Acetylated Low-Density Lipoprotein Stimulates Human
Vascular Smooth Muscle Cell Calcification by Promoting
Osteoblastic Differentiation and Inhibiting Phagocytosis
D. Proudfoot, PhD; J.D. Davies, PhD; J.N. Skepper, PhD; P.L. Weissberg, MD, FRCP; C.M. Shanahan, PhD
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Background—Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and
calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in
atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification.
Methods and Results—In situ hybridization of human atherosclerotic plaques (n⫽10) collected from patients undergoing
carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed
alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of
VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time
course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold,
whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase–polymerase chain reaction and
Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs,
and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve
as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of
nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased ⬇3-fold, implying a defect in
phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence
of acLDL.
Conclusions—These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic
removal of apoptotic bodies. (Circulation. 2002;106:●●●-●●●.)
Key Words: lipoproteins 䡲 apoptosis 䡲 muscle, smooth
V
generation of nucleation sites. In cartilage and bone, these
initiation sites are thought to be matrix vesicles, which are
membrane-bound bodies, often generated in association with
apoptosis, which bud off chondrocytes and osteoblasts.9,10
Similar structures derived from apoptotic VSMCs have been
identified in human calcified arteries.11 Like matrix vesicles,
apoptotic bodies can accumulate calcium ions and calcify in
vitro,12,13 suggesting that they may be key sites for calcium
crystal nucleation in plaques.
Calcification also requires the presence of a permissive
matrix that allows proliferation of calcium crystals. In the
normal uncalcified media, VSMCs have a contractile
phenotype and constitutively express proteins that inhibit
mineralization. These include matrix Gla protein (MGP),14
osteonectin, and osteopontin (OP).15 However, in calcified
atherosclerotic plaques, other bone-associated proteins
have been detected, including bone morphogenetic proteins, bone sialoprotein (BSP), alkaline phosphatase
(ALK), and bone Gla protein (BGP).16,17 The exact role of
ascular calcification is a common and clinically significant component of several human diseases, including
atherosclerosis, aortic stenosis, and diabetes. Its presence
correlates with an increased risk of myocardial infarction,1,2
and it impedes vascular function by decreasing vessel elasticity.3 Atherosclerotic calcification is seen as early as the
second decade of life, just after the fatty streak stage,4 and
many studies have demonstrated a clear association between
lipid accumulation and atherosclerotic calcification.4 –7 Calcification colocalizes with cholesterol crystals in human atherosclerosis5 and is present in the intima of cholesterol-fed
rabbits6 and ApoE knockout mice.7 Microscopic calcification
in aortic valves seems to form in areas in which lipoproteins
have been retained,8 and early deposits of calcification occur
in and around isolated vascular smooth muscle cells
(VSMCs) within the lipid core.4 Despite these associations,
little is known of the role lipids play in regulating vascular
calcification.
Our present understanding of the sequence of events
leading to calcium crystal formation firstly involves the
Received July 18, 2002; revision received September 13, 2002; accepted September 15, 2002.
From the Department of Medicine, University of Cambridge and Department of Anatomy, Multi-Imaging Centre, Cambridge, UK.
Correspondence to D. Proudfoot, Department of Medicine, University of Cambridge, Box 110, Addenbrooke’s Hospital, Level 6, ACCI Building, Hills
Road, Cambridge CB2 2QQ. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000041429.83465.41
1
2
Circulation
December 10, 2002
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Figure 1. In situ hybridization showing
expression of ALK, BSP, and BGP in
human carotid endarterectomy sections.
A, An area of the media at the base of a
plaque. m indicates the medial area; i,
the intima. a through f are adjacent sections showing ␣-smooth muscle-actin
staining (a), von Kossa staining (b), ORO
staining (c), dark-field photomicroscopy
demonstrating hybridization of antisense
probes to ALK (d), BSP (e), and BGP (f).
B, a through d are adjacent sections of
an enlarged area of isolated VSMCs in
the intima from panel A. Arrows indicate
VSMC outline. a, ␣-smooth muscle-actin;
b, lack of macrophages in this area by
staining for CD68; c, von Kossa staining;
and d, ORO staining. e through h are
isolated lipid-filled intimal VSMCs showing mRNA expression for ALK (e), BSP
(f), and BGP (g) (indicated by arrows). h,
Negative control for in situ hybridization.
these induced proteins in regulating mineralization is not
known, but their presence implies that osteogenic conversion of VSMCs may occur in calcified vessels. Indeed,
extensive evidence in vitro has shown that VSMCs take on
an osteo/chondrocytic phenotype.16 –18
In this study we demonstrate for the first time the
presence of subsets of VSMCs exhibiting an osteogenic
gene expression profile within atherosclerotic plaques.
