(Circulation. 1997;95:1954-1960.)
© 1997 American Heart Association, Inc.
Articles |
Calcifying Subpopulation of Bovine Aortic Smooth Muscle Cells Is Responsive to 17ß-Estradiol
From the Division of Cardiology, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, Calif.
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Abstract |
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Background Arterial calcification, common in atherosclerosis, is associated with an increased risk of clinical events such as myocardial infarction. We previously identified a subpopulation of bovine aortic medial cells, calcifying vascular cells (CVCs), that have osteoblastic characteristics and form bone mineral in vitro in the form of calcified nodules. To assess whether estrogen modulates arterial calcification as well as bone calcification, we tested CVCs for estrogen receptors and for the effect of 17ß-estradiol on formation of calcified nodules, calcium content, alkaline phosphatase activity, and osteocalcin concentration in the culture medium.
Methods and Results Estrogen receptor immunoreactivity was identified in the cytoplasm and the perinuclear region of CVCs by immunocytochemistry. CVCs were treated with 17ß-estradiol at concentrations of 0, 5, and 10 nmol/L. Twenty-one days of 17ß-estradiol treatment resulted in a significantly increased number of calcified nodules, visualized by von Kossa staining, as well as increased calcium content of the cultures. Increases in alkaline phosphatase activity, a marker for early osteoblastic differentiation, and secreted osteocalcin, a marker for late osteoblastic differentiation, were enhanced in cells treated with 17ß-estradiol compared with control cells.
Conclusions These results suggest that 17ß-estradiol promotes osteoblastic differentiation and calcification in vascular cells and that estrogen may play a regulatory role in arterial calcification.
Key Words: calcium • hormones • atherosclerosis
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Introduction |
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Arterial calcification is common in atherosclerosis, occurring in 96% of patients with coronary artery disease1 and in 83% of clinically significant lesions.2 It is associated with increased risk of clinical events such as coronary ischemia, myocardial infarction, and heart failure and serious complications of surgery and interventional procedures.3 4 5 6 7 The mechanism of arterial calcification is unknown, but it is widely considered a late and inevitable feature of atherosclerosis. However, recent studies8 9 have shown that arterial calcification is progressive, beginning as early as the second and third decades of life, often preceding arterial narrowing. It often develops histological features resembling bone,10 suggesting an organized, regulated process. We and others have provided further evidence that vascular calcification is similar to bone formation, including expression of the osteogenic differentiation factor bone morphogenetic protein-2,11 presence of the bone mineral hydroxyapatite,12 matrix vesicles,13 osteocalcin,14 and osteopontin.15 16 17 To develop an in vitro model for arterial calcification, we identified and characterized a subpopulation of bovine aortic smooth muscle cells (BASMCs) with osteoblastic characteristics that spontaneously formed bone mineral.11 18 These cells were further stimulated to calcify in vitro by transforming growth factor-ß and 25-hydroxycholesterol. This culture model of calcifying vascular cells (CVCs) has been confirmed by independent investigators.19
On the basis of epidemiological studies, estrogens are associated with a lower risk of cardiovascular disease20 21 22 and reduced bone loss in postmenopausal women.23 24 Possible mechanisms of protection against vascular disease include favorable changes in the lipoprotein profile and lipoprotein metabolism in the artery wall,25 26 27 28 inhibition of intimal cell proliferation,29 30 alteration of vascular reactivity,31 32 inhibition of platelet aggregation,33 LDL oxidation,34 35 arterial myointimal thickening after vascular injury,36 and effects on glucose and insulin metabolism.37 The positive effect of estrogen on bone mineralization may occur by direct action on the estrogen receptor in osteoblasts38 39 40 and osteoclasts41 42 and/or through production of cytokines by osteoblasts, bone marrow stromal cells, and peripheral blood monocytes.42 43 44
On the basis of the epidemiological evidence for cardiovascular protection, estrogen may be expected to inhibit vascular calcification; however, on the basis of the similarity of vascular calcification to bone formation, it would be expected to promote calcification.45 46 47 To address this issue, we tested CVCs for estrogen receptors and effects of the physiologically active estrogen metabolite 17ß-estradiol on osteoblastic differentiation in vitro. The markers for differentiation included the activity of the membrane-bound alkaline phosphatase, an indicator of early osteoblastic differentiation; levels of osteocalcin, a marker for late osteoblastic differentiation; and deposition of calcium mineral, the final stage of osteogenesis.
