Micron 67 (2014) 141–148
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Micron
journal homepage: www.elsevier.com/locate/micron
Distribution of selected elements in calcific human aortic valves
studied by microscopy combined with SR-␮XRF: Influence of lipids on
progression of calcification
Grzegorz J. Lis a,∗ , Joanna Czapla-Masztafiak b , Wojciech M. Kwiatek b , Mariusz Gajda a ,
Ewa Jasek a , Malgorzata Jasinska a , Urszula Czubek c , Manuela Borchert d , Karen Appel d ,
Jadwiga Nessler c , Jerzy Sadowski e , Jan A. Litwin a
a
Department of Histology, Jagiellonian University Medical College, Kopernika 7, 31-034 Krakow, Poland
Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Krakow, Poland
c
Department of Coronary Disease, Jagiellonian University Medical College, John Paul II Hospital, Pradnicka 80, 31-202 Krakow, Poland
d
Deutsches Elektronen-Synchrotron (DESY), Notkestraˇe 85, D-22607 Hamburg, Germany
e
Department of Cardiac and Vascular Surgery and Transplantology, Jagiellonian University Medical College, Pradnicka 80, 31-202 Krakow, Poland
b
a r t i c l e
i n f o
Article history:
Received 7 May 2014
Received in revised form 11 July 2014
Accepted 4 August 2014
Available online 12 August 2014
Keywords:
Aortic valve
Elemental composition
Lipids
Calcification
Synchrotron radiation ␮X-ray fluorescence
Histology
a b s t r a c t
Calcified heart valves display a significant imbalance in tissue content of trace and essential elements. The
valvular calcification is an age-related process and there are data suggesting involvement of lipids. We
studied elemental composition and lipid distribution in three distinct regions of calcified human aortic
valves, representing successive stages of the calcific degeneration: normal, thickened (early lesion) and
calcified (late lesion), using SR-␮XRF (Synchrotron Radiation Micro X-Ray Fluorescence) for elemental
composition and Oil Red O (ORO) staining for demonstration of lipids. Two-dimensional SR-␮XRF maps
and precise point spectra were compared with histological stainings on consecutive valve sections to
prove topographical localization and colocalization of the examined elements and lipids. In calcified
valve areas, accumulation of calcium and phosphorus was accompanied by enhanced concentrations
of strontium and zinc. Calcifications preferentially developed in lipid-rich areas of the valves. Calcium
concentration ratio between lipid-rich and lipid-free areas was not age-dependent in early lesions, but
showed a significant increase with age in late lesions, indicating age-dependent intensification of lipid
involvement in calcification process. The results suggest that mechanisms of calcification change with
progression of valve degeneration and with age.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Calcific degeneration of aortic valve is the most common type
of valvular heart disease in the Western world, causing significant
morbidity and mortality (Iung et al., 2003; Otto et al., 1999). It
shares many features with vascular atherosclerosis. Ectopic mineralization, a hallmark of the process, was previously regarded as
passive deposition of calcium salts in pathologically altered tissue
areas. Recent years brought strong evidence supporting the concept of active mechanisms participating in the pathophysiology
of the process, which is preceded by endothelial damage, inflammatory infiltration, lipid deposition and oxidation of lipoproteins
∗ Corresponding author. Tel.: +48 124227027; fax: +48 124227027.
E-mail address: [email protected] (G.J. Lis).
http://dx.doi.org/10.1016/j.micron.2014.08.002
0968-4328/© 2014 Elsevier Ltd. All rights reserved.
(O‘Brien et al., 1996; Otto et al., 1994; Kaden et al., 2005; Aikawa
et al., 2007a). It is not clear, however, whether these processes
driven by a variety of cells and cell mediators can explain all
aspects of calcific valve degeneration. Some new data suggests
that cardiovascular calcification, previously regarded as a simple
unidirectional pathway, is in fact a multi-faceted process, leading
to divergent consequences, including inhibition or even reversal
of valve destruction (Shanahan, 2007; Nicoll and Henein, 2013).
