The Role of Vitamin K in Soft-Tissue Calcification

Abstract

Seventeen vitamin K-dependent proteins have been identified to date of which several are involved in regulating soft-tissue calcification. Osteocalcin, matrix Gla protein (MGP), and possibly Gla-rich protein are all inhibitors of soft-tissue calcification and need vitamin K-dependent carboxylation for activity. A common characteristic is their low molecular weight, and it has been postulated that their small size is essential for calcification inhibition within tissues. MGP is synthesized by vascular smooth muscle cells and is the most important inhibitor of arterial mineralization currently known. Remarkably, the extrahepatic Gla proteins mentioned are only partly carboxylated in the healthy adult population, suggesting vitamin K insufficiency. Because carboxylation of the most essential Gla proteins is localized in the liver and that of the less essential Gla proteins in the extrahepatic tissues, a transport system has evolved ensuring preferential distribution of dietary vitamin K to the liver when vitamin K is limiting. This is why the first signs of vitamin K insufficiency are seen as undercarboxylation of the extrahepatic Gla proteins. New conformation-specific assays for circulating uncarboxylated MGP were developed; an assay for desphospho-uncarboxylated matrix Gla protein and another assay for total uncarboxylated matrix Gla protein. Circulating desphospho-uncarboxylated matrix Gla protein was found to be predictive of cardiovascular risk and mortality, whereas circulating total uncarboxylated matrix Gla protein was associated with the extent of prevalent arterial calcification. Vitamin K intervention studies have shown that MGP carboxylation can be increased dose dependently, but thus far only 1 study with clinical endpoints has been completed. This study showed maintenance of vascular elasticity during a 3-y supplementation period, with a parallel 12% loss of elasticity in the placebo group. More studies, both in healthy subjects and in patients at risk of vascular calcification, are required before conclusions can be drawn.

Full text

THEMATIC REVIEW SERIES: VITAMIN K
The Role of Vitamin K in Soft-Tissue Calcification
1
Elke Theuwissen,* Egbert Smit, and Cees Vermeer
VitaK, Maastricht University, Maastricht, the Netherlands
ABSTRACT
Seventeen vitamin K–dependent proteins have been identified to date of which several are involved in regulating soft-tissue calcification. Osteocalcin,
matrix Gla protein (MGP), and possibly Gla-rich protein are all inhibitors of soft-tissue calcification and need vitamin K–dependent carboxylation for
activity. A common characteristic is their low molecular weight, and it has been postulated that their small size is essential for calcification inhibition
within tissues. MGP is synthesized by vascular smooth muscle cells and is the most important inhibitor of arterial mineralization currently known.
Remarkably, the extrahepatic Gla proteins mentioned are only partly carboxylated in the healthy adult population, suggesting vitamin K insufficiency.
Because carboxylation of the most essential Gla proteins is localized in the liver and that of the less essential Gla proteins in the extrahepatic tissues, a
transport system has evolved ensuring preferential distribution of dietary vitamin K to the liver when vitamin K is limiting. This is why the first signs of
vitamin K insufficiency are seen as undercarboxylation of the extrahepatic Gla proteins. New conformation-specific assays for circulating uncarboxylated
MGP were developed; an assay for desphospho-uncarboxylated matrix Gla protein and another assay for total uncarboxylated matrix Gla protein.
Circulating desphospho-uncarboxylated matrix Gla protein was found to be predictive of cardiovascular risk and mortality, whereas circulating total
uncarboxylated matrix Gla protein was associated with the extent of prevalent arterial calcification. Vitamin K intervention studies have shown that MGP
carboxylation can be increased dose dependently, but thus far only 1 study with clinical endpoints has been completed. This study showed
maintenance of vascular elasticity during a 3-y supplementation period, with a parallel 12% loss of elasticity in the placebo group. More studies, both in
healthy subjects and in patients at risk of vascular calcification, are required before conclusions can be drawn. Adv.Nutr.3:166–173, 2012.
Introduction
In vertebrates, all extracellular body fluids are supersaturated
with respect to calcium and phosphate, resulting in a tendency
for spontaneous calcium phosphate precipitation, which is of-
ten expressed as the calcium 3inorganic phosphate product
(1). Potent inhibitors of calcium salt precipitation and crystal-
lization are therefore essential for survival, and consequently a
broad range of low and high molecular weight inhibitors are
found in the circulation. Low molecular weight inhibitors in-
clude pyrophosphate and citrate, whereas the most potent in-
hibitors are small and medium-sized proteins.
