© 1995 American Heart Association, Inc.
Articles |
ß-Glycerophosphate Accelerates Calcification in Cultured Bovine Vascular Smooth Muscle Cells
From the Second Department of Internal Medicine, Osaka City University Medical School, Japan.
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Abstract |
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Abstract Calcification is a common feature of advanced atherosclerotic lesions and is being reemphasized as a clinically significant element of vascular disease. However, the scarcity of in vitro models of vascular calcification preclude studying its molecular and cellular mechanism. In the present study, we describe an in vitro calcification system in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells (BVSMC) in the presence of ß-glycerophosphate, ascorbic acid, and insulin in a manner analogous to in vitro mineralization by osteoblasts. Calcification was confirmed by von Kossa staining and 45Ca accumulation. Factor analysis revealed that ß-glycerophosphate is the most important factor for this calcification process, suggesting that alkaline phosphatase (ALP) may be involved. As predicted, high levels of ALP expression were detected by ALP assay and Northern blot analysis. Functional significance of ALP was confirmed by demonstrating that levamisole, a specific inhibitor of ALP, inhibited BVSMC calcification in a dose-dependent manner. Bisphosphonates such as etidronate and pamidronate potently inhibited BVSMC calcification, suggesting that hydroxyapatite formation may be involved. Importantly, expression of osteopontin mRNA was dramatically increased in calcified BVSMC compared with uncalcified control cells. These data suggest that ß-glycerophosphate can induce diffuse calcification by an ALP–dependent mechanism and that this in vitro calcification system is useful for analyzing the molecular and cellular mechanisms of vascular calcification.
Key Words: bovine vascular smooth muscle cells • ß-glycerophosphate • alkaline phosphatase • vascular calcification • atherosclerosis
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Introduction |
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Calcification is a common feature in advanced atherosclerotic plaque lesions.1 Calcified arterial walls are unable to dilate or contract in response to physiological stimuli, resulting in regulatory disturbance of blood flow. Coronary artery calcification is frequently detected in patients with myocardial infarction,2 3 and its occurrence in asymptomatic young men increases the risk of clinically significant coronary artery disease.4 Moreover, it is associated significantly with arterial dissection during angioplasty.5 6 Therefore, calcification is thought to be not only an indicator of coronary artery disease but also a predictor of adverse outcomes after coronary artery intervention.
Although arterial calcification was previously thought to be a degenerative process that occurs after atherosclerosis,1 there is considerable evidence suggesting that calcification associated with atherosclerosis is an organized, regulated process similar to mineralization in bone tissue.7 Matrix vesicles, the nucleation sites for formation of hydroxyapatite, which is involved in cartilage and bone mineralization, were detected in calcified atherosclerotic lesions.8 9 BMP-2a, a potent factor for osteoblastic differentiation, was found to be expressed in the calcified lesions.10 Additionally, Gla–containing proteins such as osteocalcin and matrix Gla protein were demonstrated in advanced atherosclerotic lesions.11 12 13 14 15 Recently, several groups have reported that osteopontin, a phosphoprotein that has calcium-binding capacity, is expressed in calcified human atherosclerotic plaques and cultured VSMC.13 16 17 18 19 20 However, the molecular and cellular mechanisms of arterial calcification remain unclear. Moreover, there have been few in vitro experimental models in which the mechanism of calcification can be analyzed.21
Recently, Watson et al22 reported that BVSMC spontaneously calcify their extracellular matrix in a nodular pattern and that TGF-ß1 and 25-hydroxycholesterol stimulate this process. Additionally, they22 cloned a subpopulation of BVSMC called calcifying vascular cells, which have osteoblastic characteristics. Although these findings are novel in a qualitative sense, the rate of calcification in this system is still slow and not diffuse. Given these circumstances, we tried to establish an in vitro experimental system in which diffuse calcification, enabling biochemical studies, can be induced more easily.
