Novel Conformation-Specific Antibodies Against Matrix
?-Carboxyglutamic Acid (Gla) Protein
Undercarboxylated Matrix Gla Protein as Marker for
Leon J. Schurgers, Kirsten J.F. Teunissen, Marjo H.J. Knapen, Martijn Kwaijtaal, Rob van Diest,
Ad Appels, Chris P. Reutelingsperger, Jack P.M. Cleutjens, Cees Vermeer
Objective— Matrix ?-carboxyglutamic acid (Gla) protein (MGP), a vitamin K–dependent protein, is a potent in vivo
inhibitor of arterial calcification. We hypothesized that low endogenous production of MGP and impaired carboxylation
of MGP may contribute to the development or the progression of vascular disease.
Methods and Results—Novel conformation-specific antibodies against MGP were used for immunohistochemistry of
healthy and sclerotic arteries. In healthy arteries, MGP was mainly displayed around the elastin fibers in the tunica
media. The staining colocalized with that for carboxylated MGP, whereas undercarboxylated MGP (ucMGP) was not
detected. In atherosclerotic arteries, ucMGP was found in the intima, where it was associated with vesicular structures.
In Mo ¨nckeberg’s sclerosis of the media, ucMGP was localized around all areas of calcification. The results indicate that
ucMGP is strongly associated with vascular calcification of different etiologies. In a separate study, serum MGP
concentrations in a cohort of 172 subjects who had undergone percutaneous coronary intervention were significantly
reduced compared with an apparently healthy population.
Conclusions—These data show that impaired carboxylation of MGP is associated with intimal and medial vascular
calcification and suggest the essentiality of the vitamin K modification to the function of MGP as an inhibitor of ectopic
calcification. (Arterioscler Thromb Vasc Biol. 2005;25:1629-1633.)
Key Words: matrix Gla protein (MGP) ? vitamin K ? calcification ? atherosclerosis
exceeding the solubility product for spontaneous precipi-
tation.1However, physiological calcification is restricted
to bone and teeth, whereas soft tissue calcification is
regarded as pathological. Vascular calcification can occur
at 3 anatomic sites: the intima where it is associated with
atherosclerosis, the tunica media, and the heart valves.
Huang et al showed that coronary artery calcification does
not significantly affect stability of atheroma,2and the
involvement of calcium salt accretion on cardiovascular
disease is not known yet. However, overall vascular
calcification is regarded as one of the major complications
of cardiovascular disease and is an independent risk factor
for myocardial infarction (MI) and cardiac death.3–6There-
fore, prevention of vascular calcification is a prerequisite
for human health. Inhibition of calcification is regarded
presently as an active process in which a variety of
proteins are involved throughout the body.7In the vascu-
he extracellular fluids in the human body contain
calcium and phosphate in high concentrations, even
lature, a major calcification inhibitory factor is matrix
?-carboxyglutamic acid (Gla) protein (MGP), a vitamin
K–dependent protein synthesized by vascular smooth mus-
cle cells (VSMCs).8,9Its 5 Gla residues are formed in a
post-translational carboxylation reaction in which vitamin
K functions as an essential cofactor.10,11MGP in its
carboxylated form will be designated here as GlaMGP.
The presence of the Gla residues is critical for MGP
function, and undercarboxylated, inactive species of MGP
(designated as GluMGP) are formed during inadequate
vitamin K status or as a result of vitamin K antagonists. In
animal models, it was demonstrated that impaired MGP
synthesis12,13as well as treatment with vitamin K antago-
nists14result in arterial calcification within 2 to 4 weeks,
whereas in humans, it was demonstrated that oral antico-
agulant treatment is associated with substantially increased
heart valve calcification.15Human MGP promoter poly-
morphisms were identified and demonstrated to be associ-
ated with low MGP expression and low serum MGP
Original received March 11, 2005; final version accepted May 25, 2005.
