© 1998 American Heart Association, Inc.
Original Contributions |
Warfarin Causes Rapid Calcification of the Elastic Lamellae in Rat Arteries and Heart Valves
From the Department of Biology, University of California, San Diego, La Jolla, Calif.
Correspondence to Dr Paul A. Price, Department of Biology, 0368, University of California, San Diego, La Jolla, CA 92093-0368. E-mail pprice{at}ucsd.edu
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Abstract |
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Abstract—High doses of warfarin cause focal calcification of the elastic lamellae in the media of major arteries and in aortic heart valves in the rat. Aortic calcification was first seen after 2 weeks of warfarin treatment and progressively increased in density at 3, 4, and 5 weeks of treatment. By 5 weeks, the highly focal calcification of major arteries could be seen on radiographs and by visual inspection of the artery. The calcification of arteries induced by warfarin is similar to that seen in the matrix Gla protein (MGP)–deficient mouse, which suggests that warfarin induces artery calcification by inhibiting
-carboxylation of MGP and
thereby inactivating the putative calcification-inhibitory
activity of the protein. Warfarin treatment markedly increased the
levels of MGP mRNA and protein in calcifying arteries and decreased the
level of MGP in serum. Warfarin treatment did not affect bone growth,
overall weight gain, or serum calcium and phosphorus levels, and,
because of the concurrent administration of vitamin K, prothrombin
times and hematocrits were normal. The results indicate that the
improved warfarin plus vitamin K treatment protocol developed in this
study should provide a useful model to investigate the role of MGP in
preventing calcification of arteries and heart valves.
Key Words: matrix Gla protein • warfarin • vitamin K • artery calcification • heart valve calcification
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Introduction |
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Matrix Gla protein (MGP1 ) is a 10-kDa secreted protein that contains 5 residues of the vitamin K–dependent calcium-binding amino acid,
-carboxyglutamic acid
(Gla).1 2 MGP was originally discovered in
demineralization extracts of bone but is now known to be expressed by a
wide variety of tissues and cell types. The rat tissues with the
highest levels of MGP mRNA are cartilage, heart, kidney, and
lung,3 4 and cells known to express MGP mRNA
include osteoblasts, chondrocytes, vascular smooth muscle cells,
pneumocytes, kidney cells, and fibroblasts.3 4 5 6 7 8
Although several noncalcified tissues express MGP mRNA at a higher
level than bone, significant levels of the protein have been found only
in bone and calcified cartilage.4 9 This
observation suggests that the protein may accumulate at sites of
calcification and that much of the protein secreted by noncalcified
tissues probably escapes to plasma, where MGP is found at
concentrations of 0.3 to 1 µg/mL depending on the species. MGP is the
target of several additional posttranslational modifications in
addition to -carboxylation. Specific proteolytic cleavage at a
conserved dibasic site in the C-terminal region has been
observed in MGP isolated from human, bovine, and shark
tissues,10 and conserved
phosphorylation of 3 phosphoserine residues in the
N-terminal region has been found in MGP from shark, rat,
cow, and human tissues.11 The function of MGP was recently explored by targeted gene deletion in the mouse.12 Investigators found that MGP-deficient mice are normal at birth but that calcification of the arterial media first appears at 1 week of age and progresses rapidly to encompass the entire media by 3 weeks of age. The extensively calcified arteries are fragile, and most of the MGP-deficient mice die of exsanguination by 3 to 6 weeks of age. Surviving animals begin to show signs of bone involvement, including calcification of growth plate cartilage with growth plate closure, osteopenia, and bone fracture, after 3 weeks of age.
The objective of the current study was to determine the effect of
the vitamin K antagonist warfarin on the calcification of
soft tissues in the rat. The procedures used have their genesis in
earlier studies with the vitamin K–dependent bone Gla protein
(osteocalcin),13 14 which led to the discovery
that vitamin K cannot counteract the effect of warfarin in extrahepatic
tissues.15 This result is in marked contrast to
the well-established ability of vitamin K to counteract the effect of
warfarin on the synthesis of blood coagulation factors by the
liver.16 The fundamental dichotomy in the ability
of vitamin K to counteract the effect of warfarin in different tissues
allows continuous maintenance of animals on doses of warfarin
sufficient to severely inhibit -carboxylation of proteins
synthesized by extrahepatic tissues while maintaining normal blood
coagulation times.14 17 We report that a modified
version of the warfarin plus vitamin K protocol used in the original
studies with bone Gla protein produces a rapid, extensive calcification
of elastic lamellae in the aorta and other arteries that is quite
similar to the phenotype of the MGP-deficient mouse.