The calcification largely depends on the local tissue milieu. In
atherosclerosis and valvular calcific degeneration, involvement of
lipids and lipoproteins both locally accumulated and circulating is
well established (O‘Brien et al., 1996; Otto et al., 1994). Recent publications, however, emphasize that their mode of action seems to be
much more complex than suggested earlier and is still far from clarification (Pfanzagl, 2006; Kontush et al., 2003; Colles et al., 2001;
Demer and Tintut, 2011).
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G.J. Lis et al. / Micron 67 (2014) 141–148
Physico-biological properties and appropriate function of aortic valves depend on their structural and biochemical integrity
including elemental composition. Calcified heart valves display a
significant imbalance in tissue content of calcium, phosphorus,
iron, zinc and copper (Nystrom-Rosander et al., 2002; Tohno et al.,
2002; Sarnowski et al., 2001). There is very few data, however, on
their distribution in particular valve areas and changes in concentration accompanying the progression of valve degeneration. Up
to now, studies investigating jointly concentration and localization
of elements in different regions of the cardiovascular system have
been performed on human atherosclerotic arteries (Roijers et al.,
2011; Cichocki et al., 1985), while aortic valves have been examined only in animal models (Gajda et al., 2008). Degenerating aortic
valves are highly heterogeneous, showing areas with different levels of pathology as well as areas with normal histomorphology.
As recently proposed by Nagy et al. (2013), these morphologically
distinct areas may represent the successive stages of aortic valve
degeneration.
The biological samples, such as aortic valves, are usually studied
by histological, histochemical and immunofluorescence methods.
However, the contemporary physics offers new tools, suitable
also for the biological studies. SR-␮XRF (Synchrotron Radiation
Micro X-Ray Fluorescence) is a multielemental and highly sensitive analytical technique based on the excitation of an extremely
small sample area (a few micrometers) by the synchrotron radiation source and the resulting, element-specific X-ray fluorescence
emitted from different areas of the sample allows to examine
the concentration and lateral distribution of the elements. The
use of high-intensity focused beam obtained from synchrotron
source makes possible analysis of elements that occur in biological
material even at extremely low concentrations. Since synchrotron
delivers a beam of high flux and brightness, no preconcentration
procedure for samples is required and the concentration of different elements can be determined with high accuracy (Wang et al.,
2010; Twining et al., 2003). This technique was also successfully
implemented to the studies on distribution of selected elements
in atherosclerotic plaques in a murine model (Gajda et al., 2008,
2010).
The aim of the present study was to compare the elemental distribution in morphologically defined areas of calcifying
aortic valves and to establish the relation between the distribution of selected elements, calcification and accumulation of
lipids as hallmarks of aortic valve degeneration. Two-dimensional
maps and precise point spectra of SR-␮XRF recordings, acquired
from microscopically selected areas, were compared with histological/histochemical stainings on adjacent sections to prove
topographical co-localization of the examined elements and lipids.
Since the age is a significant factor involved in both calcification
and lipid accumulation (Stewart et al., 1997; Sell and Scully, 1965),
we also checked whether the relation between calcium and lipids
is age-dependent.
2. Materials and methods
2.1. Material harvesting and preparation
The study material comprised 15 human aortic valves. Eleven
valves were excised during routine surgery (six men and five
women, mean age 68.58 ± 8.17 yrs) due to severe stenosis
without rheumatic etiology. Four normal (non-stenotic, macroscopically unchanged) valves (three men and one woman, mean
age 49.5 ± 5.07 yrs) were collected at autopsies. The study protocol was approved by the Bioethical Committee of the Jagiellonian
University Medical College and the patients informed consent was
obtained.
2.2. Macroscopic and microscopic analysis of valve cusps
The valve cusps were first examined under Stemi 2000C stereomicroscope (Zeiss, Germany) coupled to Coolpix 990 digital camera
(Nikon, Japan).
In each valve, three distinct areas representing the successive
stages of calcific valve degeneration were analyzed: (1) normal
– thin, semitranslucent, pliable and showing unchanged microscopic structure, (2) thickened – opaque with prominent thickening
characteristic for early lesions and (3) calcified – containing focal
calcifications significantly distorting valve surface, typical for late
lesions (Fig. 1).
