Molecular Mechanisms of Vascular Calcification: Lessons Learned From The

Aorta
Jian-Su Shao, Jun Cai and Dwight A. Towler
Arterioscler Thromb Vasc Biol 2006;26;1423-1430; originally published online Apr 6,
2006;
DOI: 10.1161/01.ATV.0000220441.42041.20
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.
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 Brief Reviews

Molecular Mechanisms of Vascular Calcification

Lessons Learned From The Aorta
Jian-Su Shao, Jun Cai, Dwight A. Towler

Abstract—Vascular calcification increasingly afflicts our aging and dysmetabolic population. Once considered a passive process,
it has emerged as an actively regulated form of calcified tissue metabolism, resembling the mineralization of endochondral
and membranous bone. Executive cell types familiar to bone biologists, osteoblasts, chondrocytes, and osteoclasts, are seen
in calcifying macrovascular specimens. Lipidaceous matrix vesicles, with biochemical and ultrastructural “signatures” of
skeletal matrix vesicles, nucleate vascular mineralization in diabetes, dyslipidemia, and uremia. Skeletal morphogens (bone
morphogenetic protein-2 (BMP) and BMP4 and Wnts) divert aortic mesoangioblasts, mural pericytes (calcifying vascular
cells), or valve myofibroblasts to osteogenic fates. Paracrine signals provided by these molecules mimic the epithelial–
mesenchymal interactions that induce skeletal development. Vascular expression of pro-osteogenic morphogens is entrained
to physiological stimuli that promote calcification. Inflammation, shear, oxidative stress, hyperphosphatemia, and elastinolysis
provide stimuli that: (1) promote vascular BMP2/4 signaling and matrix remodeling; and (2) compromise vascular defenses
that limit calcium deposition, inhibit osteo/chondrogenic trans-differentiation, and enhance matrix vesicle clearance. In this
review, we discuss the biology of vascular calcification. We highlight how aortic fibrofatty tissue expansion (adventitia, valve
interstitium), the adventitial-medial vasa, vascular matrix, and matrix vesicle metabolism contribute to the regulation of aortic
calcium deposition, with greatest emphasis placed on diabetic vascular disease. (Arterioscler Thromb Vasc Biol. 2006;26:
1423-1430.)
Key Words: diabetes 䡲 vascular calcification 䡲 Wnt signaling 䡲 bone morphogenetic proteins 䡲 oxylipids

W ith advanced age, vascular inflammation, hyperten-

sion, and certain metabolic disorders, calcium accu-
mulates in the arterial macrovasculature.1 Calcification of
aortic valve leaflets and atherosclerotic plaques have long
been recognized as clinically important.2 However, medial
artery calcification (MAC) also portends mortality and
amputation risk.1,3–5 Studies of vascular calcified tissue
metabolism significantly lag behind those of skeletal
metabolism. Executive cell types familiar to bone biolo-
gists are seen in calcifying aortic specimens.1,6 As in bone,
endothelial, mesenchymal, and hematopoietic cell lineages
control vascular mineral accumulation, with cellular activ-
ities entrained to morphogenetic, metabolic, inflammatory,
and mechanical demands placed on each vascular
segment.1
We provide a brief overview of vascular calcification,
emphasizing how paracrine osteogenic signals recruited by
dysmetabolic insults promote aortic calcium deposition in
diabetic vascular disease. We point to emerging evidence that
inflammation, mechanical, and metabolic oxidative stresses
not only provide stimuli that induce vascular osteogenic
morphogens but also compromise defense mechanisms that
limit vascular calcium deposition.1
Aortic MAC
MAC is a highly characteristic feature of diabetes and chronic
kidney disease (CKD).3,4 Although diabetes is the major
cause of CKD, hyperglycemia conveys independent risk for
vascular calcification.5,7 Aortic calcium scores, but not coro-
nary calcium scores, are linearly related to fasting blood
glucose.8 MAC has emerged as an exceptionally strong
predictor of lower extremity amputation and mortality in
patients with type II diabetes;4,9 mechanisms are still unclear
but may relate to abnormal aortofemoral Windkessel physi-
ology that generates systolic hypertension, increases myocar-
dial workload, and perturbs normal microvascular tissue
perfusion. Aortic pulse wave velocity, an index of vascular
stiffness, is highly correlated with the prevalence of aortic
calcification and diabetes in CKD5.10 Increases in aortic
stiffness convey the impact of diabetes-enhanced cardiovas-
cular mortality.11 Thus, a better understanding of the mech-
anisms controlling aortic MAC is required to address the
burgeoning unmet clinical needs of diabetic vascular disease
and CKD.
