© 2000 American Heart Association, Inc.
Integrative Physiology |
Apoptosis Regulates Human Vascular Calcification In Vitro
Evidence for Initiation of Vascular Calcification by Apoptotic Bodies
From the Department of Medicine, University of Cambridge, Addenbrooke’s Hospital (D.P., L.H., M.R.B., C.M.H., P.L.W) and Department of Anatomy, Multi-Imaging Centre (J.N.S.), Cambridge, UK.
Correspondence to D. Proudfoot, Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Level 6, ACCI Building, Hills Rd, Cambridge CB2 2QQ, UK. E-mail dp{at}mole.bio.cam.ac.uk
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Abstract—The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after
28 days. Apoptosis occurred before the onset of
calcification in VSMC nodules and was detected by several methods,
including nuclear morphology, the TUNEL technique, and external display
of phosphatidyl serine. Inhibition of apoptosis with the caspase
inhibitor ZVAD.fmk reduced calcification in nodules by 40%, as
measured by the cresolphthalein method and alizarin red staining. In
addition, when apoptosis was stimulated in nodular cultures with
anti-Fas IgM, there was a 10-fold increase in calcification.
Furthermore, incubation of VSMC-derived apoptotic bodies with
45Ca demonstrated that, like matrix
vesicles, they can concentrate calcium. These observations provide
evidence that apoptosis precedes VSMC calcification and that apoptotic
bodies derived from VSMCs may act as nucleating structures for calcium
crystal formation.
Key Words: calcification • vascular smooth muscle • apoptosis • apoptotic bodies
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Introduction |
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Intimal calcification is a common and early event in the pathogenesis of atherosclerosis. Calcification also occurs in the media of the vessel wall, particularly in aging, diabetes, and uremia. The presence of calcification in the coronary arteries correlates with an increased risk of myocardial infarction1 2 3 and predisposes to coronary dissection after angioplasty.4 Vascular calcification is also a potent predictor of future cardiovascular events in asymptomatic patients.5 The calcium deposits exist mainly in the form of calcium apatite, which is the type of mineral normally found in bone.6 Recently, the discovery that bone-associated genes can be expressed in the blood vessel wall strongly suggests that vascular calcification is an actively regulated process.7 8 9 However, the mechanisms that generate nucleation sites for calcium crystal formation in the vasculature have yet to be determined.
Matrix vesicles are thought to initiate calcification in forming bone and mineralizing cartilage.10 These are membrane-bound vesicles that are produced by budding from chondrocytes, osteoblasts, and odontoblasts and contain the necessary calcium-binding proteins and phosphatases for nucleation of hydroxyapatite. Matrix vesicle-like structures have also been found in calcified arteries and heart valves.11 12 Kockx et al13 showed that in advanced carotid atherosclerotic plaques, these structures were derived from vascular smooth muscle cells (VSMCs) and contained BAX protein, a proapoptotic member of the bcl-2 family, indicating that they may be remnants of apoptotic cells. It is thought that cell death may lead to matrix vesicle generation,10 14 and Hashimoto et al15 recently demonstrated that chondrocyte apoptotic bodies have similarities with matrix vesicles.
We have previously shown that human VSMCs spontaneously form
multicellular nodules and deposit calcium crystals after 28 days in
culture and that, by using electron microscopy, matrix vesicle-like
structures can be identified within the
nodules.16 In the present
study, we have established that apoptosis occurs in VSMC nodules and
have tested the hypothesis that apoptosis initiates calcification by
inhibiting VSMC apoptosis with the cell-permeable caspase-inhibitor
ZVAD.fmk. We demonstrate that inhibiting apoptosis also inhibits
calcification. In addition, stimulation of apoptosis with a combination
of anti-Fas IgM and cycloheximide increased nodule calcification.
Furthermore, we have shown that apoptotic bodies derived from cultured
human VSMCs can concentrate and crystallize calcium. These studies
provide experimental evidence for the first time, to our knowledge, to
show that apoptosis precedes calcification and that apoptotic bodies
are capable of initiating vascular
calcification.