Fetuin-A is a 48-kD protein synthesized in the liver and se-
creted into the circulation; it is the most important systemic in-
hibitor of soft-tissue calcification. Transgenic fetuin-A–deficient
mice display a severe diffuse systemic calcification phenotype
with punctuate calcified lesions in most tissues (2). Besides
fetuin-A, a number of small vitamin K–dependent proteins
have been discovered acting as potent calcification inhibitors.
Examples are osteocalcin (OC), also known as bone Gla pro-
tein, matrix Gla protein (MGP), and possibly also the newly
discovered Gla-rich protein (GRP), which are all proteins be-
tween 5 and w10 kD. In contrast to fetuin-A, these proteins
are local inhibitors of calcification, i.e., they are synthesized in
the tissues in which they exert their function. OC is synthe-
sized by the osteoblasts in bone, and transgenic OC-deficient
mice show increased bone mineral content (3). MGP is pri-
marily synthesized by chondrocytes and vascular smooth mus-
cle cells, and MGP-deficient mice show impaired growth
(resulting from excessive growth plate calcification) and mas-
sive calcification of the arterial tunica media (4). An interesting
question is why fetuin-A alone is unable to stop calcification of
these tissues. Recently, a hypothesis was presented that pro-
vided a possible answer to this question. In their paper, Price
et al. (5) demonstrate that with its molecular mass of 48 kD,
fetuin-A is too large to penetrate the luminal space within col-
lagen and elastin fibrils. But if no other inhibitors are present,
these fibrils will rapidly calcify. Only calcification inhibitors
that are small enough to penetrate the collagen and elastin fi-
brils, i.e., OC and MGP, will be able to move freely into the fi-
bril and prevent mineral growth inside. This explains why the
elastin fibrils are prone to calcification, especially during
1
Conflicts of interest: E. Theuwissen, E. Smit, and C. Vermeer, no conflicts of interest.
* To whom correspondence should be addressed: E-mail: e.theuwissen@vitak.com.
2
Abbreviations used: AS, aortic stenosis; CAC, coronary artery calcification; cGMP, carboxylated
matrix Gla protein; CKD, chronic kidney disease; CVD, cardiovascular disease; dp-cMGP,
desphospho-carboxylated matrix Gla protein; dp-ucMGP, desphospho-uncarboxylated matrix
Gla protein; GRP, Gla-rich protein; HD, hemodialysis; HF, heart failure; MGP, matrix Gla protein;
MK-7, menaquinone-7; OC, osteocalcin; t-ucMGP, total uncarboxylated matrix Gla protein;
ucMGP, uncarboxylated matrix Gla protein; VKA, vitamin K antagonist.
166 ã2012 American Society for Nutrition. Adv. Nutr. 3: 166–173, 2012; doi:10.3945/an.111.001628.
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vitamin K insufficiency, and demonstrates the vital impor-
tance of vitamin K in the prevention of soft-tissue calcification.
In this paper, we review the current knowledge in this field,
especially with respect to the role of MGP in the regulation
of soft-tissue calcification, which may have implications for
the risk of cardiovascular and other diseases.
Current status of knowledge
Vitamin K status and insufficiency
Animal models. OC was the firstGlaproteindiscoverednotto
be involved in blood coagulation and also the first Gla protein
found to be synthesized outside the liver (6–8). Remarkably, the
molecular role of OC has remained obscure for >30 y. In vitro
experiments with purified OC showed that it is a strong inhib-
itor of calcium salt precipitation from supersaturated solutions
(9). OC-deficient animals show increased bone size and high
bone mineral content (3). Therefore, OC was described as a
negative regulator of bone growth. Only after the hypothesis
of mineralization by inhibitor exclusion was put forward did
it become clear how OC may contribute to the controlled depo-
sition of hydroxyapatite in bone (5).
In contrast to OC, the phenotype of MGP-deficient mice
immediately showed the function of MGP (4). MGP
2/2
ani-
mals were smaller than their heterozygous littermates, resulting
from calcification of the epiphysis that blocked longitudinal
growth. Moreover, all large arteries rapidly calcified, resulting
in death at 6–8 wk after birth. Tissue-specific expression of
MGP in the vascular smooth muscle cells completely pre-
vented arterial calcification; hepatic MGP expression did result
in high circulating MGP levels, but not in the prevention of ar-
tery calcification (10). These data clearly showed that MGP is a
local inhibitor of calcification and thus complementary to the
systemic action of fetuin-A.