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Methods |
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Materials
Vitamin K1 (phylloquinone) and warfarin were purchased from Sigma Chemical Co. For injections, stock solutions of vitamin K1 (10 mg/mL) were prepared and stored in sterile, foil-wrapped containers at 4°C. Stock solutions of sodium warfarin (50 mg/mL) were prepared and stored at 4°C. Simonsen albino rats (derived from Sprague-Dawley rats) were purchased from Simonsen Laboratories (Gilroy, Calif).
Methods
Calcified tissues were dried, weighed, and extracted with a
10-fold excess (wt/vol) of 10% formic acid for 16 hours at 4°C. MGP
levels in the acid extracts and serum were determined by
radioimmunoassay as described previously.18
Calcium levels in acid tissue extracts were determined
colorimetrically by using cresolphthalein complex 1
(Sigma), and phosphate levels were determined
colorimetrically as described
previously.19 Tissue sectioning and staining were
performed by Biological Testing Service. For radiographs, the carotid
arteries and abdominal aorta were removed at necropsy, cleaned of
nonvascular tissue, and photographed with a Hewlett-Packard model 4380N
Flexitron x-ray system. Prothrombin times were determined by using the
thromboplastin time with calcium reagents and following the
manufacturer's procedure (Sigma).
For Northern blot analysis, total RNA was isolated from thoracic aortas and normal rat kidney cells3 with an RNA STAT-60 kit (Tel-Test B).Forty micrograms of total RNA from each sample was fractionated on a 1% formaldehyde-agarose gel in MOPS buffer and transferred onto a Hybond-N membrane (Amersham). After 3 hours of prehybridization in 50% formamide, 5x SSC, 5x Denhardt's solution, and 100 µg/mL denatured salmon sperm DNA at 42°C, the blot was hybridized with a randomly primed, 32 P-labeled cDNA probe for rat MGP20 for 16 hours. The membrane was washed 3 times for 1 hour each with 0.1x SSC containing 0.1% SDS at 65°C, and then exposed to x-ray film.
Maintenance of Animals
Study procedures were reviewed and approved by the University of
California, San Diego, Animal Subjects Committee before initiation.
Male Sprague-Dawley rats were given free access to rodent diet 5001
(Purina Mills), which is 0.67% phosphorus and 0.95% calcium by
weight. This diet contains 500 µg phylloquinone/kg and has no added
menadione. All injections were administered subcutaneously in the back
of animals essentially as described
previously.13 14 15 Twenty-four and 48 hours before
the first warfarin injection, all rats received doses of 1.5 mg vitamin
K1/100 g body weight. Previous studies have shown
that this loading dose of vitamin K is necessary to prevent bleeding
during the first week of warfarin treatment.15
The first warfarin dose, 15 mg/100 g, and 1.5 mg vitamin
K1/100 g were administered at 8 AM
when rats reached 42 days of age. A second 15 mg/100 g dose of warfarin
was administered at 8 PM with no accompanying vitamin K.
This routine was maintained every day until termination of the
experiment. To reduce trauma at injection sites, 25-gauge needles were
used for all injections, and subcutaneous injection sites were rotated
between the 4 quadrants of the back. Animals were killed by
exsanguination while they were under metofane anesthetic, and selected
tissues were removed and fixed in 10% buffered formalin or frozen at
-70°C for later studies.
The first 5-week study was performed with 10 rats in the warfarin treatment group and 10 rats in the control group; rats in the control group received daily injections of the same dose of vitamin K1 but no warfarin. In a subsequent study, 2 rats were maintained on warfarin for 1 week and 2 for 2 weeks. In a third study, 2 rats were maintained on warfarin for 3 weeks, 2 for 4 weeks, and 2 for 5 weeks. Finally, in the course of developing the dose regimen for the revised warfarin protocol, we maintained 4 animals on the standard dosage of 15 mg warfarin/100 g every 12 zhours for 3 weeks with either the standard 1.5 mg vitamin K1/100 g per day or one half of this dose.
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Results |
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Modification of Warfarin Plus Vitamin K Protocol
In the initial study of the effect of experimentally induced vitamin K deficiency on arterial calcification, 42-day-old male rats were treated for 6 weeks with the daily dosages of warfarin and vitamin K, which, in previous studies,14 lowered levels of bone Gla protein in bone to 2% of normal levels. The presence of mineralization in the arteries of warfarin-treated rats was not detected, either as an increase in calcium levels of the aorta or as the presence of von Kossa staining in histological sections of the aorta, femoral artery, or carotid artery (data not shown). One possible explanation for the failure to find an increase in arterial calcification in these warfarin-treated rats is that the protocol developed to reduce levels of bone Gla protein in bone to 2% of normal levels did not produce a comparably significant defect in
-carboxylation of MGP. Another possibility is that even
2% of normal MGP -carboxylation may be sufficient to prevent
significant calcification in the arteries of warfarin-treated rats. In
either case, the solution to the arterial calcification
problem would be to further reduce the level of MGP -carboxylation.
