Original Contributions |
From the Division of Cardiology, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, Calif.
Correspondence to Karol E. Watson, 47-123 Center for Health Sciences, UCLA, School of Medicine, Los Angeles, CA 90095. E-mail kwatson{at}medicine.medsch.ucla.edu
| Abstract |
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5
integrins significantly inhibited the fibronectin-mediated increases in
alkaline phosphatase activity, indicating that integrin-based signaling
may be involved. These data suggest that matrix composition can
regulate development of arterial calcification and that a
subpopulation of vascular cells preferentially produces positively
regulating matrix components.
Key Words: atherosclerosis calcification extracellular matrix osteogenesis
| Introduction |
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Vascular calcification may represent a process by which cells
that normally exhibit a smooth muscle phenotype differentiate
into cells that exhibit an osteoblast-like phenotype. Recently,
we isolated such osteoblast-like cells from the artery wall and have
termed them calcifying vascular cells (CVCs).24
CVCs were initially cloned from subpopulations of bovine aortic smooth
muscle cells, and they exhibit several osteoblastic features in
vitrospecifically, aggregation into nodules, spontaneous formation of
hydroxyapatite mineral after nodulation, and production of bone
differentiation factors.25 CVCs also produce
extensive extracellular matrix that allows for in vitro assessment of
matrix components. In the current study, we have used cloned CVCs to
assess the role of extracellular matrix components on in vitro
calcification. Our results reveal that 2 of the matrix components known
to be important in bone formation, fibronectin and collagen
I,26 27 are also important in promoting
mineralization of vascular cells, and that the matrix component
collagen IV inhibits mineralization parameters.
Furthermore, blocking antibodies to
5 integrins inhibit the
fibronectin-mediated increases in alkaline phosphatase activity,
indicating that integrin-based signaling may be involved. These data
suggest that specific extracellular matrix molecules are capable of
promoting arterial calcification and that a subpopulation
of vascular cells preferentially produces these matrix components.
These data also indicatea potential role for the
5 integrin and
integrin-based signaling in the promotion of in vitro vascular
calcification. Further research on the interplay between vascular
cells, their extracellular matrix, and cellular integrins may lead to
new therapeutic approaches to modify atherosclerotic calcification.
| Methods |
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-SM
actin staining, and negative factor VIIIrelated antigen staining.
Mineralizing medial cell clones were a subpopulation of BASMCs termed
CVCs. CVCs were isolated from primary cultures of BASMCs in which
multicellular nodules spontaneously appeared. From these nodule-forming
cultures, CVCs were cloned by limiting dilution and single-cell
harvesting, and clonal lines were identified as CVCs by their positive
staining with monoclonal antibody 3G5, as previously
described,24 and by their ability to form
calcified nodules in tissue culture. The standard tissue culture media
for all cells was Dulbecco's modified Eagle's medium (Irvine
Scientific) with 15% FBS (Hyclone Laboratories) supplemented with
L-glutamine (2 mmol/L), sodium pyruvate (1
mmol/L), penicillin (100 U/mL), Fungizone (0.25 µg/mL), and HEPES
buffer adjusted to pH 7.25, all from Irvine Scientific. Cells were
plated at a density of 10 000 cells/cm2 with
media changes every 72 hours.
Preparation of Cell-Synthesized Extracellular Matrix
Cell-synthesized extracellular matrix was prepared by allowing
cells to grow on tissue culture plastic for 14 days followed by
detachment of the cell monolayer with 25 mmol/L
NH4OH. For detachment, the monolayers were
incubated with NH4OH for 10 minutes, followed by
extensive washing with calcium- and magnesium-free PBS (Gibco) and
microscopic inspection of the dishes to ensure complete cell removal.
If cells remained after the initial incubation, dilute alkali treatment
was repeated to complete cell removal. Cell-free extracellular matrixes
were maintained in tissue culture incubators with PBS for 2 weeks to
ensure that no cells remained.