In diabetes and CKD, MAC proceeds via matrix vesicle-
nucleated mineralization,12–14 with apatitic calcium phosphate
deposition in the tunica media occurring in the absence of

Original received February 6, 2006; final version accepted March 22, 2006.
From the Washington University School of Medicine, Department of Medicine, St. Louis, Mo.
Correspondence to Dwight A. Towler, MD, PhD, Washington University School of Medicine, Campus Box 8301, 660 South Euclid Ave, St. Louis,
MO 63110. E-mail dtowler@im.wustl.edu
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000220441.42041.20

1423
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1424 Arterioscler Thromb Vasc Biol. July 2006
atheroma and neointima. (Of note, this differs from calcific
uremic arteriolopathy, an uncommon disorder in which fibro-
proliferative occlusion and medial calcification of arterioles
cause skin and sometimes intestinal necrosis15). The concen-
tric nature of MAC stands in stark contrast to the eccentric,
calcified atherosclerotic plaque.1,16 At least 2 types of lipid
vesicles have been identified to date that nucleate vascular
calcification: (1) the apoptotic bodies (250-nm diameter) of
dead and dying cells; and (2) mineralizing matrix vesicles
(100 nm diameter) actively extruded by viable vascular
smooth muscle cells (VSMCs) and calcifying vascular cells
(CVCs).12–14,17 The latter resembles the mineralization of
membranous bone,13,17 is intensely procalcific,14 and appears
predominate in aortic calcification.12,14,17
Mechanisms controlling MAC in type II diabetes are
beginning to be understood. High-fat diets that induce obe-
sity, insulin-resistant diabetes, and dyslipidemia promote
aortic MAC and valve calcification in male low-density
lipoprotein receptor (LDLR)– deficient mice.18,19 An aortic
bone morphogenetic protein-2 (BMP2)–muscle segment ho-
meobox homolog (Msx2) signaling cascade is activated by
mural oxidative stress and inflammatory cytokines18,19 (Fig-
ure 1). Because Msx2-dependent gene expression is critical
for craniofacial bone formation,20 this suggested that similar
signals participate in diabetic MAC. Intriguingly, a subset of
myofibroblasts in the fibrofatty aortic adventitia and aortic
valve interstitium, but not the tunica media, elaborated this
early BMP2–Msx2 response.18,19
Emerging evidence indicates that vascular osteogenic sig-
nals, initiated by adventitial BMP2–Msx2 actions, are con-
centrically conveyed to the calcifying tunica media via the
vasa vasorum18,19,21,22 (Figure 1). Diabetes causes low-grade
adventitial and medial inflammation, with adipocyte-laden
expansion and associated mural neoangiogenesis23,24(Figure
1). Primary vasa, arising from overt branch points in the
arterial tree, sprout and meander through the adventitia, then
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Figure 1. Evolving model of diabetic MAC. Diabetes
and dyslipidemia induce oxidative stress, low-grade
inflammation, and angiogenesis in the adventitia of dia-
betic arteries. Glucose, reactive oxygen species, and
TNF-␣ upregulate BMP2/4 production by pericytes and
endothelial cells in the vessel wall; this promotes
adventitial Msx2–Wnt signaling. Subsequently,
enhanced adventitial Wnt production (increased Wnt3a
and Wnt7a, decreased Dkk1) augments medial nuclear
␤-catenin accumulation, ALP activity, and osteogenic
differentiation. The mural CVC, a macrovascular myofi-
broblast related to the microvascular pericyte, is
thought to be the resident osteoprogenitor. Adventitial
Sca1⫹ mesenchymal progenitors are present in murine
aortas, contribute to medial and intimal disease pro-
cesses, and can undergo osteogenic differentiation in
response to BMP2–Wnt signaling. However, the lineage
relationship between Sca1⫹ progenitors and the CVC
is currently unknown; speculation based on studies of
aortic mesoangioblast development suggests that
CVCs arise from Sca1⫹ cells.35 Hyperphosphatemia, a
common feature of diabetes in the setting of CKD, pro-
motes osteo/chondrogenic “trans-differentiation” of aor-
tic VSMCs via Runx2.37 The relative extent to which
CVC recruitment vs VSMC “trans-differentiation” mech-
anisms contribute to the osteogenic calcification in dia-
betic MAC has yet to be determined.
