© 2000 American Heart Association, Inc.
Vascular Biology |
Warfarin-Induced Artery Calcification Is Accelerated by Growth and Vitamin D
From the Department of Biology, University of California, San Diego, La Jolla.
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—The present studies demonstrate that growth and vitamin D treatment enhance the extent of artery calcification in rats given sufficient doses of Warfarin to inhibit
-carboxylation of matrix Gla protein, a calcification
inhibitor known to be expressed by smooth muscle cells and
macrophages in the artery wall. The first series of experiments
examined the influence of age and growth status on artery calcification
in Warfarin-treated rats. Treatment for 2 weeks with Warfarin caused
massive focal calcification of the artery media in 20-day-old rats and
less extensive focal calcification in 42-day-old rats. In contrast, no
artery calcification could be detected in 10-month-old adult rats even
after 4 weeks of Warfarin treatment. To directly examine the importance
of growth to Warfarin-induced artery calcification in animals of the
same age, 20-day-old rats were fed for 2 weeks either an ad libitum
diet or a 6-g/d restricted diet that maintains weight but prevents
growth. Concurrent treatment of both dietary groups with Warfarin
produced massive focal calcification of the artery media in the ad
libitum–fed rats but no detectable artery calcification in the
restricted-diet, growth-inhibited group. Although the explanation for
the association between artery calcification and growth status cannot
be determined from the present study, there was a relationship
between higher serum phosphate and susceptibility to artery
calcification, with 30% higher levels of serum phosphate in young, ad
libitum–fed rats compared with either of the groups that was
resistant to Warfarin-induced artery calcification, ie, the
10-month-old rats and the restricted-diet, growth-inhibited young rats.
This observation suggests that increased susceptibility to
Warfarin-induced artery calcification could be related to higher serum
phosphate levels. The second set of experiments examined the
possible synergy between vitamin D and Warfarin in artery
calcification. High doses of vitamin D are known to cause calcification
of the artery media in as little as 3 to 4 days. High doses of the
vitamin K antagonist Warfarin are also known to cause
calcification of the artery media, but at treatment times of 2 weeks or
longer yet not at 1 week. In the current study, we investigated the
synergy between these 2 treatments and found that concurrent Warfarin
administration dramatically increased the extent of calcification in
the media of vitamin D–treated rats at 3 and 4 days. There was a close
parallel between the effect of vitamin D dose on artery calcification
and the effect of vitamin D dose on the elevation of serum calcium,
which suggests that vitamin D may induce artery calcification through
its effect on serum calcium. Because Warfarin treatment had no effect
on the elevation in serum calcium produced by vitamin D, the synergy
between Warfarin and vitamin D is probably best explained by the
hypothesis that Warfarin inhibits the activity of matrix Gla protein as
a calcification inhibitor. High levels of matrix Gla
protein are found at sites of artery calcification in rats treated with
vitamin D plus Warfarin, and chemical analysis showed that the
protein that accumulated was indeed not -carboxylated. These
observations indicate that although the -carboxyglutamate residues
of matrix Gla protein are apparently required for its function as a
calcification inhibitor, they are not required for its
accumulation at calcification sites.
Key Words: Warfarin • vitamin K • vitamin D • artery calcification • matrix Gla protein
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Introduction |
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The current studies were performed to identify those factors that influence the extent of artery calcification in rats treated with Warfarin, a vitamin K antagonist that inhibits the formation of the calcium-binding amino acid,
-carboxyglutamic acid (Gla), in specific proteins. The target for
Warfarin treatment in these investigations is matrix Gla protein (MGP),
a vitamin K–dependent protein that inhibits artery calcification and
that is secreted by vascular smooth muscle cells and
macrophages in the artery.
