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© 1999 American Heart Association, Inc.
Vascular Biology |
Dexamethasone Enhances In Vitro Vascular Calcification by Promoting Osteoblastic Differentiation of Vascular Smooth Muscle Cells
From the Second Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan.
Correspondence and reprint requests to Atsushi Shioi, MD, Second Department of Internal Medicine, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail as{at}msic.med.osaka-cu.ac.jp
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Abstract |
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Abstract—Vascular calcification is often associated with atherosclerotic lesions. Moreover, the process of atherosclerotic calcification has several features similar to the mineralization of skeletal tissue. Therefore, we hypothesized that vascular smooth muscle cells might acquire osteoblastic characteristics during the development of atherosclerotic lesions. In the present study, we investigated the effect of dexamethasone (Dex), which is well known to be a potent stimulator of osteoblastic differentiation in vitro, on vascular calcification by using an in vitro calcification model. We demonstrated that Dex increased bovine vascular smooth muscle cell (BVSMC) calcification in a dose- and time-dependent manner. Dex also enhanced several phenotypic markers of osteoblasts, such as alkaline phosphatase activity, procollagen type I carboxy-terminal peptide production, and cAMP responses to parathyroid hormone in BVSMCs. We also examined the effects of Dex on human osteoblast-like (Saos-2) cells and compared its effects on BVSMCs and Saos-2 cells. The effects of Dex on alkaline phosphatase activity and the cAMP response to parathyroid hormone in BVSMCs were less prominent than those in Saos-2 cells. Interestingly, we detected that Osf2/Cbfa1, a key transcription factor in osteoblastic differentiation, was expressed in both BVSMCs and Saos-2 cells and that Dex increased the gene expression of both transcription factors. These findings suggest that Dex may enhance osteoblastic differentiation of BVSMCs in vitro.
Key Words: alkaline phosphatase • core-binding factor-
1 • atherosclerosis
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Introduction |
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Calcification is a common feature of advanced atherosclerotic lesions.1 Atherosclerotic plaque calcification, especially in the coronary arteries, is associated with clinical complications such as myocardial infarction, impaired vascular tone, dissection in angioplasty, poor surgical outcome, and coronary insufficiency due to loss of aortic recoil.2 3 4 5 Although the mechanism of vascular calcification remains to be established, recent evidence suggests that it has several features similar to mineralization in skeletal tissue, including the expression of bone morphogenetic protein-2 (BMP-2), osteocalcin, and osteopontin and the presence of the bone mineral hydroxyapatite and matrix vesicles.6 7 8 9 10 11
To clarify the mechanism of vascular calcification, we developed an in vitro calcification system in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells (BVSMCs) in the presence of ß-glycerophosphate (ß-GP).12 In this model, alkaline phosphatase (ALP), which is 1 of the markers for osteoblastic differentiation, is critical for vascular calcification and the expression of osteopontin mRNA, which increases during the development of calcification. Furthermore, we identified a local calcium-regulating system in which parathyroid hormone (PTH) –related peptide plays an important role as an autocrine/paracrine regulator of vascular calcification.13 Through recent evidence demonstrated by us and other investigators,5 14 we hypothesized that VSMCs might acquire osteoblastic characteristics during the development of atherosclerotic lesions.