The effects of MGP deficiency could be mimicked by fee-
ding rodents a diet containing a mixture of warfarin and
phylloquinone. The warfarin + phylloquinone model is based
on the preferential distribution of phylloquinone to the liver.
By combining a high dose of warfarin with a relatively low
dose of phylloquinone, hepatic synthesis of blood coagulation
factors can be maintained at a level that prevents bleeding,
and severe vitamin K deficiency will be evoked in all extrahe-
patic tissues. The phenotype of this model is comparable to
that of the MGP-deficient mouse showing calcification of
the epiphysis and severe arterial calcifications. Initially, the re-
ported model required daily injections with phylloquinone
(11,12), but in our laboratory, a regimen was developed in
which both warfarin and phylloquinone could be given in
the diet (13). The calcifications (visualized in black by von
Kossa staining) in a mouse on the warfarin + phylloquinone
diet are shown in Figure 1. Next to arterial calcifications, se-
vere calcifications were found in other tissues, such as the
liver, kidney, and heart.
Human equivalents of animal models. MGP deficiency in
humans is known as the Keutel syndrome, a rare autosomal re-
cessive disease that was first described in 1971 (14). Affected
subjects exhibit severe cartilage calcifications, brachytelephalan-
gism, peripheral pulmonary artery stenosis, hearing loss, and
facial abnormalities (i.e., midface hypoplasia and a depressed
nasal bridge). Keutel syndrome was linked to mutations in
the gene coding for MGP leading to the absence or nonfunc-
tional forms of MGP (15,16). A striking difference with
MGP-deficient animals is that effects on the vasculature
are less prominent, and an explanation may be that GRP
forms a backup system for MGP in humans and not in
mice. GRP was discovered in the cartilage of sturgeons
(17), but is also found in mammals, including humans
(18). Its function has not yet been established unequivocally,
but seems to be related to calcification inhibition as well
(19).
Figure 1 Calcification induced by the
warfarin + phylloquinone regimen. Calcifications
(visualized by von Kossa staining) induced by
feeding a DBA2 mouse warfarin (3 mg/g) and
phylloquinone (1.5 mg/g) for 6 wk. A, liver; B,
kidney; C, heart; D, aorta. The arrows indicate
areas of calcifications. TA, tunica adventitia; EC,
endocardium; H, hepatocytes; G, glomerulus; L,
lumen; MC, myocardium; T, tubulus; TM, tunica
media. (C. Vermeer et al., VitaK, Maastricht
University, the Netherlands, unpublished results).
MGP, vitamin K, and calcification 167
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Human equivalents of the warfarin + phylloquinone animals
are patients using oral anticoagulants [also known as vitamin K
antagonists (VKA)], including warfarin, acenocoumarol, and
phenprocoumon. These drugs are widely prescribed to subjects
with an increased thrombosis risk and are often taken for many
years or lifelong. Obviously, their mode of action is systemic,
and all vitamin K-dependent calcification inhibitors (including
OC, MGP, and GRP) will remain uncarboxylated and thus in-
active during VKA treatment. The first study demonstrating
strongly increased calcification of the aortic valves in patients
taking oral anticoagulants (20) has been confirmed by many
other researchers. Koos et al. (21) demonstrated that subjects
taking anticoagulants had a significantly higher degree of arterial
and aortic valve calcification than control subjects. In a case re-
port, Hristova et al. (22) described a kidney transplant recipient
exhibiting massive arterial calcification after the initiation of war-
farin therapy. Distal subcutaneous necrosis ultimately led to the
patient’s death. Rennenberg et al. (23) showed rapid calcification
of the femoral artery when treating patients with oral anticoagu-
lants, with an OR of 8.5 for calcification in patients compared
with controls. Similarly, patients taking oral anticoagulants
showed significantly increased levels of coronary calcification
(24). From these and other data, we conclude that the use of
VKA results in decreased carboxylation of MGP and probably
also GRP, which may form a strong risk factor for calcification
of the vasculature and heart valves.
Triage theory. Preferential distribution of phylloquinone to
the liver is consistent with the triage theory proposed by
McCannandAmes(25).Natureensuresthatatsuboptimal
supply, vitamins and minerals are primarily used for functions
required for short-term survival. Because carboxylation of the
most essential Gla proteins is localized in the liver and that of
the less essential Gla proteins in the extrahepatic tissues, a
transport system has evolved ensuring preferential targeting
to the liver to preserve coagulation when dietary vitamin K is
inadequate. Only at hepatic vitamin K sufficiency, particularly
the long-chain menaquinones, are transported to extrahepatic
tissues. This is why the first signs of vitamin K insufficiency are
seen as incomplete carboxylation of extrahepatic Gla proteins.