From the Cardiovascular Research Institute (L.J.S., K.J.F.T., M.H.J.K., M.K., A.A., C.P.R., C.V.), VitaK (L.J.S., K.J.F.T., C.V.), and EURON,
European Graduate School of Neuroscience (R.vD.), Maastricht University, the Netherlands; and the Department of Pathology (J.P.M.C.), University
Hospital Maastricht, the Netherlands.
Correspondence to Dr L.J. Schurgers, Department of Biochemistry, University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands.
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000173313.46222.43
levels.16In addition to animal models for impaired MGP
expression, polymorphisms in humans were demonstrated
to be associated with an increased risk for MI in humans.17
Calcification of vascular tissue has been observed in a
number of conditions, including aging, diabetes, and renal
disease.18Arterial calcification is associated with MI and
ischemia in peripheral vascular disease, especially in diabetes
and end-stage renal disease. Arterial calcification can occur in
the intima (atherosclerosis), where it is associated with
macrophage- and lipid-rich atherosclerotic lesions; it may
also occur in the media (Mo ¨nckeberg’s sclerosis), indepen-
dently of atherosclerosis and almost exclusively associated
with VSMCs.19The independence and unique features of
these conditions is suggestive of different etiologic pathways.
Mo ¨nckeberg’s sclerosis gained increasing attention because
of findings linking it with mortality, cardiovascular disease in
diabetes, advanced renal disease, lower extremity amputation,
and poor outcomes in peripheral arterial disease.4
Using conformation-specific antibodies, Sweatt et al reported
recently the presence of poorly carboxylated MGP in the
calcified vasculature of aging rats.20In another study, Price et al
induced arterial calcification in rats by vitamin D plus warfarin
treatment and demonstrated by protein sequencing that the MGP
that accumulated at the sites of calcification was poorly carbox-
ylated.21Because only carboxylated MGP has calcification-in-
hibitory activity, we hypothesized that there are 2 independent
conditions contributing to the development or progression of
cardiovascular calcification associated with MGP: low constitu-
tive MGP synthesis in the vasculature and incomplete MGP
carboxylation as a result of poor vitamin K status. Phase I of the
present study was performed in surgical specimens obtained
from our department of pathology. These specimens were used
to study the localization and carboxylation status of MGP using
a panel of novel monoclonal antibodies. Study phase II was
facilitated by our access to a study in which the effects of
exhaustion on angioplasty patients were evaluated on new
coronary events.22In this cohort, we tested this hypothesis by
measuring MGP in serum from healthy subjects and cardiovas-
Materials and Methods
Materials and Methods can be found in the online supplement,
available at http://atvb.ahajournals.org.
Study Phase I: Measurement of MGP Species
Sections of carotid arteries with various degrees of athero-
sclerosis from 5 different donors were compared with normal,
nonaffected carotid arteries (also 5 donors; Table I, available
online at http://atvb.ahajournals.org). In normal vascular
tissue, neither calcification nor lipid infiltration was ob-
served. Staining with CD68 for macrophage infiltration was
negative (data not shown). In Figure 1, immunohistochemical
localization of MGP species during different stages of ath-
erosclerosis is shown. In all healthy arteries (Figure 1), total
MGP (tMGP) was not well visible in the intima, but most of
it had accumulated around the medial elastin fibers (Figure
1A); GluMGP was not found at this site (Figure 1B). With the
antibody specifically recognizing GlaMGP (Figure 1C), we
found staining coinciding with tMGP, suggesting that the
elastin-associated MGP in the healthy vasculature mainly
occurs in its carboxylated form. In early atherosclerotic
lesions (Figure 2; stage II to III), substantial intima thickening
was observed; calcification was seen occasionally as isolated
stippling, and lipid accumulation was evident from oil red-O
staining (data not shown). Also, CD68 staining was positive,
demonstrating macrophage infiltration. In these preparations
Figure 1. Immunohistochemical localization of MGP species in
an apparently healthy arterial vessel wall. A represents total
MGP; B, GluMGP; and C, GlaMGP staining. The GluMGP pro-
tein seems absent at this stage, whereas total MGP and
GlaMGP are present. Arrows indicate the elastin fibers in the
media. M indicates media; In, intima; Lu, lumen. The hematoxy-
lin/eosin (H/E) staining demonstrates no signs of abnormalities.