MGP is a 10-kDa secreted protein that contains 5 residues of
-carboxyglutamic acid.1 2 MGP was originally discovered
in demineralization extracts of bone, but it 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 do express MGP mRNA at a higher level than bone,
significant levels of the protein itself have only been found 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 0.3 to 1 µg/mL, depending on the
species. MGP is the target of several other posttranslational
modifications in addition to -carboxylation. Specific proteolytic
cleavage at a conserved dibasic site in the C-terminal
region has been observed for MGP isolated from human, bovine, and shark
tissues,9 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
Recent genetic and biochemical studies have established MGP as the
first protein known to act as a calcification inhibitor in
vivo. In humans, defects in the MGP gene that predict a nonfunctional
MGP protein have been shown to be responsible for Keutel
syndrome.12 This syndrome is a rare, inherited disease
characterized by abnormal calcification of cartilages, including
costal, nasal, auricle, tracheal, and growth plate cartilage; by nasal
hypoplasia and brachytelephalangia; and by multiple
peripheral pulmonary artery
stenoses.13 14 In mice, targeted deletion of the
MGP gene causes rapid calcification of the elastic lamellae
of the arterial media that begins at birth and is
sufficiently extensive by 3 to 6 weeks of age that the arteries become
rigid tubes that fracture, causing death by exsanguination in most of
the affected mice by 6 weeks of age.15 MGP-deficient
mice also display abnormal calcification of growth plate and tracheal
cartilage. Finally, treatment of rats with the vitamin K
antagonist Warfarin at doses that inhibit the
-carboxylation of MGP causes rapid calcification of elastic lamellae
of arteries and of aortic heart valves and increased expression of MGP
mRNA in the calcifying artery.16
Calcification is a common finding in the pathophysiology of the aging
human artery and heart valve and is associated with several
cardiovascular disease states. Calcification is almost
universally associated with atherosclerotic plaques, and calcification
at plaque sites is typically so extensive as to be rigid and bonelike
in consistency.17 18 19 Calcification also
occurs in human aortic heart valves20 21 22 23 and is usually
seen in valves removed in the course of valve replacement surgery. The
human cardiovascular system is the site of 2 additional
kinds of calcification. In some individuals, arteries become rigidly
calcified in a linear fashion, to an extent such that the artery
resembles a rigid tube. This calcification, which has been termed
Monckeberg’s syndrome, is confined to the media of the artery and is
not necessarily associated with atherosclerotic plaque
formation.24 25 In all individuals, there is a less
obvious, diffuse calcification of the artery, a calcification that is
confined to the artery media and that does not result in artery
rigidity.26 27 28 29 30 31 Such diffuse calcifications of the artery
can be detected as early as the second decade of life and accumulate
with age, becoming 
15% of the dry weight of the media by the eighth
decade of life.31 We have analyzed the MGP levels
in all of these cardiovascular calcifications, and in
each instance we have found MGP at levels higher than those found in
any normal human tissue, including bone (personal observation,
manuscript in preparation).
A major objective of our research is to understand the role of MGP in
human cardiovascular calcifications. The current
investigations are 1 of several designed to identify the factors that
act synergistically with Warfarin to accelerate artery calcification in
the rat, with the goals of further understanding the role of MGP in
this process and of identifying the physiological
circumstances in which it might, in future studies, be appropriate to
search for the possible association between Warfarin treatment and
increased risk of cardiovascular calcification in
humans. In the first series of experiments presented here, we
have determined the effect of age and growth rate on artery
calcification in the Warfarin-treated rat. These studies reveal that
Warfarin-induced artery calcification occurred only in the growing
animal and was not seen either in older, nongrowing rats or in young
rats whose growth was temporarily arrested by a calorically restricted
diet. In the second set of experiments, we examined the effect of
concurrent Warfarin treatment in an animal model in which some degree
of artery calcification had been induced by high doses of vitamin D.
Treatment with high doses of vitamin D has been known for many years to
induce artery calcification in humans, rats, and other
animals.32 33 34 35 36 37 This vitamin D–induced calcification is
confined to the media of arteries and closely resembles the pattern of
calcification seen in Monckeberg’s syndrome in
humans.24 25 In the vitamin D treatment regime used for
the current studies, vitamin D induced marked artery calcification
within 4 days of treatment.33 We report here that
concurrent Warfarin treatment accelerated artery calcification in the
vitamin D–treated rat in this 4-day treatment interval, even though
Warfarin treatment alone did not cause detectable artery calcification
at treatment times of 1 week or less. The vitamin D/Warfarin model
described here has advantages for future studies of the mechanism by
which MGP normally inhibits artery calcification, because it is now
possible to examine the difference in the nature of the MGP interaction
with mineral in a situation in which MGP is -carboxylated and
mineralization is inhibited and in a situation in which MGP is not
-carboxylated and mineralization is accelerated.