Results suggest that estrogen has a positive regulatory role in arterial calcification by direct effects on CVCs.
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Methods |
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Tissue Culture
CVCs were cloned from primary BASMC cultures and identified as previously described.11 18 CVCs and BASMCs were cultured in DMEM (Irvine Scientific) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories), penicillin (100 U/mL), sodium pyruvate (1 mmol/L), streptomycin (100 U/mL), fungizone (0.25 µg/mL, Gibco BRL), L-glutamine (2 mmol/L), and 25 mmol/L HEPES buffer adjusted to pH 7.25. CVC clones were trypsinized before formation of nodules, plated in 12-well or 24-well tissue culture dishes (Costar) at a density of 16 000 cells/cm2, and grown for
28 days.
Treatments
Twenty-four hours after seeding, 17ß-estradiol (water-soluble,
Sigma Chemical Co) dissolved in PBS was added to the cultures to a
final physiological concentration of 5 or 10
nmol/L. Control additions included (1) PBS alone, (2)
physiologically inactive 17
-estradiol
(Sigma) in 0.01% ethanol (final concentration in medium), and (3)
0.01% ethanol vehicle alone. 17ß-Estradiol–containing media as well
as control media were changed every 3 days; however, the estradiol was
replenished every 24 hours because of its short half-life. In addition,
the estradiol solution was dissolved in PBS immediately before adding
it to the media. Phenol red was used as a pH indicator in the media at
a concentration of 27 µmol/L (10 mg/L) for alkaline phosphatase
experiments. Phenol red–free medium was used for Western blot
analysis. Although this agent may have estrogenic effects in
some cell lines,48 no effect was found in CVCs.
Immunocytochemistry
Cells were cultured in two-well chamber slides (Costar) until
70% to 80% confluence. Immunocytochemistry was performed on
permeabilized cells with a monoclonal antibody against
the estrogen receptor (Affinity Bioreagents Inc) at a dilution of 1:100
and the Vectastain Elite ABC kit (Vector Laboratories). This antibody
is reactive with the estrogen receptor from calf, human, rat, and
mouse.49 A monoclonal antibody for CD19 (DAKO Corp) at the
same dilution was used as a negative control. Staining was performed by
use of the peroxidase chromagen method (AEC kit; Biomeda
Corporation).
Von Kossa Calcium Stain
Cultured cells were fixed for 30 minutes in 0.1%
glutaraldehyde and incubated with 5% silver nitrate
for 30 minutes at room temperature in the dark. The cells were washed
gently with double-distilled water, exposed to ultraviolet light for 30
minutes, and counterstained with 0.1% eosin for 30 seconds. Calcium
mineral stained black. Calcified nodules in each well were counted
twice by use of phase-contrast microscopy by two different individuals
blinded to treatment conditions.
Calcium Determination
Calcium bound to extracellular matrix, calcium mineral, and
cellular proteins (collectively termed "insoluble calcium") was
determined by the method of Webster.50 CVCs were cultured
in 12-well plates. The medium was changed to serum-free medium 12 hours
before calcium determination. For calcium determination, the cells were
washed three times with PBS without calcium, trypsinized, and carefully
scraped in 0.5 mL of PBS without calcium by use of a cell scraper. The
cell samples were incubated at 38°C for 30 minutes, disrupted by
three freeze/thaw cycles, incubated at 38°C for 30 minutes, sonicated
for 3 minutes with a sonicator probe, and centrifuged for 30
minutes at 3000g. The pellet (insoluble calcium) was
suspended in 100 µL of 6N HCl, incubated at 100°C for 30 minutes,
and neutralized with 100 µL of 6N NaOH. The calcium determination was
performed in duplicate wells with 200-µL samples, and the remaining
steps were performed as previously described.50
Cell Counting
CVCs were dispersed with collagenase (type I, Sigma)
0.5 mg/mL in DMEM for 45 to 60 minutes at 37°C, then trypsin (Sigma)
0.1% in 2 mmol/L EDTA in PBS to ensure that the cells were
dispersed. Trypsinization alone did not disperse cells in nodules. The
cells were counted by hemocytometry. Each estimated cell number is a
mean of four determinations.