Serial 10 ␮m thick unfixed frozen sections of the abovementioned valve cusp areas were thaw-mounted on 3 ␮m-thick Mylar
foil, immediately air-dried, stored in tightly sealed dishes at room
temperature and subjected to SR-␮XRF measurements. Adjacent
sections were used for histology and histochemistry.
2.3. Histology and histochemistry
Sections were fixed in 4% buffered formaldehyde for 5 min
and stained with hematoxylin and eosin for general morphology, with Oil Red O (ORO) for lipids and with von Kossa
method for calcifications. They were examined under bright
field/fluorescence Olympus BX50 microscope (Olympus, Japan).
Images were recorded using DP-71 digital CCD camera (Olympus,
Japan) coupled to IBM PC-class computer equipped with AnalySISFIVE® (Soft Imaging System GmbH, Münster, Germany) image
analysis system. Histochemically stained sections served to determine the locations of lipid-rich and calcified areas in the adjacent
sections used for SR-␮XRF measurements.
2.4. SR-XRF
Precise point spectra and two-dimensional maps of phosphorus,
sulphur, calcium, iron, strontium, copper and zinc were performed
using SR-␮XRF at the bending magnet beamline L of the synchrotron radiation source DORIS III (DESY, Hamburg, Germany). The
experiment was conducted in the air, at room temperature. The
samples were mounted on a remotely controlled stage equipped
with high-precision stepping motors allowing micrometric movement in xyz space. Samples were fixed at an angle of 45◦ to the
incident beam. The primary photon energy was set to 17.5 keV by
a multilayer double monochromator, that enables the analysis of
elements with atomic number between 14 (Si) and 38 (Sr). The Vortex SDD semiconductor detector was used for spectral acquisition.
2D maps were acquired from the studied areas of the valves (normal, early lesions and late lesions), with the resolution of 15 ␮m,
for 3 s/pixel. They were used for qualitative assessment of element distribution in the valves. Point spectra from these areas
(10 points per area in each valve) were recorded with the resolution of 15 ␮m, for 300 s/point and used for quantitative analysis of
the elemental content. The obtained spectral data were analyzed
by AXIL software and peak areas in XRF spectra were calculated,
including background subtraction and integration of the area under
the peak of the K␣ line of the selected elements. For each element, the peak areas were normalized to the incident beam flux
and to the peak area under the Compton peak to compensate for
the differences in thickness and density of valve sections and the
inhomogeneity of sample matrix composition that would influence the beam penetration depth. The concentrations of studied
elements were expressed in arbitrary units, proportional to the
number of counts.
G.J. Lis et al. / Micron 67 (2014) 141–148
143
Fig. 1. Valve leaflet areas representing the successive stages of aortic valve degeneration seen macroscopically (a) and their histomorphology. Left panel (b, e and h) normal
area; central panel (c, f and i) thickened area representing early lesion; right panel (d, g and j) heavily calcified area representing late lesion. Hematoxylin and eosin (HE)
staining (b–d); Oil Red O (ORO) staining for lipids (e–g); von Kossa staining for calcifications (h–j). Bars = 100 ␮m.
2.5. Statistical analysis
The variables were expressed as median [lower–upper quartile
values] or mean ± SD depending on their type and distribution. The
Kolmogorov–Smirnov test was used to assess conformity with the
normal distribution. The statistical analysis included Student’s ttest for variables with normal distribution, Mann–Whitney U-test
if departure from normality was found and Wilcoxon matched pairs
test for comparison of the elemental content in pairs of microscopically selected areas in the valves. The Kruskal–Wallis test followed
by Dunn’s post hoc test was used for multiple group comparison.
Spearman rank correlation was employed to assess the association between continuous variables. Statistical analysis related to
elemental content was based on SR-␮XRF precise point spectra
measurements. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc., USA). In all tests,
two-tailed p values < 0.05 were considered statistically significant.