These VSMCs were located in lipid-rich areas of the
plaque, and studies in vitro demonstrated that spontaneous
lipid accumulation occurred in osteogenic VSMCs and
preceded their calcification. Moreover, addition or removal of lipoproteins altered the time course of osteogenic
differentiation and calcification of VSMCs. Additional
analysis of the mechanisms involved in accelerated calcification revealed a novel mechanism whereby modified
lipoproteins act via the scavenger receptor SRA1 to inhibit
VSMC phagocytosis of apoptotic bodies, thereby creating
additional nidi for calcification.
Methods
Cell Culture and Materials
VSMCs were cultured from explants of human aorta (from males
ranging in age from 15 to 70 years) in 20% FCS/Medium 199, as
described previously.18 Lipoprotein-deficient serum (LPDS) and
low-density lipoprotein (LDL) were a gift from Dr C. Fitzsimmons,
Cambridge, and were prepared by ultracentrifugation.19 Acetylated
LDL (acLDL) was prepared by addition of 1.5 ␮L acetic anhydride
per milligram of LDL in 50% ice-cold saturated sodium acetate over
a 1-hour period at 4°C. The acLDL was dialyzed against PBS/0.3
mol/L EDTA and once against PBS and gave a single band on
agarose electrophoresis with an REM value of 5.0, ie, pure, fully
acetylated LDL.
In Situ Hybridization
Carotid endarterectomy samples from 10 patients were snap-frozen
and mounted in Tissue-Tek OCT embedding compound (Miles
Ames Division, Inc). Sections (8 to 10 ␮m) were mounted onto
gelatinized slides and processed for in situ hybridization as previously described.15 Sense and antisense 35S-labeled cRNA probes
were transcribed from cRNA of human ALK, BSP, and BGP, cloned
into PCRII (Invitrogen). Slides were exposed for 3 weeks, devel-
Proudfoot et al
Lipids and Vascular Calcification
3
Measurement of Calcification
Calcification was measured in cultures at day 28 either by staining
with Alizarin red or by the cresolphthalein method as described
previously.13
Reverse-Transcription Polymerase Chain Reaction
Figure 2. Lipid staining in 7-day VSMC nodular cultures. A,
ORO staining revealed that lipid was only associated with the
nodule, not the monolayer. B, A maximum intensity projection of
a whole nodule incubated with Nile red (shown as green) and
Hoechst (blue) generated by confocal microscopy. The small
arrow indicates intracellular lipid droplets, whereas the larger
arrow indicates a pool of extracellular lipid.
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
oped, stained with H&E, and mounted. Immunohistochemistry was
performed with an avidin-biotin immunoperoxidase kit (Dako).
VSMCs were identified with a mouse monoclonal antibody (Dako
M815, dilution 1:25) to human ␣-smooth muscle actin, and macrophages were identified with a mouse monoclonal antibody to CD68
(Dako macrophage EMB11, dilution 1:20). Calcification and lipid
were demonstrated by von Kossa and oil red O (ORO) staining,
respectively.
Detection of Lipid
VSMC cultures were fixed in 4% formaldehyde in PBS for 15
minutes and stained with ORO solution. Alternatively, cells were
incubated with Nile red (500 ng/mL in PBS) for 30 minutes, rinsed
briefly with bisbenzimide (Hoechst, 2 ␮g/mL in PBS, Sigma), and
washed in PBS before observation with a Leica TCS-SP-MP
confocal microscope. DiI-labeled acLDL (DiI-acLDL, Biomedical
Technologies, Inc) uptake by nodules was assessed using the
manufacturer’s protocol.
RNA and cDNA were prepared from monolayer and nodular VSMCs
and analyzed by reverse-transcription polymerase chain reaction
(RT-PCR) as described previously.15,18 Briefly, to ensure that each
PCR reaction was performed within the linear range of amplification,
test reactions were performed for each primer pair at 20, 25, 30, 35,
and 40 cycles. Southern blots were performed on these reaction
products and hybridized and counted. Plots of these were used to
establish linearity and final quantitation, and normalization of PCR
products was performed in real time using an Instant Imager.15 The
oligonucleotide primers were designed from published human sequences in the Genbank/European Molecular Biology databases,
with the size of the product and sequencing verifying correct gene
amplification.
