2,6,38 Conversely, calcium resorption is enhanced by osteoclast Nox2 or Nox4-derived ROS15,23 and inhibited by nitric oxide.16
Although antioxidant LA effects have been reported in several preparations25 and in humans,26 precise mechanisms of LA effects are unclear. LA reacts with peroxynitrite only at low rates,27 and probably does not scavenge hydrogen peroxide directly. Although LA might owe its antioxidant activity to the reduced form dihydrolipoic acid, LA itself can also scavenge species such as hypochlorous acid thanks to the unique reactivity of its 2,3-dithiolane ring.28 Moreover, LA shares with distinct antioxidants such as resveratrol, but not 
-tocopherol or ascorbic acid, the capacity to induce heme oxygenase-1 via Nrf-2.29
Tempol-mediated increase in calcification and hydrogen peroxide levels provide further evidence that ROS accelerate AV calcification in vivo. Central to this question is the mechanism of tempol effects in our system. Similarly to other reports,30 tempol decreased superoxide levels and nitrotyrosine staining, in line with its proposed mechanisms of action, namely SOD-mimetic activity30 and shift of nitroxidative stress toward nitric oxide.31 Tempol-induced hydrogen peroxide increase was previously reported in endothelial32 or tumor cells33 and increased at mmol/L tempol concentrations or high glucose levels. Our EPR assessment of plasma tempol concentration yielded values 5 µmol/L (supplemental Figure X), well within or even below usual. Explanation for those effects is yet unclear, but it is unlikely that hydrogen peroxide increase is the usual outcome of possible SOD-like tempol effect, which in itself is debatable.34 Mitochondrial function impairment attributable to tempol was reported.32 In addition, while reacting poorly with superoxide anion radical, tempol reactivity strongly enhances at lower pHs via direct reaction with hydroperoxyl radical (OOH·), the protonated uncharged form of superoxide.35 Thus, at least in our in vivo model, an unusually strong SOD-like effect might have occurred because of decreased pH caused by the carbonic anhydrase activity of osteoclast, which promotes intra- and extracellular acidification.36 This is consistent with lack of toxicity of high tempol concentrations in intact cells30 and with tempol-induced oxidant stress in the acidic renal medulla.37 Also, we observed higher cell loss around calcifying foci with tempol. Increased progression of valve calcification with tempol may be clinically relevant, considering that this nitroxide antioxidant is already undergoing prospective clinical studies for several conditions.30
Collectively, our observations point to a role of redox processes, particularly those resulting in hydrogen peroxide increase, possibly related to NAD(P)H oxidase activity, in the progression rate of AV calcification. These results further a link between pathogenesis of AV stenosis and atherosclerosis, and pave the way to clinically effective interventions capable of slowing such progression, as suggested by protective LA effects.
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Acknowledgments |
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We thank Carlos Rochitte for aortic specimen computed tomography, Leonora Loppnow, Victor Debbas, and Ana Lucia Garippo for technical assistance, and Léa Demarchi and Solange Consorti for tissue processing.
Sources of Funding
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Redoxoma Millenium Institute), Financiadora de Estudos e Projetos.
Disclosures
None.
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Footnotes |
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Original received September 25, 2007; final version accepted December 12, 2007.
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