The amount of calcium-deficient hexagonal hydroxyapatite in aortic valves is influenced by gender and associated with genetic polymorphisms in patients with severe calcific aortic stenosis
Jan R Ortleppa,*,
Fabian Schmitza,
Vera Mevissena,
Stefan Weißb,
Jürgen Husterb,
Richard Dronskowskib,
Georg Langebartelsc,
Rüdiger Autschbachc,
Klaus Zerresd,
Christian Webere,
Peter Hanratha and
Rainer Hoffmanna
a Medical Clinic I, University Hospital of Aachen, Aachen University of Technology, Pauwelsstrasse 30, 52152 Aachen, Germany
b Institute of Inorganic Chemistry, Aachen University of Technology, Aachen, Germany
c Clinic for Cardiac Surgery, University Hospital of Aachen, Aachen University of Technology, Aachen, Germany
d Institute for Human Genetics, University Hospital of Aachen, Aachen University of Technology, Aachen, Germany
e Division of Molecular Cardiovascular Research, Medical Clinic I, Aachen University of Technology, Pauwelsstrasse 30, 52057 Aachen, Germany
Received March 25, 2003;
revised August 9, 2003;
accepted September 4, 2003
* Corresponding author. Tel.: +49-241-8089300/8088660; fax: +49-241-8082414
E-mail address: jrortlepp{at}ukaachen.de
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Abstract
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Aims The study evaluated the relationship between cardiovascular risk factors (CRF), gene polymorphism, calcification and fibrosis of stenotic aortic valves.
Methods and results The calcium content of 187 excised stenotic aortic valves was determined using atomic absorption spectroscopy. Hydroxyproline content was quantified. Left-heart catheterization was performed. CRF and genotypes of the interleukin 10, connective tissue growth factor (CTGF) and chemokine receptor 5 (CCR5) polymorphisms were assessed. Calcification consisted of Ca-deficient hexagonal hydroxyapatite, Ca10x(HPO4)x(PO4)6x(OH)2x; with
. Calcification (quintiles) was positively associated with the mean gradient across the aortic valve (44±14, 52±17, 54±16, 60±15, 68±19 mmHg;
). Males (
) had a higher degree of calcification (26.1±8.9 vs 20.8±9.2 mass%;
), despite the same mean gradient across the aortic valve (56±17 vs 56±19 mmHg;
). CRF were not, whereas interleukin 10 polymorphisms 1082, 819, and 592 were significantly associated with the degree of calcification. Furthermore, if certain allele carriers had additionally the rare CCR5 or CTGF allele the degree of calcification was higher.
Conclusion Calcification of stenotic aortic valves consists of Ca-deficient hexagonal hydroxyapatite. Gender and genetic polymorphisms have an impact on the degree of aortic valve calcification.
Key Words: Calcification Degenerative calcific aortic stenosis Gene polymorphisms Cardiovascular risk factors Mass spectroscopy
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Introduction
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Several risk factors for coronary artery disease (CAD) have been defined and their evaluation has been implemented into clinical practice. 1,2 Calcific aortic stenosis (AS) is the most frequent acquired valvular disease.35 The same traditional cardiovascular risk factors associated with CAD have been related to AS.57 Sclerosis of the aortic valve is a potential precursor of aortic stenosis and a surrogate marker for atherosclerosis. Aortic sclerosis is associated with a 50% increased risk of cardiovascular mortality and myocardial infarction.8 Recent studies showed evidence for the association of LDL-cholesterol and aortic valve calcification.911 Thus, AS and CAD share similarities. However, though calcification of the aortic valve is closely linked to CAD, CAD is not closely linked to AS, hence most patients with CAD do not have AS. This indicates that despite similarities both disease entities must have different pathogenesis. At least partially due to the link of AS to CAD, AS is associated with cardiovascular risk factors. Still, the impact of these risk factors on the degree of calcification in severely stenotic aortic