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Materials and Methods |
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Cell Culture
VSMCs were prepared from medial explants of human aortic tissue and cultured in 20% FCS in M199 medium (Sigma) and formed multicellular nodules, as described previously.16
Induction of Apoptosis and Apoptotic Body
Preparation
Induction of apoptosis in VSMCs was achieved by
either addition of anti-Fas IgM (100ng/mL, clone CH11, Upstate
Biotechnology) and cycloheximide (10 µg/mL, Sigma) in serum-free M199
to primary human VSMCs for 24 hours or by serum starvation of a human
coronary plaque cell line HASMC 66 (human coronary artery smooth
muscle cell).17 Apoptotic
bodies (ABs) were harvested from these cultures by centrifugation of
cell supernatants at 2500 rpm.
Detection of Apoptosis
Nuclear Morphology
Nodules were cultured in 35-mm dishes in M199
containing 20% FCS and monitored live at 37°C. Bisbenzimide 2
µg/mL (final concentration, Hoechst No. 33258, Sigma) was added to
the medium, and the nodules were monitored by confocal microscopy
(Leica TCS-MP) so that the whole nodule could be imaged. Apoptotic
cells were quantified by counting fragmented or condensed nuclei by
capturing serial optical sections through the nodules. For this
analysis, the glow-over mode was used, because this gave the best
contrast between saturated and nonsaturated
fluorescence.
TUNEL Labeling
TUNEL labeling was performed on nodule sections
pretreated with citric acid, as described
previously.18
Phosphatidyl Serine Exposure
Nodules were treated as described above while being
monitored by time-lapse confocal microscopy. Annexin V-FITC (4 µL/mL,
Clontech) and propidium iodide (500 nmol/L, Clontech) were added to the
culture medium and monitored for initiation of annexin V
binding.
Detection of Calcification in Multicellular
Nodules
VSMCs were grown in 12-well plates fixed in 4%
formaldehyde in PBS for 45 minutes at 4°C. The cultures were then
washed in distilled water and exposed to alizarin red (2% aqueous,
Sigma) for 5 minutes and then washed again with distilled water.
Alternatively, the calcified material in each well was extracted from
nonfixed cell layers with 0.1 mol/L HCl overnight at room temperature
and quantified using
cresolphthalein.19 20
45Ca Accumulation
into ABs
To measure calcium accumulation in ABs, the method of
Hashimoto et al15 was used
and modified minimally. The calcifying reaction mixture contained
45Ca (50 000 cpm) and 40 µg of ABs,
and the samples were incubated at 37°C for 24 hours. The samples were
then centrifuged at 6500 rpm for 10 minutes, and washed pellets were
dissolved in 0.1 mol/L HCl and then placed in HiSafe scintillation
fluid. Disodium ATP (100% pure, Roche) and Nonidet P-40 (NP-40, Sigma)
were included in some experiments. A synthetic cartilage lymph
calcification buffer was also used in some
experiments.21
Energy Dispersive X-ray Microanalysis
Adherent ABs mounted on thermonox discs were rinsed
in distilled water, quench-frozen in melting propane, and freeze-dried.
Elemental content of ABs was performed by energy dispersive x-ray (EDX)
in a Philips XL30-FEG system equipped with an Oxford Instrument ISIS,
GEM spectrometer.
Calcium Detection by Confocal
Microscopy
ABs were cultured in 35-mm dishes and incubated with
calcein-AM (2 µmol/L, Molecular Probes) for 30 minutes. ABs were then
washed 3 times in serum-free medium, and green fluorescence was
examined using the confocal microscope.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Human VSMC Nodular Cultures
VSMCs cultured from explants of human aortic media formed the distinct hill- and valley-type of morphology, with cells retracting from some areas and grouping into multicellular aggregates or nodules (Figures 1A
and 1B). In cross section, nodules contained
concentric layers of cells arranged peripherally with many small cells
in the middle of the nodule
(Figure 1C). As nodules developed in culture, they contained
fewer cells, which were mainly located around the periphery of the
nodule with central areas of acellularity
(Figure 1D). Therefore, with time, cells were lost from the
center of the nodules.
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Detection of Apoptosis in Nodules
To find out whether apoptosis occurred in VSMC nodules,
several different methods were used. Stains including hematoxylin and
eosin (H&E) and Hoechst revealed many nuclei with condensed or
fragmented nuclei within nodules
(Figures 2A through 2C). The TUNEL method was also used, which
detects DNA breaks characteristic of apoptosis. Several nuclei stained
positively by this method
(Figure 2D), indicating that they are likely to have been
apoptotic. However, because TUNEL can potentially detect nonapoptotic
cells,22 23 we
also investigated exposure of phosphatidyl serine (PS) by cells in
nodules. Several PS-exposing cells were observed within and on the
periphery of the nodule, and many cells had already died, as indicated
by propidium iodide staining
(Figure 2F). In addition, in nodules monitored by time-lapse
video microscopy, we observed cells within nodules undergoing surface
membrane blebbing, characteristic of apoptosis (data not shown).