In the present study, we describe an in vitro calcification system in which diffuse calcification can be induced by culturing BVSMC in the presence of ß-glycerophosphate, ascorbic acid, and insulin, analogous to in vitro mineralization by osteoblasts.23 24 Calcification of BVSMC was confirmed by von Kossa staining and 45Ca accumulation. We demonstrate that BVSMC abundantly express ALP and that l-tetramisole (levamisole), a specific inhibitor of ALP,25 inhibits this calcification in a dose-dependent manner. By using 45CaCl2, we also demonstrate that bisphosphonates such as etidronate (EHDP) and pamidronate (APD) inhibit BVSMC calcification. Importantly, we found that osteopontin mRNA is expressed exclusively in calcified BVSMC as compared with uncalcified control cells. These results suggest that cultured BVSMC with high levels of ALP expression have a capacity to calcify their extracellular matrix and that this in vitro calcification system is useful for analyzing the molecular and cellular mechanism of VSMC calcification.
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Methods |
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Reagents
ß-Glycerophosphate, porcine insulin, and ascorbic acid were obtained from Sigma Chemical Co. EHDP was kindly provided by Sumitomo Pharmaceuticals Co, Ltd, and APD was kindly provided by Ciba-Geigy, Ltd. l-Tetramisole (levamisole) was purchased from Sigma Chemical Co. Unless otherwise stated, all other reagents were obtained from Wako Pure Chemical Industries, Ltd.
Cell Culture
BVSMC were obtained by an explant method originally described by
Ross.26 Briefly, medial tissue was separated from segments
of bovine aorta. Small pieces of tissue (1 to 2 mm3) were
placed in a 10-cm culture dish and cultured for several weeks in DMEM
containing 4.5 g/L of glucose supplemented with 15% FBS (GIBCO) and 10
mmol/L sodium pyruvate (GIBCO) at 37°C in a humidified atmosphere
containing 5% CO2. Cells that had migrated from the
explants were collected and maintained in DMEM containing 15% FBS
supplemented with 10 mmol/L sodium pyruvate. For calcification
experiments, the cells up to passage 8 were used.
In Vitro Calcification of BVSMC
BVSMC were cultured in DMEM containing 10 mmol/L sodium pyruvate
supplemented with 15% FBS (growing medium). After confluence, the
cells were inoculated in DMEM containing 10 mmol/L sodium pyruvate
supplemented with 15% FBS in the presence of 10 mmol/L
ß-glycerophosphate, 10–7 mol/L
insulin, and 50 µg/mL of ascorbic acid for 14 days (calcification
medium). The medium was replaced with fresh medium every 3 days. In
certain experiments, only ß-glycerophosphate was used to induce
BVSMC calcification. Moreover, in the time-course experiments,
before being cultured in calcification medium (defined as day 0), cells
were selected to be used as controls.
Cytochemical Staining for ALP and von Kossa Staining
The expression of ALP was visualized by incubating
formalin-fixed cells at 37°C for 30 minutes with 0.1 mol/L
Tris-HCl (pH 8.5) containing 2 mg/mL of disodium naphthol AS-BI
phosphate (Sigma Chemical Co) and 0.6 mg/mL of fast violet B salt
(Sigma Chemical Co). Mineral deposition was assessed by von Kossa
staining (30 minutes, 5% silver nitrate).
Assay of ALP Activity
After the cells were washed three times with PBS,
cellular proteins were solubilized with 1% Triton X-100 in 0.9% NaCl
and centrifuged, and the supernatants were assayed for ALP
activity as described previously.27 One unit was defined
as the activity producing 1 nmol of p-nitrophenol for 30
minutes. Protein concentrations were determined by Bradford's
method.28
Immunofluorescence Microscopy and
Immunoblot Analysis of 
-Actin
Expression
For immunofluorescence microscopy, cells
were cultured on coverslips (15 mm in diameter) (Matsunami) for 4 days,
fixed with 4% formalin in PBS, treated for 60 minutes at 25°C with
monoclonal anti--smooth muscle actin antibody (1A4) (Sigma
Chemical Co) diluted with PBS 1:400, and then stained for 30 minutes
with FITC–conjugated rabbit anti-mouse IgG polyclonal antibody
(Dako Japan) diluted with PBS 1:40. Mouse nonimmune IgG was used as a
control for the primary antibody. For immunoblot
analysis, cellular protein was solubilized in 1% Triton X-100
in PBS containing 1 mmol/L PMSF and microcentrifuged.