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Methods |
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Materials
Vitamin K1 (phylloquinone), vitamin D3 (cholecalciferol), and Warfarin were purchased from Sigma Chemical Co. For injections, stock solutions of vitamin K1 were prepared at 10 mg/mL and stored in sterile, foil-wrapped containers at 4°C. Stock solutions of sodium Warfarin were prepared at 50 mg/mL in 0.15 mol/L NaCl and stored in sterile, foil-wrapped containers at 4°C. The stock solution of vitamin D was prepared for subcutaneous injection by dissolving 33 mg of vitamin D3 (1.32x106 IU) in 200 µL of absolute ethanol and mixing this solution with 1.4 mL of emulphor (alkamuls EL-620, Rhone-Poulenc) for 15 minutes. Water (18.4 mL) containing 750 mg of dextrose was then added, and the final solution was mixed for an additional 15 minutes, placed in foil-wrapped containers, and stored at 4°C. Fresh vitamin D solution was prepared for each 3-day injection cycle. Simonsen albino rats (Sprague-Dawley derived) were purchased from Simonsen Labs (Gilroy, Calif).
Methods
For measurement of mineral and MGP accumulation in arteries,
each tissue was removed within 30 minutes of death and immediately
frozen. Tissues were subsequently washed by continuous mixing with 1 mL
of wash solution (100 mmol/L CaCl2, 20
mmol/L HEPES [pH 7.4], 0.15 mol/L NaCl, and 0.02%
NaN3) for 24 hours at 37°C. The wash solution
was exchanged for fresh solution, and the wash was continued for an
additional 24 hours. Washed aortas were briefly patted with a dry
tissue and demineralized with 1 mL of 10% formic acid for 24 hours at
room temperature. The MGP levels in the acid extracts and in serum were
determined by radioimmunoassay as described previously38 ;
this assay uses polyclonal antiserum and reacts identically with native
MGP and with MGP in which Gla residues have been heat-decarboxylated to
Glu. Calcium levels in acid tissue extracts and serum were determined
colorimetrically by using cresolphthalein complexone
(Sigma), and phosphate levels were determined
colorimetrically as described.39 Tissue
sectioning and staining were performed by Biological Testing Service,
Inc (Sorrento Valley, Calif).
To purify MGP for determining the -carboxylation status of the
protein, aortas were dissected at sacrifice from 3 rats treated with
300 000 IU/kg of vitamin D at 0, 24, and 48 hours and with Warfarin
every 12 hours, beginning with the first vitamin D injection, for a
total Warfarin treatment time of 96 hours. Arteries were rinsed with
20 mmol/L HEPES [pH 7.4], 0.15 mol/L NaCl and dried. The dried
aortas were then extracted with 4 changes of 20 mL of 6 mol/L
guanidine-HCl with 20 mmol/L HEPES, pH 7.4, for 48 hours at room
temperature; rinsed with 20 mmol/L HEPES, pH 7.4, with 0.15 mol/L
NaCl and 100 mmol/L CaCl2; and demineralized
for 2 hours with 1.5 mL of 0.15 mol/L HCl at 4°C. A 200-µL aliquot
of the acid extract was then adsorbed to a polyvinylidene
difluoride (PVDF) membrane by using a Prosorb device
(Perkin-Elmer) and subjected to N-terminal protein
sequencing. MGP was also purified from rat bone as
described,10 40 adsorbed onto PVDF, and sequenced.
The degree of -carboxylation at the glutamic acid residue at
position 2 in the MGP sequence was calculated from a comparison of the
recovery of glutamic acid at residue 2 to the value predicted from a
repetitive yield analysis of the recovery of amino acids in the
first 10 residues of the protein.41
Maintenance of Animals
Male Sprague-Dawley rats were fed ad libitum with rodent diet
5001 (Purina Mills Inc), a diet that is 0.67% phosphorus and 0.95%
calcium by weight. This diet contains 500 µg/kg of phylloquinone and
has no added menadione. All injections were administered subcutaneously
into the backs of the animals essentially as described in earlier
studies16 . At 24 and 48 hours before the first Warfarin
injection, all rats received doses of 1.5 mg vitamin
K1 per 100 g of body weight. Previous
studies had shown that this loading dose of vitamin K was necessary to
prevent bleeding during the first week of Warfarin
treatment.42 On the initial day of Warfarin treatment, the
first Warfarin dose, 15 mg/100 g of body weight, and 1.5 mg of vitamin
K1 per 100 g, were administered at 8
AM, and the second identical dose of Warfarin was
administered at 8 PM with no accompanying vitamin K. This
routine was maintained every day until termination of the experiment.