ramify to form secondary vasa that circumferentially pene-
trate and percolate the aortic tunica media25 (Figure 1). The
vasa vasorum is most evident in larger mammals25 and
becomes grossly manifest in dyslipidic mice.26,27
What molecules convey vascular osteogenic signals? Re-
cent data from our laboratory19 and the Rajamannan labora-
tory28 have shown that Wnts are important. Wnts are secreted
polypeptides that bind specific LDLR-related protein (LRP)/
frizzled heterodimers, activate LRP5- and LRP6- signaling
cascades, and augment gene expression via nuclear ␤-catenin
in the canonical pathway.19,28 Cultured Msx2-expressing
mesenchymal cells secrete an osteogenic activity that is
antagonized by Dickkopf homolog (Dkk1), an inhibitory
ligand of LRP5/6 and paracrine Wnt signaling.19 These
results were confirmed in vivo using cytomegalovirus pro-
moter/immediate early enhancer–Msx2 transgenic mice,19 a
model validated previously in studies of ectopic calvarial
bone formation. Whereas Msx2 accumulates in the aortic
adventitia, alkaline phosphatase (ALP) induction occurs in
the tunica media with concomitant MAC.19 Importantly, the
Msx2 transgene selectively upregulated galactosidase (LacZ)
in the tunica media of TOPGAL mice (T-cell factor/lympoid
enhancer binding factor optimal promoter-galactosidase re-
porter mouse; demarcates canonical Wnt actions in vivo).19
The vector of mural microvascular flow is from adventitia to
media25 (Figure 1); therefore, we posited that paracrine Wnt
signals were elaborated by Msx2-expressing cells of the
adventitia, and that these Wnt signals programmed concentric
mineralization via the CVCs29 of the tunica media.19 Surgical
stripping of the adventitia significantly reduces MAC in rats
fed high-fat diets, consistent with this notion.21
How does induction of vascular BMP contribute to activa-
tion of this osteogenic signal? In craniofacial osteoblasts,
BMP2 is a key stimulus for Msx2 expression and enhances
Wnt signaling.30 Aortic Msx2–Wnt signaling is also stimu-
lated by BMP2 (Figure 2). Intraperitoneal BMP2 administra-
 Shao et al Molecular Mechanisms of Vascular Calcification 1425

Figure 2. BMP2 activation of aortic Msx2–Wnt signaling and MAC in vivo. A and B, TOPGAL⫹;LDLR⫹/⫺ mice were intraperitoneally
injected with either vehicle (VEH) or 50 ng/gm recombinant human BMP2 (R&D Systems) for 3 days. A, Aortic tissues were harvested
for analysis of mRNA accumulation by fluorescence RT-qPCR normalized to 18S signal. Data are presented as percentage of vehicle-
treated controls. BMP2 upregulated aortic Msx2 and canonical Wnt signaling, the latter indicated by the accumulation of LacZ mRNA.