valves is uncertain. Most patients with cardiovascular risk factors may develop CAD but do not develop calcific aortic stenosis. The extent of calcification in severely stenotic valves is likely to be controlled by other mechanisms than those responsible for the development of atherosclerosis. It is known that pathological alterations of the calcium metabolism like in renal disease or hyperparathyroidism are associated with calcification of the aortic valve.1214 Furthermore, there is evidence that elevation of the C-reactive protein is associated with AS.15 Thus, inflammation may represent a contributing factor in the pathogenesis of aortic stenosis. Genes encoding products regulating inflammation are candidate genes for aortic valve calcification. Promotor polymorphisms of the interleukin 10 gene are associated with an elevated expression of the gene product16 and therefore are of functional importance. A 32-bp deletion polymorphism of the chemokine receptor 5 (CCR5) gene is associated with an altered susceptibility and immune response to virus infection17,18 as well as improved renal transplant survival19 due to an inactive receptor.20 The connective tissue growth factor (CTGF) stimulates proliferation of osteoblasts.21 A recently described promoter polymorphism22 is an intriguing candidate for calcification. Thus, the polymorphisms of the CCR5 and CTGF gene seem to be functionally relevant by enhancing antiinflammation or osteogenesis. The aim of this study was to describe the kind of calcification in stenotic aortic valves and to evaluate the impact of cardiovascular risk factors and genetic polymorphisms on the degree of aortic valvular calcification and fibrosis determined by calcium atomic absorption spectroscopy and hydroxyproline quantification.
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Methods
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Study population
Consecutive patients with symptomatic calcific aortic stenosis were included in the study. Inclusion criteria were elective diagnostic left-heart catheterization and indication for aortic valve replacement due to calcific aortic stenosis (severe symptomatic aortic stenosis or patients with demand for bypass surgery and concomitant moderate to severe calcific aortic stenosis). Exclusion criteria were bicuspid aortic valves, history of rheumatic fever, status post endocarditis, concomitant severe aortic regurgitation or other relevant valvular heart disease and coexistence of cardiomyopathies. All patients gave written informed consent. The study was approved by the local ethical committee (Ethik-Kommission des Universitätsklinikums Aachen, EK712 from 1998, addenda in 1998, 2000 and 2002).
Left-heart catheterization
Coronary angiography including at least four views of each coronary artery was performed in all patients. Coronary angiograms were analyzed by two independent cardiologists and classified as demonstrating: either (a) non-significant CAD; i.e., clear coronary arteries or coronary sclerosis (signs of atherosclerosis with stenosis less than 50%) or (b) CAD (stenosis greater than 50% in at least one vessel). The gradient across the aortic valve was measured by pull back of the catheter from the left ventricle into the ascending aorta. The mean gradient was calculated by a computer assisted program (Metek, Roetgen, Germany).
Cardiovascular risk factors
Cardiovascular risk factors were defined as hypercholesterolemia (cholesterol level >220 mg/dl or medically treated), arterial hypertension (blood pressure >140/90 mmHg or medically treated), diabetes mellitus (overnight fasting serum glucose >126 mg/dl on at least two occasions or medically treated), and smoking (the definition of smoking in this study was "do you smoke yes or no?". Pack years were not systematically analyzed). Morning fasting cholesterol, triglycerides, and glucose were measured the day before or on the day of left-heart catheterization between 8.00 and 11.00 a.m. Laboratory results were available in 177 of 187 patients.