We therefore increased the warfarin dose by a factor of 2 and
administered this dose every 12 hours rather than every 24 hours. The
total daily dose of warfarin was thereby increased 4-fold.
Rats maintained for 5 weeks on this revised warfarin plus vitamin K
protocol were quite healthy. As shown in Figure 1
, the modified warfarin treatment
protocol had no effect on the rate at which the initially 6-week-old
rats gained weight over the 5 weeks of the experiment. There was also
no effect of 5 weeks of warfarin treatment on tibial length, which was
3.9 cm for both the warfarin-treated and control rats, and there was no
radiographic evidence of growth plate fusion (radiographs
not shown). The absence of an effect of warfarin on bone growth is in
agreement with results of earlier studies, which showed that warfarin
causes growth plate fusion only in rats 4 months of age or
older.21 Serum chemistry tests, performed after 4
weeks of treatment, did not detect a significant difference between the
warfarin-treated and control groups in any of the measured
parameters, including serum calcium and phosphorus levels.
As in earlier experiments,14 15 concurrent
treatment of animals with high doses of vitamin K was able to
counteract the effect of warfarin on the carboxylation of blood
coagulation factors by the liver, as shown by the fact that prothrombin
times and hematocrits remained normal throughout the experiment and
that there was no single instance of uncontrolled bleeding at a site of
injection.
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Effect of Warfarin on Calcification of Arteries
The time course of arterial calcification was
investigated in 42-day-old male rats. The 10 experimental animals were
given warfarin every 12 hours and vitamin K every 24 hours; the 10
control rats were given vitamin K alone every 24 hours. Two animals
were killed from each group every week, and the aorta, heart, lungs,
kidneys, and carotid and femoral arteries were fixed in formalin. No
calcification was detected by von Kossa staining of abdominal aorta
sections after 1 week of warfarin treatment. By 2 weeks of treatment,
however, longitudinal sections of the abdominal aorta contained darkly
stained regions of calcification that were confined to linear
structures within the aortic media (Figure 2). These linear structures were
identified as the elastic lamellae of the aortic media by staining
serial sections of the abdominal aorta for elastin and mineral. By 3
weeks of treatment, the number of calcified regions in the abdominal
aorta had increased significantly (Figure 2). Each calcified region was
focal, with areas of intense von Kossa staining for mineral
interspersed with regions of the aortic media apparently free of any
calcification. By 4 and 5 weeks of treatment, the intensity of von
Kossa staining in regions of calcification increased further, and some
calcified regions eventually became so hard that there was
fragmentation during sectioning (Figure 2). A similar time course and
pattern of medial calcification were also observed in von
Kossa–stained sections of the coronary, carotid, and femoral
arteries of warfarin-treated rats (micrographs not shown).
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We found that the time course of warfarin-induced arterial
calcification is remarkably reproducible. In subsequent experiments, we
examined the extent of arterial calcification in rats
maintained on warfarin for 1, 2, 3, 4, or 5 weeks, and the results of
these experiments, performed over a 4-month period with 2 rats per time
point, were indistinguishable from the time course depicted in Figure 2
. In a separate experiment, we investigated the effect of reducing the
maintenance dose of vitamin K by a factor of 2 on the extent of
calcification at 3 weeks of warfarin treatment. Four animals were
maintained on warfarin, 2 with the standard dose of vitamin K and 2
with half of this dose. Calcification of the abdominal aorta in the 2
groups was indistinguishable, as might have been anticipated from the
previously documented inability of vitamin K to counteract the effect
of warfarin in extrahepatic tissues.15 Because
prothrombin times were prolonged 1.2-fold to 1.6-fold in the group
receiving half the standard dose of vitamin K, we used the standard
dose of 1.5 mg vitamin K/100 g body weight in all other experiments
reported here.