Animals were killed by exsanguination while under metofane anesthetic,
and selected tissues were removed and fixed in 10% buffered formalin
or frozen at -20°C for later studies.
Experiments on the Effects of Age and Growth Status on
Warfarin-Induced Artery Calcification
To determine the effect of age on the susceptibility to Warfarin
induced artery calcification, male Sprague-Dawley rats were treated
with Warfarin beginning at 20 and 42 days of age and at 10 months of
age. The 4 animals in each age group received injections of Warfarin
every 12 hours and vitamin K every 24 hours (see detailed procedure
above) until the end of the experiment. The animals in the 20- and
42-day groups were killed by exsanguination after 2 weeks of Warfarin
treatment, and the animals in the 10-month group were killed after 4
weeks of Warfarin treatment. This experiment has been repeated several
times with consistent results for animals in each age
group.
To evaluate the effect of growth rate on artery calcification in 20-day-old rats, pilot experiments were carried out to establish the measured weight of the diet that allowed maintenance of body weight without allowing bone growth (as evidenced by a failure to increase tibia and femur length). These experiments established 6 g/d as a suitable maintenance diet. In the first experiment, 12 weanling, male Sprague-Dawley rats, 20 days of age, were caged individually and fed either ad libitum (6 rats) or a measured 6 g/d (6 rats) throughout the experiment. Three rats in each group received Warfarin every 12 hours and vitamin K every 24 hours from the start of the experiment, and the other 3 were not injected. All rats were killed by exsanguination 14 days after the beginning of Warfarin treatment and dietary manipulation.
In the second experiment, 8 male rats 20 days of age were caged individually and fed a measured 6 g/d for 2 weeks. Four of the animals were continued on the 6 g/d diet for an additional 2 weeks, and 4 were placed on an ad libitum diet for an additional 2 weeks. All 8 animals received Warfarin every 12 hours and vitamin K every 24 hours for weeks 3 and 4 only, and all rats were killed 28 days after the start of the experiment, at the end of week 4.
Experiments on the Synergy Between Warfarin and Vitamin D in
Artery Calcification
Previous studies on artery calcification in vitamin D–treated
rats have used oral gavage to deliver the vitamin.33 In
our first series of experiments, we used this gavage method to examine
the effects of Warfarin on artery calcification in rats treated with
300 000 IU/kg of vitamin D at 0, 24, and 48 hours. For subsequent
studies, we developed a subcutaneous injection vehicle (see above) for
delivering vitamin D to ensure greater uniformity in vitamin D dose. In
the initial pilot experiment with the injection method, we examined the
effects of Warfarin on rats treated with 300 000 IU/kg of vitamin D at
0, 24, and 48 hours. At the 96-hour time point examined, an identical
degree of artery calcification was found for animals treated with
vitamin D by the gavage and injection methods, which indicates that
vitamin D is taken up to a similar extent from the gut and from the
subcutaneous injection site. The subcutaneous injection vehicle
developed for the present studies may be useful in future studies
of this vitamin in animal models.
In the initial survival study, 24 seven-week-old, male Sprague-Dawley rats received subcutaneous doses of 300 000 IU vitamin D per kg of body weight at 0, 24, and 48 hours. Starting at t=0, 15 of these animals received injections of Warfarin every 12 hours and of vitamin K every 24 hours (see detailed procedure above), and 9 animals received injections of vitamin K alone every 24 hours. Survival was noted every 12 hours.
In the vitamin D dose-response study, 30 seven-week-old, male Sprague-Dawley rats were divided into 5 groups of 6 rats each. Each group was given subcutaneous injections of a different dose of vitamin D (100 000, 200 000, 300 000, or 500 000 IU vitamin D per kg) or of vehicle at t=0, 24, and 48 hours. Starting at t=0, half of the animals in each group received injections of Warfarin every 12 hours and of vitamin K every 24 hours, and the remaining animals in each group received injections of vitamin K alone every 24 hours. The 3 Warfarin-treated rats in the 500 000 IU vitamin D group died between 72 and 84 hours. All surviving animals were killed by exsanguination at 96 hours.