Indirect Immunofluorescent Staining
Immunofluorescent staining was performed on cultures of
nonmineralizing, slowly mineralizing, and rapidly mineralizing cells.
Cells were grown in chamber slides on tissue culture plastic for 24
hours before staining. The cultures were then fixed in 4%
paraformaldehyde at room temperature for 5 minutes,
followed by incubation with a blocking solution containing 5% BSA
(vol/vol) and 3% nonimmune goat serum (vol/vol). Primary antibodies
were applied for 2 hours, followed by "quenching" of
autofluorescence with 0.2% NaHB4 for 15
minutes. FITC-conjugated secondary antibodies (Sigma Chemical Co) were
then applied (1:30 dilution) for 30 minutes. After the slides were
washed, mounting medium and a coverslip were applied, and the cells
were examined by fluorescence microscopy (Axioscope 20; Carl
Zeiss, Inc). Primary antibodies used were rabbit anti-bovine collagen
I, rabbit anti-bovine collagen IV, rabbit anti-rat laminin, and rabbit
anti-chicken fibronectin antibodies, all at a dilution of 1:50 (all
antibodies obtained from Chemicon).
Immunoblotting
Cellular and extracellular matrix proteins were extracted from
confluent monolayers of cells cultured for 14 days on tissue culture
plastic. The proteins were extracted with lysis buffer containing
10 mmol/L HEPES, 200 mmol/L NaCl, 2 mmol/L
CaCl2, 1.5% Triton X-100, 1 mmol/L EDTA,
0.05% leupeptin, 0.07% pepstatin, and 0.2 mmol/L PMSF. The
extracted proteins were separated on an 8% acrylamide gel
(Novex X-Cell Mini-Cell) loaded with 12 µg protein per lane, followed
by transfer onto 0.45-µm nitrocellulose membranes (Bio-Rad).
Immunoblotting was performed with the ABC method
(Vector Laboratories) and the following antibodies: rabbit anti-bovine
collagen I, rabbit anti-bovine collagen IV, rabbit anti-rat laminin,
and rabbit anti-chicken fibronectin (all antibodies obtained from
Chemicon and diluted 1:100). Quantification of
immunoblotting data was accomplished by calculating
relative densitometric units for each band. This procedure consisted of
multiplying the mean band density by the band area by using National
Institutes of Health (NIH) Image software. For each blot, the band with
the lowest densitometric unit was assigned a reference value of 1.0.
Values obtained by densitometry were verified as being linearly related
to the amount of protein on the blots by constructing standard curves
for each matrix protein. Three separate immunoblots from 3
separate extractions were performed for each matrix molecule.
Experiments Performed on Purified Matrix Molecules
Slowly mineralizing cells were plated at a density of 10 000
cells/cm2 onto purified collagen I, collagen IV,
and fibronectin, or onto tissue culture plastic (all matrix molecules
obtained from Sigma and coated at 10 µg/cm2)
and cultured for 40 days. Cultures were observed for mineralized nodule
formation and alkaline phosphatase activity. Quantification of
mineralization was accomplished at the end of the 40-day culture period
by multiplying the mean nodule density by the nodule area in each
culture with the use of NIH Image software. Alkaline phosphatase
determinations were performed every 4 days throughout the 40-day
culture period. For alkaline phosphatase determinations, cells were
lysed in buffer containing 0.2% NP40 in 1 mmol/L
MgCl2 at 4°C for 10 minutes. The cells were
then scraped into an Eppendorf tube and sonicated for 10 seconds.
Alkaline phosphatase activity was quantified by measuring release of
p-nitrophenol from p-nitrophenyl phosphatase
(alkaline phosphatase assay; Sigma Diagnostics) and
normalized to total cell number as determined by DNA content. Three
separate experiments were performed.