Collectively, these results confirm that many cells within the nodule
undergo apoptosis by day 7 of nodule culture.
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We previously observed that calcification was not detected
in nodules until day 28 of
culture.16 Because apoptosis
was evident before the onset of calcification, we investigated the
relationship between apoptosis and calcification by estimating the
apoptotic indices in nodules over the 28-day culture. To obtain an
accurate measure of the whole nodule apoptotic events, nodules were
optically sectioned using the confocal microscope. Apoptotic cells were
visualized using Hoechst nuclear morphology, ie, fragmented or
condensed nuclei were counted and an index was generated as the
percentage of apoptotic cells compared with total nodular cell number.
This analysis showed that the apoptotic index did not significantly
change between days 7 and 21 but increased at day 28
(Figure 3). Therefore, apoptosis occurs before calcium
crystals are deposited, and the higher rates of apoptosis coincide with
the onset of calcification.
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The apoptotic indices measured in nodules at various time
points were relatively high (>20%), which would predict that older
nodules would eventually become acellular. However, nodules cultured
for 8 weeks still contained viable cells, mainly peripherally
(Figure 1D
). By observing nodules with time-lapse
videomicroscopy, we found that nodules recruit cells from the
surrounding monolayer (data not shown). Therefore,
although the rate of apoptosis in nodules is relatively high, migration
of VSMCs into nodules accounts for the maintenance of
cellularity.
Effect of ZVAD.fmk on Calcification
To confirm that the caspase inhibitor ZVAD.fmk had an
antiapoptotic effect in VSMC nodules, its effects on nodule apoptosis
were measured using Hoechst, as in Figure 3. ZVAD.fmk (100 µmol/L)
decreased the apoptotic index in day-7 nodules, from 39.1±7.5% in the
control nodules to 28.0±9.6% in treated nodules (n=6,
P=0.03). Examples of optical
sections of nodules treated with or without ZVAD.fmk are shown in
Figure 4. To test the possibility that ZVAD.fmk affected
cell proliferation, Ki-67 staining was performed on nodule sections,
but no difference was seen between control and ZVAD.fmk-treated groups
(see the online data supplement, available at
http://www.circresaha.org.). Also, we did not observe an effect of
ZVAD.fmk on necrotic cell death in VSMCs (data not shown).
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After verification of the antiapoptotic effects of ZVAD.fmk
on nodule apoptosis, ZVAD.fmk (100 µmol/L) was added throughout the
28-day culture period, and its effects on subsequent calcification were
measured. Treatment with ZVAD.fmk reduced the amount of calcification
in nodular cultures, as assessed by alizarin red staining
(Figures 5A and 5B) as well as calcium content in the nodular
cultures
(Figure 5C). It is important to note that the presence of
ZVAD.fmk had no effect on the total number of nodules. Therefore,
inhibition of apoptosis in nodules with ZVAD.fmk reduced the resulting
nodule calcification.
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Effect of Enhancing Apoptosis in
Nodules
The role of apoptosis was investigated additionally by
exposure of the nodular cultures to apoptotic stimuli: a combination of
anti-Fas IgM and cycloheximide. Treatment of VSMC nodular cultures with
anti-Fas IgM and cycloheximide significantly increased the total amount
of calcium deposited in cultures by 10-fold
(Figure 6).
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Accumulation of Calcium by VSMC-Derived
ABs
To find out whether ABs derived from VSMCs could
accumulate calcium in a similar manner to chondrocyte ABs and matrix
vesicles,15 HASMC 66 cells
and primary VSMCs were used to generate ABs
(Figure 7A). When VSMC-derived ABs were incubated in
calcifying medium, they accumulated 45Ca
from solution
(Figure 7B).45Ca accumulation was
not stimulated by ATP (1 mmol/L), but ABs pretreated with the detergent
NP-40 to permeabilize the AB membrane failed to accumulate
45Ca
(Figure 7B). These studies showed that ABs can concentrate
45Ca by a mechanism that requires an intact
AB membrane.