Alkaline Phosphatase Assay
Cells were plated in 24-well tissue culture trays and grown in
standard culture medium with 10% fetal calf serum and 0 or 10 nmol/L
of 17ß-estradiol or 17-estradiol for 4, 7, 11, 14, 22, and 28
days. Triplicate wells were used for each time point. Alkaline
phosphatase activity was quantified in whole-cell
homogenate with the use of a modification of the alkaline
phosphatase assay kit from Sigma. The assay was designed for use in a
24-well culture tray. The culture media were removed, and each well was
rinsed twice with 1.0 mL of PBS. Two wells without cells were used as
blanks and were treated in the same manner as the test wells throughout
the assay. The cells were placed on ice, and 200 µL of lysis buffer
(0.2% NP-40 in 1 mmol/L magnesium chloride) was added to each
well. The lysed cells were scraped with a cell scraper, incubated for
10 minutes on ice, and sonicated for 10 seconds with a sonicator probe
directly in the wells. After sonication, 1 mL of buffer A was added to
each well. Buffer A was made by adding stock substrate solution to
alkaline buffer solution 221 (Sigma) in a ratio of 1:1. Stock substrate
was made by adding the contents of a 100-mg capsule of Sigma 104
phosphate substrate (from the Sigma alkaline phosphatase assay kit)
into 25 mL of doubly distilled water. After addition of buffer A, the
dishes were removed from ice and incubated at 37°C for 30 minutes.
The reaction was stopped by the addition of 12 µL of 1N NaOH to each
well. Reaction mixture (220 µL) from each well was transferred in
duplicates to individual wells of a 96-well plate. The absorbance was
read in a Vmax kinetic microplate reader (Molecular Devices Corp) at
405 nm. Calibration standards consisted of dilutions of
p-nitrophenol stock on 0.02N sodium hydroxide (Sigma). The
results are given in Sigma units normalized to protein content, where 1
Sigma unit is equivalent to the enzyme activity required to release 1
mol of p-nitrophenol per hour. Blank wells were subtracted
from the optical density reading.
Alkaline Phosphatase Histochemical Staining
Staining solution was prepared by adding 46 µL of nitroblue
tetrazolium solution (4-nitroblue tetrazolium chloride, 75 mg/mL in
70% [vol/vol] dimethylformamide [DMF], from the Genius nucleic
acid detection system, Boehringer-Mannheim) and 35 µL of
X-phosphate solution (50 mg/mL in DMF; Boehringer-Mannheim) to
10 mL of 2 mmol/L Tris-HCl, pH 9.5. Cells to be stained were
rinsed twice with 2 mmol/L Tris-HCl, pH 9.5, and then incubated
with the staining solution in the dark at 37°C until color developed
(30 minutes to 24 hours). After development, the staining solution was
aspirated, and the cells were fixed with 4%
paraformaldehyde for 5 minutes at room temperature. The
cells were washed with PBS and mounted with a water-based mounting
medium.
Osteocalcin Western Blot Analysis
CVCs plated in 24-well tissue culture dishes were treated with 0
or 10 nmol/L 17ß-estradiol for up to 28 days. One milliliter of
fresh, phenol red–free medium with the appropriate 17ß-estradiol
concentration was added to each well and incubated for 72 hours.
Estradiol was replenished every 24 hours as described above. Medium was
recovered and centrifuged for 10 minutes at 500g to
remove any remaining cells. Media with the same serum content, not
exposed to cells, with or without hydroxyapatite adsorption, were used
as controls. Hydroxyapatite adsorption was performed by adding 20 µL
of a 50% slurry of hydroxyapatite (BioGel HTP, DNA grade; Bio-Rad
Laboratories) in DMEM to 100 µL of concentrated medium. The
hydroxyapatite was removed by low-speed centrifugation,
and the supernatant was analyzed. Serum-free medium was not
used for these experiments because CVCs did not tolerate this condition
for prolonged periods.
The medium was concentrated using Centriprep concentrators (Amicon, Inc), and dried with the Speed Vac system (Savant Instruments, Inc). The protein concentration was determined by use of the Bradford reagent (Bio-Rad Laboratories). Media proteins were separated on a 10% to 20% gradient tricine, SDS-polyacrylamide gel (Novex X-cell Mini-Cell), loaded with either equal amounts of protein per lane (0.25 µg) or equal volumes of concentrated, conditioned medium per lane. Proteins were then transferred onto enhanced chemiluminescence (ECL) nitrocellulose membranes (Hybond membranes, Amersham) with a Trans Blot electrophoretic transfer cell (Bio-Rad Laboratories). Western blot analysis was performed with a monoclonal antibody to osteocalcin (Biodesign International) recognizing bovine osteocalcin. The antibody was diluted 1:500 and detected by use of the ECL system.