Structural identification of calcification
An example of the macroscopic view of calcification deposits is given in Fig. 1A (aortic valve excised by the heart surgeon). A small sample (few mg) of seemingly single-phase, calcified, solid material was ground finely and prepared as a flat sample for X-ray powder diffraction. The diffraction pattern was recorded in transmission geometry between 6° and 70° in
using a DebyeScherrer powder diffractometer (Stoe STADI P2), monochromatized Cu K
1 radiation (focusing germanium monochromator), and a linear proportional counter (6400 steps, 12 s/step). The crystalline phase was identified by comparison with the corresponding entry from the crystallographic literature.23,24
Analysis of the calcification in aortic valves (atomic absorption analysis)
Patients were referred to the Clinic for Cardiac Surgery and underwent aortic valve replacement. The valves were excised by the surgeons and frozen at 80 °C. Valves were than homogenized in liquid nitrogen. After evaporation of the liquid nitrogen samples were frozen. For analysis, the samples were given to 60 ml of concentrated sulfuric acid. The samples were transferred into beakers and held, until completely dissolved, at a temperature of 150 °C. The solutions were allowed to cool to room temperature and then refilled to volumes of 250 ml. After further dilution, the individual calcium contents were determined by means of an atomic absorption analysis (Shimadzu AA-6200) at a wavelength of 422.7 nm using an air-acetylene flame. At first the amount of calcification was determined as the total Ca mass. According to the X-ray diffraction measurements, the calcification consists of Ca-deficient hexagonal hydroxyapatite, the chemical formula of which can be approximately shortened to Ca5(PO4)3OH hydroxyapatite, with a molar mass of 502.32 g/mol. The mass contribution of all Ca atoms in this crystalline phase is five times the atomic mass of Ca, that is five times 40.08 g/mol, namely 200.4 g/mol. Thus, the mass percentage of Ca in hydroxyapatite is 100 times 200.4/502.32 =39.89%. In order to mathematically switch from Ca mass to hydroxyapatite mass, the conversion factor therefore is 1 divided by 0.3989=2.507. Since the calcification in the valves is Ca-deficient hydroxyapatite Ca10x(HPO4)x(PO4)6x(OH)2x; with
(see Results), the calculated calcification is below the real calcification mass and should be considered as surrogate marker of valve calcification. In a pilot study analysis of five macroscopic uncalcified aortic valves was also performed.
Hydroxyproline analysis
Hydroxyproline was quantified in duplicates from 50 mg of valvular homogenized tissue. The tissue was dissolved in 4 ml of 6 mol/L HCl and hydrolyzed at 110 °C for 16 h. Hydrolysates were filtered. Methanol (10 µl), to resolve residual HCl, was given to 30 µl aliquots. The 40-µl was evaporated in a Reacti-Therm III System (Pierce Biotechnology, Rockford, USA) under nitrogen blowing and warming of the tubes (60 °C). The residue was dissolved in 1.0 ml of 50% isopropanol and incubated with 0.2 ml of 0.84% chloramine-T (in 42 mmol/L sodium acetate, 2.6 mmol/L citric acid, 39.5 ml isopropanol, pH 6.0) for 10 min at room temperature, followed by the addition of a solution of 0.248 g of p-dimethylamino-benzaldehyde in 0.27 ml of 70% perchloric acid and 0.73 ml isopropanol, incubated at 50 °C for 90 min. Absorption was read at 558 nm against a reagent blank. Hydroxyproline concentrations were determined from a standard curve using 05.0 µg high-purity hydroxyproline (Merck, Hohenbrunn, Germany). Probes with high absorptions that reached the nonlinear range of the standard curve were further diluted. The hydroxyproline content was given as µg/mg. The amount of "anticipated" collagen was calculated by multiplying the hydroxyproline content by 7.46.
Genotyping
Genomic DNA of patients was extracted from whole blood or value tissue using the InVitek Isolation Kit (InVitek, Berlin, Germany). Genotype analysis was performed with allele-specific fluorogenic oligonucleotide probes in an assay combining polymerase chain reaction (PCR) and the
nuclease reaction (TaqMan technique; Applied Biosystems, Weiterstadt, Germany). Primers were used to amplify a sequence containing the polymorphic site. Allele-specific labelled probes were used to identify the polymorphic site. FAM, i.e., 6-carboxy-fluorescein, and VIC (Applied Biosystems, Weiterstadt, Germany; patent pending) were the fluorogenic dyes used to accomplish allelic discrimination. The 2-step thermocycling procedure consisted of 40 cycles of denaturation at 92 °C for 15 s and primer annealing and extension at 60 °C for 1 min. Table 1 shows the primer and probe sequences.