McCann and Ames concluded that long-term micronutrient
insufficiencies are a risk factor for the development of a wide
variety of age-related diseases, such as osteoporosis, cardiovas-
cular disease (CVD), and cancer.
The extrahepatic Gla proteins OC and MGP, for which
conformation-specific tests have been developed, exhibit sub-
stantial undercarboxylation in subjects not taking vitamin K
supplements. Recently, a conformation-specific test for un-
carboxylated GRP was described, showing that this protein
also partially occurs in a noncarboxylated form (M. Herfs,
E. Smit, C. Viegas, S. Simes, C. Vermeer, VitaK, Maastricht
University, the Netherlands, unpublished results). At this
time, there is no example of an extrahepatic Gla protein dem-
onstrated to be fully carboxylated in nonsupplemented
healthy subjects. We conclude that the Western diet contains
insufficient vitamin K to ascertain the requirements of extra-
hepatic tissues, such as bone and the vascular wall.
MGP, vitamin K status, and calcification
MGPexistsasvariousdistinctspeciesaccordingtoitsstateof
phosphorylation and/or carboxylation, including phosphory-
lated carboxylated MGP (cGMP), phosphorylated ucMGP
(p-ucMGP), desphospho-carboxylated matrix Gla protein
(dp-cMGP), and desphospho-uncarboxylated matrix Gla pro-
tein (dp-ucMGP). Unfortunately, there are currently no assays
available to measure each individual circulating MGP species
or even the total circulating MGP pool. An overview of pub-
lished papers in which circulating MGP species—dp-ucMGP,
dp-cMGP, total uncarboxylated matrix Gla protein (t-
ucMGP)—have been used to explore their potential diagnostic
utility is given in the following (see also TABL E S 1 and 2).
dp-ucMGP. Uncarboxylated species of MGP have been de-
tected in the healthy population and may be a useful marker
of vascular vitamin K status and disease. The most robust as-
say available to date is that for circulating MGP lacking both
posttranslational modifications, i.e., serine phosphorylation
and g-glutamate carboxylation. This fraction of total circulat-
ing MGP is dp-ucMGP. As for uncarboxylated OC, changes in
systemic vitamin K status resulting from the use of vitamin K
supplementation or vitamin K antagonists are reflected in cir-
culating levels of dp-ucMGP. Circulating dp-ucMGP levels
were shown to correlate with age in healthy adults, with sig-
nificantly higher levels in the elderly (65 y and older) (26).
Case-control studies showed higher dp-ucMGP values among
patients at risk of vascular calcification, including those with
rheumatoid arthritis, aortic valve disease, aortic stenosis (AS),
heart failure (HF), chronic kidney disease (CKD), and pa-
tients taking VKA (26–30). Extremely high dp-ucMGP values
were measured in dialysis patients with median dp-ucMGP
levels that were 3–5 times higher than in the corresponding
healthy controls. It was further shown in 107 CKD patients
that plasma dp-ucMGP levels increased progressively with
CKD stage (31). High circulating dp-ucMGP could reflect a
low dietary intake of vitamin K, resulting in lower inhibition
of artery calcification.