An overview of the pattern of arterial calcification in the
warfarin-treated rat can be obtained either by visual inspection of the
tissues with a dissecting microscope or by examination of radiographs
of the arteries. Visual detection of calcification is easiest in the
carotid artery, which normally has a translucent aspect. As shown in
Figure 3, calcification of the carotid
artery in warfarin-treated rats appears as discontinuous, whitish
deposits against a background of otherwise translucent tissue. These
white regions often form irregular bands around the artery that appear
to be hard when the tissue is manipulated. After the carotid artery
shown in Figure 3 was fixed and sectioned longitudinally, calcification
appeared as focal deposits similar to those shown in Figure 2. We
therefore conclude that focal calcification seen in longitudinal
sections can, in some instances, represent cross-sectional cuts
through circumferential bands of calcification. Calcification can
easily be detected radiographically in the aorta and
carotid artery by 5 weeks of warfarin treatment (Figure 4). In each instance, the calcification
seen in the radiograph is markedly discontinuous, with areas of
calcification separated by apparently uncalcified regions of the
artery. Radiographs of the carotid artery and aorta also show that
calcification often appears as irregular bands rather than as punctate
foci.
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Chemical analyses were performed on the aorta and selected
additional tissues from rats maintained for 4 weeks on the revised
warfarin plus vitamin K protocol. As shown in Table 1, warfarin treatment increased the total
level of calcium in acid extracts of the aorta by 
4-fold. Phosphate
levels in the acid extracts were increased to a similar extent (data
not shown). Warfarin treatment also caused a 4-fold increase in the
level of calcium in the kidney (Table 1).
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Effect of Warfarin on MGP Expression
The effect of warfarin treatment on MGP expression in arteries was
evaluated in 2 ways. Northern blot analysis of MGP mRNA levels
was performed on RNA isolated from the aortas of rats treated for 4
weeks with warfarin and vitamin K or vitamin K alone. As shown in
Figure 5, warfarin treatment
significantly increased the level of MGP mRNA expression in the aorta
of warfarin-treated rats. MGP antigen levels were also determined by
radioimmunoassay of the acid demineralization extracts of the aorta of
rats maintained for 4 weeks on the warfarin and control protocols. As
shown in Table 1, warfarin treatment increased the level of MGP in the
aorta of the warfarin-treated animals by 13-fold.
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The dramatic accumulation of MGP antigen in the calcified aortas of the
warfarin-treated rats suggests that -carboxylation of the protein
may not be essential for its accumulation in calcified tissue. To test
this hypothesis further, we examined the level of MGP in the segment of
the metaphysis that had formed in the course of bone elongation during
the 4 weeks of warfarin treatment. As shown in Table 1, MGP antigen
accumulated in the regions of bone calcification formed during the
interval of warfarin treatment, but the level of MGP per unit of
mineral calcium was reduced by 5-fold compared with the level in
control bone tissue. A similar reduction in MGP levels was also seen in
SDS gels of acid-extracted proteins from this region of the metaphysis
of rats treated with warfarin for 4 weeks (data not shown).
Because warfarin-induced calcification increases the level of MGP mRNA
and antigen in vascular tissues, it might be anticipated that there
would also be an increase in the level of MGP antigen in blood. As
shown in Table 2, however, the level of
serum MGP was actually almost 3-fold lower in warfarin-treated rats.
This decrease is serum MGP did not change during the 5 weeks of
warfarin treatment and was therefore unrelated to arterial
calcification. It seems more likely that the decrease in serum MGP
levels was due either to increased clearance of the noncarboxylated
protein or to a warfarin-induced decrease in the secretion of MGP from
cells similar to the warfarin-induced decrease in the secretion of
prothrombin reported in rat
hepatocytes.22 23
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Effect of Warfarin on Calcification of Heart, Lung, and
Kidney
Because the highest levels of MGP mRNA have been found in the
heart, lung, and kidney of rats of this age,3 it
seems logical to conclude that each of these tissues may, under some
circumstances, be prone to calcify. To evaluate the effect of warfarin
treatment on the calcification of these tissues, the heart, lungs, and
kidneys were removed at necropsy from rats treated for 4 weeks with
warfarin and vitamin K or vitamin K alone. Sections of each tissue were
then subjected to von Kossa staining to detect areas of mineralization.
In kidney, calcification was found in the elastic lamellae of arteries
in the renal hilum but not in any other structure (not shown). In
heart, calcification was found only in the elastic lamellae of the
coronary artery (not shown) and aortic heart valve (Figure 6). In lung, calcification was found in
the elastic lamellae of a number of arteries but not in any other
structures. Although we did not evaluate, by
histological methods, the calcification of any other
soft tissues in warfarin-treated rats, it should be noted that such
calcifications, if present, were not sufficiently extensive to be
detected in whole-animal radiographs of rats treated with warfarin for
5 weeks.