In the time-course study, 24 seven-week-old, male Sprague-Dawley rats received subcutaneous doses of 300 000 IU vitamin D per kg at t=0, 24, and 48 hours. Starting at t=0, 12 of these animals received injections of Warfarin every 12 hours and of vitamin K every 24 hours, and 12 animals received injections of vitamin K alone every 24 hours. Two animals from each group were killed at 48 hours, 4 from each group at 72 hours, and 6 from each group at 96 hours. All animal experiments were approved by the University of California at San Diego animal subjects committee.
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Results |
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Effect of Age on Artery Calcification in Warfarin-Treated Rats
Age has a dramatic effect on artery calcification in the Warfarin-treated rat. As shown in Figure 1
, 20-day-old rats treated with Warfarin
for 2 weeks had a similar focal pattern of aortic calcification to that
seen earlier in 42-day-old rats treated with Warfarin for 2
weeks.16 The primary differences between the 20- and
42-day-old animals are that the younger animals had a greater number of
areas with medial calcification and the intensity of von Kossa staining
in these areas was more intense. In contrast, 10-month-old rats proved
to be remarkably resistant to Warfarin-induced artery
calcification, with no detectable von Kossa staining in the aorta
(Figure 1), carotid artery, or aortic heart valves after 4 weeks
of Warfarin treatment.
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Serum calcium, phosphate, and MGP levels are shown in Table 1 for 20-day-old, 42-day-old, and
10-month-old Warfarin-treated rats. Serum phosphate levels were similar
for 20- and 42-day-old rats but are significantly lower for the
10-month-old rats (P<0.001). Serum calcium, phosphate, and
MGP levels were also determined in age-matched, vitamin K–replete
control rats (data not shown). These measurements showed that Warfarin
treatment did not affect serum calcium and phosphate levels at any age
but did reduce serum MGP levels by 3-fold in the 20- and 42-day-old
rats and by 2.5-fold in the 10-month-old rats. A similar reduction in
circulating levels of MGP has been seen previously in Warfarin-treated
rats, but it is presently unknown whether the lower levels of this
serum protein are due to increased clearance from the blood or
decreased release into the bloodstream from sites of MGP synthesis.
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Effect of Growth on Warfarin-Induced Artery Calcification in
20-Day-Old Rats
To separate the effects of age and growth on the susceptibility to
calcification in the Warfarin-treated rat, 20-day-old rats were placed
on the same diet and fed either ad libitum or 6 g/d, a restricted food
intake that allows maintenance of body mass but permits little
or no net growth, as evidenced by the failure of long bones to increase
in length. As shown in Figure 2, experiment 1, 2 weeks of Warfarin treatment produced the expected
extensive calcification of the artery media in the ad libitum–fed
animals. In contrast, no von Kossa staining could be detected in the
aorta (Figure 2), carotid arteries, or heart valves of the
calorically restricted weanling rats after 2 weeks of Warfarin
treatment. Analysis of the acid demineralization extracts of
the carotid arteries at the end of the experiment showed that the
levels of mineral phosphate and calcium in the ad libitum–fed group
were 7-fold above control levels, whereas the levels of mineral
phosphate and calcium in the restricted diet–fed rats were at control
levels (data not shown). The molar ratio of calcium to phosphate in the
carotid arteries of the ad libitum–fed, Warfarin-treated rats was
1.50:1.
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Serum calcium, phosphate, and MGP levels were determined at the end of
the 2-week period of ad libitum or restricted diet feeding, and the
values are shown in Table 2. As shown,
rats fed the calorically restricted diet had significantly lower levels
of serum phosphate (P<0.001) compared with rats fed ad
libitum. Warfarin treatment had no effect on serum calcium and
phosphate levels in either the ad libitum–fed or restricted diet
groups but did reduce serum MGP levels by >2-fold.