Solubilized proteins were electrophoresed on 10% sodium
dodecyl sulfate polyacrylamide gel electrophoresis
under reducing conditions, then transferred by electrophoresis
to polyvinylidene difluoride membrane (Millipore Corp).
Membranes were blocked with blocking buffer (50 mmol/L Tris-HCl, 500
mmol/L NaCl, 10 mg/mL bovine serum albumin, and 0.1% Tween 20)
for 30 minutes at 25°C, washed, and incubated with 1A4 at 1:500
dilution for 60 minutes. After washing, blots were incubated with
horseradish peroxidase–conjugated sheep anti-mouse IgG
antibody (Amersham) at 1:1000 dilution, washed, and visualized by use
of ECL detection reagents (Amersham) according to the manufacturer's
instructions.
Calcification Assay
Calcification was assessed by 45Ca accumulation in
the cell layer as described previously,29 with slight
modifications. Cells were incubated in calcification medium containing
0.5 µCi/mL of 45CaCl2 (1 µCi per well) for
48 hours at specific times in 6-well plates. To examine the effects of
various agents on calcification, agents were added at the beginning of
the assay. After a 48-hour incubation period with
45CaCl2, the medium was removed and
the cell layer was washed five times with PBS, scraped into
borosilicate tubes containing 0.5 mL of perchloric acid, and spun
vigorously. Then, 0.5 mL of hydrogen peroxide was added and the
suspensions were incubated for 60 minutes at 80°C. After incubation,
the mixture was dissolved in 1.0 mL of ethylene glycol monoethyl ether
and spun vigorously, and radioactivity was measured by liquid
scintillation counting with 10 mL of ACS-II (Amersham).
Preparation of cDNA Probes
Bovine osteopontin cDNA probe containing a 416-base pair
fragment (corresponding to 265 to 680 in the coding
region)30 was obtained by reverse transcription of an mRNA
from BVSMC, followed by polymerase chain reaction and subcloning into
TA cloning vector (Invitrogen). Sequences of the obtained cDNA were
confirmed by dideoxy sequencing method. Human ALP
(liver/bone/kidney–type) cDNA probe was obtained from Japanese
Cancer Research Resources Bank.31
RNA Isolation and Northern Hybridization
Total RNA was isolated from BVSMC by extraction with acid
guanidium thiocyanate-phenol-chloroform. Twenty micrograms of
total RNA were electrophoresed on 1% agarose gels containing
formaldehyde and transferred to a nylon filter (Hybond N, Amersham).
Blots were prehybridized at 37°C for 24 hours in a buffer containing
50% formamide, 3x SSC (1x SSC; 0.15 mol/L NaCl and 0.015 mol/L
sodium citrate, pH 7.4), 50 mmol/L Tris-HCl (pH 7.5), 0.1% sodium
dodecyl sulfate, 20 µg/mL tRNA, and 1x Denhardt's solution
and then hybridized at 37°C for 48 hours with cDNA probes for human
ALP or bovine osteopontin, which were labeled with
[-32P]dCTP (3000 Ci/mL; New England Nuclear) by use of
a random priming method (Megaprime cDNA labeling system, Amersham).
Blots were washed and autoradiographed with x-ray film at
-70°C. The amounts of RNA were quantified by densitometric
scanning and normalized by comparison with GAPDH.32
Statistics
In certain experiments, data were analyzed for
statistical significance by ANOVA with post hoc analysis,
unless otherwise stated. These analyses were performed with the
assistance of a computer program (SUPERANOVA version 1.1,
Abacus Concepts).