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Comparison of the Effect of Warfarin in Rats With the Phenotype of the MGP-Deficient Mouse
As noted above, targeted deletion of the MGP gene in mice causes calcification of the major arteries that can first be detected at 1 week of age.12 This calcification begins within the elastic lamellae of the arterial media and progresses to the point that the arteries become rigid tubes that are fragile and rupture, causing death by exsanguination in most MGP-deficient mice by 3 to 6 weeks of age. Tracheal ring cartilage also calcifies in MGP-deficient mice by 2 to 3 weeks of age. After 3 weeks of age, surviving mice grow more slowly, partly because of the abnormal calcification of growth plate cartilage, and eventually have osteopenia and bone fractures.12
Most of the salient phenotypic features that appear early in
MGP-deficient mice are also found to a somewhat muted degree in
warfarin-treated rats. The same arteries also calcify in
warfarin-treated rats, and calcification also begins within the elastic
lamella of the aortic media. Calcification progresses more slowly in
warfarin-treated rats, however, and is focal and discontinuous in
appearance, in contrast to the calcification seen in MGP-deficient
mice, which forms a continuous calcification sheet that encompasses the
entire arterial media. We believe that the muted extent of
the calcification induced by warfarin in rats is due to the fact that
-carboxylation of MGP was not completely inhibited by the warfarin
doses we used. It is also possible, however, that the protein is in
fact completely non– -carboxylated at these warfarin doses and that
the non– -carboxylated protein retains some residual activity as a
calcification inhibitor. The major difference between the
phenotype of the MGP-deficient mouse and the warfarin-treated
rat is the absence of the reduced bone growth, osteopenia, and
fractures in the warfarin-treated rat that are features of the mouse
phenotype after 3 weeks of age. One possible explanation for
this difference is that the defect in MGP -carboxylation produced by
warfarin has no effect on bone metabolism. It is also
possible that rats the age of those used in the current study are more
similar to mice in the first 3 weeks of life, a period in which bone
growth abnormalities were not observed in the MGP-deficient
mouse.12 This possibility is supported by results
of earlier studies of rats maintained on warfarin, which found that
warfarin treatment causes abnormal calcification of growth plate
cartilage and attendant cessation of longitudinal bone growth that are
essentially identical to those seen in MGP-deficient mice but that
occur only in rats 4 months of age or
older.14 21
A puzzling feature of both the MGP-deficient mouse phenotype and the warfarin-treated rat syndrome is the absence of evidence of calcification at other tissue sites known to express MGP at a high rate. One possibility is that rodents maintained on carefully designed diets in the absence of stresses they would be expected to encounter in nature may have very little tendency to calcify most soft tissues. One test of this hypothesis will be to examine the calcification of selected soft tissues in warfarin-treated animals subjected to dietary manipulations, such as altered levels of calcium and phosphate. In this context, it is worth noting that kidney calcification has long been a problem with rodent chows and that the elimination of this problem in diets such as the one used here has required careful adjustment of the calcium and phosphate contents of the diet.24 25 26 It is therefore possible that diets with different calcium and phosphate contents, and different ratios of calcium to phosphate, will lead to accelerated calcification of kidneys and other soft tissues in the warfarin-treated rat. Another test of the hypothesis that MGP functions as a calcification inhibitor in tissues other than arteries will be to subject warfarin-treated rats to stresses known to cause systemic or focal calcification at nonvascular sites in humans, such as uremia, tissue trauma, and cancer.
Arterial Calcification and the Mechanism of MGP
Action
Previous studies with MGP-deficient mice and the current study
with warfarin-treated rats provide strong evidence that MGP functions
in vivo as a calcification inhibitor. To our knowledge, no
other protein has been established to have this function in vivo, and
no other protein has a targeted deletion that causes increased
calcification of any tissue. The fact that calcification of arteries is
so extensive in animals that either do not express this gene or cannot
-carboxylate this protein argues strongly against the hypothesis
that there is an equivalently active inhibitor of
calcification in vascular tissue and that MGP has a mere backup
function in preventing calcification of this tissue. It seems more
likely that MGP is central to the process by which the calcification of
arteries is normally inhibited in vivo.
The origin of calcification within the elastic lamellae of the media of arteries in MGP-deficient mice and warfarin-treated rats suggests that elastic lamellae of arteries are prone to calcify in vivo. This hypothesis is supported by studies showing that the elastic lamellae are the first sites of arterial calcification in humans and that the accumulation of calcification within these elastic lamellae begins by the second decade of life and increases progressively with age.27 28 29 30 31 32 Elastin has also been shown to be a potent and reproducible initiator of calcification in vitro.33 34 35 36 37 38 39 In vitro calcification of elastin occurs in serum and neutral buffered solutions at physiological concentrations of calcium and phosphate. This finding is in marked contrast to results of most studies on macromolecule- or cell culture– induced calcification, which relied on supraphysiological concentrations of calcium and phosphate or the agent ß-glycerophosphate to achieve calcification.