Osteoblastic differentiation is a multistep process, proceeding through
defined stages of maturation from a committed progenitor cell of
mesenchymal origin capable of proliferation to a postproliferative
osteoblast expressing bone phenotypic markers.15 However,
the molecular basis of osteoblast-specific gene expression and
differentiation remains unclear. Recently, a key regulatory
transcription factor in osteoblastic differentiation,
osteoblast-specific transcription factor-2/core-binding factor-
subunit 1 (Osf2/Cbfa1), has been identified. The
Osf2/Cbfa1 gene generates 2 types of transcripts,
osteoblast-specific and T cell–specific
isoforms.16 17 18 In the mouse, the osteoblast isoform
is different from the T-cell isoform in that the former contains a
unique 87–amino acid sequence at its amino-terminal end. However, the
precise roles of the 2 transcripts in osteoblastic differentiation
still remain unclear. The homozygous Osf2/Cbfa1
(–/–) mouse shows a total lack of bone and a retention of the
partially calcified cartilaginous skeleton.18 In
humans, mutations of this gene cause cleidocranial dysplasia, an
autosomal dominant skeletal disorder.17 19 Moreover,
overexpression of the osteoblast isoform in nonosteoblastic cells
induces expression of the principal osteoblast-specific genes, such as
1(I) procollagen, osteopontin, bone sialoprotein, and
osteocalcin.16 Therefore, Osf2/Cbfa1
(osteoblast isoform) is thought to be 1 of the "master genes" for
osteoblastic differentiation.
Dexamethasone (Dex), a potent, synthetic glucocorticoid, is well known to promote differentiation of progenitor cells, such as bone marrow stromal cells, to the osteoblastic phenotype.20 Chronic treatment of osteoblast cultures with Dex increases the number of mineralized bone nodules in primary fetal rat calvarial osteoblast cultures.21 22 Glucocorticoid promotes phenotypic markers of osteoblast differentiation, such as ALP, cAMP responses to PTH, osteopontin, bone sialoprotein, and osteocalcin, while it depresses production of insulin-like growth factor-I and type I collagen by osteoblasts.20 23 24 25 26 27 28 29 30
Although glucocorticoids have been shown to inhibit in vitro proliferation of VSMCs and prevent the development of atherosclerosis in experimental animals,31 32 33 34 35 retrospective studies including pathological findings obtained at autopsy have suggested that glucocorticoids adversely affect atherogenesis in humans.36 Moreover, the involvement of glucocorticoids in atherogenesis is supported by the strong correlation between an increased serum cortisol level in humans and the extent of coronary artery disease.37 Furthermore, long-term administration of glucocorticoids induces several metabolic and pathophysiological complications, such as insulin resistance, hypertension, and hyperlipidemia, which are thought to be coronary risk factors. However, the mechanism of glucocorticoid action in atherogenesis is poorly understood. Considering the fact that calcification is a common feature of advanced atherosclerotic lesions, it is important to evaluate the effect of glucocorticoids on vascular calcification.
In this study, we investigated the effect of Dex on vascular calcification by using an in vitro calcification model. We first demonstrated that Dex increased calcium deposition in a time- and dose-dependent manner. In this process, Dex increased ALP activity, its mRNA expression, and procollagen type I C-peptide (PICP) production and influenced cAMP responses to PTH. Finally, we demonstrated that Dex promoted expression of the Osf2/Cbfa1 gene. These results suggest that Dex may stimulate vascular calcification by promoting osteoblastic differentiation of VSMCs.
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Methods |
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Reagents
Media, FCS, and sodium pyruvate were purchased from GIBCO. ß-GP and Dex were obtained from Sigma Chemical Co. Unless otherwise mentioned, all other reagents were obtained from Wako Pure Chemical Industries, Ltd.
Cell Culture and In Vitro Calcification
BVSMCs were obtained from the media of aortas by an explant
method as previously described.12 Cells that had migrated
from the explants were collected and maintained in Dulbecco's modified
Eagle's medium (DMEM; high glucose [4.5 g/L]) containing 15% FCS
and 10 mmol/L sodium pyruvate supplemented with 100 U/mL
penicillin and 100 µg/mL streptomycin (growing medium) at 37°C in a
humidified atmosphere containing 5% CO2. The
cells up to passage 8 were used for experiments. Human osteoblast-like
cells (Saos-2) and Jurkat cells were obtained from the American Type
Culture Collection (Manassas, Va) and maintained in DMEM supplemented
with 15% FCS. Calcification of BVSMCs and Saos-2 cells was induced as
previously described.12 After reaching confluence, the
cells were incubated in DMEM containing 15% FCS in the presence of
10 mmol/L ß-GP. The medium was replaced with fresh medium every
2 days. In the time-course experiments, the beginning day of culture in
the calcification medium was defined as day 0.