45Calcium Incorporation
Slowly mineralizing cells were plated at a density of 10 000
cells/cm2 onto purified collagen I, collagen IV,
and fibronectin, or onto tissue culture plastic. After 7 days, 1
µCi/mL of 45CaCl2 was
added and the cells cultured for an additional 48 hours. After this
time, the medium was removed and the cells washed with PBS and scraped
into scintillation vials. Perchloric acid (0.2 mL) and 0.4 mL of 3%
H2O2 were added to the
vials, followed by incubation at 80°C for 60 minutes. After
incubation, the mixture was dissolved in 0.6 mL of ethylene glycol
monoethyl ether and counted in a scintillation counter.
Anti-Integrin Experiments
Anti-integrin
5 antibodies were purchased from Becton
Dickinson and used at a concentration of 1 µg/mL. Slowly mineralizing
cells were plated at a density of 10 000
cells/cm2 onto either collagen I or fibronectin,
and 24 hours later anti-integrin treatments began. Treatments were
carried out every 3 days for 9 days total. Control antibodies used were
anti-CD3 antibodies (Dako Corporation) at an equivalent total protein
concentration.
Statistics
All data are presented as mean±SD. Intergroup
comparisons were performed by unpaired Student's t test.
Probability values of 0.05 or less were required for assumption of
statistical significance.
| Results |
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Effect of Cell-Synthesized Extracellular Matrix on In Vitro
Calcification (Matrix Swapping)
We evaluated the rate of mineralization of vascular cell clones
when cultured on extracellular matrixes synthesized by different cells.
Matrix synthesized by the 3 different types of clones was obtained as
described above in Methods. Each clone was then plated either back onto
its own matrix or onto matrix synthesized by 1 of the other cell types
(matrix swapping).
(1) When nonmineralizing clones were plated onto any of the 3 matrix types, the cells remained nonmineralizing.
(2) When slowly mineralizing clones were plated onto either
nonmineralizing matrix or onto slowly mineralizing matrix, they
remained slowly mineralizing; however, when plated onto rapidly
mineralizing matrix, such cells became rapidly mineralizing, with the
time necessary for nodule formation to occur decreasing from 33±3.0 to
7.8±1.3 days (Table 1
). Furthermore, by
21 days these nodules were heavily calcified, as demonstrated by von
Kossa staining (Figure 1
).
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(3) When rapidly mineralizing clones were plated on any of the 3 matrix types, the cells remained rapidly mineralizing.
Thus, a change in the rate of mineralization occurred only when slowly mineralizing cells were plated onto rapidly mineralizing matrix. These results suggest that mineralization can occur only when cells capable of mineralization are paired with a "permissive" extracellular matrix and that the rate of mineralization is determined, at least in part, by the composition of the matrix. A permissive matrix either can be supplied exogenously (as in the case of slowly mineralizing cells plated on rapidly mineralizing matrix) or can be produced by the cells themselves (as in the case of rapidly mineralizing cells plated on nonmineralizing matrix).
Analysis of Matrix Molecules Produced
The composition and amount of matrix produced by each class of
medial cell clone were next determined by immunofluorescent
staining and immunoblotting. The molecules
analyzed by immunofluorescent staining were laminin,
collagen I, fibronectin, and collagen IV. Laminin was nearly
undetectable in rapidly mineralizing clones (Figure 2C
), as well as in slowly mineralizing
and nonmineralizing clones (data not shown); thus, this molecule was
not studied further. Collagen I, fibronectin, and collagen IV were all
present in detectable amounts in rapidly mineralizing clones
(Figure 2A
, 2B
, and 2D
), as well as in slowly mineralizing and
nonmineralizing clones (data not shown). The greatest amounts of
collagen I and fibronectin were present in rapidly mineralizing
clones, as determined by immunoblotting (Figure 3
). Rapidly mineralizing clones produced
3 times more collagen I than did either nonmineralizing or slowly
mineralizing clones (Table 2
). Rapidly
mineralizing clones also produced
3 times more fibronectin than did
slowly mineralizing clones and 16 times more fibronectin than did
nonmineralizing clones (Table 2
). Collagen IV revealed an opposite
pattern of abundance, with nonmineralizing clones producing the highest
amounts (Table 2
). Nonmineralizing clones produced 70% more collagen
IV as did rapidly mineralizing clones.