The focal nature of calcification in the aorta of warfarin-treated rats may provide insight into the mechanism by which MGP normally inhibits arterial calcification in vivo. Focal mineralization is not observed in MGP-deficient mice, presumably because there is so little ability to inhibit calcification that all of the many crystal nuclei generated by elastin can grow and thereby form a solid sheet of medial calcification. In warfarin-treated rats, the ability to inhibit calcification would presumably remain sufficient enough to inactivate most crystal nuclei, probably by a process that involves direct MGP binding to the mineral surface. A few nuclei would, however, grow and attain the critical calcification size at which the generation of seed crystals exceeds the ability of the residual MGP activity to inhibit the calcification chain reaction, and calcification would spread focally within and eventually between the elastic lamellae at this site.
The detailed MGP mechanism that best fits the available data is one in which the protein binds tightly and selectively to hydroxyapatite crystal nuclei and prevents their growth and ability to seed daughter crystals. It is also possible that the putative MGP-coated crystals may be recognized by cells in the vicinity of elastin nucleation sites and that cells may clear some of these coated crystals and dissolve them within the cell. The calcification-inhibitory activity of MGP is likely to be highly regulated, which may explain the carboxyl-terminal processing of MGP10 and the presence of phosphoserine at 3 conserved sites of partial phosphorylation.11 This inhibitory activity must be driven by an unusually strong association of the protein with the mineral nuclei. We speculate that this association includes strong lateral interactions between adjacent, mineral-bound MGP molecules and that this lateral interaction reflects the ability of MGP to self-associate. We previously showed that MGP is highly insoluble in vitro because of its tendency to self-associate and that this property of the protein is conserved in MGP from species ranging from shark to human.1 9
Effect of Warfarin on MGP Expression in Arteries
The current study demonstrated that warfarin causes an increased
level of MGP mRNA expression and accumulation of MGP antigen in aorta.
The most likely explanation for the increase in MGP mRNA in the aorta
is increased synthesis of the protein by vascular smooth muscle cells
in response to calcification of the aortic media. Vascular smooth
muscle cells are known to express MGP mRNA in cell
culture6 and at sites of calcification in
atherosclerotic plaques.40 41 In the
anatomy of the rat aortic media, vascular smooth muscle cells
are interspersed between each elastic lamella. Because calcification
begins within the elastic lamella, it is possible that vascular smooth
muscle cells sense the presence of nearby regions of calcifying elastin
and respond with increased MGP expression to help arrest further growth
of the calcification. Studies are needed to correlate cellular levels
of MGP mRNA in smooth muscle cells of the artery with the proximity of
these cells to actively calcifying elastic lamellae and to identify the
mechanisms by which cells sense nearby sites of ongoing calcium
accretion.
The dramatic 13-fold increase in the level of MGP antigen in aorta
after 4 weeks of warfarin treatment probably arose from MGP secreted by
vascular smooth muscle cells in the aorta, and MGP was probably bound
to the calcification itself. This latter hypothesis is supported by the
fact that MGP can be released from the aorta only by demineralization
of the tissue. If MGP in fact accumulates on the surface of crystals
during the course of arterial calcification, it may be more
insightful to express MGP content of the aorta per unit of mineral
calcium rather than per milligram dry weight. When expressed this way,
the MGP content of calcifying aorta is >20-fold higher than that of
bone (Table 1) or any other tissue examined to
date.4 The simplest explanation for this
observation is that the local concentration of MGP may be maintained at
a far higher level near sites of arterial mineralization
than is the case in bone.
The fact that MGP accumulates in the bone metaphysis and aorta of
the warfarin-treated rat (Table 1) despite the probable
under–-carboxylation of the protein indicates that
-carboxylation of MGP is not required for binding to calcifying
tissues and possibly to the apatite in these tissues. In support of
this hypothesis, we found that thermal decarboxylation of MGP in
vitro does not affect the ability of the protein to bind to
hydroxyapatite from serum. If non–-carboxylated MGP does accumulate
in the calcifying aorta because of its ability to bind to
hydroxyapatite in this tissue, it is unclear why the
non–-carboxylated protein fails to arrest the growth of
mineral.