Statistical Analysis
The number of nodules was displayed as mean±SD, and the means
were compared by use of Student's t test, with a level of
significance of P<.05.
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Results |
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Immunoreactivity to anti–estrogen receptor antibody was identified in cloned CVCs by immunocytochemistry with the use of an antibody recognizing the bovine estrogen receptor. Staining was observed in the cytoplasm and in the perinuclear region in cells grown to 60% to 70% confluence (Fig 1
). Clones of
noncalcifying vascular medial cells, presumably smooth muscle cells,
also expressed the receptor, which confirms previous
studies51 52 of mixed medial cell populations. The
measurable effects of treatment with 17ß-estradiol on bone
differentiation markers, described below, suggest that these are
functional estrogen receptors.
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Calcium mineral deposits formed by cultured CVCs were assessed by the
silver nitrate method (von Kossa staining). 17ß-Estradiol treatment
of two distinct CVC clones resulted in a significant increase in the
number of calcified nodules per well at 14 (Fig 2A) and
21 days (Fig 2B) compared with untreated wells. The number of calcified
nodules increased in a dose-dependent manner with increasing
concentrations of 17ß-estradiol (Fig 2C). Noncloned BASMCs, used as
control cells, formed no or only rare calcified nodules with and
without 17ß-estradiol treatment in this time period (Fig 2C).
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Insoluble calcium (matrix-bound and protein-bound calcium) was quantified in mineralized CVCs after 21 days of 17ß-estradiol treatment by use of the method of Webster and was normalized for total protein. Insoluble calcium varied with cell passage number and the specific clone used. The variation in mineral production among different clones has not been quantified. On the basis of visual assessment of von Kossa staining, we estimated a 40% variation among rapidly mineralizing clones and a greater difference between rapidly and slowly mineralizing clones. Most dramatically, 17ß-estradiol increased insoluble calcium by 183% and 708% at concentrations of 5 and 10 nmol/L, respectively, in rapidly mineralizing CVCs. The results suggest that 17ß-estradiol increases the amount of insoluble calcium deposited by CVCs, and they are consistent with the above finding of an estradiol-induced increase in the number of calcified nodules.
The number of cells per well, counted by enzymatic dispersion and
hemocytometry after 21 days of treatment, did not differ between 0, 5,
and 10 nmol/L of 17ß-estradiol (Table). Similarly,
total protein per well did not differ between treatments (results not
shown).
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As in bone cells, alkaline phosphatase activity of CVCs increased
between days 14 and 22 in nontreated CVCs (Fig 3A). With
17ß-estradiol treatment, this alkaline phosphatase activity increased
even further (Fig 3A). These results are consistent with the
increased formation of calcified nodules after 21 days of
17ß-estradiol treatment (shown above). Control experiments using
17-estradiol showed no enhancement of the alkaline phosphatase
induction (results not shown). Effects of 17-estradiol on calcium
deposition could not be assessed because prolonged treatment (>15
days) with 17-estradiol had toxic effects on CVCs, evidenced by
formation of vacuoles and increased friability. The high level of
alkaline phosphatase activity in calcifying cultures was localized in
the nodules (Fig 3B).
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Osteocalcin concentration in the CVC media was determined by Western
blot analysis, which revealed an immunoreactive protein band
migrating with an apparent molecular weight of 
10 kD (Fig 4A), consistent with previous
reports.53 After 72 hours of incubation, osteocalcin
levels were initially similar to those of control medium not exposed to
cells but containing osteocalcin present in the fetal bovine serum
(Fig 4B). In both control and treated cultures, the osteocalcin level
in the supernatant increased over time with a greater increase in the
treated cultures (Fig 4A and 4B). Results were similar regardless of
the gel-loading technique: equal volumes of concentrated medium (Fig 4A) or equal amounts of protein (Fig 4B). In the latter method,
osteocalcin levels were calculated on the basis of the dilutions used
to achieve equal protein loading.
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To determine whether some of the secreted osteocalcin could be
lost from the supernatant by binding to hydroxyapatite in the calcified
matrix, osteocalcin levels were measured in nonconditioned medium
before and after direct addition of commercially prepared
hydroxyapatite. There was a 64% decline in the osteocalcin level of
the supernatant after hydroxyapatite addition (Fig 4B
).