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Effects of Purified Extracellular Matrix Molecules on Mineralized
Nodule Formation, Calcium Incorporation, and Alkaline Phosphatase
Activity
To further investigate the role of matrix on in vitro
calcification, slowly mineralizing clones were plated onto the purified
matrix molecules collagen I, fibronectin, and collagen IV to determine
whether purified matrix molecules could reproduce the results obtained
with the cell-synthesized extracellular matrix. Slowly mineralizing
clones were plated onto the various matrixes, cultured for a total of
40 days, and assessed for mineralized nodule formation and calcium
incorporation, as well as alkaline phosphatase activity as a marker of
osteoblastic differentiation.
Mineralized nodule formation was enhanced by growth on either collagen
I or fibronectin and inhibited by growth on collagen IV, compared with
tissue culture plastic. Cells grown on tissue culture plastic formed
small, discrete nodules during the culture period as previously
described24 (Figure 4A
). When these cells were grown on
either collagen I or fibronectin, much larger nodules were formed that
appeared to be connected by large ridges of cells and extracellular
material (Figure 4B
and 4C
). In contrast, growth on collagen IV
significantly inhibited mineralized nodule formation (Figure 4D
). When
the area of mineralization was calculated in each culture, cells grown
on collagen I or fibronectin produced 3.1±1.1 times as much
mineralization as did cells grown on tissue culture plastic, whereas
cells grown on collagen IV produced only 0.21±0.18 times the
mineralization as cells grown on tissue culture plastic (Figure 5A
).
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Radiolabeled calcium incorporation also increased significantly. Cells
grown on collagen I or fibronectin incorporated 2.2±0.5 times the
calcium as did cells grown on plastic, and cells grown on collagen IV
incorporated only 0.05±0.01 times the calcium as did cells grown on
plastic (Figure 5B
). All calcium incorporation experiments were
performed at confluence. At confluence, there was no difference in cell
number, regardless of the matrix on which the cells were plated.
Alkaline phosphatase activity was also enhanced by growth on either
collagen I or fibronectin. Slowly mineralizing clones grown on collagen
I or fibronectin expressed up to 8 times greater alkaline phosphatase
activity compared with clones grown on either collagen IV or tissue
culture plastic. This increase began at 6 days of culture (Figure 6A
), and this relationship held even when
alkaline phosphatase activity was controlled for cell number (Figure 6B
). When rapidly mineralizing cells were grown on the various matrix
molecules, there was a high level of nodule formation, alkaline
phosphatase activity, and calcium incorporation, and none of these
parameters was altered by growth on specific matrix
molecules, with the exception that calcium incorporation decreased on
collagen IV (data not shown). This finding again is consistent
with the hypothesis that mineralization occurs when cells capable of
mineralization are paired with a permissive extracellular matrix. The
permissive matrix can either be supplied exogenously or produced by the
cells themselves (rapidly mineralizing clones).
|
Anti-Integrin Treatment of Slowly Mineralizing Cells
Treatment of slowly mineralizing clones with blocking
antibodies to
5 integrins resulted in a decrease in alkaline
phosphatase activity over a 7-day treatment period. For cells grown on
fibronectin, which is an
5 ligand, the decrease was 77±33% and
statistically significant (P=0.013) (Figure 7
). For cells grown on collagen I, which
is not an
5 ligand, the decrease was 34±14% and not statistically
significant. This decrease in alkaline phosphatase activity when cells
grown on fibronectin were treated with blocking antibodies to the
5
integrin suggests that integrin-based signaling is involved in the
matrix modulation of alkaline phosphatase activity.