Quantification of Calcium Deposition
The cells were decalcified with 0.6N HCl for 24 hours. The
calcium content was determined by measuring the concentrations of
calcium in the HCl supernatant by the o-cresolphthalein
complexone method (Calcium C-test Wako; Wako Pure Chemical Industries).
After decalcification, the cells were washed 3 times with PBS and
solubilized with 0.1N NaOH/0.1% SDS. The protein content was measured
with a bicinchoninic acid protein assay kit (Pierce). The calcium
content of the cell layer was normalized to protein content.
ALP Assay
After the cells were washed twice with PBS, the 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.12 One unit was defined as the
activity producing 1 nmol of p-nitrophenol for 30 minutes.
Protein concentrations were determined with a bicinchoninic acid
protein assay kit (Pierce).
PICP Assay
The cells were plated into 24-well plates and grown to
confluence. The medium was replaced with phenol red–free DMEM
containing 0.2% FCS in the presence of Dex. After the indicated period
of incubation, the supernatants were collected and stored at -20°C
until assay. PICP secreted into the culture medium by BVSMCs and Saos-2
cells was assessed by measuring the PICP content of the culture
supernatant with an enzyme immunosorbent assay kit (PIP EIA kit,
Takara). The data were normalized to the protein content of the cell
layer.
Measurement of cAMP
cAMP responses to PTH were assessed by measuring intracellular
cAMP. The cells were plated into 24-well plates and grown to confluence
before treatment with either vehicle or various concentrations of Dex
(10-10 to 10-7 mol/L).
After 4 days of treatment with Dex, the cells were washed twice with
PBS and preincubated for 10 minutes with DMEM containing 0.1% BSA and
1.0 mmol/L 3-isobutyl-1-methylxanthine at 37°C. Human PTH(1–34)
(10-7 mol/L) was then added to the medium, and
the cells were incubated for an additional 15 minutes at 37°C.
Thereafter the medium was removed, and the cell layer containing cAMP
was extracted with 500 µL of 5% trichloroacetic acid.
One-hundred-microliter aliquots of these samples were washed 3 times
with 5 volumes of water-saturated ethyl ether and then dried. The
extract was analyzed for cAMP by utilizing a cAMP
radioimmunoassay kit (Yamasa Shoyu).
Preparation of cDNA Probes
The human ALP (liver/bone/kidney type) cDNA probe was obtained
from the Japanese Cancer Research Resources Bank, Osaka, Japan.
Human Osf2/Cbfa1 cDNA probes (585-bp fragments) were obtained by
reverse transcription of an mRNA from Saos-2 cells, followed by
polymerase chain reaction and subcloning into the TA cloning vector
(Invitrogen). Sequences of the obtained cDNA were confirmed by the
dideoxy sequencing method. The Osf2/Cbfa1 cDNA probe, which contains
the carboxy terminus of the coding region (684 to 1268 bp of human
Osf2/Cbfa1 cDNA), can recognize both isoforms of
Osf2/Cbfa1 gene transcripts, ie,
osteoblast-specific and T cell–specific isoforms, as confirmed by
Northern blot analysis with polyA+ RNA of Saos-2 and of Jurkat
(human T-cell line) cells, respectively.
RNA Isolation and Northern Blot Analysis
Total RNA was prepared using the
acid-guanidinium-isothiocyanate-phenol-chloroform extraction method.
PolyA+ RNA was obtained by use of an mRNA isolation kit (Microfast
Track kit, Invitrogen) by using oligo(dT) cellulose for adsorption.