B, Histochemical staining for LacZ (blue, nuclear red counterstain) in thoracic aortas of BMP2-treated animals confirmed that Wnt sig-
naling is enhanced in the tunica media. C, LDLR⫺/⫺ mice fed high-fat Western diets (HFD) were treated 3 days per week with either
vehicle (HFD⫹VEH) or 50 ng/gm BMP2 (HFD ⫹ BMP2) for 4 weeks. At death, aortic calcium content was measured. BMP2 significantly
increased aortic calcification (Student 2-sided t test). D, Alizarin red staining revealed that calcification occurred within the aortic tunica
media. A diet-induced factor, potentially an oxylipid-containing matrix vesicle,29,64,65,85 is required to robustly elaborate MAC in BMP2-
treated, Msx2 transgenic, and uremic murine disease models.19,84 Methods have been detailed previously.19

tion upregulates aortic Msx2 and LacZ mRNAs in TOPGAL
mice (Figure 2A). LacZ histochemistry localizes enhanced
canonical Wnt signaling to the aortic tunica media (Figure
2B). Moreover, thrice-weekly BMP2 treatment for 4 weeks
augments aortic calcium 2-fold in LDLR⫺/⫺ mice fed
high-fat diabetogenic diets (Figure 2C). Calcium accumula-
tion (alizarin red stain) again localizes to the aortic tunica
media (Figure 2D). Thus, BMP2 can activate an aortic Wnt
signaling cascade that drives osteogenic mineralization of
vascular progenitors via processes that resemble craniofacial
membranous bone formation.19,31 The concentric medial cal-
cification of diabetes is proposed to arise in part from the
vasa-dominated relationship between: (1) BMP2-stimulated
cells of the periaortic adventitia that express Msx2 and
elaborate Wnts;19 and (2) the CVCs in the tunica media6,29
(Figure 1). It remains possible that the osteogenic potential of
vascular progenitors is programmed within the adventitia but
is elaborated only when these progenitors migrate with the
vasa into the tunica media.22,32
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What are the origins of aortic osteogenic cells and Msx2-
expressing adventitial cells? At least 2 aortic mesenchymal
cell types can contribute to the ectopic osteogenic programs
of vascular calcification: (1) multipotent vascular mesenchy-
mal progenitors that are recruited to form the mural CVCs;
and (2) VSMCs that can undergo osteo/chondrogenic trans-
differentiation in response to hyperphosphatemia. Demer first
described the aortic CVCs.6,29 The CVC is a macrovascular
myofibroblast subtype related to the microvascular pericyte.33
Of note, pericytes from multiple vascular beds function as
osteoprogenitors in vitro.33 It is highly probable that CVCs
arise from local mesenchymal progenitors recruited during
vascular injury responses.22,29,32,33 Markers for pericytes and
CVCs are few but include 3G5, smooth muscle ␣-actin, and
Stro1 (human).33 Importantly, an abundant Sca1⫹ (stem cell
antigen) cell population resides within the aortic adventitia in
dyslipidemic apolipoprotein E⫺/⫺22 and LDLR⫺/⫺ (our
unpublished data, 2005) mice that contributes to medial and
intimal injury responses. During vertebrate development, a
1426 Arterioscler Thromb Vasc Biol. July 2006
Sca1⫹ CD34⫹ mesenchymal progenitor, the mesoangio-
blast,34,35 resides in the dorsal aorta that is programmed by
Msx2.36 In response to BMP2, mesoangioblasts upregulate
ALP and differentiate into mineralizing osteoblasts.34 Be-
cause neoangiogenesis generates bipotential endothelial cell,
VSMC progenitors resembling the mesoangioblast,34,36 our
working model posits that dysmetabolic signals that expand
the adventitial vasa simultaneously expand the mural pool of
Sca1⫹ mesenchymal progenitors. In addition, many labora-
tories have demonstrated that aortic VSMCs can undergo a
type of phenotypic modulation: “trans-differentiating” into
mineralizing VSMCs that elaborate markers of the osteo/
chondrogenic lineage.1,37 The hyperphosphatemia of CKD is
an important stimulus for this process,38 signaling through
cell surface Na/phosphate cotransporter Pit1/Glvr1.37 Ele-
vated extracellular phosphate upregulates VSMC expression
of Runx2/Cbaf1, the prototypic osteo/chondrogenic transcrip-
tion factor.37 Moreover, hyperphosphatemia, a common met-
abolic insult in patients with CKD5,38 enhances production of
apoptotic bodies and matrix vesicles that nucleate vascular
mineral deposition.14 Intriguingly, apoptotic bodies simulta-
neously upregulate the expression of stromal cell– derived
factor (SDF)-1␣/CXCL12;32 because SDF-1␣ mediates vas-
cular homing of Sca1⫹ progenitors, medial VSMC vesicula-
tion14 could help recruit adventitial osteoprogenitors.