In CKD patients, increased dp-ucMGP levels (>921 pmol/L)
were associated with aortic calcification and overall mortality
(31). The association with mortality was, however, lost after cor-
rection for age and other confounders, such as CKD stage, he-
moglobin, and the aortic calcification score. In this patient
group, patients on VKA therapy presented significantly higher
levels of dp-ucMGP. A separate study in 19 VKA patients found
a significant correlation between plasma dp-ucMGP levels and
femoral artery calcification (30). The relationship between dp-
ucMGP and vascular calcification could not be confirmed in a
recently studied cohort of healthy elderly (32). Elevated levels of
dp-ucMGP (>950 pmol/L) were also associated with overall
mortality (adjusted HR >7) in 147 AS patients (27). Again, a
positive association was found between the use of VKA
and dp-ucMGP levels. In the non-VKA users in this patient
group, circulating dp-ucMGP was significantly correlated
with neurohormonal and hemodynamic markers of HF se-
verity. Plasma dp-ucMGP levels were shown to increase with
disease severity, as assessed by clinical, neurohormonal, and
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Table 1. Overview of associations between circulating MGP (dp-ucMGP, dp-cMGP, t-ucMGP) and calcification
1
Population, age MGP species MGP categories Calcification score Statistics
96 HT patients, 53 y
2
t-ucMGP(nmol/L) 4361 61111 Vascular calcification (scored as
present/not present by
renal angiography)
Correlation analysis (P= 0.164)
438 adults, 68 y (32) dp-ucMGP (pmol/L) 237 (52–330)
403 (335–462)
524 (464–599)
826 (604–2994)
149 650
263 648
243 649
235 647
(CAC)
ANCOVA (P= 0.55)
188 dialysis patients, 59 y (29) dp-cMGP (pmol/L) 12% lower levels in high
score group
0–6 (low)
7–15 (high)
Total calcification
(pelvis + hands + carotids)
ttest (P= 0.011)
36 HT patients, 53 y (30) t-ucMGP (nmol/L) 3471 (2031–4260)
4708 (4351–5215)
6126 (5416–9603)
145 (0–1546)
111 (1–3866)
36 (0–5951)
(CAC)
Kruskal-Wallis (NS)
19 OAC patients, 48 y (23) dp-ucMGP (pmol/L) 1439 6481 Femoral artery calcification Correlation analysis (r= 0.59, P,0.001),
MV regression analysis
3
(P,0.05, b= 0.68)
107 CKD patients, 67 y (31) dp-ucMGP (pmol/L) G921
.921
2.4 62.9
4.4 63.1
(AC)
ttest (P,0.001)
615 CVD patients without DM, 68 y (36) t-ucMGP (nmol/L) 3287 61178 Mitral annular calcification Logistic regression (OR = 0.64, P,0.001;
MV
4
OR = 0.73, P= 0.03)
221 CVD patients with DM, 68 y (36) t-ucMGP (nmol/L) 3287 61178 Mitral annular calcification Logistic regression (OR = 1.29, P= 0.08;
MV
4
OR = 1.89, P= 0.001)
191 AVD patients, 71 y (35) t-ucMGP (nmol/L) 320
400
1800 (OAC use)
400 (no OAC use)
(CAC )
ND
40 HD patients,67 y (34) t-ucMGP (nmol/L) 237 666
174 646*
171 666**
#103.0 (low)
103.1–600.0
$600.1
(CAC)
Fisher exact (*P= 0.022 and **P= 0.021
compared with low CAC score)
1
AC, aortic calcification; AVD, aortic valve disease; CAC, coronary artery calcification; CKD, chronic kidney disease; CVD, cardiovascular disease; DM, diabetes mellitus 2; dp-cMGP, desphospho-carboxylated matrix Gla protein; dp-ucMGP, desphos-
pho-uncarboxylated matrix Gla protein; HD, hemodialysis; HT, hypertensive; MGP, matrix Gla protein; MV OR, multivariable adjusted OR; ND, not determined; OAC, oral anticoagulant; t-ucMGP, total uncarboxylated matrix Gla protein; ucMGP,
uncarboxylated matrix Gla protein.
2
R. Rennenberg, L. Sckungers, B. Van Varik, E. Magdeleyns, C. Vermeer, P. de Leeuw, A. Kroon. Maastricht University Medical Center, Maastricht, the Netherlands, unpublished results.
3
Adjusted for age, fasting glucose, LDL cholesterol, estimated creatinine clearance, and calcium phosphate product.
4
Adjusted for age, sex, race, BMI, hypertension, smoking, blood pressure, albumin, total cholesterol, HDL, C-reactive protein, and epidermal growth factor receptor.
MGP, vitamin K, and calcification 169
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hemodynamic measurements, in a separate cohort of 179
HF patients (28). Moreover, high levels of dp-ucMGP
were associated with mortality due to progression of HF (ad-
justed HR >5). Also in this cohort, VKA use influenced
plasma dp-ucMGP levels. In conclusion, all published data
show that dp-ucMGP is inversely correlated with vitamin
K intake/status, and in most studies, this marker is also in-
versely correlated with survival and life expectancy.
dp-cMGP. Theoretically, this marker forms the mirror image
of dp-ucMGP, and 2 studies have been published investigating
the association with survival. Ueland et al. (27) found that AS
patients with above the median levels of dp-cMGP had a higher
unadjusted mortality rate, but in the multivariate model the sig-
nificance of these associations were lost. Schlieper et al. (29)
studied a cohort of 188 hemodialysis (HD) patients and found
an inverse association between dp-cMGP and both cardiovascu-
lar (HR >2) and overall mortality (adjusted HR >2). Circulating
dp-cMGP levels were 12% lower in the HD patents with more
extensive calcifications (total calcification score of 7–15) com-
pared with the HD patients with fewer calcifications (total cal-
cification score of 0–6). When interpreting these data, it should
be realized that the dp-cMGP assay became available only re-
cently and has only been used on a limited scale; also its sensi-
tivity is less than that of the dp-ucMGP assay.