Western Analysis
Nodules were separated from monolayers by trypsinization and
subsequent filtration through a 70-␮m sieve (Falcon), which retained
the nodules. Monolayer, nodules, or human peripheral blood monocytes (prepared as described previously18) were lysed, resuspended
in sample buffer containing 100 mmol/L ␤-mercaptoethanol, and
resolved by 6% SDS-PAGE after the protocol of Gough et al.20 The
mouse polyclonal SRA antibody (a gift from D. Greaves, Oxford20)
was used at a dilution of 1:2000, and binding was detected with a
peroxidase-conjugated anti-mouse IgG (Amersham, 1:1000) and
visualized by chemiluminescence (ECL, Amersham).
Apoptosis/Apoptotic Body Measurement
Apoptosis was measured in nodules as described previously.13
Apoptotic bodies were quantified by counting small, intensely
Figure 3. Analysis of lipid and calcification in 28-day nodules treated with 20%
LPDS, native LDL (nLDL, 100 ␮g/mL), or
acLDL (100 ␮g/mL). A, ORO and Alizarin
red staining are much weaker in LPDStreated nodules compared with either
nLDL- or acLDL-treated nodules. B,
Measurement of calcification using the
cresolphthalein method. Controls were
incubated with 20% FCS/M199 alone.
4
Circulation
December 10, 2002
Figure 4. A, RT-PCR analysis of gene
expression in monolayer and nodular
VSMCs at days 3, 7, and 14. ␤micro indicates ␤2-microglobulin control. B, Western
blot analysis for SRA protein in monolayer
and nodular VSMCs. 10 ␮g of human peripheral blood-derived monocyte/macrophage lysates (hPBM, positive control) and
20 ␮g of either day-14 monolayer or nodular cell lysates were separated under
reducing conditions on 6% SDS-PAGE. A
75-kDa monomer of SRA was detected in
hPBM and nodular VSMCs.
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Hoechst-stained structures in nodules by acquiring serial optical
sections through whole nodules using confocal microscopy.
Apoptotic Body Binding Assay
The number of apoptotic bodies bound to VSMCs was assessed by
a modified method of Bennett et al.21 Briefly, VSMCs were
dispersed from nodules as described previously18 and plated in
4-well chamber slides at 10 000 cells/well. Apoptotic bodies were
prepared by serum-starving HASMC 66 cells13 and collected by
centrifugation at 2500 rpm. The bodies were incubated with 2 ␮g/mL
Hoechst and washed with medium before incubating with VSMCs in
chamber slides at 200 000 bodies/well for 2 hours at 37°C. The cells
were then washed 3 times in PBS and fixed in 4% formaldehyde.
Apoptotic body binding was measured by counting fluorescent
bodies bound to underlying VSMCs. Approximately 50 VSMCs
were counted from a random area of at least 8 different wells. In
some experiments, cells were incubated with a goat SRA polyclonal
antibody (Chemicon).
Statistical Analysis
ANOVA was used to test for differences within groups of samples.
Student’s t test was used to compare two means. Results are
expressed as mean⫾SD
Results
VSMC Expression of Osteogenic Markers in
Atherosclerotic Plaques
days.15,18 Using ORO, we demonstrated that these nodules
accumulated lipid, even at early stages before calcification
occurred (Figure 2A). Confocal microscopy using Nile red to
image lipid within nodules revealed that the lipid droplets were
present both intracellularly and extracellularly (Figure 2B).
Requirement of Lipid/Lipoproteins for Calcification
To test whether lipoproteins were required for calcification,
VSMCs were incubated in medium containing LPDS for 28
days. The nodules that formed in the presence of LPDS
stained less intensely with ORO and Alizarin red compared
with native LDL controls (Figure 3A). LPDS also significantly inhibited nodule formation (control, 22.5⫾4.2 nodules/
well; LPDS, 2.4⫾0.4 nodules/well; n⫽4; P⬍0.001) and
calcification (Figure 3B). Conversely, addition of lipid in the
form of acLDL stimulated calcification 3-fold (Figure 3B)
and nodule formation (control, 22.5⫾4.2 nodules/well;
acLDL, 37.1⫾4.3 nodules/well; n⫽4, P⫽0.003). Calcification and lipid accumulation were confined to the nodules after
acLDL treatment; the monolayer cells did not accumulate
lipid (Figure 3A). Thus, lipoproteins (or lipoproteinassociated molecules) are required for nodule formation and
calcification, and this process is stimulated by acLDL.