Statistical analysis
Continuous data are presented as mean±SD and categorical data are presented as frequencies. Differences in continuous data were analyzed by ANOVA. The effects of genotypes, haplotypes and cardiovascular risk factors on aortic valvular calcification and fibrosis were assessed by means of univariate analyses. For categorical/binary variables, this was done by means of an ANOVA. For continuous variables, this was done first by dividing the data into quintiles and testing the difference between quintiles (using ANOVA). Then linear regression analyses were performed to investigate the direct effect of the continuous characteristics on calcification. After univariate analysis we also performed a multiple regression analysis including the genetic status (combined carriage of interleukin 10 haplotype associated with calcification and CCR5/CTGF rare alleles) and the non-genetic status of age, smoking, diabetes mellitus, hypercholesterolemia, and hypertension. Calculations were performed using SPSS for windows version 10.0. Significant differences were defined as a two sided p-value <0.05. Linear regression analysis was performed to define the association of cardiovascular risk factors with valvular calcification. The null hypothesis was that calcification of the aortic valve is not different between the different genotypes nor between patients with different cardiovascular risk factors.
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Results
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Study population
The clinical characteristics are summarized in Table 2. The study population represents a normal cross-section of patients with calcific aortic stenosis.
Crystal-chemical form of calcification
The X-ray powder diffraction pattern is given in Fig. 1. Despite some diffuse background due to amorphous material, the sample is fairly crystalline, and the comparatively sharp Bragg peaks are characteristic for the hexagonal form of hydroxyapatite, Ca5(PO4)3OH;23 a refinement of the lattice constants based on 29 indexed reflections yields
pm and
pm. Since it has been noted24 that pure hydroxyapatite Ca10(PO4)6(OH)2 (=2Ca5(PO4)3OH) never occurs in biological systems, the crystalline component is likely to be calcium-deficient hydroxyapatite, the approximate chemical composition of which is Ca10x(HPO4)x(PO4)6x(OH)2x (with
). This nonstoichiometric material always contains Ca defects; accordingly, the AAS determination of the Ca mass content (3233%) of two independent samples turned out to be about one fifth lower than the ideally stoichiometric mass content (39.9%). Amazingly, the crystal structure of the "mother" compound, namely hexagonal Ca5(PO4)3OH, is still adapted by the nonstoichiometric phase. Because of the slight nonstoichiometry explained above and for reasons of simplicity, calcification contents are given both as Ca mass percents and also expressed as pure Ca5(PO4)3OH mass% as a surrogate marker for valve calcification, which indicates the minimum of calcification, in hydroxyapatite.
Degree of calcification of the aortic valve
In a pilot study analysis of five macroscopic totally uncalcified aortic valves revealed calcium mass of less than 1% (data not shown). The mean mass percentage of crude Ca in stenotic valves was 9.5±3.8 mass% corresponding with 23.7±9.4 mass% Ca5(PO4)3OH. As given in Fig. 2 males had a higher degree of calcification than females (26.1±8.9 vs 20.8±9.2 mass%;
), despite having the same mean gradient across the aortic valve (56±17 vs 56±19 mmHg;
).

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Fig. 2 (A) The difference in the extent of aortic valve calcification between both genders. In contrast to the degree of aortic valve calcification (crude Ca5(PO4)3OH in mass%) the mean gradient across the aortic valve was identical between males and females (56±17 vs 56±19 mmHg; , (B)).
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Relation of calcification and fibrosis with severity of the aortic stenosis
The degree of calcification (given as 1st, 2nd, 3rd, 4th, and 5th quintile) was positively associated with the mean gradient across the aortic valve (44±14, 52±17, 54±16, 60±15, 68±19 mmHg;
; linear regression:
,
,
). This positive association is illustrated in Fig. 3A. The degree of fibrosis assessed by quantification of collagen (given as 1st, 2nd, 3rd, 4th, and 5th quintile) was inversely associated with the mean gradient across the aortic valve (63±16, 56±18, 55±15, 57±21, 49±17 mmHg;
; linear regression:
,
,
), as presented in Fig. 3B. As given in Fig. 3C the ratio of fibrosis to calcification was highest (1:6.4) in very severely stenotic valves compared to less stenotic valves (1:2.7) (gradient given as 1st, 2nd, 3rd, 4th, 5th quintile).