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Results |
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To confirm the obtained cells were VSMC, the expression of
-smooth muscle actin in BVSMC was examined by
immunofluorescence. -Smooth muscle actin was
stained in a filamentous manner in all of the BVSMC (data not shown),
indicating that the cells preserve the character of vascular smooth
muscle. By culturing BVSMC in calcification medium, granular deposits
were developed in the cell layer within a few days (Fig 1a
). These deposits coalesced into
nodules, finally resulting in diffuse calcification of cell layer by
the 14th day (Fig 1b), whereas in the control culture,
no deposits were found during the culture period (data not shown).
Calcification was confirmed by von Kossa staining (Fig 1c). Black-stained particles were diffusely
scattered throughout the cell layer. Nodular calcification was also
encountered as described by Watson et al.22 To further
confirm BVSMC calcification and facilitate its quantitative
analysis, we used a calcification assay to evaluate calcium
accumulation into the cell layer by using
45CaCl2. We first examined the time course of
calcium deposition induced by calcification medium (Fig 2). Calcium deposition was initiated just
after culturing in calcification medium. Calcium deposition rate peaked
after 72 hours and then decreased, suggesting that it is necessary to
change the medium every 3 days to achieve extensive calcification.
Maximum percent incorporation of total added radioactivity was 52.2%.
In the control culture, on the other hand, only 0.2% was incorporated
into the cell layer.
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We next examined which factors of calcification medium are necessary in
BVSMC calcification, using the calcification assay (Table 1). Factor analysis revealed that
ß-glycerophosphate was most important for inducing calcium
deposition. In the absence of ß-glycerophosphate, no
significant calcification was induced. Additionally, ascorbic acid and
insulin may also affect calcification irrespective of the presence of
ß-glycerophosphate. In the following studies, we used only
ß-glycerophosphate to induce calcification.
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The important role of ß-glycerophosphate in calcification
suggests that ALP may be involved in the process. To prove this
hypothesis, we examined the expression of ALP in BVSMC. Calcified BVSMC
were positively stained with cytochemical staining for ALP, and high
levels of ALP expression were detected around calcified deposits (Fig 1d
). To further confirm high levels of ALP expression
in BVSMC and its relevance to calcification, we measured ALP activity
during the calcification process. After initiation of calcification,
ALP activity was increased in a time-dependent manner and slightly
decreased late in the process (Fig 3).
Gene expression of ALP (liver/kidney/bone-type) in BVSMC was also
confirmed by Northern blot analysis (Fig 4). As a positive control, human
osteoblast-like cells (SaOS2), which are known to express ALP at
high levels, were used.33 On the other hand, no band was
detected in rat fetal VSMC (A7r5), which seem to have the synthetic
phenotype.34 The level of mRNA expression was also
increased after initiation of calcification and was decreased late in
the process (Fig 5). Since it is known
that ß-glycerophosphate increases the expression of ALP in
osteoblasts to some degree,35 36 it is likely that the
induction of ALP in BVSMC may be a direct effect of
ß-glycerophosphate. Therefore, we monitored levels of gene
expression and activity in the absence of ß-glycerophosphate as
uncalcified controls. Even in the absence of ß-glycerophosphate,
the induction of ALP was observed to the same degree as in its presence
(data not shown). Thus, this induction is thought to be a
time-dependent phenomenon such as that observed in the
differentiation process of osteoblasts.37
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To clarify the functional significance of ALP expression in BVSMC
calcification, we examined the effect of levamisole, a specific
inhibitor of ALP,25 on BVSMC calcification.