Warfarin Dose and Artery Calcification
The dramatic difference between the complete absence of artery
calcification with a warfarin dose given once every 24 hours and the
extensive and rapid calcification of arteries with twice this dose
administered every 12 hours may be due in part to the rapid clearance
of warfarin from plasma in the rat. Previous studies have shown that
warfarin is cleared from the plasma of rats with a half-time of 5 hours
and that the half-time for warfarin clearance is not affected by the
age or sex of the animal.42 The half-time for
warfarin clearance is also not affected by warfarin
dose,43 and the R and S
enantiomers of warfarin are cleared at about the same
rate.44 Because of the rapid clearance of
warfarin from plasma, the concentration of warfarin achieved shortly
after a single subcutaneous injection of the drug will fall to 20% of
this value at 12 hours and to <4% of this value by 24 hours. A daily
injection schedule, as used in our earlier version of the warfarin plus
vitamin K protocol,13 14 15 21 will therefore
produce marked oscillations in the plasma concentration of
the drug. If the effect of warfarin on -carboxylation of proteins in
extrahepatic tissues is proportional to the plasma concentration of the
drug, the daily injection protocol will produce severe impairment of
-carboxylation shortly after warfarin administration, followed by
substantial recovery by the time of the next injection at 24 hours. The
12-hour injection protocol used here would, in contrast, cause a
12-hour oscillation between the warfarin concentration
shortly after injection and a concentration that is 20% of this level
and therefore would allow substantially less time at the lower plasma
warfarin levels for recovery of extrahepatic protein -carboxylation
status.
An argument can be made that continuous growth of calcification nuclei
into large mineral deposits is in fact more dependent on the minimum
level of warfarin in plasma during each 24-hour period than on the
maximum level. This is because the process of mineralization is an
autocatalytic process, much like a chain reaction, which needs a
continuous rate of crystal nuclei generation to sustain growth. When
the level of MGP -carboxylation rises to the point that the activity
of MGP secreted near the site of calcification is sufficient to reduce
the level of crystal nuclei generation below that needed to sustain the
mineralization chain reaction, the growth of the mineral phase will
cease. Because normally -carboxylated MGP would now presumably coat
and thereby inactivate mineral surfaces, a subsequent
warfarin dose would be unable to restart mineral growth at this site.
The daily dose of warfarin required to cause arterial
calcification would therefore be determined by the need to keep the
plasma concentration of drug above a given value throughout each
24-hour period.
Vitamin K Dose, Blood Coagulation, and Artery
Calcification
The change from a given warfarin dose administered every 24 hours
to twice this dose given every 12 hours affects the dose of vitamin K
needed to counteract the effect of warfarin on blood coagulation. When
animals were maintained on the new, higher warfarin dose regimen and
the former vitamin K dose of 0.75 mg/100 g, blood coagulation times
were longer than normal and there were occasional incidents of bleeding
around subcutaneous injection sites. Doubling this vitamin K dose
restored normal coagulation times and eliminated bleeding at injection
sites. The ability of vitamin K to counteract the effect of warfarin on
the -carboxylation of blood coagulation factors by the liver was
established in earlier studies.15 16
In contrast, doubling the vitamin K dose did not significantly affect the extent of artery calcification produced by the new, higher warfarin dose regimen. The inability of vitamin K to counteract the effect of warfarin on the synthesis of bone Gla protein by osteoblasts in bone was noted in previous studies,15 and it has been speculated that vitamin K can counteract the effect of warfarin only at sites of blood coagulation factor synthesis in hepatocytes.15 The results of the current study support this hypothesis and indicate that arteries and bone cells share an epoxide reductase recycling mechanism that is inhibited by warfarin in a way that cannot be affected by even high doses of vitamin K.
Does the Defective Carboxylation of MGP Accelerate Soft Tissue
Calcification in Humans?
Results of the current study indicate that it is possible
that a defect in the -carboxylation of MGP in humans could
accelerate calcification of arteries and heart valves. The possible
importance of vitamin K intake to arterial calcification is
supported by a recent report that lower dietary intake of vitamin K is
correlated with increased aortic calcification in
humans.45 To further test the hypothesis that
defective -carboxylation of MGP contributes to arterial
calcification and to establish the vitamin K intake required to repair
this defect, we are currently developing assays to measure the
-carboxylation status of plasma MGP analogous to the
hydroxyapatite-binding assay we developed earlier for determining the
-carboxylation status of bone Gla protein in
serum.13
|
Acknowledgments |
|---|
This work was supported in part by the US Public Health Service (grant AR27029). We thank Kien Trinh for assistance with the Northern blot procedures and Amanda Wallace for help with animal treatments.
Received December 9, 1997; accepted March 18, 1998.