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Fig. 3 The relation of the extent of aortic valve calcification (A) and hydroxyproline content (B) (each given in quintiles) with the mean gradient across the aortic valve and the ratio of fibrosis of calcification in dependence of the severity of stenosis (C) (given in quintiles of the invasively determined mean gradient).
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Relation of traditional cardiovascular risk factors and age with the degree of calcification and fibrosis
There was no association between the presence of each traditional cardiovascular risk factor and the degree of valve calcification or fibrosis except in smokers, who had a slightly higher content of collagen (see Table 3). In a general linear regression analysis neither cholesterol (
;
;
) nor triglycerides (
;
;
) nor glucose (
;
;
) were associated with the degree of calcification. Age was also not associated with the degree of calcification (
;
), whereas age was negatively associated with fibrosis (
;
) in a linear regression model, indicating that younger patients had more fibrosis than older patients.
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Table 3 Association of cardiovascular risk factors with calcification of the aortic valve at the time of valve replacement
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Genotypes and allele frequencies of the interleukin 10, CCR5, and CTGF polymorphisms
The tested genetic polymorphisms were not different from HardyWeinberg equilibrium. Interleukin 10 1082 G/A: 41 patients (22%) were homozygous for the G allele, 54 patients (29%) were homozygous for the A allele and 92 patients (49%) were heterozygous. Interleukin 10 819 C/T: 106 patients (57%) were homozygous for the C allele, 7 patients (4%) were homozygous for the T allele and 74 patients (40%) were heterozygous. Interleukin 10 592 C/A: 105 patients (56%) were homozygous for the C allele, 7 patients (4%) were homozygous for the A allele and 75 patients (40%) were heterozygous. CCR5
: 39 patients (21%) were heterozygous for the 32-bp deletion. No patient was homozygous for the 32-bp deletion allele. CTGF 447 G/C: 14 patients carried the rare 447 C allele (heterozygous). No patient was homozygous for the rare C allele.
Relation of genotypes with degree of calcification and fibrosis
As given in Table 4, the interleukin 10 genotypes were significantly associated with the degree of calcification (1082:
, 819:
,
:
). Combining the genotypes of the interleukin 10 promoter region calcification was significantly associated with the haplotype (ANOVA comparison between seven different haplotypes
). In the whole study population patients carrying the CCR5
or CTGF 447 C allele had a higher degree of calcification (CCR5/CTGF vs Wildtype
). Combining both interleukin 10 haplotypes and rare CCR5/CTGF alleles was associated with an additive effect (
). These findings are also illustrated in Fig. 4. The genotypes were not associated with different amounts of valvular hydroxyproline (data not shown). In a multiple regression analysis including the genetic status, age and the cardiovascular risk factors only the genetic status remained significant (
;
), whereas the non-genetic factors did not (hypertension
,
; hypercholesterolemia
,
; diabetes mellitus
,
; smoking
,
; age
,
).
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Table 4 Association of Interleukin 10, CCR5, and CTGF polymorphism with the degree of aortic valve calcification determined by atomic absorption analysis in native human aortic valves excised for aortic valve replacement due to symptomatic aortic stenosis in 187 consecutive patients
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Fig. 4 (A) The extent of aortic valve calcification in dependence of interleukin 10 gene promoter haplotypes. (B) Patients carrying certain interleukin haplotypes plus the CCR5 or CTGF rare allele had the highest extent of aortic valve calcification.
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Discussion
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Recent studies
Traditional cardiovascular risk factors are associated with CAD and AS. However, the exact pathogenesis of AS remains insufficiently understood. Recent studies showed evidence that cholesterol-lowering therapy with statins reduces calcification of the aortic valve.1011 It is uncertain if this effect is due to the direct lowering of cholesterol or the anti-inflammatory potential of statins. The influence of genetic factors on AS remains undetermined.