Levamisole inhibited calcification in a dose-dependent manner (Fig 6), and its potency was confirmed by its
inhibitory effect on ALP activity (data not shown). The
median inhibitory dose (ID50) for
calcification was almost the same as that for ALP activity
(ID50=10–5 mol/L). In
addition, since BVSMC lost their calcifying capacity by repeated
passages, we compared ALP activities between early- and
late-passaged cells. The early-passaged cells (passage No. 6)
actively calcified their extracellular matrix, and the level of ALP
activity was high, whereas the late-passaged cells (passage No. 13)
did not calcify, and the level of ALP was as low as that of mouse bone
marrow stromal cells (ST2) (39.3±13.2 versus 45.3±8.5 U/mg protein,
mean±SEM), which do not calcify even in the presence of
ß-glycerophosphate (Fig 7). These
results suggest that a high level of ALP expression in BVSMC is
necessary in the calcification process.
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Because it has been reported that hydroxyapatite formation may be
involved in vascular calcification, we examined the effects of
bisphosphonates (etidronate [EHDP] and pamidronate [APD]),
inhibitors of hydroxyapatite formation, on in vitro BVSMC
calcification. EHDP inhibited calcium accumulation in a
dose-dependent manner (Fig 8),
and maximum inhibition (95.2%) was achieved at a dose of 100 µg/mL.
APD also inhibited BVSMC calcification (74% inhibition) at a dose of
10 µg/mL (Table 2). These data suggest
that hydroxyapatite formation may be involved in calcification.
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Because it is possible that during the calcification process, BVSMC
could lose their vascular smooth muscle characteristics and acquire a
calcifying capacity, we examined the expression of -smooth
muscle actin in BVSMC by immunoblot. During this process,
BVSMC still expressed -smooth muscle actin, and the level of
expression was rather increased as calcification progressed (Fig 9a), suggesting that the nature of
vascular smooth muscle is preserved during the process. Furthermore,
because it has been suggested that osteopontin may be involved in
vascular calcification, we examined the expression of osteopontin in
uncalcified and calcified BVSMC. After confluence, BVSMC were cultured
in the absence or presence of ß-glycerophosphate for 14 days, and
gene expression of osteopontin was examined by Northern blot
analysis. Osteopontin mRNA was exclusively expressed in
calcified BVSMC compared with uncalcified control (Fig 9b). This result suggests that the expression of
osteopontin is regulated in this calcification system and that
osteopontin may contribute to vascular calcification.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present study, we have shown that ß-glycerophosphate accelerates in vitro calcification by BVSMC and induces extensive calcium deposition (Fig 1a
and 1c) in a manner analogous to in vitro mineralization by
osteoblasts.23 24 Because it is thought that
ß-glycerophosphate is a substrate for ALP that is degraded,
releasing inorganic phosphate to raise the local concentration of
phosphorus,23 38 accelerated calcification may be caused
by a high level of ALP expression in BVSMC in the presence of
ß-glycerophosphate. In this context, we have demonstrated its
high level of expression and functional relevance to BVSMC
calcification (Figs 3, 4, 5, 6, and 7).
The capacity of VSMC to calcify their extracellular matrix in vitro was
demonstrated by Watson et al.22 They cloned calcifying
vascular cells that can form calcified nodules. However, it took more
than 14 days for the cells to calcify, calcification was localized only
in the nodules, and the number of nodules was very low. These problems
preclude the investigation of the molecular mechanism of calcification.
In our system, diffuse calcification was induced by
ß-glycerophosphate within 14 days, which is based on high levels
of ALP expression (Fig 2). Moreover, it is possible to
examine the effects of various agents such as bisphosphonates on BVSMC
calcification by quantifying calcium deposition (Fig 8),
suggesting that this model system is suitable for analyzing the
mechanism of vascular calcification.
Vascular calcification, especially coronary artery calcification, is being reemphasized as a clinically significant element of vascular disease.4 5 6 Moreover, it has been proposed that vascular calcification is an organized and regulated process instead of a passive precipitation or adsorption.7 Putative regulators of vascular calcification have been suggested: Gla-containing proteins such as osteocalcin11 12 15 and matrix Gla protein,13 14 osteopontin,13 16 17 18 19 20 and BMP-2a.10 These proteins are expressed exclusively in calcified atherosclerotic lesions. However, regulation of the expression of these proteins in relation to calcification is not fully understood.