t-ucMGP. In contrast to both assays described above, the
principle of the t-ucMGP assay is not that of a sandwich
ELISA, but that of a competitive, single antibody assay. The
fraction recognized by this assay is the sum of p-ucMGP
and dp-ucMGP. Because the observed plasma levels are
>1000-fold higher than those of dp-ucMGP, we assume that
the t-ucMGP mainly consists of phosphorylated ucMGP spe-
cies. The phosphoserines equip the molecule with strong cal-
cium-binding groups irrespective of the Gla content, and the
data observed can best be explained by assuming that the ma-
jority of this fraction has a strong affinity for vascular calcifi-
cations. This is illustrated by the fact that low t-ucMGP levels
have been found in patients prone to vascular calcification, in-
cluding patients with rheumatoid arthritis, dialysis patients,
and patients with CVD defined as myocardial infarction, cor-
onary artery disease, angioplasty, AS, and calciphylaxis
(26,33). In particular, dialysis and calciphylaxis patients were
characterized by extremely low values; nearly all patients had
values well below the healthy adult range. In healthy adults, se-
rum t-ucMGP levels were shown to be similar among the in-
dicated age groups: younger than 25 y, 25–65 y, and older than
65 y (26). Nevertheless, the circulating t-ucMGP value could
discriminate between healthy controls and subjects with mod-
erate to severe hypertension (R. Rennenberg et al., Maastricht
University Medical Center, Maastricht, the Netherlands, un-
published results).
In studies in which arterial calcification was quantified, low
circulating t-ucMGP levels were found to parallel high calcifi-
cation scores, but there are insufficient data to suggest a causal
relationship, for instance, by direct binding of t-ucMGP to the
sites of calcification. Lower circulating t-ucMGP levels were as-
sociated with higher calcification scores in 40 HD patients (34),
Table 2. Overview of associations between circulating MGP (dp-ucMGP, dp-cMGP, t-ucMGP) and mortality
1
Population, age MGP species
MGP
categories
Statistics, CV
mortality
Statistics, all-cause
mortality
179 chronic HF patients,
56 y (28)
dp-ucMGP (pmol/L) 22SD
21SD
+1 SD
+2 SD
Kaplan-Meier (P= 0.001)
MV Cox regression
2
(HR = 5.62, P= 0.001)
Kaplan-Meier (P= 0.002)
188 dialysis patients, 59 y (29) dp-cMGP (pmol/L) ,6139
.6139
Kaplan-Meier (P= 0.003)
MV Cox regression
3
(HR = 2.74, P= 0.015)
Kaplan-Meier (P= 0.008)
MV Cox regression
3
(HR = 2.16, P= 0.027)
147 AS patients, 74 y (27) dp-ucMGP (pmol/L) ,950
.950
ND Kaplan-Meier (P,0.001)
MV Cox regression
4
(HR = 7.29, P= 0.002)
dp-cMGP (pmol/L) ,2400
.2400
ND Kaplan-Meier (P,0.001),
MV analysis (NS)
833 CVD patients, 67 y (36) t-ucMGP (nmol/L) ,2757 (ref)
2757–3649
.3649
Kaplan-Meier (NS
5
)
MV Cox regression
6
(HR = 0.65, NS
5
)
Kaplan-Meier (P,0.05)
MV Cox regression
6
(HR = 0.48, P,0.05)
107 CKD patients, 67 y (31) dp-ucMGP (pmol/L) #921
.921
ND Kaplan-Meier (P= 0.006)
MV Cox regression
7
(HR = 1.57, NS)
1
AS, aortic stenosis; CKD, chronic kidney disease; CV, cardiovascular; CVD, cardiovascular disease; dp-cMGP, desphospho-carboxylated matrix Gla protein; dp-ucMGP, desphospho-
uncarboxylatedmatrixGlaprotein;HF,heartfailure;MV,multivariate;ND,notdetermined;t-ucMGP,totaluncarboxylatedmatrixGlaprotein.
2
Adjusted for New York Heart Association functional class II–IV, etiology, previous myocardial infarction, creatinine, C-reactive protein, N-terminal pro–B-type natriuretic peptide, warfarin,
aspririn use.
3
Adjusted for age, calcium, phosphate, high-sensitivity C-re active protein.