Twenty micrograms of total RNA and 1 microgram of polyA+ RNA were
denatured and separated by electrophoresis on 1% agarose gels
containing formaldehyde and transferred to a nylon filter (Hybond N,
Amersham). Blots were prehybridized for 24 hours at 37°C in a buffer
containing 50% formamide, 3x SSC (1x SSC is 0.15 mol/L NaCl and
15 mmol/L sodium citrate, pH 7.4), 50 mmol/L Tris-HCl (pH
7.5), 0.1% SDS, 20 µg/mL denatured salmon sperm DNA, and 1x
Denhardt's solution and then hybridized at 37°C for 48 hours with
cDNA probes for human ALP and Osf2/Cbfa1, which had been 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 mRNA were quantified by densitometric scanning
and normalized by comparison with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Measurements of DNA Synthesis
DNA synthesis of BVSMCs was evaluated by
[3H]thymidine incorporation assays. The cells
were grown in 24-well plates until confluent and then incubated in
serum-free DMEM for 48 hours. After 48 hours, the medium was changed to
DMEM containing 15% FCS or platelet-derived growth factor
(PDGF)-BB (10 ng/mL) in the presence or absence of the indicated
concentrations of Dex. The cells were subsequently incubated for 21
hours and then labeled with 1 µCi/mL
[3H]thymidine (6.7 Ci/mmol, New England
Nuclear) for an additional 3 hours.
[3H]Thymidine incorporated into DNA was
evaluated by trichloroacetic acid precipitation and counting in a
scintillation counter (Beckman Instruments).
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 (StatView version 4.11, Abacus
Concepts).
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Results |
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We first examined the effect of Dex on BVSMC calcification. As previously described, ß-GP induced calcium deposition in a time-dependent manner. In the presence of ß-GP, Dex (10-7 mol/L) significantly increased calcium deposition compared with calcified controls at each time point (Figure 1A
). The calcium deposition in the
Dex-treated group increased to 175% of the calcified control value on
day 6. Likewise, Dex promoted calcium deposition in a dose-dependent
manner on day 4, and the calcium deposition increased to 322% of the
calcified control value at 10-7 mol/L (Figure 1B). These results suggest that Dex increases BVSMC
calcification.
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ALP is known to be 1 of the phenotypic markers of osteoblastic
differentiation. Because we reported that ALP plays an important role
in this calcification system, we next examined the effect of Dex on ALP
activity in BVSMCs. As a positive control of Dex's effect, we utilized
human osteoblast-like (Saos-2) cells. In the absence of ß-GP, Dex
(10-7 mol/L) enhanced ALP activity in a
time-dependent manner, and ALP activity had increased to 222% of
controls on day 6 (Figure 2A). On day 4,
Dex dose-dependently increased ALP activity in the absence of ß-GP,
and the maximal effect (236% of control) was observed at
10-7 mol/L (Figure 2B). In the presence
of ß-GP, Dex also increased ALP activity in BVSMCs, but the response
to Dex was less prominent than that in its absence (Table 1). Furthermore, a greater response was
observed in Saos-2 cells, both in the absence and presence of ß-GP
(Table 1). Next, we examined the effect of Dex on expression of
the ALP gene in BVSMCs. Dex dose-dependently promoted the
expression of ALP mRNA at 48 hours, and the maximal effect was observed
at 10-7 mmol/L (180% increase of control;
Figure 2C). Taken together, these results suggest that Dex may
accelerate BVSMC calcification partially through enhancing expression
of the ALP gene and its activity.
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Type I collagen is 1 of the early phenotypic markers for osteoblastic
differentiation and may play an important role in this process. We
utilized PICP as an index for type I collagen synthesis. We measured
PICP contents of the culture supernatants to investigate whether Dex
affects type I collagen synthesis by BVSMCs. In the absence of ß-GP,
Dex (10-7 mol/L) increased PICP secretion by
BVSMCs in a time-dependent manner up to day 6, and on day 6, PICP
production by Dex-treated BVSMCs had reached 241% of untreated
BVSMCs (Figure 3A). On day 4, Dex
dose-dependently increased PICP production, and at
10-7 mol/L the maximal effect was observed
(313% increase of control; Figure 3B). Interestingly, Dex
exerted no stimulatory effect on PICP production by BVSMCs in
the presence of ß-GP (Table 1
). Furthermore, Dex did not
affect PICP production by Saos-2 cells, irrespective of ß-GP
(Table 1).