Mechanism of VSMC Nodule Lipid Accumulation
In situ hybridization of human carotid atherosclerotic plaques
demonstrated that VSMCs in lipid-rich, calcified regions at
the base of plaques expressed BSP, BGP, and ALK (Figure
1A). These genes were not expressed by normal medial
VSMCs (data not shown17). In addition, isolated, lipid-filled
VSMCs in the intima also coexpressed these bone markers
(Figure 1B). Thus, human VSMCs in lipid-rich atherosclerotic lesions undergo osteogenic differentiation and elaborate
a bone-like matrix.
We next compared the monolayer and nodular VSMCs for
expression of lipoprotein and scavenger receptors using
RT-PCR. Monolayer and nodular cells expressed similar
levels of CLA1, CD68, LDL receptor, LDL–related protein
receptor, and the very low-density lipoprotein receptor (Figure 4A), whereas SRAII, as well as CD36 and LOX-1, were
not detected (data not shown). However, scavenger receptor
class AI (SRAI) mRNA was absent in monolayer cells, but its
expression increased as nodules developed (Figure 4A). This
phenomenon was confirmed by Western analysis (Figure 4B).
Lipid Is Associated With VSMC Calcification In Vitro
Effects of Blocking SRA on Calcification
In vitro, human VSMCs spontaneously express boneassociated proteins and form nodules that calcify at ⬇28
To test the involvement of SRA1 in calcification, VSMC
cultures were incubated with acLDL in the presence of
Proudfoot et al
Lipids and Vascular Calcification
5
Effects of acLDL on Apoptosis
Nodules treated with acLDL had similar apoptotic indices to
native LDL controls (Figure 7A). However, there was a
greater abundance of apoptotic bodies within nodules treated
with acLDL (Figure 7B). This implied that acLDL might
interfere with phagocytic removal of apoptotic bodies. This
was tested using an apoptotic body binding assay in which
apoptotic bodies derived from VSMCs were labeled with
Hoechst and incubated with SRA1-positive VSMCs dispersed from nodules (Figure 8A). This assay showed that
VSMC-apoptotic body binding was significantly reduced in
the presence of either acLDL or SRA antibodies (Figures 8B
and 8C). AcLDL had no effect on apoptotic body binding to
SRA1-negative monolayer VSMCs (data not shown).
Discussion
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Figure 5. A, Effect of polyinosinic acid (PI) on acLDL-induced
calcification. VSMCs were treated with either acLDL (100
␮g/mL), PI (100 ␮g/mL), acLDL⫹PI, or no additions (control) for
28 days and calcification measured by counting numbers of
nodules positive for Alizarin red staining. PI had no effect alone
but significantly inhibited the acLDL-induced calcification. B,
Effect of PI on acLDL uptake into nodules. DiI-acLDL (10 ␮g/mL)
was incubated with nodular cultures⫾PI (100 ␮g/mL) for 4
hours, and uptake was qualitatively estimated using fluorescence microscopy. Uptake of DiI-acLDL was reduced in the
presence of PI. The lower panel shows corresponding Hoechststained nodules.
polyinosinic acid (PI), a ligand for SRA. PI was found to
block the stimulatory effect of acLDL on nodule calcification
(Figure 5A). To confirm the interaction of PI with SRA,
nodules were incubated with or without PI and assessed for
acLDL uptake using DiI-acLDL. Nodules incubated with PI
accumulated less DiI-acLDL (Figure 5B).
Effects of acLDL on Osteogenic Differentiation
of VSMCs
Human VSMCs were treated with either LPDS, native LDL,
or acLDL for up to 28 days, and expression of osteogenic
markers was assessed by RT-PCR (Figure 6). As previously
reported, human VSMCs default to an osteo/chondrocytic
phenotype in vitro, and their expression of bone markers
changes as calcification occurs.17 Compared with native
LDL-treated controls, LPDS-treated cells expressed higher
levels of OP and BGP. Treatment with acLDL increased
expression of ALK and BSP by day 21. AcLDL and LPDS
did not alter the expression of MGP or osteonectin mRNA
(data not shown). Therefore, lipoproteins influence the expression of a subset of bone marker genes by VSMCs in vitro.