|
| Discussion |
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Mineralization is usually confined to specific locations in vivo, and only specialized cells are able to secrete a permissive matrix. When calcification is uncontrolled, it may contribute to the pathogenesis of a variety of diseases, including atherosclerosis. Ectopic calcification is a prominent feature of atherosclerosis, and until recently, the cells responsible for this mineralization were not known. We have recently discovered a subpopulation of cells isolated from the media of aortas that have osteoblast-like properties and that in vitro are capable of forming a calcified matrix.25 In the current studies, we have begun to characterize the regulatory role of the extracellular matrix produced by these vascular cells that calcify, and our results indicate that CVCs secrete a complex extracellular matrix that can influence the rate and amount of calcification produced in vitro. The predominant matrix components produced by mineralizing cells, collagen I and fibronectin, were found to promote calcification, whereas collagen IV was inhibitory.
Collagen I and fibronectin are known to be critical in the formation of calcified structures and are associated with osteoblast differentiation.26 27 Collagen I comprises over 90% of the organic compartment of mature bone, and type I collagen fibrils are believed to be the "scaffolding" necessary for mineralization. During embryogenesis, formation of the core of limb buds coincides with a transient expression of collagen I and fibronectin.26 27 As mesenchymal precursors differentiate into osteoblasts, collagen I, fibronectin, and alkaline phosphatase are upregulated.31 32 In addition, fibronectin is present in the condensing core of chick limb bud mesenchyme during cartilage and bone differentiation but is completely absent from the adjacent muscle.33
The role of extracellular matrix in atherosclerosis is increasingly recognized,9 10 11 12 13 14 15 16 34 35 36 and atherosclerotic calcification represents an extreme case of matrix alteration. Vascular cells are known to synthesize their own complex extracellular matrix, and this synthesis is affected by the pre-existing matrix as well as the state of differentiation of the cell.37 There are 2 major classes of matrix in the vessel wall: the basal lamina and the interstitial matrix. The basal lamina includes collagen IV, laminin, and heparan sulfate proteoglycans, whereas the interstitial matrixes include collagen I, collagen III, fibronectin, elastin, and chondroitin sulfate proteoglycans. Numerous changes in the extracellular matrix occur in atherosclerosis, and alterations in the composition of artery wall matrix contributes to atherogenesis.9 10 11 12 13 14 15 16 Just as in the development of calcified structures, collagen I and fibronectin have also been specifically implicated in the development of atherosclerotic lesions. Intimal sclerosis and lesion progression involve increased expression of collagen I,13 and investigators have also demonstrated that phenotypic modulation of vascular cells occurs when the cells are grown on collagen I or fibronectin but that this modulation is inhibited when cells are grown on elastin or laminin.38 39
In addition to direct effects of matrix molecules on the cells, matrix-bound growth factors may also modulate calcification. Extracellular matrix specifically binds cytokines and growth factors, affecting both their availability and biological activity.40 One growth factor that may be particularly important in vascular calcification is transforming growth factor-ß1 (TGF-ß1). TGF-ß1 is bound by a variety of matrix molecules, including fibronectin,41 42 and expression of the TGFß-1 gene is regulated by extracellular matrix.43 TGFß-1 is thought to be a major contributor to the sclerosis and fibrosis seen in atherosclerosis, as well as the end-stage fibrosis of parenchymal organs such as the liver, lungs, and kidneys.44 It has been proposed that many of the biological effects of TGFß-1 are mediated by its ability to regulate the synthesis of extracellular matrix, and TGF-ß1 is known to greatly increase the production of collagen I and fibronectin.45 We have previously shown TGFß-1 to greatly increase the formation of calcified nodules by CVCs.25 The current results suggest that the mechanism of this effect may be through alterations in matrix synthesis or composition.