3. Results
3.1. Aortic valve morphology
Microscopic examination of stenotic valve leaflets confirmed
the occurrence of the three areas differing in histomorphology. The
pathological changes: lipid-rich areas and focal calcifications were
mostly located in the superficial fibrosa layer. Prominent lipid-rich
areas were observed in early and late lesions. The early lesions
showed the presence of fine granular calcifications, while the late
lesions contained massive calcified deposits. In normal regions of
stenotic valves (as well as in nonstenotic valves) lipid accumulations and calcifications were absent. In early lesions, lipid-rich areas
were located superficially, along generally unaltered fibrosa. In late
lesions, lipids were located within focal calcifications and in their
vicinity (Fig. 1).
3.2. Elemental content of the examined areas of stenotic valves
Analysis of stenotic valves revealed significant differences in
the elemental content of normal areas, early lesions and late
lesions (Fig. 2). Even though microscopic examination of normal
valves and macroscopically normal parts of stenotic valves did not
reveal significant differences, elemental analysis showed higher
calcium and lower potassium content in normal areas of stenotic
valves as compared to normal valves (64.51 [54.26–74.93] vs 34.80
[33.44–50.12]; p = 0.001 and 1.07 [0.32–1.97] vs 3.64 [1.34–6.06];
p = 0.006). There were no significant differences in the content of
other elements between these groups.
Generally, the examined elements showed a tendency to accumulate in early and late lesions, as compared to normal areas of the
valves. Significantly higher concentrations of calcium, copper and
sulphur were observed in early lesions, and of all studied elements
except iron in late lesions. In case of calcium, phosphorus, strontium and zinc, late lesions showed significantly higher content of
these elements than early lesions (Fig. 3).
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G.J. Lis et al. / Micron 67 (2014) 141–148
Fig. 2. Merged representative SR-␮XRF point spectra obtained from normal area, early lesion (ls) and late lesion of the stenotic aortic valve. Counts are normalized to the
beam flux. XRF K␣, L␣ and K␤ (when indicated) lines of elements are presented.
Since the normal valves were collected from significantly
younger individuals than stenotic valves (49.5 ± 5.07 vs
68.58 ± 8.17; p = 0.001), we checked if lower calcium levels in
normal valves as compared to morphologically unaltered regions
of stenotic valves can be related to age differences. Indeed, calcium
content was positively correlated with age in both normal areas
of stenotic valves and in early lesions but not in late lesions. A
strong calcium/age correlation was also found when normal areas
of stenotic valves and nonstenotic valves were analyzed jointly
(Fig. 4a).
3.3. Lipids and elemental content in early lesions
The representative distribution of the studied elements in
early lesion is shown in Fig. 5. The ORO-positive lipid-rich areas
contained significantly more calcium, phosphorus, potassium,
Fig. 3. Relative concentrations of the examined elements in normal areas, early lesions and late lesions of degenerating aortic valves. Data presented as box-and-whisker
plots showing median, lower-upper quartile and outliers. [aU] – arbitrary unit, ls – lesions; *p < 0.05; **p < 0.01; ***p < 0.001 for the Kruskal–Wallis test followed by Dunn’s
post hoc test.
G.J. Lis et al. / Micron 67 (2014) 141–148
145
Fig. 4. Progression of aortic valve calcification with age and the involvement of lipids. (a) Comparison of calcium/age relation in normal areas, early lesions and late lesions.
ls means lesions; (b and c) comparison of calcium content/age relation in lipid-rich (ORO+) and lipid-free (ORO−) areas of early (b) and late (c) lesions; (d and e) changes
in proportion of calcium content in lipid-rich (ORO+) areas to calcium content in lipid-free (ORO−) areas of early (d) and late (e) lesions in relation to age. Points represent
median values. For each graph, Spearman’s rank correlation coefficient (r) and p values are specified.
copper and strontium as compared to ORO-negative, lipid-free
areas (Table 1). There was a strong correlation in calcium content between these areas (r = 0.84, p = 0.001). The accumulation of
lipids did not influence positive calcium/age correlation which was
observed in both, lipid-rich and lipid-free areas, although the progression of calcification was more substantial in the former ones
(Fig. 4b).