Major new findings
This study thoroughly characterized the type and degree of valvular calcification using atomic absorption spectroscopy for precise analysis of valvular calcification. In addition, the crystalline component of the calcification (Ca-deficient hexagonal hydroxyapatite) was crystal-chemically identified by its characteristic Bragg peaks through X-ray powder diffraction. Calcification consists of Ca-deficient hexagonal hydroxyapatite. It was positively related to the mean gradient across the aortic valve, whereas fibrosis assessed by the amount of hydroxyproline was negatively associated. The ratio of fibrosis to calcification was lowest in the valves with least stenotic severity, indicating that fibrosis is a major component of earlier stages of stenotic valve disease, while calcification was high in valves with high stenosis severity indicating that calcification is dominating in the later stages.
Cardiovascular risk factors were not significantly associated with the degree of calcification at the time of valve replacement. This does not exclude that cardiovascular risk factors have an important role in the pathogenesis of aortic valve sclerosis. Cardiovascular risk factors may initiate a process in the aortic valve resulting in aortic valve sclerosis similar to atherosclerosis, but further factors are likely to be necessary for the progression towards severe calcification and stenosis. As we did not investigate quantitatively the amount of calcification in the early stages of aortic sclerosis and stenosis, we might have missed an association between cardiovascular risk factors and calcification. In previous studies hypercholesterolemia was thought to be related with aortic calcification. Furthermore, recent studies have shown therapy with statins to be associated with lowering valve calcification.1011 However, this potentially beneficial effect of statins1011 seen in small numbers of subjects in non-randomized retrospective analysis could have also been due to their anti-inflammatory effects. There is clear evidence that aortic valve lesions contain lipoproteins, and cholesterol is a major component of aortic valve calcification.2526 But elevation of LDL-cholesterol does not seem to promote this calcification in a simple way, hence most patients with hypercholesterolemia develop CAD without valve calcification. There has to be an additional stimulus which promotes calcification of early lesions of degenerative valvular aortic stenosis.
While no significant impact of cardiovascular risk factors for valvular calcification could be demonstrated in this study, based on the results of this study it may be suggested that other factors are important for the calcification of the aortic valve. For instance males had a higher degree of calcification than females. The finding of a difference in the amount of calcification between both genders is a sign of genetically controlled calcification. Of the investigated polymorphisms surprisingly the genotypes with a functional anti-inflammatory potential were positively associated with the amount of pure Ca mass from calcium-deficient hydroxyapatite. Thus, genetically determined anti-inflammatory reactions might trigger valvular calcification. It remains uncertain whether these anti-inflammatory reactions are reactions to exogenous infective agents such as virus or bacterial infections and whether these reactions are primarily local or systemic. However, these findings indicate that the components leading to valvular stenosis (calcification, inflammation and proliferation of extracellular matrix) might be multifactorial with a polygenic basis. We speculate that several genetic components are involved in the development of calcification.
Limitation
The limitation of this study is that it is a cross-sectional study without analyzing the progression of aortic stenosis. However, because of the accurate method applied in the study it should have had the power to detect small effects of cardiovascular risk factors on the degree of calcification. We cannot exclude that cardiovascular risk factors especially hypercholesterolemia911 promote calcification of the aortic valve. But at the time of valve replacement we could not detect an association between serum cholesterol level and valve calcification. The number of patients in this study is relatively small for a genetic association study. The association of genotypes with the degree of calcification has to be confirmed in larger studies. However, the relatively small patient number reflects the complexity of the phenotypic analysis.
Conclusion
Calcification of the aortic valve consists of Ca-deficient hexagonal hydroxyapatite and is strongly associated with the severity of AS, whereas the amount of fibrosis is lowest in severely stenotic valves. Cardiovascular risk factors are not associated with the amount of calcification at the time of valve replacement, whereas gender and genetic polymorphisms are associated with the extent of calcification, indicating that AS might be a multifactorial disease with an at least partial polygenic basis. Further investigation on the pathogenesis of calcific aortic stenosis should therefore focus on the role of inflammatory genes and their genetic variants. Analysis of cardiovascular risk factor might not be sufficient to explain the severity of valve calcification.
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Acknowledgments
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This study was supported by the START program of the University Hospital of Aachen and is part of the doctoral thesis of Fabian Schmitz.
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