4
Adjusted for age, sex, BMI, epidermal growth factor receptor, N-terminal pro–B-type natriuretic peptide, C-reactive protein, hypertension, diabetes mellitus, left ventricular ejection
fraction.
5
Significant when modeled continuously (instead of tertile-based modeling).
6
Adjusted for age, sex, race, waist-to-hip ratio, smoking, hypertension, diabetes mellitus, blood pressure, epidermal growth factor receptor, total cholesterol, HDL cholesterol, C-reactive
protein, ejection fraction, peak exercise capacity, use of aspirin, b-blockers, statins, anticoagulants.
7
Adjusted for age, calculated propensity score.
170 Theuwissen et al.
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191 patients with aortic valve disease (35), and >800 patients
with stable CVD (36). Among the patients with stable CVD,
this inverse association was limited to patients without diabetes
(n= 615). The mechanism responsible for the effect modifica-
tion by diabetes state could, however, not be explained. Also,
more calcification was seen in 36 hypertensive patients with
thelowestvaluesoft-ucMGP,butthetrendacrossthet-
ucMGP tertiles was not significant (study 1) (30). In another
hypertensive patient group (n= 96), no significant correlation
was found between circulating t-ucMGP levels and the pres-
ence of calcification, as determined during renal angiography
(study 2) (R. Rennenberg et al., Maastricht University Medical
Center, Maastricht, the Netherlands, unpublished results). The
lack of statistical significance may be explained by the small pa-
tientpopulation(study1)orbytheatypicalmethodusedto
determine calcification (study 2). In 200 healthy postmenopau-
sal females, the inverse relationship between t-ucMGP and cal-
cification was only found in women with clear coronary artery
calcification (CAC), i.e., CAC score >0 (G. Dalmeijer et al., Ju-
lius Center for Health Sciences and Primary Care, University
medical Center Utrecht, the Netherlands, unpublished results).
Circulating t-ucMGP levels were significantly lower in those
with CAC (n= 75) compared with those without CAC (n=
125, CAC score <10).
Like dp-ucMGP and dp-cMGP, circulating t-ucMGP was
also associated with mortality risk (36). Compared with
CVD patients in the lowest t-ucMGP tertile, CVD patients
in the highest tertile had ~50% lower risk of mortality in
models adjusted for age, sex, and race. This association re-
mained after adjustment for traditional CVD risk factors,
kidney function, C-reactive protein, ejection fraction, peak
exercise capacity, and medication use, but was limited to
persons without diabetes (n= 610). The association of
t-ucMGP with CVD events was in the same direction, but
of lesser magnitude. When t-ucMGP was evaluated as a con-
tinuous predictor variable, each 1000-nmol/L higher
t-ucMGP level was associated with a 22% lower risk of mor-
tality and a 16% lower risk of CVD events.
Taken together, the data that have been published thus far
suggest that dp-ucMGP may become a risk marker for cardio-
vascular disease and mortality, whereas t-ucMGP, through its
calcium-binding capacity, may become a marker for vascular
calcification. At present, we are developing an assay for
p-cMGP, which on a theoretical basis has the highest cal-
cium-binding capacity and would therefore be the marker
of choice for monitoring vascular calcifications.
Immunohistochemical staining. Conformation-specificanti-
MGP antibodies have also been used for immunohistochemical
staining of healthy and atherosclerotic vessels. In healthy arter-
ies, there is little accumulation of either cMGP or ucMGP. In
calcified atherosclerotic lesions as well as in Mönckeberg’sme-
dia sclerosis, however, both forms of MGP accumulate, proba-
bly because of increased expression. It was reported that
elevated levels of calcium trigger MGP expression in cultures
vascular smooth muscle cells (37). As shown in Figure 2,
cMGP is the most predominant form in early lesions, whereas
ucMGP is mainly found around atherosclerotic lesions.
Vitamin K status and supplementation
Animal studies. Using the warfarin + phylloquinone model for
arterial calcification in rodents, Spronk et al. (13) showed that
menaquinone-4 and not phylloquinone inhibited warfarin-
induced arterial calcification.Thisisexplainedbythedifferent
transport and tissue distribution of menaquinones, resulting in
a much more prominent protective effect of menaquinones
(compared with phylloquinone) on arterial calcification. Later
studies, in which the long-chain menaquinone-7 (MK-7)
was used to counteract warfarin-induced calcification, showed
that this form of menaquinone was even more potent than
menaquinone-4. This is consistent with population-based
Figure 2 Carboxylated matrix Gla protein
(cMGP) and uncarboxylated matrix Gla protein
(ucMGP) in the arterial wall. Early and advanced
lesions in the arterial wall were stained (red) for
cMGP and ucMGP. A, cMGP in early lesion. B,
cMGP in advanced lesion. C, ucMGP in early
lesion. D, ucMGP in advanced lesion. TI, tunica
intima; TM, tunica media. (C. Vermeer et al.,
VitaK, Maastricht University, the Netherlands,
unpublished results).