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References |
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M. R. Davies, R. J. Lund, and K. A. Hruska BMP-7 Is an Efficacious Treatment of Vascular Calcification in a Murine Model of Atherosclerosis and Chronic Renal Failure J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1559 - 1567. [Abstract] [Full Text] [PDF]
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 M. Y. Speer, M. D. McKee, R. E. Guldberg, L. Liaw, H.-Y. Yang, E. Tung, G. Karsenty, and C. M. Giachelli Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein-deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor of Vascular Calcification In Vivo J. Exp. Med., October 21, 2002; 196(8): 1047 - 1055. [Abstract] [Full Text] [PDF]
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P.-J. Lin, D.-Y. Jin, J.-K. Tie, S. R. Presnell, D. L. Straight, and D. W. Stafford The Putative Vitamin K-dependent gamma -Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site J. Biol. Chem., August 2, 2002; 277(32): 28584 - 28591. [Abstract] [Full Text] [PDF]
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P. A. Price, G. R. Thomas, A. W. Pardini, W. F. Figueira, J. M. Caputo, and M. K. Williamson Discovery of a High Molecular Weight Complex of Calcium, Phosphate, Fetuin, and Matrix gamma -Carboxyglutamic Acid Protein in the Serum of Etidronate-treated Rats J. Biol. Chem., February 1, 2002; 277(6): 3926 - 3934. [Abstract] [Full Text] [PDF]
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 P. A. Price, J. R. Buckley, and M. K. Williamson The Amino Bisphosphonate Ibandronate Prevents Vitamin D Toxicity and Inhibits Vitamin D-Induced Calcification of Arteries, Cartilage, Lungs and Kidneys in Rats J. Nutr., November 1, 2001; 131(11): 2910 - 2915. [Abstract] [Full Text] [PDF]
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M. A Engelse, J. M Neele, A. L.J.J Bronckers, H. Pannekoek, and C. J.M de Vries Vascular calcification: expression patterns of the osteoblast-specific gene core binding factor {alpha}-1 and the protective factor matrix gla protein in human atherogenesis Cardiovasc Res, November 1, 2001; 52(2): 281 - 289. [Abstract] [Full Text] [PDF]
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P. A. Price, H. H. June, J. R. Buckley, and M. K. Williamson Osteoprotegerin Inhibits Artery Calcification Induced by Warfarin and by Vitamin D Arterioscler Thromb Vasc Biol, October 1, 2001; 21(10): 1610 - 1616. [Abstract] [Full Text] [PDF]
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 B. Newman, L. I. Gigout, L. Sudre, M. E. Grant, and G. A. Wallis Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival J. Cell Biol., August 6, 2001; 154(3): 659 - 666. [Abstract] [Full Text] [PDF]
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 L. J. Schurgers and C. Vermeer Corn oil-induced decrease in arterial thrombosis tendency may be related to altered plasma vitamin K transport J. Lipid Res., July 1, 2001; 42(7): 1120 - 1124. [Abstract] [Full Text] [PDF]
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P. A. Price, S. A. Faus, and M. K. Williamson Bisphosphonates Alendronate and Ibandronate Inhibit Artery Calcification at Doses Comparable to Those That Inhibit Bone Resorption Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 817 - 824. [Abstract] [Full Text] [PDF]
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 A Farzaneh-Far, D Proudfoot, C Shanahan, and P L Weissberg Vascular and valvar calcification: recent advances Heart, January 1, 2001; 85(1): 13 - 17. [Full Text]
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 A. Farzaneh-Far, C. Iribarren, and W. S. Browner Origins and Consequences of Vascular Calcification JAMA, September 27, 2000; 284(12): 1515 - 1516. [Full Text] [PDF]
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 A. Joshi, W. E. Berdon, C. Ruzal-Shapiro, and R. J. Barst CT Detection of Tracheobronchial Calcification in an 18-Year-Old on Maintenance Warfarin Sodium Therapy: Cause and Effect? Am. J. Roentgenol., September 1, 2000; 175(3): 921 - 922. [Full Text] [PDF]
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L. A. J. L. M. Braam, P. Dissel, B. L. M. G. Gijsbers, H. M. H. Spronk, K. Hamulyak, B. A. M. Soute, W. Debie, and C. Vermeer Assay for Human Matrix Gla Protein in Serum : Potential Applications in the Cardiovascular Field Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1257 - 1261. [Abstract] [Full Text] [PDF]
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A FARZANEH-FAR, C SHANAHAN, D PROUDFOOT, and P. WEISSBERG Mobile intracardiac calcinosis: risk of thromboembolism in patients with haemodialysed end stage renal disease Heart, May 1, 2000; 83(5): 582a - 582. [Full Text]
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P. A. Price, S. A. Faus, and M. K. Williamson Warfarin-Induced Artery Calcification Is Accelerated by Growth and Vitamin D Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 317 - 327. [Abstract] [Full Text] [PDF]
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