The other elements did not show significant correlation with age
except from strontium which was positively correlated with age in
lipid-free areas (r = 0.68, p = 0.02).
In both areas the calcium content was positively correlated
with phosphorus and strontium (lipid-rich: r = 0.74, p = 0.01 for
both elements; lipid-free: r = 0.73, p = 0.01 and r = 0.85, p = 0.0008,
respectively). Correlation of calcium with zinc and copper in lipidrich areas was close to significance (r = 0.6, p = 0.05 and r = 0.58,
p = 0.06 respectively).
3.4. Lipids and elemental content in late lesions
The representative distribution of elements in late lesion is
shown in Fig. 5. In such lesions, lipid-rich areas contained more
calcium, phosphorus, potassium and strontium (Table 1), but in
contrast to early lesions, calcium content in both, lipid-rich and
lipid-free areas did not correlate significantly with each other
(r = 0.49, p = 0.13). Likewise, positive calcium/age correlations seen
in both areas of early lesions were not found in late lesions (Fig. 4c).
Fig. 5. Corresponding areas of degenerating aortic valve showing localization of Oil Red O (ORO) stained lipids and relative concentrations of elements (SR-␮XRF maps) in
early lesion (upper panel) and late lesion (lower panel). *Nodular calcification in late lesion.
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G.J. Lis et al. / Micron 67 (2014) 141–148
Table 1
Distribution of elements in lipid-rich and lipid-free areas of early and late lesions.
Lipid-free areas
p value
Early lesions
2.18 [1.22–3.36]a
P
S
7.45 [6.77–9.22]
K
1.7 [1.02–4.67]
123.7 [79.9–174.0]
Ca
Fe
2.95 [1.9–4.32]
0.31 [0.22–0.43]
Cu
14.44 [10.25–15.66]
Zn
1.05 [0.63–1.49]
Sr
Lipid-rich areas
1.21 [0.7–1.45]
4.51 [3.17–11.05]
1.09 [0.48–2.15]
93.5 [66.1–121.8]
3.28 [2.28–10.05]
0.21 [0.11–0.39]
14.62 [6.65–16.85]
0.83 [0.65–0.95]
0.0001
0.1
0.02
0.007
0.2
0.01
0.8
0.03
Late lesions
P
S
K
Ca
Fe
Cu
Zn
Sr
1.86 [1.42–4.69]
7.70 [3.89–8.81]
1.77 [0.83–2.86]
173.6 [122.7–363.9]
5.69 [2.23–20.21]
0.31 [0.24–0.41]
19.39 [15.74–20.68]
1.03 [0.75–2.25]
0.009
0.8
0.04
0.006
0.9
0.1
0.2
0.001
5.62 [3.04–9.07]
5.58 [5.17–7.64]
4.71 [4.4–5.37]
524.4 [469.1–743.7]
3.97 [1.72–9.93]
0.23 [0.15–0.40]
20.33 [17.57–23.68]
3.41 [2.47–3.99]
a
Median [lower–upper quartile], arbitrary units, p value calculated for Wilcoxon
matched pairs test.
In both areas of late lesions, calcium content was positively
correlated with phosphorus and strontium (lipid-rich: r = 0.93,
p = 0.0001 for both elements, lipid-free: r = 0.79, p = 0.004; r = 0.97,
p < 0.0001 respectively). Additionally, in lipid-rich areas calcium
correlated positively with potassium, copper and zinc (r = 0.77,
p = 0.009; r = 0.79, p = 0.007 and r = 0.68, p = 0.03 respectively).
3.5. Calcification develops preferentially in lipid-rich areas
Wilcoxon matched pairs test employed for comparison of calcium concentration in lipid-rich and lipid-free areas of early and
late lesions showed not only higher concentration of calcium in
lipid-rich areas as compared to lipid-free ones (Table 1), but also a
significant increase in calcium content in lipid-rich regions of late
lesions as compared to lipid-rich regions of early lesions (524.4
[469.1–743.7] vs 123.7 [79.9–174.0], p = 0.004). Such increase was
not observed in lipid-free areas (173.6 [122.7–363.9] vs 93.5
[66.1–121.8], p = 0.2).