Previous studies have demonstrated a link between lipids and
vascular calcification and implied that lipids may have a role
in nucleating calcium crystals5,22 or stimulating osteogenic
differentiation.23,24 We have extended these studies in human
VSMCs and investigated the expression of several bone
marker genes as well as the role of lipoproteins in apoptosis
and apoptotic body clearance. This study demonstrates, for
the first time, that an osteogenic phenotype of VSMCs exists
in atherosclerotic lesions in association with sites of lipid
accumulation and calcification.
The mechanisms linking lipid, osteogenic gene expression,
and calcification observed in vivo were investigated using a
model of VSMC calcification where postconfluent VSMCs
form nodules that calcify within 28 days. These studies
demonstrated that lipid accumulation occurred spontaneously
in nodules and in the absence of lipoproteins, fewer nodules
formed, less lipid accumulated, and less calcification occurred. In the presence of acLDL, nodule formation, lipid
accumulation, and calcification were stimulated above the
native LDL controls. The mechanism by which lipid accumulates in VSMCs is not known, but we showed that human
VSMCs and nodules expressed several lipoprotein and scavenger receptors. In particular, the acLDL receptor, SRA1,
which is not normally expressed by cultured VSMCs, was
specifically induced in nodular cells. These studies raised the
question of why SRA1 is induced in VSMCs, and one
possibility is that in the apoptotic environment of the VSMC
nodule, oxidative stress would be expected, which is a known
inducer of expression of SRA in VSMCs.25 Treatment of
nodules with PI inhibited acLDL-induced stimulation of
calcification. Although PI is not a specific SRA ligand, these
results suggest that acLDL may interact with SRA1 to
stimulate calcification.
Treatment of VSMC cultures with LPDS or acLDL
changed their osteogenic gene expression profile. However,
the extent to which changes in bone-marker gene expression
affect calcification is not clear because of the poor knowledge
of the function of many of the proteins involved. ALK and
BSP are thought to be procalcification factors, whereas BGP
and OP have been demonstrated as anticalcification factors.26,27 VSMCs exposed to LPDS expressed higher levels of
BGP and OP mRNA than calcifying nodular cultures, in
keeping with the inhibitory action of these proteins. Con-
6
Circulation
December 10, 2002
Figure 6. A, Southern blots and agarose
gel electrophoresis showing RT-PCR
analysis of gene expression in VSMC
cultures at days 14, 21, and 28 treated
with LPDS, control medium (nLDL, 100
␮g/mL), or acLDL, 100 ␮g/mL. B,
Graphs represent quantification of data
from 6A showing relative amounts of
ALK, BSP, and BGP mRNA normalized
to control (␤-microglobulin).
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
versely, acLDL increased expression of ALK and BSP, which
might stimulate nodular calcification. Indeed, increased expression of ALK in nodular cells by acLDL could potentially
stimulate production of apoptotic bodies with increased ALK
activity, which is known to be a key regulator/initiator of
matrix vesicle calcification via generation of local phosphate
ions.9 These findings are supported by other studies in which
minimally oxidized LDL and isoprostanes stimulated ALK
activity in calcifying vascular cells.24
Figure 7. A, Effect of acLDL on nodule apoptosis. Nodular cultures were incubated with nLDL or acLDL (both 100 ␮g/mL) for
7 days and then incubated with 2 ␮g/mL Hoechst. Serial optical
sections through nodules were imaged using confocal microscopy. Condensed or fragmented nuclei were counted in both
groups, but no significant difference was found. B, Effect of
acLDL on apoptotic body number in nodules. The nodules in A
were additionally analyzed for content of apoptotic bodies by
counting small, condensed, and isolated fluorescent structures
and presented relative to the total amount of apoptosis. The
acLDL-treated nodules contained significantly more apoptotic
bodies than the nLDL-treated nodules (*P⫽0.01, n⫽6).