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As another marker of osteoblastic differentiation, we investigated the
effect of Dex on cAMP production in response to PTH
stimulation. Dex stimulated cAMP responses to PTH in BVSMCs both in the
presence and absence of ß-GP (Table 2).
The stimulatory effect of Dex in the absence of ß-GP was greater than
that in its presence. Moreover, Dex exerted greater responses in Saos-2
cells compared with BVSMCs (Table 2). These data suggest that
Dex promotes cAMP responsiveness to PTH in BVSMCs as well as in Saos-2
cells.
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We next assessed the effect of Dex on gene expression of
Osf2/Cbfa1 in BVSMCs. The mRNA of
Osf2/Cbfa1 expressed in BVSMCs was compared with
that in Saos-2 and Jurkat cells (Figure 4A). The transcript in BVSMCs was larger
than that in Saos-2 and Jurkat cells. The transcripts detected in
Saos-2 and Jurkat cells are thought to be human osteoblast– and T
cell–specific isoforms, respectively. Additionally, Dex
(10-7 mol/L) increased Osf2/Cbfa1 mRNA
expression 24 hours after treatment in Saos-2, but the presence of
ß-GP did not affect its expression (Figure 4B). In BVSMCs, Dex
also enhanced mRNA expression in a time-dependent manner (Figure 4C). Furthermore, the presence of ß-GP exerted no apparent
effect on expression at 24 hours (data not shown).
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Because it is likely that Dex may promote expression of osteoblastic
markers in BVSMCs by inhibiting their proliferative capacity, we
finally examined the effect of Dex on DNA synthesis in BVSMCs. The
cells were incubated in DMEM containing 15% FCS or 10 ng/mL PDGF-BB in
the presence or absence of the indicated concentrations of Dex for 24
hours. Both FCS and PDGF-BB stimulated DNA synthesis in BVSMCs, and Dex
inhibited the stimulatory effect of PDGF-BB on DNA synthesis (Figure 5A). However, Dex did not affect DNA
synthesis stimulated by FCS (Figure 5B), suggesting that Dex may
not exert a direct effect on BVSMC proliferation in this calcification
model.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Because glucocorticoids are well known to be potent stimulators of osteoblastic differentiation,20 21 22 38 we utilized Dex, a potent synthetic glucocorticoid, to induce BVSMCs to acquire osteoblastic characteristics. As shown in this study, Dex enhanced not only in vitro calcification but also several phenotypic markers for osteoblastic differentiation in BVSMCs, such as ALP expression, type I collagen production, and cAMP responsiveness to PTH (Figures 1
, 2, and 3). Moreover, we confirmed the potency
of Dex on osteoblastic differentiation by utilizing Saos-2 cells as a
positive control. Regarding ALP activity and the cAMP response to PTH,
the responsiveness of BVSMCs to Dex was less prominent than that of
Saos-2 cells (Tables 1 and 2). The less potent effect of
Dex on BVSMCs may be ascribed to a heterogeneous population
of cells in the BVSMC culture. Interestingly, Dex exerted no effect on
PICP production by Saos-2 cells, whereas Dex increased PICP
secretion by BVSMCs in the absence of ß-GP (Table 1). Early
phenotypic markers for osteoblastic differentiation such as PICP may
not be affected by Dex in well-differentiated osteoblastic cells.
However, the precise mechanism by which such differences of
responsiveness to Dex are induced remains to be clarified.