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Elemental Analysis of VSMC ABs
To investigate the type of calcium deposited in ABs,
ABs were incubated in calcifying medium for 24 hours at 37°C and
analyzed for their elemental content by EDX
(Figures 8A and 8B). In both preparations there was a large
signal for Ca2+, confirming that ABs
concentrate calcium, but a low signal for phosphate. The backscattered
image suggests that the calcium is in a concentrated, crystallized form
and, from the elemental profile, is most likely to consist of calcium
carbonate. Thus, VSMC-derived ABs are capable of concentrating and
crystallizing calcium, which is distributed throughout the AB with
occasional voids
(Figure 8C).
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Association of Apoptosis With Calcification
The link between cell death and vascular calcification has been noted in pathological studies.14 24 In this study, we provide experimental evidence that implicates a role for apoptosis in the initiation of vascular calcification.
The occurrence of apoptosis in our in vitro calcification
model was confirmed by several methods, including ultrastructural
characteristics, TUNEL, and PS exposure in VSMC nodular cells. At the
day-7 stage of nodule development, there was a mixture of apoptotic
cells, viable cells, and cells with damaged cell membranes. The
propidium iodide–positive cells at this stage may have been the result
of apoptosis (ie, secondary necrosis) or primary necrosis within the
nodules. Apoptosis occurred early in VSMC nodules, but we have
previously shown that calcium crystal deposition is not detected until
day 28 by von Kossa staining or
EDX.16 At the 28-day time
point, the apoptotic index increased, coinciding with detection of
calcification. These observations show that apoptosis precedes the
onset of calcium crystal formation but that if apoptosis initiates
calcification, its effects are delayed in early nodules, which would
imply that the calcification process in nodules is regulated. Possible
calcification-limiting factors produced by VSMCs in the nodule are
mineralization-regulating proteins, such as matrix Gla
protein.16 Another
possibility is that if ABs are the initiators of calcification, the
cells within the early nodules would be expected to recognize and
phagocytose the ABs.25 Older
nodules may contain less-efficient phagocytes, allowing the ABs to
stimulate calcium crystal growth. In support of a role for apoptosis in
calcification, other studies in cultured cells have shown associations
with apoptosis and calcification. Apoptosis occurred in cultures of
chick embryonal limb bud mesenchymal cells, which were used as a model
of chondrocyte
differentiation,26 and Lynch
et al27 have shown that
apoptosis is an integral part of osteoblast differentiation and
calcification in fetal rat calvarial osteoblast cultures.
To test whether apoptosis was actually required for calcification to occur, apoptosis was inhibited in nodules by the caspase inhibitor ZVAD.fmk. Alizarin red staining and calcium quantitation clearly showed that ZVAD.fmk inhibited calcification in VSMC nodules. ZVAD.fmk is a broad-spectrum inhibitor of caspases, and we confirmed its antiapoptotic effects in VSMC nodules. Other studies28 have shown that the mechanism of action of ZVAD.fmk is to prevent completion of the apoptotic program, which may not involve a delay in the onset of apoptosis. Caspase inhibition can also inhibit the release of ABs from cells.29 The role of apoptosis in our in vitro calcification model was then additionally examined by stimulating apoptosis in nodules with anti-Fas IgM and cycloheximide. This treatment resulted in a 10-fold stimulation of calcification, which strongly supports the role of apoptosis in calcification.
Role of VSMC-Derived ABs in Initiating
Calcification
Because a lack of clearance of ABs was a potential
mechanism of induction of calcification in VSMC nodules, we were
tempted to speculate that ABs derived from VSMCs could initiate
calcification in a similar manner to chondrocyte matrix vesicles or
ABs.15 We demonstrated that
VSMC-derived ABs accumulated calcium via a mechanism that involved an
intact AB membrane, because when it was permeabilized with NP-40, no
calcium accumulation was observed. The induction of calcium
accumulation in chondrocyte-derived matrix vesicles and ABs was
dependent on the presence of
ATP.15 However, ATP had no
effect on VSMC AB calcium uptake. In fact, the role of ATP in
calcification is not clear, because in different studies, ATP had
stimulatory effects on
calcification,15 was not
necessary for matrix vesicle
calcification,21 or
inhibited calcification.30
The lack of dependency on ATP for calcium uptake into VSMC ABs suggests
the following: calcium is taken up into VSMC ABs by an ion channel or
calcium-binding protein that does not require ATP for its activity; ABs
may contain sufficient ATP to accumulate calcium, and by adding
exogenous ATP, there is no additive effect; and VSMC ABs may not
contain the necessary enzymes for ATP hydrolysis, which are present in
matrix vesicles.31 PS
exposure by ABs generates a potential calcium-binding
site32 as well as a membrane
surface suitable for hydroxyapatite
deposition.33 However,
confocal images of cross sections of ABs loaded with calcein suggested
that calcium was concentrated throughout the AB rather than bound at
the membrane.