Our previous studies have shown that apoptosis occurs
spontaneously at a relatively high rate in nodules (⬇20%) and
that alteration of the apoptotic rate influences calcification.13
These studies also showed that apoptotic bodies derived from
VSMCs can serve as a nidus for calcification. Apoptotic
bodies are normally rapidly phagocytosed in vivo, but their
detection in atherosclerotic plaques10,11 implies that phagocytosis is inhibited, which would promote calcification in an
environment with sufficient local Ca2⫹, PO43⫺, and appropriate matrix. Given the importance of apoptosis in calcification
and because other studies have demonstrated that modified
lipoproteins can stimulate apoptosis,28 we tested the effects of
acLDL on nodule apoptosis. There was, however, no effect of
acLDL on nodule apoptosis, but we did find a greater number
of apoptotic bodies in acLDL-treated nodules. This implied a
defect in phagocytosis of apoptotic bodies by nodular
VSMCs in the presence of acLDL, which was confirmed in
an apoptotic body binding assay. SRA antibodies also inhibited apoptotic body binding, which provided a mechanism to
explain how acLDL might stimulate calcification; ie, acLDL
competes with apoptotic bodies for SRA binding. Other
studies have shown that oxidized LDL competes with apoptotic bodies for macrophage binding,29 and SRA is thought
to be important in this process.30 This raises the possibility
that other oxidized lipids that are known to stimulate calcification could also act via inhibition of phagocytosis. Thus, our
studies suggest that VSMC as well as macrophage phagocytosis is likely to be inhibited in the setting of the lipid-rich
atherosclerotic plaque and may explain why apoptotic bodies
are detected in these plaques.31 In relation to this, a lack of
phagocytic function in plaques may also contribute to the
finding of vascular-derived apoptotic vesicles in circulating
blood of atherosclerosis-prone patients.32
In conclusion, these studies highlight a new role for
lipoproteins and VSMCs in the development of calcification
in atherosclerosis.
Acknowledgments
This study was supported by a British Heart Foundation program
grant (RG/200004).
Proudfoot et al
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Figure 8. A, Demonstration of apoptotic body binding to
VSMCs. VSMC-derived apoptotic bodies were labeled with
Hoechst and incubated with VSMCs dispersed from nodules for
2 hours. After extensive washing with PBS and fixing, the cultures were analyzed by fluorescence microscopy. AcLDL 100
␮g/mL (B) and SRA polyclonal antibody 1:200 (C) significantly
inhibited the number of apoptotic bodies bound to VSMCs
(*P⬍0.0002, n⫽12; *P⬍0.0001; n⫽8, respectively).
References
1. Beadenkopf WG, Daoud AS, Love BM. Calcification in the coronary
arteries and its relationship to arteriosclerosis and myocardial infarction.
Am J Roentgenol Radium Ther Nucl Med. 1964;92:865– 871.
2. Farb A, Burke AP, Tang AL, et al. Coronary plaque erosion without rupture into
a lipid core: a frequent cause of coronary thrombosis in sudden coronary death.
Circulation. 1996;93:1354–1363.
3. Farzaneh-Far A, Proudfoot D, Shanahan C, et al. Vascular and valvar calcification: recent advances. Heart. 2001;85:13–17.
4. Stary HC. Atlas of Atherosclerosis: Progression and Regression. New York, NY:
Parthenon; 1999.
5. Sarig S, Weiss TA, Katz I, et al. Detection of cholesterol associated with calcium
mineral using confocal fluorescence microscopy. Lab Invest. 1994;71:782–787.
6. Kramsch DM, Chan CT. The effect of agents interfering with soft tissue calcification and cell proliferation on calcific fibrous-fatty plaques in rabbits. Circ Res.
1978;42:562–571.
Lipids and Vascular Calcification
7
7. Qiao JH, Xie PZ, Fishbein MC, et al. Pathology of atheromatous lesions in inbred
and genetically engineered mice: genetic determination of arterial calcification.
Arterioscler Thromb. 1994;14:1480–1497.
8. O’Brien KD, Reichenbach DD, Marcovina SM, et al. Apolipoproteins B (a) and
E accumulate in the morphologically early lesion of “degenerative” valvular
aortic stenosis. Arterioscler Thromb Vasc Biol. 1996;16:523–532.
9. Anderson HC. Molecular biology of matrix vesicles. Clin Orthop Rel Res.
1995;314:266–280.
10. Kim KM. Apoptosis and calcification. Scanning Microsc. 1995;9:1137–1178.
11. Kockx MM, DeMeyer GRY, Muhring J, et al. Apoptosis and related proteins in
different stages of human atherosclerotic plaques. Circulation. 1998;97:
2307–2315.
12. Hashimoto S, Ochs RL, Rosen F, et al. Chondrocyte-derived apoptotic bodies and
calcification of articular cartilage. Proc Natl Acad Sci U S A. 1998;95:
3094–3099.