Figure 2. Immunohistochemical localization of MGP species in
stage II to III atherosclerotic lesion. A represents total MGP; B,
GluMGP; and C, GlaMGP staining. The predominant form is
GluMGP, mainly around vesicular structures. The von Kossa
stain reveals that these vesicular structures contain calcium. The
inset in B demonstrates that the rounded structures are annexin
V positive, thus, they most likely expose PS. Arrows indicate
vesicular structures (size ?30 to 850 nm). M indicates media; In,
intima; Lu, lumen.
1630 Arterioscler Thromb Vasc Biol.
staining for tMGP showed MGP accumulation in vesicular
structures (Figure 2A and 2D) in the intima and similar patterns
for GluMGP (Figure 2B). However, staining for GlaMGP was
poor (Figure 2C). Moreover, von Kossa staining for calcification
revealed small calcified spots (arrows). The diameter of these
rounded structures was estimated at 30 to 850 nm, which is
similar to that of apoptotic bodies, lipid debris, or cellular
remnants. The mean vesicular size?SD was 246?13 nm. The
size was measured using a microscope coupled to a computer-
ized morphometry system (Quantimed 570; Leica; data not
shown). More evidence that the vesicular structures were of
cellular origin came from staining with annexin V (Figure 2B,
inset). Annexin V is known to bind phosphatidyl serine (PS)–
exposing cells or apoptotic (PS positive) bodies. Together, these
data suggest substantial undercarboxylation of MGP in vesicular
structures formed during the progression of atherosclerosis. In
type Vb plaques (characterized by calcium salt precipitates and
bone formation; Figure 3), calcification was demonstrated by
von Kossa staining. A positive staining for apoptosis was
observed at the interface between tissue and calcium crystals. At
this stage, substantial amounts of MGP had accumulated around
the mineralized areas, and comparable intensities for tMGP
(Figure 3A) and GluMGP (Figure 3B) staining were observed.
The staining for GlaMGP was poor at this stage, suggesting that
most of the MGP was present in its undercarboxylated nonactive
form (Figure 3C). Also, at the interface of calcium crystal and
surrounding tissues, vesicular structures were present, mainly
colocalizing with GluMGP (Figure 3B and von Kossa, inset).
Mo ¨nckeberg’s Sclerosis
To investigate MGP localization in medial calcification, we
(n?6; Table I). Staining with oil red-O and CD68 antibodies
confirmed the absence of inflammation and lipid infiltration
(data not shown). Figure 4 shows MGP localization in an
example of a peripheral artery from a diabetic patient. This is a
typical form of media sclerosis (Mo ¨nckeberg’s sclerosis) with
major calcifications starting around the elastin fibers in the
tunica media (von Kossa). Most tMGP was localized in the
noncalcified areas (Figure 4A), whereas GluMGP was almost
exclusively found to be associated with areas of calcification
(Figure 4B). More advanced stages of media calcification were
still devoid of lipids and macrophages but contained the vesic-
ular GluMGP-rich structures as observed in atherosclerosis.
GlaMGP was also found around the elastic fibers, although less
specific associated with calcified areas (Figure 4C). Together,
the immunohistochemical data demonstrate that in atheroscle-
rotic intima sclerosis and diabetic media sclerosis, GluMGP is
abundantly present, suggesting local vitamin K deficiency and
impaired protection against calcification attributable to poor
Study Phase II: MGP Measurement in Serum
Circulating MGP-related antigen and vitamin K status were
measured in an apparently healthy reference population
(Table IIa, available online at http://atvb.ahajournals.org) as
well as in a group after percutaneous coronary intervention
(PCI; Table IIb). Table IIa shows the demographic and
medical characteristics of the healthy control group and Table
IIb that of the patient population. Figure 5 shows the serum
MGP distribution in the reference (mean ? SD; 11.5?4.0)
and the patient group (5.0?2.1 and 6.23?1.9, respectively).
