Vascular calcification and osteoporosis—from clinical observation ...

254 Osteoporos Int (2007) 18:251–259
The passive hypothesis is based on the concept that—in
an appropriate microenvironment—calcium and phosphate
physiochemically precipitate in areas of advanced tissue
degeneration or necrosis within the vascular wall when the
physiological calcium phosphate solubility threshold is
exceeded [11]. This largely acellular extracellular process
that occurs in association with membrane debris is further
enhanced by a deficient macrophage-mediated clearance
mechanism [31]. There are several potential calcification
inhibitors that may act either locally or systemically [11].
MGP is a vitamin K-dependent inhibitor of calcification
that acts locally, while fetuin-A is a circulating factor that
prevents mineral precipitation and serves as an opsonin,
thus enhancing phagocytosis of mineral precipitates [11]. In
addition, pyrophosphate is a potent inhibitor of vascular
calcification, as exemplified by a syndrome termed generalized
arterial calcification of infancy that is caused by lossof-function
mutations of ecto-nucleotide pyrophosphatase/
phosphodiesterase-1 (ENPP1), the enzyme that generates
pyrophosphate [32]. Several lines of evidence indicate that
active and passive mechanisms occur in parallel and are not
mutually exclusive [10].
Table 2 Knockout mice with a combined skeletal and vascular
phenotype or vascular calcification
Gene References Phenotype
Matrix Gla
protein
[33] Enchondral ossification defects,
arterial calcification
Osteopontin [39] Protection against ovariectomyinduced
osteoporosis
[44] Aggravation of arterial calcification
in MGP-deficient mice
Fetuin-A [47] Secondary hyperparathyroidism due
to chronic renal failure, arterial
calcification, calciphylaxis and
extensive ectopic calcification a
Smad6 [50] Heart valve hyperplasia, arterial
ossification b
Klotho [13] Osteoporosis, atherosclerosis,
arterial calcification, other agingrelated
disorders c
Osteoprotegerin [14] Osteoporosis, arterial calcification
a Ectopic calcification precedes renal failure in calcification-prone
DBA/2 mice; b no skeletal abnormalities; c including gonadal and
skin atrophy, growth hormone deficiency, physical inactivity and
pulmonary emphysema
Lessons from the skeletal and vascular phenotype
of knockout mice
The generation and characterization of knockout mice with
targeted deletions of bone-related genes have provided
important mechanistic insights in bone metabolism, and
surprisingly, some of these animal models display a
combined skeletal and vascular phenotype (Table 2). The
physiological function of these genes and the skeletal and
vascular phenotypes of six distinct knockout mice will be
briefly discussed.
Matrix Gla protein (MGP)
MGP is a member of a family of mineral-binding proteins
that includes several coagulation factors (factors VII and
IX), anti-coagulation factors (proteins C and S) and
osteocalcin, a constituent of bone matrix that inhibits bone
formation [12]. Like other family members, MGP contains
γ-carboxy-glutamic acid (Gla) residues, which account for
its high affinity for hydroxyapatite that forms via γ-
carboxylation, a process that is inhibited by warfarin [11].
The crucial role of MGP for bone and cartilage
metabolism is underlined by the phenotype of MGPdeficient
mice. These mice exhibit tachycardia, short stature
and die prematurely at 2 months [33]. The direct cause of
death in these animals is severe hemorrhage due to a
ruptured thoracic or abdominal aorta. Detailed analysis
using alizarin red staining for detection of mineralized
tissues revealed extensive vascular calcification in mice as
early as 2 to 3 weeks postnatally [33]. The elastic lamellae
of the aorta and the internal elastic lamina of the coronary
arteries were calcified as detected by von Kossa staining. In
addition, the aorta of MGP-deficient mice displayed
features of cartilaginous metaplasia, with the presence of
chondrocytes and a metachromic cartilaginous matrix,
including type II collagen and proteoglycans [33]. These
changes reduced vascular compliance with stiffening of the
vascular wall and a higher propensity for rupture and
thrombus formation.
The skeletal phenotype of MGP-deficient mice is characterized
by calcifications of cartilage in the proliferating
chondrocyte zone, which is not present in wild-type mice
[33]. Moreover, proliferating chondrocytes of MGP-deficient
mice are not organized in columns, and hypertrophic
chondrocytes are absent, indicating a severe enchondral
ossification defect at the growth plate that resulted in short
stature and osteopenia [33].
Osteopontin
Osteopontin is an abundant acidic non-collagenous bone
matrix glycoprotein that binds to integrins, including α v β 3 -
integrin, which is the major integrin type on the osteoclastic
cell surface [34, 35]. Integrins are crucial for osteoclast
migration to resorption sites, attachment to bone and
formation of the sealing zone [34, 35]. Osteopontin bound
to substrate enhances attachment of osteoclasts [34, 35]. By