We demonstrated for the first time that in vitro expression of osteopontin is dramatically increased in calcified BVSMC in contrast to uncalcified control, which is consistent with previously reported in vivo data.14 16 17 18 19 20 Recent investigations revealed that the expression of osteopontin in VSMC is strongly associated with cell proliferation39 and that growth factors for VSMC such as TGF-ß, angiotensin II, and basic fibroblast growth factor can increase its expression.16 Since it is likely that long-term culturing up to 14 days after confluence may decrease the growth capacity of BVSMC, the expression of osteopontin is assumed to be decreased, as is seen in uncalcified BVSMC culture. However, expression in calcified BVSMC was dramatically increased. This finding suggests that the expression of osteopontin is directly correlated with calcification. Detailed studies should be performed to confirm this hypothesis. Recently, it has been reported that dephosphorylation of osteopontin by osteoclastic tartrate-resistant acid phosphatase may modulate its adhesive character to osteoclasts,40 suggesting that the extent of phosphorylation of osteopontin may affect its adhesive function. Because it has been suggested that ALP may dephosphorylate osteopontin,41 it is also possible that the calcium-binding capacity of osteopontin may be modulated by ALP.
ALP is believed to be one of the phenotypic markers of osteoblasts and is indispensable for bone mineralization.42 Although possible roles of ALP in bone mineralization have been proposed, it is still unclear how ALP functions in calcification, even in bone tissue. It is true that ß-glycerophosphate enhances in vitro mineralization by osteoblasts, but this effect is not specific for osteoblasts. Rather, it simply reflects the presence of ALP.43 Furthermore, even in the absence of ß-glycerophosphate, certain osteoblast-like cells can actively calcify their extracellular matrix.44 Additionally, the actual substrate for ALP has not been identified.45 Therefore, BVSMC calcification in the presence of ß-glycerophosphate may simply represent high levels of ALP expression in the cells. In human atherosclerotic lesions, the existence of ALP was cytochemically demonstrated in matrix vesicles.9 However, its functional significance in calcification is not clear. How the expression of ALP is regulated in VSMC and in vivo atherosclerotic lesions remains to be clarified.
Although ß-glycerophosphate can increase ALP activity in
osteoblasts,35 the time-dependent induction of ALP
expression in BVSMC is thought to be correlated with increased cell
density irrespective of its presence. The inverse relation between the
level of ALP activity and proliferative capacity was reported in
various cells, including osteoblasts.36 46 Moreover,
passage-dependent expression of ALP in BVSMC as demonstrated in Fig 7 is compatible with this relationship, because it is
possible to assume that late-passaged cells (with a low level of
ALP activity) have a greater capacity to proliferate, whereas
early-passaged cells (with a high level of ALP activity) grow more
slowly. This interpretation also seems applicable to the relationship
between atherosclerosis and vascular calcification.
Early in the formation of atherosclerotic lesions, VSMC with synthetic
phenotype actively proliferate and secrete extracellular
matrix, whereas late in the formation of these lesions, VSMC become
quiescent and may undergo "redifferentiation" into the cells with
osteoblastic phenotype, resulting in high levels of ALP
expression. To prove this hypothesis, it will be necessary to
demonstrate the redifferentiation event in vitro.
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Selected Abbreviations and Acronyms |
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-carboxyglutamic acid
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Acknowledgments |
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The authors thank Sumitomo Pharmaceuticals Co, Ltd, and Ciba-Geigy, Ltd, for providing etidronate (EHDP) and pamidronate (APD), respectively.
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Footnotes |
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Reprint requests to Atsushi Shioi, MD, Second Department of Internal Medicine, Osaka City University Medical School, 1-5-7, Asahi-machi, Abeno-ku, Osaka 545, Japan.
Received March 10, 1995; accepted September 13, 1995.
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