13. Proudfoot D, Skepper JN, Hegyi L, et al. Apoptosis regulates human vascular
calcification in vitro: evidence for initiation of vascular calcification by apoptotic
bodies. Circ Res. 2000;87:1055–1062.
14. Luo GDP, McKee MD, Pinero GJ, et al. Spontaneous calcification of arteries and
cartilage in mice lacking matrix Gla protein. Nature. 1997;386:78–81.
15. Shanahan CM, Cary NR, Salisbury JR, et al. Medial localization of
mineralization-regulating proteins in association with Monckeberg’s sclerosis:
evidence for smooth muscle cell-mediated vascular calcification. Circulation.
1999;100:2168–2176.
16. Bostrøm K, Watson KE, Horn S, et al. Bone morphogenetic protein expression in
human atherosclerotic lesions. J Clin Invest. 1993;91:1800–1809.
17. Shanahan CM, Proudfoot D, Tyson KL, et al. Expression of mineralisationregulating proteins in association with human vascular calcification. Z Kardiol.
2000;89(suppl 2):63–68.
18. Proudfoot D, Skepper JN, Shanahan CM, et al. Calcification of human vascular
cells in vitro is correlated with high levels of matrix Gla protein and low levels of
osteopontin expression. Arterioscler Thromb Vasc Biol. 1998;18:379–388.
19. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of
ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:
1345–1354.
20. Gough PJ, Greaves DR, Suzuki H, et al. Analysis of macrophage scavenger
receptor (SR-A) expression in human aortic atherosclerotic lesions. Arterioscler
Thromb Vasc Biol. 1999;19:461–471.
21. Bennett MR, Gibson DF, Schwartz SM, et al. Binding and phagocytosis of
apoptotic vascular smooth muscle cells is mediated in part by exposure of
phosphatidyl serine. Circ Res. 1995;77:1136–1142.
22. Cotmore JM, Nichols G, Wuthier RE. Phospholipid-calcium phosphate complex:
enhanced calcium migration in the presence of phosphate. Science. 1971;172:
1339–1341.
23. Watson KE, Bostrøm K, Ravindranath R, et al. TGF-␤1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994;93:
2106–2113.
24. Parhami F, Morrow AD, Balucan J, et al. Lipid oxidation products have opposite
effects on calcifying vascular cell and bone cell differentiation: a possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler
Thromb Vasc Biol. 1997;17:680–687.
25. Mietus-Snyder M, Gowri MS, Pitas RE. Class A scavenger receptor
up-regulation in smooth muscle cells by oxidized low density lipoprotein:
enhancement by calcium flux and concurrent cyclooxygenase-2 up-regulation.
J Biol Chem. 2000;275:17661–17670.
26. Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcindeficient mice. Nature. 1996;382:448–452.
27. Wada T, McKee MD, Steitz S, et al. Calcification of vascular smooth muscle cell
cultures: inhibition by osteopontin. Circ Res. 1999;84:166–178.
28. Okura Y, Brink M, Itabe H, et al. Oxidized low-density lipoprotein is associated
with apoptosis of vascular smooth muscle cells in human atherosclerotic plaques.
Circulation. 2000;102:2680–2686.
29. Chang MK, Bergmark C, Laurila A, et al. Monoclonal antibodies against
oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidation-specific epitopes
mediate macrophage recognition. Proc Natl Acad Sci U S A. 1999;96:
6353–6358.
30. Platt N, Suzuki H, Kurihara Y, et al. Role for the class A macrophage scavenger
receptor in the phagocytosis of apoptotic thymocytes in vitro. Proc Natl Acad Sci
U S A. 1996;93:12456–12460.
31. Hegyi L, Skepper JN, Cary NR, et al. Foam cell apoptosis and the development
of the lipid core of human atherosclerosis. J Pathol. 1996;180:423–429.
32. Mallat Z, Benamer H, Hugel B, et al. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients
with acute coronary syndromes. Circulation. 2000;101:841–843.
Acetylated Low-Density Lipoprotein Stimulates Human Vascular Smooth Muscle Cell
Calcification by Promoting Osteoblastic Differentiation and Inhibiting Phagocytosis
D. Proudfoot, J. D. Davies, J. N. Skepper, P. L. Weissberg and C. M. Shanahan
Circulation. published online November 11, 2002;
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2002 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/early/2002/11/11/01.CIR.0000041429.83465.41.citation
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/