Obviously, serum MGP was below the normal range at 1.5 and
7.5 months after PCI. The fact that it remained low even at 7.5
months after PCI is suggestive for low constitutive MGP
expression, not related to the surgical intervention. In a subse-
Figure 3. Immunohistochemical staining of MGP species in
stage Vb atherosclerotic lesion. A represents total MGP; B,
GluMGP; and C, GlaMGP staining. GluMGP staining is the pre-
dominant staining, colocalizing with von Kossa staining. In the
insets can be seen that at the interface of calcium crystal (Bo)
and surrounding tissue, vesicular structures are present. These
rounded structures are heavily calcified (von Kossa staining).
Arrows indicate vesicular structures. M indicates media; Lu,
lumen; Bo, bone.
Figure 4. Immunohistochemical localization of MGP species in
Mo ¨nckeberg’s sclerosis of the media. Four subsequent sections
from a peripheral diabetic artery were stained in different ways.
Because the sections are slightly tilted from left to right, the
massive calcified area at top left to middle may be used for ori-
entation. A represents total MGP; B, GluMGP; and C, GlaMGP
staining. It can be seen clearly from these pictures that Glu–
MGP completely colocalizes where calcification is present.
Arrows indicate calcified areas of the elastic lamina. M, indi-
cates media; In, intima; Lu, lumen; Ad?adventitia.
Schurgers et alUndercarboxylated Matrix Gla Protein as Marker for Vascular Calcification
quent analysis, we tested whether the medication used might
affect the circulating MGP concentration. Cross-tables with
quartile distribution of MGP were compared with baseline
medication, and the significance of differences between users
and nonusers was tested with the Pearson ?2test. There was no
effect of medication on the serum MGP concentration.
In this article, we demonstrated that low circulating MGP and
an impaired ?-carboxylation of the protein at its tissue site of
expression are associated with the development and progres-
sion of cardiovascular disease.
In a first approach, we investigated by immunohistochemistry
whether undercarboxylation (and thus suboptimal protein activ-
To this aim, we developed conformation-specific antibodies
exclusively recognizing GluMGP and GlaMGP (Figure I, avail-
able online at http://atvb.ahajournals.org). In healthy arteries,
devoid of calcification and lipid or macrophage infiltration, total
MGP was deposited mainly along the elastin fibers. It is
noteworthy that at this stage, almost no GluMGP was found.
Apparently, most of the MGP is active in the healthy vascula-
ture, which is demonstrated by the staining with anti-GlaMGP.
The abundance of active, carboxylated MGP is consistent with
in vitro cell culture data, showing that the vitamin K antagonist
warfarin induces calcification, whereas vitamin K has a protec-
tive effect.28Stage III atherosclerosis is associated with lipid
infiltration and inflammation, as defined by Virmani et al.25
Substantial amounts of MGP accumulated in vesicular struc-
tures, which may be apoptotic bodies or lipid debris originating
from cell death. Staining for GluMGP revealed that a major part
of MGP associated with these structures was undercarboxylated,
suggesting that at these increased expression levels, the vitamin
K–dependent carboxylation machinery loses its ability to
?-carboxylate all the MGP synthesized by the vessel wall. The
most likely explanation for this impaired carboxylation is that
there are insufficient vitamin K reserves in the vessel wall to
cope with the increased MGP expression, but other explanations
cannot be ruled out. The abundance of inactive undercarboxy-
lated MGP in vesicular structures is consistent with in vitro cell
culture data, showing that matrix vesicles and apoptotic bodies,
which are thought to be the nucleation site for vascular calcifi-
cation, have an increased tendency to calcify if they originate
from cells treated with warfarin.29Moreover, previous studies
showed that apoptosis is involved in calcification of VSMC
ic bodies and vesicles29could calcify in a similar manner to
chondrocyte-derived matrix vesicles.31,32It is noteworthy that
MGP is involved in calcification and apoptosis of VSMCs and
chondrocytes. In stage Vb atherosclerosis, calcification and bone
formation were accompanied by MGP accumulation, predomi-
nantly in the contact area between mineral and tissue; also, in
this case, a substantial part of the MGP occurred in an under-
carboxylated form, and the staining for carboxylated MGP in a
corresponding section was poor. This is consistent with studies
in young rats showing that warfarin causes vascular calcification
within 2 weeks because of inactivation of MGP,14,21and with
studies in aging rats showing that vascular calcifications are
associated with undercarboxylated MGP.20In a second ap-
proach, we explored whether our findings in atherosclerotic
lesions were also applicable to Mo ¨nckeberg’s sclerosis, a form
of vascular calcification of completely different etiology. In this
condition, MGP was associated primarily with the elastin fibers.