Elemental analysis of the bodies in calcifying solutions
revealed that they contained abundant calcium but very low phosphate.
The EDX spectra suggested that calcium carbonate may be the form of
calcium present in ABs. Calcium carbonate comprises 9% of the total
calcium crystals in human atherosclerotic
lesions,7 and carbonate
apatite is found in bone as well as ectopic
calcification.34 Calcium
carbonate can also act as a precursor to carbonate apatite formation
under certain conditions.35
These observations suggest that VSMC-derived ABs can concentrate and
crystallize calcium in a form that is found in vivo. Therefore, VSMC
ABs have similarities with chondrocyte-derived matrix vesicles but
produce a different type of calcium crystal in vitro. This may be
attributable to differences in in vitro culture conditions or perhaps
to intrinsic differences in protein expression.
It is interesting to note that not all chondrocyte matrix vesicles calcify, only those at specific sites in the cartilage matrix.21 Matrix vesicles in vitro will only calcify if they are preincubated in ascorbate- and phosphate-rich medium, which generates matrix vesicles enriched with annexin V (which can act as a Ca2+ channel) and alkaline phosphatase.21 These observations suggest that matrix vesicles are not all equivalent and that only tissues normally engaged in mineralization produce mineralization-competent vesicles. However, it is also possible that nonmineralizing tissues produce inhibitors of matrix vesicle function to block mineralization.21 Therefore, one can hypothesize that a lack of production of inhibitors of matrix vesicle calcification may lead to the development of pathological calcification.
Relevance of Apoptosis to Calcification in
Disease
Some studies have implied that ABs in atherosclerotic
plaques are similar to matrix vesicles and that these may initiate
calcification.23 24 36
In an electron microscopic study by
Stary,37 atherosclerotic
plaques were described as containing lipid-laden VSMCs, which shed
calcifying membrane-bound vesicles. In addition, although calcification
in atherosclerosis has been detected mainly in association with
extracellular structures such as matrix vesicles and extracellular
matrix, intracellular calcification has also been
observed.12 37
These may be calcified cell organelles or calcified structures that
have been engulfed by VSMCs.
If the matrix vesicle-like structures in plaques are apoptotic remnants, they should be rapidly cleared by adjacent phagocytic macrophages or VSMCs. In the largely acellular lipid core, phagocytosis may be impaired because of the presence of oxidized lipids that have been shown to compete with ABs for binding to phagocytes.38 Nonphagocytosed ABs would either undergo secondary necrosis or could proceed to calcify, depending on their local environment. Therefore, the presence of efficient phagocytic cells in atherosclerotic lesions is important for effective scavenging and, thereby, regulation of calcium deposition. This concept was tested experimentally by Kim,39 who demonstrated that rat aortic segments calcified when placed in Millipore chambers in the peritoneal cavity, but when aortic segments were grafted and contained inflammatory cells, calcification was minimal. Finally, in support of the role of apoptosis in calcification in vivo, mice lacking matrix Gla protein or osteoprotegerin develop medial vascular calcification and both proteins have potential roles in apoptosis.40 41
In summary, we have shown that apoptosis precedes human vascular calcification in vitro and that VSMC-derived ABs can concentrate and crystallize calcium. Therefore, what remains to be tested is whether a lack of phagocytosis of ABs leads to the progression of vascular calcification.
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Acknowledgments |
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This work was supported by a British Heart Foundation program grant. Confocal microscopy and EDX were carried out in the Multi-Imaging Center in Cambridge, UK, which was established with funding from the Wellcome Trust (grant 055203/Z/98).
Received July 5, 2000; revision received October 18, 2000; accepted October 18, 2000.
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