Several key factors in bone mineralization have been demonstrated in
calcified lesions of arterial walls, such as matrix
vesicles, BMP-2, osteopontin, matrix Gla protein, osteocalcin, and type
I collagen.6 7 8 9 10 11 39 We previously demonstrated the
significance of ALP, osteopontin, and PTH-related peptide in an in
vitro model of vascular calcification by utilizing
BVSMCs.12 13 Recently, a key regulatory factor in
osteoblastic differentiation, Osf2/Cbfa1, has been identified. BMP-7
induces expression of the osteoblastic isoform, followed by its
enhancement of the osteocalcin gene in nonosteoblastic
cells.16 Therefore, the Osf2/Cbfa1
gene is thought to be 1 of the "master genes" of as well as a
molecular marker for osteoblastic differentiation. In this study, we
showed the presence of the Osf2/Cbfa1 gene in
cultured BVSMCs as well as in Saos-2 cells (Figure 4A and 4B).
Additionally, we cloned a 5' partial sequence of the bovine
osteoblast–specific Osf2/Cbfa1 transcript by
reverse transcription–polymerase chain reaction by using total RNA
from BVSMCs in preliminary experiments (K.M. et al, unpublished data,
1998). This evidence suggests that cultured VSMCs may be committed to
differentiate into osteoblastic cells under certain conditions.
However, whether the transcript detected in BVSMCs is the
osteoblast-specific isoform remains to be confirmed. Furthermore, Dex
enhanced the gene expression of Osf2/Cbfa1 in a
time dependent manner in BVSMCs (Figure 4C). Therefore, it is
likely that Dex may promote osteoblastic differentiation of VSMCs by
increasing the expression of the Osf2/Cbfa1
gene.
Linkage of phenotypic gene induction to the downregulation of
proliferation is the hallmark of differentiation in numerous cell
types.40 Some agents inhibiting the proliferation of
osteoblast-lineage cells, such as hydroxyurea, can induce osteoblastic
differentiation. It is therefore possible that antiproliferative agents
of VSMCs may induce osteoblastic differentiation under certain
conditions. Moreover, 17ß-estradiol has been reported to promote
osteoblastic differentiation of bovine vascular cells and in vitro
calcification without affecting cell growth.41 In this
study, we examined the hypothesis that Dex may inhibit the
proliferative capacity of BVSMCs, resulting in osteoblastic
differentiation. Because Dex did not affect DNA synthesis in the
presence of 15% FCS (Figure 5B), Dex may directly induce
osteoblastic differentiation of BVSMCs without affecting their
growth.
The mechanisms of glucocorticoid action on atherogenesis remain to be evaluated. When applied as anti-inflammatory drugs at high doses, glucocorticoids suppress the development of atherosclerosis in experimental animals, despite enhancement of hypertriglyceridemia and hypercholesterolemia.32 33 34 35 Glucocorticoids have also been shown to inhibit the proliferation of cultured VSMCs and the thrombin-induced expression of growth factors.31 42 On the other hand, glucocorticoids are capable of decreasing the expression of hepatic LDL receptors, stimulating the net synthesis of apoB-100 and apoB-48 and decreasing their intracellular degradation.43 These changes are potentially atherogenic, and the strong correlation between an increased serum cortisol level in humans and the extent of coronary artery disease has also been documented.37 In this study, we have shown that Dex increases in vitro calcification by promoting osteoblastic phenotypes in BVSMCs. Taking into consideration that chronic treatment with glucocorticoids induces osteoporosis and that vascular calcification is often associated with osteoporosis, it is suggested that Dex may develop and exacerbate vascular calcification. Further studies are necessary to clarify the long-term effect of glucocorticoid administration on the development of vascular calcification, especially calcified atherosclerotic plaque lesions.
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Acknowledgments |
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This work was supported in part by grants-in-aid 08671177 (to Y.N.) and 09770793 (to A.S.) for scientific research from the Ministry of Education, Science, and Culture of Japan.
Received April 7, 1998; accepted February 4, 1999.
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