Integrins are a large family of heterodimeric transmembrane receptors
that mediate attachment of cells to the extracellular
matrix.46 The integrin receptor family is
composed of distinct
and ß subunits that heterodimerize to form
receptors with characteristic binding specificities. As receptors for
the extracellular matrix, integrins mediate a variety of signals that
regulate several important events, including
differentiation.47 The
5 integrin subunit
heterodimerizes with the ß1 subunit to form a receptor for
fibronectin.48 In the current studies, blocking
antibodies to the
5 subunit inhibited the fibronectin-mediated
increase in alkaline phosphatase by CVCs. When CVCs were plated on a
collagen I substrate, there was a slight (nonsignificant) decrease in
alkaline phosphatase, and because cells plated on collagen I may
continue to produce their own fibronectin, some decrease in alkaline
phosphatase activity might be expected. The decrease seen in alkaline
phosphatase activity when CVCs plated on fibronectin were treated with
the blocking antibody to the
5 integrin was much greater and
suggests that integrin-based signaling is involved. Because the
anti-
5 antibody has not previously been shown to be neutralizing in
bovine cells, however, the possibility of a specific effect of this
antibody unrelated to integrins cannot be excluded.
Several diseases as well as normal physiological processes are known to be modulated by changes in the extracellular matrix. The current results reveal that extracellular matrix changes also modulate in vitro vascular calcification. Further research on the interplay between vascular cells, their extracellular matrix, and cellular integrins may lead to new therapeutic approaches to treat atherosclerotic calcification.
| Acknowledgments |
|---|
Received February 17, 1998; accepted July 27, 1998.
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M. D. Frame, R. J. Rivers, O. Altland, and S. Cameron Mechanisms initiating integrin-stimulated flow recruitment in arteriolar networks J Appl Physiol, June 1, 2007; 102(6): 2279 - 2287. [Abstract] [Full Text] [PDF] |
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I. Z. Jaffe, Y. Tintut, B. G. Newfell, L. L. Demer, and M. E. Mendelsohn Mineralocorticoid Receptor Activation Promotes Vascular Cell Calcification Arterioscler Thromb Vasc Biol, April 1, 2007; 27(4): 799 - 805. [Abstract] [Full Text] [PDF] |
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K. Subburaman, N. Pernodet, S. Y. Kwak, E. DiMasi, S. Ge, V. Zaitsev, X. Ba, N. L. Yang, and M. Rafailovich Templated biomineralization on self-assembled protein fibers PNAS, October 3, 2006; 103(40): 14672 - 14677. [Abstract] [Full Text] [PDF] |
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K. Schapira, E. Lutgens, A. de Fougerolles, A. Sprague, A. Roemen, H. Gardner, V. Koteliansky, M. Daemen, and S. Heeneman Genetic Deletion or Antibody Blockade of {alpha}1{beta}1 Integrin Induces a Stable Plaque Phenotype in ApoE-/- Mice Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1917 - 1924. [Abstract] [Full Text] [PDF] |
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L. L Demer Vascular calcification and osteoporosis: inflammatory responses to oxidized lipids Int. J. Epidemiol., August 1, 2002; 31(4): 737 - 741. [Full Text] [PDF] |
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T. B. Drueke Foreword: Extraskeletal calcifications in patients with chronic renal failure Nephrol. Dial. Transplant., February 1, 2002; 17(2): 330 - 331. [Full Text] [PDF] |
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A. F. Zebboudj, M. Imura, and K. Bostrom Matrix GLA Protein, a Regulatory Protein for Bone Morphogenetic Protein-2 J. Biol. Chem., February 1, 2002; 277(6): 4388 - 4394. [Abstract] [Full Text] [PDF] |
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A Farzaneh-Far, D Proudfoot, C Shanahan, and P L Weissberg Vascular and valvar calcification: recent advances Heart, January 1, 2001; 85(1): 13 - 17. [Full Text] |
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C. Iribarren, S. Sidney, B. Sternfeld, and W. S. Browner Calcification of the Aortic Arch: Risk Factors and Association With Coronary Heart Disease, Stroke, and Peripheral Vascular Disease JAMA, June 7, 2000; 283(21): 2810 - 2815. [Abstract] [Full Text] [PDF] |
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