Most MGP was found in the noncalcified areas, whereas the
GluMGP fraction was almost exclusively present in the exten-
sively calcified parts of the tunica media. Although other
explanations are possible, these data are consistent with the
hypothesis that local poor vitamin K tissue status is a risk factor
for vascular calcification.
Subsequently, we addressed the question whether the
putative low-tissue MGP expression and poor tissue vitamin
K status (as deduced from GluMGP) can be monitored in
serum. A broad range for serum MGP was found in an
apparently healthy reference population. Possible reasons for
variation in this group include promoter polymorphisms
affecting MGP expression16,17and subclinical levels of ath-
erosclerosis and vascular calcification (known to be common
in all adults),33which may induce MGP expression to
variable extents. It should be noted that the reference popu-
lation was not screened for vascular disease and presumably
contained subjects with thus far undetected arterial calcifica-
tion. About one third of the reference population had serum
MGP levels ?15 nmol/L, whereas in the patient group, high
serum MGP levels were not present, and 90% of all values
were ?10 nmol/L. The median value for the reference group
was substantially and significantly higher than that for the
patient group at both time points measured. This is consistent
with a recent study from Japan, in which an inverse correla-
tion was observed between coronary artery calcification and
circulating MGP.34Unfortunately, the GluMGP antibody did
not lend itself for testing in serum because this antibody only
recognizes ucMGP after fixation or denaturation.
One key question arising from this study is: does the
measurement of serum MGP have any diagnostic utility?
From the limited data presently available, it seems that the
assay may be useful as an extra marker for cardiovascular risk
assessment, complementary to existing tests such as serum
cholesterol and blood pressure. The clinical utility of the
assay in the diagnosis of cardiovascular patients remains
unclear. During the development of atherosclerosis, vascular
MGP synthesis may be triggered locally,8,35but whether this
Figure 5. Serum MGP in health and disease. Left, Reference
population. Middle, PCI patients at 1.5 months after surgery.
Right, Same cohort at 7.5 months after surgery.
1632Arterioscler Thromb Vasc Biol.
is reflected by an increase of circulating MGP to levels above Download full-text
the normal range is currently unknown.
It is noteworthy that accumulation of GluMGP in calcified
arteries was identified as a second variable associated with
cardiovascular disease. The most probable mechanism under-
lying this observation is incomplete MGP carboxylation
resulting in suboptimal inhibition of arterial calcification.
This hypothesis is strengthened by preliminary data from our
group showing that patients with severe calcifications have an
overall vitamin K deficiency, as measured by the ratio of
undercarboxylated osteocalcin to carboxylated osteocalcin
and plasma vitamin K concentrations.
Unlike low MGP expression, incomplete MGP carboxylation
may be amenable to treatment by increasing vitamin K intakes.
To follow such treatment, a serum-based assay for undercar-
fraction of total MGP that circulates as ucMGP and the response
of this fraction to increased vitamin K intakes. Such an assay
would provide a better insight of the relationship of MGP
activity to vitamin K status in cardiovascular disease.
The work described in this article was supported by grant 2001.033
of the Netherlands Heart Foundation.
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Schurgers et al Undercarboxylated Matrix Gla Protein as Marker for Vascular Calcification