Whether Sca1⫹ aortic adventitial cells differentially express
Msx2, elaborate canonical Wnts, or contribute to the CVC
lineage has yet to be determined. Moreover, the relative
contribution of Sca1⫹ progenitor recruitment22,29,32 versus
VSMC “trans-differentiation”14,37 to the birth of vascular
osteogenic cells has yet to be examined in diabetic MAC and
may change if CKD ensues.5,7
What signals recruit vascular BMP2 signaling in diabetes?
High glucose concentrations upregulate BMP2 production in
pericytes and mesangial myofibroblasts.18,39 In response to
tumor necrosis factor-␣ (TNF-␣), peroxides, and shear stress,
the endothelial cell also produces BMP240 and BMP4.41
Adipose tissue itself is an endocrine gland that produces
TNF-␣, interleukin-6, and adipokines.24 The inflammatory
fibrofatty adipose tissue expansion in the periaortic adventi-
tia18,21,22,24 and aortic valve interstitium42 before vascular
calcification is likely to play an important role in disease
initiation (Figure 1). The oxidative stress, inflammation, fatty
connective tissue expansion, and neovascularization of the
diabetic adventitia24 can all serve to stimulate aortic produc-
tion of BMPs31,43 that exert paracrine influence on regional
mesenchymal progenitors (Figure 1).
Why don’t all vascular beds calcify in response to meta-
bolic insult and BMP signaling? The answer lies in the
number of defense mechanisms that prevent tissue mineral-
ization. Inorganic pyrophosphate (PPi), matrix Gla protein
(MGP), and fetuin are chief among these. PPi is a VSMC-
generated organic anion that inhibits mineralization and is a
physiological substrate that matrix vesicle-associated ALP
must hydrolyze to promote calcium deposition.12 Extracellu-
lar PPi is generated by 2 mechanisms. First, the membrane
transporter ankylosis (ank) directs secretion of PPi.44 Second,
the enzyme ectonucleotide pyrophosphatase/phosphodiester-
ase I (NPP1) cleaves extracellular NTPs to generate PPi. Loss
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of extracellular PPi from either NPP1 or ank deficiency
predisposes to massive aortic calcification.44 PPi is required
to stabilize the VSMC phenotype; VSMCs that cannot gen-
erate a PPi-replete extracellular milieu undergo osteo/chon-
drogenic trans-differentiation.44 MGP is a calcium-binding
matrix protein that binds and inhibits BMP2 induction of
ALP.45 In addition, carboxylated MGP produced by VSMCs
binds matrix elastin and inhibits calcification.46,47 Mice lack-
ing MGP develop profound panarterial vascular calcification
(endochondral ossification) and die from aortic rupture.48 The
diverse roles of BMP2 and BMP4 during vasculogenesis and
development are regulated by a diverse cadre of vascular
BMP inhibitors.45,49 Intracellular defenses to osteogenic vas-
cular BMP signaling also exist; Smad6, an inhibitory vascular
Smad, attenuates BMP2 activation of receptor Smad trans-
activators.49 Mice lacking Smad6 develop aortic valve and
outflow tract ossification.49 Fetuin is an important humoral
inhibitor of soft tissue mineralization that controls the metab-
olism of vascular matrix vesicles50 (vide infra). Deficiencies