MGP, vitamin K, and calcification 171
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studies in which only the long-chain menaquinones had car-
dioprotective effects.
Human intervention studies. In a limited number of 3-mo
clinical trials, it was demonstrated that vitamin K–containing
supplements result in a strong and significant decrease of cir-
culating dp-ucMGP in healthy adults (26,38,39). Thus far,
most data have been obtained with MK-7; MK-7 was required
in doses between 45 mg/d and 90 mg/d to find a statistically
significant effect on MGP carboxylation. Based on its shorter
half-life and different tissue-distribution pattern, it is to be ex-
pected that higher doses will be required for phylloquinone.
Thus far, only one 3-y clinical trial has been published with
clinical endpoints. In this 3-y study, 181 postmenopausal
women received either phylloquinone (1 mg/d) or placebo
against a background of cholecalciferol (8 mg/d). Although
Yo u n g ’s elasticity modulus (a marker for vascular stiffening)
decreased by 12% in the placebo group, vascular characteris-
tics remained unchanged in the vitamin K group (40).
It is well-known that CKD patients are at high risk of vas-
cular calcification, and each year 10% of the HD population
will die of cardiovascular complications. We have found that
circulating dp-ucMGP levels in these patients are higher than
in any other patient group, in many cases even higher than
in those taking oral anticoagulants. This is suggestive of a
very poor vitamin K status of the vascular wall. Obviously,
this group is an ideal target for testing whether vitamin K sup-
plements can normalize the circulating dp-ucMGP levels and
whether the change in vascular vitamin K status will result in
an improved clinical outcome. Westenfeld et al. (41) demon-
strated in HD patients that indeed menaquinone supplements
induce a dose-dependent decrease in plasma dp-ucMGP levels.
Recently, we entered into collaborative studies with the
Maastricht University Medical Center (Maastricht, the Nether-
lands) and the RWTH Aachen University (Aachen, Germany)
in which clinical endpoints will be monitored in 2 indepen-
dent trials among CVD patients. In 1 trial, the effect of mena-
quinones (MenaQ7, 360 mg/d) on the progression of CAC is
investigated in Dutch patients with established coronary artery
disease. In the second trial, the effect of phylloquinone (2 mg/
d) on the progression of aortic valve calcification is monitored
in German patients with preexisting valve calcification. The
potential of vitamin K as a modifiable risk factor is especially
exciting because of its simplicity: with the aid of food supple-
ments or fortified foods, vitamin K consumption can be in-
creased without major changes in the dietary pattern.
CONCLUSIONS
The triage theory predicts that, with limited intake, vitamins
are transported to those tissues where they are important for
immediate survival of the organism. For vitamin K, this im-
plies a preferential transport to the liver, the organ where the
coagulation factors are synthesized. Without doubt, bleeding
caused by an impaired hemostatic system is the most life-
threatening consequence of vitamin K deficiency. This
mechanism explains why vitamin K insufficiency will occur
sooner and be more pronounced in extrahepatic tissue than
in the liver and is consistent with the relatively high levels of
circulating uncarboxylated OC and dp-ucMGP observed in
healthy subjects. The question is whether the undercarboxy-
lation (10–40%) of circulating OC, MGP, or GRP, as seen in
the healthy adult population, is associated with an increased
risk of age-related diseases, such as soft-tissue calcification.
Only long-term vitamin K intervention studies in healthy sub-
jects may answer this question. The very high levels of dp-
ucMGP found in certain patient groups warrant studies inves-
tigating a beneficial clinical outcome of vitamin K supplemen-
tation. Notably, HD patients form an excellent model for
apparent vascular vitamin K insufficiency and concomitant
arterial calcification. A second target group for vitamin K sup-
plementation studies is formed by subjects taking high doses
of supplemental calcium. It has been reported that the in-
creased calcium load is a risk factor for vascular calcification.
Finally, it should be mentioned that most vitamin K interven-
tion studies have used high doses of vitamin K (both phyllo-
quinone and menaquinones). Benefits of increased vitamin K
intake at nutritionally relevant doses in healthy subjects await
the outcomes of clinical trials currently in progress.
Acknowledgments
All authors have read and approved the manuscript.
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