3.6. Involvement of lipids in calcification related to age is
different in early and late lesions
We analyzed the influence of age on the proportion of calcium
located in lipid-rich and lipid-free areas (Fig. 4d and e). In early
lesions, calcium concentration ratio was not significantly related
to age, while in late lesions it significantly increased with patients’
age (r = 0.38, p = 0.25) and (r = 0.69, p = 0.02, respectively).
4. Discussion
Our study has demonstrated that changes in valve histomorphology reflecting the successive stages of the calcific degeneration
are accompanied by substantial changes in the content of tissuebound elements in the altered valves. In early lesions, characterized
by valve thickening without macroscopically detectable calcification, SR-␮XRF revealed not only substantial accumulation of copper
and sulphur but also significant increase in calcium, reflecting the
initial phase of diffuse calcification observed microscopically. In
late lesions showing the presence of large focal mineral deposits, a
strong increase in the concentration of calcium, phosphorus, strontium as well as zinc was found.
Increased content of sulphur in early lesions can reflect
matrix remodeling leading to accumulation of highly sulphated
proteoglycans. Increased proteoglycan and decreased collagen content in early valve lesions were found in mouse model of aortic valve
disease (Krishnamurthy et al., 2012).
The increase in copper concentration in early lesions suggests
significance of this trace element in early valve matrix remodeling. Formation of late lesions, however, was not associated
with further accumulation of copper, suggesting a limited (if any)
role of this element in the progression of valve degeneration.
Decreased copper content in heavily calcified human aortic valves
as well as atherosclerotic lesions in cholesterol-fed rabbits was
found by others (Nystrom-Rosander et al., 2002; Rajendran et al.,
2007).
Since various forms of calcium phosphates including hydroxyapatites typical for bone are the predominant components of
cardiovascular calcifications, the observed increase in calcium and
phosphorus in late lesions can be expected. In both early and late
lesions, calcium and phosphorus colocalized with strontium and
zinc. Such colocalization was also observed in calcified atherosclerotic plaques of apoE/LDLR-double knockout mice (Gajda et al.,
2008). Strontium mimics calcium and can substitute this element
in the mineralization processes (Davis et al., 2000).
The role of zinc seems to be more complex. This essential trace
element is required for matrix mineralization in various tissues
(Kwun et al., 2010; Yusa et al., 2011). Zinc was demonstrated
to colocalize with calcifications in athersoclerotic arteries and its
involvement in early deposition of calcium salts was suggested
(Roijers et al., 2011). There are, however, also contradictory results,
showing decreased concentration of zinc in atherosclerotic lesions
(Minqin et al., 2003). We found that zinc correlates with calcification in lipid-rich areas. Zinc can be involved in prevention
of lipid peroxidation, since it is not only a structural component
of superoxide dismutase, but also competes with redox-active
iron and copper, indirectly limiting their peroxidative activity
(Bettger, 1993). On the other hand zinc is present in matrix metaloproteinases (MMPs), endopeptidases able to degrade all organic
components of extracellular matrix and proved to participate in
stenotic valves remodeling (Kaden et al., 2005; Soini et al., 2001). It
is thus possible that zinc in lipid-free areas is involved in different
processes, not related directly to calcification.
Macroscopically undetectable (diffuse) calcifications have been
demonstrated by various modalities in early lesions of aortic valves
(Aikawa et al., 2007a; Lis et al., 2003). This form of calcification has
the tendency to increase with age, even in healthy aortic valves
and aorta, as detected by compositional analyses based on a dryweight basis (Tohno et al., 2002; Ohnishi et al., 2003). Indeed,
we have demonstrated that in normal areas and early lesions of
stenotic valves calcium concentration increases with age and that
fine calcifications as well as lipids are present in early lesions, what
supports the notion that both calcifications and lipids are involved
in valve degeneration in the initial phase of the process (O‘Brien
et al., 1996; Otto et al., 1994). Lipids present on the surfaces of cells
and matrix components can act as calcium nucleators during calcification of aortic valves (Ortolani et al., 2010). Since in this study
higher amounts of calcium were found to colocalize with lipids in
both early and late lesions, our results suggest a pathophysiological link between lipid accumulation and calcification in human
aortic valves. This supports the lipid hypothesis of cardiovascular
calcification proposed by Demer (1997).