in serum fetuin arising from genetic, inflammatory, or meta-
bolic insult promote widespread tissue calcium deposition
(eg, heart and lung) that curiously spares the aorta in mouse
models.51 Other molecules such as osteopontin have more
complex roles, inhibiting calcification but also promoting
calcium egress via extracellular matrix acidification.52 Pro-
calcific vascular cytokine signaling is held in check by
osteoprotegerin, most probably via inhibition of RANKL.1,53
Thus, in addition to the upregulation of pro-osteogenic
signals, inhibitors of mineral accumulation must be inacti-
vated to permit robust aortic calcification. Both regulatory
arms are profoundly perturbed in CKD. The mechanisms
controlling aortic MAC in CKD overlap those of diabetes;
however, the hyperphosphatemia, reduced serum PPi and
fetuin, and secondary hyperparathyroidism of CKD accentu-
ate aortic calcium accumulation.38 Hyperphosphatemia pro-
motes VSMC matrix vesicle formation.14 Intriguingly, matrix
vesicles may either promote or inhibit calcium deposition,
dependent on whether fetuin is recruited.50 Moreover, fetuin
promotes “phagocytotic clearance” of pro-osteogenic matrix
vesicles.50 Thus, in the setting of CKD, reduced serum fetuin
levels contribute to the vascular procalcific milieu.50
Electron microscopy studies of human postmortem speci-
mens demonstrated early on that aortic calcium deposition in
both MAC and atherosclerosis initiates at lipid vesicles
located along and between elastic laminae but not within the
elastin fibers.12,13,17 However, aortic calcification in associa-
tion with primary alterations in elastin matrix metabolism
represents a unique entry point in a feed-forward cycle of
MAC. The most aggressive drug-induced animal models of
MAC combine either nicotine, a stimulus for elastinolysis,54
or warfarin, a mechanism for inhibiting MGP– elastin inter-
action,46,47 with excessive vitamin D. Ex vivo, devitalized
aortic valves and aortas depleted of VSMCs and myofibro-
blasts, the sources of matrix vesicles, can calcify by elastin-
mediated nucleation.55–57 However, matrix-bound lipids still
play a role because ethanolic delipidation inhibits ex vivo
calcium deposition of devitalized aortic valves.55 In vivo,
aberrant elastin organization and metabolism is characterized
by aortic root dilatation and MAC, as evident in Marfan
 Shao et al
syndrome. Primary fibrillin 1 insufficiency causes abnormal
adventitial microfibrillar matrix organization;58 secondary
changes in elastin metabolism impair medial VSMC terminal
differentiation59 and promote elastin-nucleated medial calci-
fication.58 Direct elastin-nucleated calcification also occurs in
pseudoxanthoma elasticum;60 electron microscopy confirms
calcium deposition along elastin fibers in the absence of
matrix vesicle formation.60 Thus, although mechanisms are
still being elucidated, altered elastin matrix metabolism
enhances aortic calcium deposition and VSMC phenotypic
drift.58,59 Because calcium phosphate mineral deposition sup-
presses VSMC production of tropoelastin,61 elastin matrix
metabolism no doubt contributes to the progression of vas-
cular calcium load in all forms of MAC.