Four different, representative clones of CVCs were used for these experiments, and the results were consistent for all the clones.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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These results indicate that CVCs from the bovine aortic media that spontaneously form calcified nodules in vitro significantly increase their formation of calcified nodules in response to 17ß-estradiol. This response resembles that of bone-derived, osteoblastic cells and raises the question of whether estrogen treatment may contribute in the long term to vascular calcification. Specifically, this study suggests that functional estrogen receptors are present in CVCs and that 17ß-estradiol enhances in a stereospecific manner the rise in both alkaline phosphatase activity and secreted osteocalcin as well as the calcium mineral deposition associated with osteoblastic differentiation in CVCs.
Estrogen receptors have been demonstrated in cells from mixed vascular smooth muscle cells,51 52 osteoblast-like cells,38 39 40 osteoclasts,41 42 and bone endothelial cells.54 Estrogen-binding sites have also been demonstrated in human and rabbit aortas.55 The stereospecific effects of 17ß-estradiol on CVCs suggest that the estrogen receptors are functional and that the observed effects most likely are mediated through gene expression rather than through direct membrane signaling.
Osteoblastic differentiation entails a sequential expression of bone-related genes that eventually results in deposition of calcium mineral.56 A transient increase in membrane-bound alkaline phosphatase has been considered an important marker for the onset of osteogenic differentiation in vivo and in vitro,57 whereas a transient increase in osteocalcin expression represents one of the final stages of osteogenic differentiation. Deposition of calcium mineral occurs simultaneously with the subsequent decline in osteocalcin expression. Except for platelets, osteoblastic cells are the sole known source of osteocalcin.
The effects of estrogen on bone-derived cells have been studied extensively. Estrogen increases mineralization and calcium content in cultured bones58 as well as in bone implants in vivo.59 Cancellous bone formation in vivo increases with estrogen and decreases with estrogen antagonists.60 17ß-Estradiol increases enzymatic activity and mRNA for alkaline phosphatase in several osteoblastic cell lines61 62 63 64 though not in others.65 The effects were dose dependent and enhanced by earlier treatment.62
The effect of estrogen on the expression of bone-related factors and
mineralization has not been studied in vascular cell populations to the
best of our knowledge. 17ß-Estradiol accelerates osteoblastic
differentiation in CVCs on the basis of alkaline phosphatase activity
and the degree of calcification nodules. 17ß-Estradiol also enhances
the later stage of osteoblastic differentiation on the basis of
osteocalcin concentration in the media. Unexpectedly, the level of
osteocalcin in CVC supernatant decreased below control at 4 and 7 days
and again at 28 days of culture (lower curve in Fig 4B). The early
decline may be due to degradation or cellular metabolism of
the osteocalcin present in the fetal bovine serum without an
equivalent amount of synthesis by the cells. The later decline may be
due to binding of osteocalcin to matrix calcium deposits, as occurs in
bone. This possibility is supported by our experiments in which
hydroxyapatite was added directly to the nonconditioned medium. In
postmenopausal women and ovariectomized rats, serum osteocalcin is
inversely related to estrogen levels.66 67 68 69 With estrogen
replacement, osteocalcin returns to its original lower level. The
postmenopausal increase in osteocalcin has been related to increased
bone turnover and may result from release of osteocalcin from
mineralized matrix, to which it binds avidly.53
In bone cell cultures, osteoblastic differentiation is usually associated with a decrease in proliferation; however, the estrogenic effect on proliferation varies in different model systems.48 62 65 There was no effect of 17ß-estradiol on cell number in the present study, which is consistent with previous evidence that estrogen has a greater effect on differentiation than on growth.56 61 63
This report provides evidence that osteoblast-like CVCs from the bovine aorta respond to 17ß-estradiol in a manner similar to the response of bone-derived osteoblastic cells. These results raise the concern that estrogen replacement at doses that promote bone mineralization may also promote vascular calcification in the long term. It would also be important to assess the effects of combined estrogen and progesterone replacement therapy on vascular calcification.
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Acknowledgments |
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This work was supported in part by NIH grant HL-30568, the Streisand Research Fund established by the Lincy Foundation, the Oberkotter Research Fund, and the Stein/Oppenheimer Endowment Fund. The authors are grateful to Dr Stanley Korenman for assistance in obtaining antibodies and to Paul Yeghiayan and Amethyst Vu for superb technical assistance.
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Footnotes |
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Reprint requests to Linda L. Demer, MD, PhD, Division of Cardiology, UCLA School of Medicine, Box 951679, Room 47-123 CHS, Los Angeles, CA 90095-1679.
Received September 9, 1996; revision received November 26, 1996; accepted November 27, 1996.
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