Since potassium is the main intracellular cation, its higher concentration in lipid-rich areas indicates that calcification progresses
in a milieu rich in cell debris (e.g. apoptotic vesicles) and/or matrix
vesicles, both influencing calcification (Jian et al., 2003; Kim, 1976;
Galeone et al., 2013). Highly oxidized lipids are cytotoxic for cells
(Colles et al., 2001; Gajda et al., 2008), although their impact critically depends on local pH and lipid concentration (Pfanzagl, 2006;
Brodeur et al., 2008).
G.J. Lis et al. / Micron 67 (2014) 141–148
We showed that in early lesions calcification increased with age
in both lipid-rich and lipid-free areas and that proportion of calcium
deposited in these areas was not significantly modified by age. This
may suggest that although in early phase of the disease lipids facilitate calcification, the process is regulated mainly by other factors,
such as inflammatory cells and their mediators (Otto et al., 1994;
Aikawa et al., 2007b). This thesis can be supported by reports on
the use of statins in the treatment of aortic stenosis. Their antiinflammatory activity seems to be independent of lipid-lowering
effect and indeed, promising results are expected rather in early
but not in advanced phase of the disease (Dimitrow and Jawien,
2010; Antonini-Canterin et al., 2008).
In late lesions, total calcium concentration as well as calcium
content in lipid-rich and lipid-free areas was not significantly
related to age, but proportion of calcium content in lipid-rich and
lipid-free areas significantly increased with age, pointing to a lesser
involvement of lipids in the calcification process in younger individuals. This is a seemingly confusing result, since age-dependent
increase in calcium proportion should be accompanied by a similar increase in calcium concentration, at least in lipid-rich areas. It
cannot be excluded that this discrepancy results from much higher
dispersion of calcium concentration values in advanced lesions,
requiring larger number of valve samples to achieve a reliable statistical evidence of the tendency for its increase with age, observed
in lipid-rich areas. Moreover, calcium in lipid-free areas did not
show such tendency at all (Fig. 4c).
These differences between early and late lesions suggest that
in advanced lesions either new players are involved or the tissue
response to factors acting in early lesions is substantially different.
The latter possibility can be supported by results of Aikawa et al.
(2007b) who demonstrated that in mouse model of atherosclerosis, calcification was positively correlated with inflammatory
macrophages in early lesions, but negatively in more advanced
ones. The role of lipids can also be different in early and late lesions.
Lipid oxidation products enhance formation of osteoclasts (Tintut
et al., 2004) which occur in stenotic valves (Nagy et al., 2013; Lis
et al., 2014) and are able to decalcify bone tissue. Hence, it cannot be excluded that the role of lipids in late lesions is bimodal: on
one hand they promote calcification, being important components
of the permissive matrix, on the other hand they may facilitate
mobilization of mechanisms inhibiting calcification.
In conclusion, we have demonstrated that aortic valve degeneration leading to formation of focal calcifications is associated with
significant changes in the elemental content and that calcifications
are preferentially located in lipid-rich areas. In early lesions, lipids
seem to be less intimately associated with the progression of calcification than in the late ones, where (especially in older patients)
lipid-rich areas accumulate much higher proportion of calcium.
This suggests that calcification mechanisms are modulated with
the progression of calcific aortic valve stenosis and with age.
Acknowledgements
The research program including this study has received funding from the European Commission Framework Programme 7
(FP7/2007–2013) project D-II-20100089 EC at HASYLAB, Hamburg,
Germany and was supported by Statutory grant (K/ZDS/000987 to
G.J.L.) from the Jagiellonian University Medical College.
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