Atherosclerotic Aortic Calcification
Mechanisms of aortic atherosclerotic calcification are over-
lapping yet distinct from those of MAC. This form of aortic
calcium deposition, the type Vb atherosclerotic plaque,62 has
been described excellently1 and will be considered only
briefly. Atherosclerotic calcification is intimally oriented,
eccentric, initiating at the base of necrotic fibrofatty plaques
via apoptotic vesicles arising from dead and dying
VSMCs.1,62 Adjacent chondrogenic and osteogenic processes
are recruited by CVC activation and contribute to procalcific
matrix remodeling.63 As in endochondral bone formation,
ALP induction, Cbfa1/Runx2 and Msx2 expression, type II
and type I collagen deposition, and angiogenic invasion are
salient components.63 The initiating stimuli are inflammatory
and redox dependent,64 and bone morphogens are recruited
with disease progression.6 Indeed, Demer first identified
vascular BMP2 expression within calcifying atherosclerotic
plaques.6 Oxidation of cholesterol-laden lipoprotein deposits
generate bioactive oxysterols that synergize with vascular
BMP2 to promote ectopic osteogenic gene regulatory
programs.65
Major features of atherosclerotic calcification that differ
from diabetic MAC include abundant fibrosis, extensive
cellular necrosis, apoptotic body formation, and cholesterol
crystal accumulation that can support some epitaxial calcium
phosphate deposition.1,14,50,66 By histology, endochondral
bone formation very commonly ensues; in advanced disease,
this ectopic bone can support hematopoietic marrow ele-
ments.6 The high level of Cbfa1/Runx2 observed in calcifying
atherosclerotic plaques is particularly important.38,48,63 In
addition to promoting ALP expression, Runx2: (1) strongly
promotes expression of type I collagen, and (2) upregulates
the expression of vascular endothelial growth factor, the
prototypic osteogenic–angiogenic coupling factor.67 Karsenty
demonstrated that sustained ectopic expression of dermal
ALP with the regional type I collagen deposition was suffi-
cient to drive heterotopic dermal mineralization.68 However,
this type of ectopic dermal mineralization was not associated
with matrix vesicles or apoptotic bodies that characterize
MAC or atherosclerotic calcification.12–14,17,50 Thus, drawing
on lessons learned from skeletal development, synergistic
interactions between paracrine BMP and vascular endothelial
growth factor signaling with neoangiogenesis, robust aortic
expression of Runx2, matricrine cues provided by type I
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Molecular Mechanisms of Vascular Calcification 1427
collagen accumulation, and vascular lipidaceous matrix ves-
icle production likely combine to drive ectopic bone forma-
tion in advanced type Vb plaques.1
Aortic Valve Calcification
Approximately 30% of patients ⱖ65 years of age have
echocardographic evidence of aortic sclerosis, with 2% over-
all exhibiting aortic stenosis.69 Calcium deposition is a
particularly ominous feature of aortic valve disease; in
patients with asymptomatic aortic stenosis, moderate to
severe valve calcification is the single most significant
determinant of clinical disease progression.70 In a cross-
sectional study, Otto described the histopathologic progres-
sion of aortic valve calcification.42 Degenerative lipid accu-
mulation, fatty expansion of the valve fibrosus,
neoangiogenesis, and stippled interstitial calcium deposition
are accompanied by macrophage and T-cell infiltrates at the
earliest stages of disease.42 Neoangiogenesis is also a key
concomitant of valve calcification.71,72 In many ways, the
early changes of the aortic valve interstitium42 are reminis-
cent of those described in the tunica adventitia with diabetes
and dyslipidemia (vide supra). Similar inflammatory histol-
ogy occurs in calcifying bicuspid aortic valves in the com-
plete absence of atherosclerosis.73
Thus, aortic valve calcification occurs in response to
mechanical stressors, inflammation, and the metabolic chal-
lenges of diabetes, dyslipidemia, and uremia.1,38,74,75 During
disease progression, histological and molecular analyses
clearly demonstrate that a phase of active osteogenic mineral
deposition contributes to vascular calcium accumulation. By
histology, this appears to occur principally via nonendochon-
dral processes in aortic valves,75 although the chondrogenic
transcription factor Sox9 is upregulated in both calcifying and
noncalcifying diseased valves.76 In advanced disease, woven
bone formation is histologically evident in 13% of cases.77 At
the molecular level, active BMP–Msx2-Wnt signaling is
detectable in virtually all calcifying aortic valves76,77 (D.A.
Towler, unpublished data, 2002). However, by histology,
massive concretions of acellular amorphous calcium phos-
phate are also seen, suggesting that profound epitaxial min-
eral deposition occurs once cell-based mineralization has
initiated.77 The disappointing effects of statins on the pro-
gression of established aortic valve calcification may reflect
this fact.78
Mechanisms controlling initiation and progression of aortic
valve calcification are poorly understood, largely because of
the limitations of current animal models. Rajamannan first
demonstrated that aortic myofibroblasts undergo osteogenic
trans-differentiation.76,79 Pharmacological doses of statins
prevented osteogenic trans-differentiation of aortic valve
myofibroblasts in culture.79 Recently, she has identified the
mechanistic underpinnings; Wnt/LRP5/␤-catenin signaling, a
signaling cascade absolutely required for osteoblast differen-
tiation in the skeleton,80 is activated by oxidized LDL
cholesterol in valve myofibroblasts and is inhibited by statin
administration.28 This elegant work provides robust evidence
that mineral deposition directed by aortic valve myofibro-
blasts is osteogenic in nature and is potentially preventable
via pharmacological intervention.
1428 Arterioscler Thromb Vasc Biol. July 2006
Summary
Over the past 25 years, we have learned much concerning the
biology of aortic calcification. Active mineralization mecha-
nisms clearly resembling those of skeletal endochondral and
membranous ossification participate in vascular calcium ac-
cumulation. However, the endocrine physiology of vascular
calcium deposition and its turnover is poorly understood and
will depend on the histoanatomic, mechanical, developmen-
tal, matricrine, and metabolic features of the diseased vascu-
lar segment. Many fundamental questions remain to be
addressed. The field of vascular matrix vesicle metabolism is
in its infancy; mechanisms whereby cells shed and endocy-
tose vascular matrix vesicles14,50 and how osteogenic mor-
phogens and matrix control these processes are poorly char-
acterized. The origins of vascular osteoprogenitors must be
clearly delineated. Although circulating progenitors may
contribute,32 most mineralizing osteoprogenitors appear lo-
cally recruited in response to paracrine osteogenic cues 19,22,34
or hyperphosphatemic stimulation.14,37,38 The “paracrinol-
ogy” of vascular adventitial–medial signaling and its meta-
bolic regulation is poorly characterized. Although canonical
Wnt signals are capable of mediating these interactions,19,28
the specific LRP5 and LRP6 ligands that participate in
vascular Wnt signaling cascades have yet to be determined.
Novel therapeutics are needed.78 Dependent on disease
stage and setting, the metabolic, endocrine, inflammatory,
elastinolytic, and mechanical insults will differ in relative
contribution to the extent of aortic calcification. Stage-
specific mechanisms contributing to calcific aortic disease
must be carefully considered as clinical studies are designed
and therapeutic strategies crafted. Given the benefits of
sevelamer on aortic calcification in dialysis patients,81 this
phosphate- and sterol-binding resin also holds promise for
diabetic patients with declining renal function. 38 Although
statins cannot treat established aortic valve calcification,78
early treatment with statins may prevent valve mineraliza-
tion,28,79 particularly in high-risk patients after bioprosthetic
valve implantation.1 Small studies suggest that bisphospho-
nates can inhibit aortic mineral deposition in extant disease;82
mechanisms are unknown, and the potential role of Pit137 as
a pyrophosphate/bisphosphonate receptor44 has yet to be
explored. Whether soluble inhibitors of the BMP2–Msx2-
Wnt signaling cascade 19 alter the progression of aortic
calcium accumulation will soon be tested. As the mechanisms
that initiate and propagate vascular pro-osteogenic morpho-
gen signaling are better understood, other potential therapeu-
tic strategies will emerge.1 Antagonism of the endothelin A
receptor, a receptor that enhances vascular BMP2 signaling,43
has been shown to promote regression of medial calcium
deposition.83 BMP7, a unique BMP that stabilizes VSMC
phenotype and inhibits vascular BMP2 signaling, holds prom-
ise for ameliorating uremic vasculopathy.84 The absence of a
robust murine model of calcific aortic stenosis remains a
major scientific shortcoming. This limits our ability to exam-
ine how strategies targeting inflammation, oxidative stress,
morphogen signaling, angiogenesis, matrix metabolism, and
epitaxial mineral deposition may differentially influence ini-
tiation versus progression phases of aortic stenosis. Never-
theless, given the blossoming field of cardiovascular endo-
Downloaded from atvb.ahajournals.org by on March 13, 2010
crinology, the near future holds tremendous promise that new
pharmacotherapies will emerge to help address the unmet
clinical need in calcific aortic disease.
Sources of Funding
This work was supported by the National Heart, Lung, and Blood
Institute and the Barnes-Jewish Hospital Foundation.
Disclosures
None.
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