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A second growth study was carried out to determine whether rats that
were initially fed a calorically restricted diet would become
susceptible to Warfarin-induced artery calcification when subsequently
fed ad libitum. All 20-day-old rats were first fed a calorically
restricted diet for 2 weeks and then divided into 2 groups, 1 of which
was continued on the restricted diet for 2 weeks and the other of which
was placed on the ad libitum diet for 2 weeks. All rats were treated
with Warfarin for the last 2 weeks only. The calorically restricted
diet used in these studies essentially prevents weight gain in weanling
rats, with a weight gain of only 3% in the final 2 weeks of feeding
with this diet. In marked contrast, the rats that were first fed the
restricted diet for 2 weeks and then placed on the ad libitum diet for
the final 2 weeks had a 2.7-fold increase in body weight in the final 2
weeks (169% increase in body weight). The effect of treating these
animals with Warfarin during this final 2-week period of ad libitum
feeding was to induce a similar pattern of aortic calcification (Figure 2
, experiment 2) to that seen in the ad libitum–fed, 20-day-old
rats treated for 2 weeks with Warfarin (Figures 1 and 2,
experiment 1). In contrast, rats maintained on the restricted diet for
the full 4 weeks and treated with Warfarin for the final 2 weeks only
had no evidence of von Kossa staining in the aorta (Figure 2, experiment 2), carotid arteries, or aortic heart valves.
Serum calcium and phosphate levels were determined at the end of the 4-week experiment. Compared with the 4 rats fed ad libitum for the final 2 weeks of the experiment, the 4 rats fed the restricted diet throughout the 4-week experiment had significantly lower levels of serum phosphate (7.5±1.1 versus 10.8±0.8 mg/dL, P<0.005) and slightly lower levels of serum calcium (10.4±0.2 versus 11.8±0.2 mg/dL).
Effect of Warfarin Treatment on the Survival of Vitamin
D–Treated Rats
To explore the possible synergy between vitamin D and
Warfarin in artery calcification, we selected a vitamin D dosage
regimen that has been shown to induce significant artery calcification
without causing death.33 Rats were treated with 300 000
IU of vitamin D per kg of body weight at 0, 24, and 48 hours and every
12 hours with Warfarin or vehicle, starting with the first vitamin D
injection. In agreement with earlier studies,33 there was
no mortality in the rats treated with vitamin D alone (Figure 3). In contrast, all rats treated with
Warfarin plus vitamin D died within 9 days. This effect of Warfarin
appears to be an exacerbation of the known lethal effects of high
vitamin D doses, since previous studies had shown that treatment with
500 000 IU/kg of vitamin D alone at 0, 24, and 48 hours caused a 34%
mortality by 120 hours,32 33 and even higher vitamin D
doses are known to be lethal. The cause of death in rats treated with
massive doses of vitamin D alone was not established in earlier
studies, and we could not establish the cause of death in rats treated
with lower vitamin D doses together with Warfarin. We noted a marked
15% to 20% weight loss in the period preceding death. This weight
loss, which has been noted previously in rats treated with lethal doses
of vitamin D alone,35 may be a factor in mortality, but
the cause of this weight loss is unknown. It should be noted that the
mortality observed in rats treated with vitamin D plus Warfarin is not
due to an effect of Warfarin on blood coagulation, since coagulation
times remained normal throughout the time course of treatment with
vitamin D and Warfarin, and there was no evidence of bleeding in any of
the animals.
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Effect of Warfarin on Artery Calcification in Vitamin
D–Treated Rats
To establish the effect of concurrent Warfarin treatment on artery
calcification in the vitamin D–treated rat, rats were treated with the
300 000 IU/kg dose of vitamin D at 0, 24, and 48 hours and every 12
hours with Warfarin or vehicle for 96 hours beginning with the first
vitamin D injection. In agreement with earlier studies, treatment with
vitamin D plus vehicle caused calcification of the media of the aorta
(Figure 4), carotid, and other arteries.
Warfarin treatment dramatically increased the extent of medial
calcification in the aorta (Figure 4) and other arteries
compared with the calcification seen in rats treated with vitamin D
alone, but there was no evidence for a Warfarin effect on medial
thickness. In agreement with earlier studies, there was no evidence of
von Kossa staining of mineral in the arteries of rats treated for 96
hours with Warfarin alone (not shown). In rats treated with vitamin D
plus Warfarin, calcification appeared to occur throughout individual,
circumferential, lamellar sheets of elastin, resulting in a ribbon-like
histological pattern of von Kossa staining for mineral
(Figure 4). Hematoxylin/eosin staining of adjacent sections of
arteries from the rats treated with vitamin D alone or with vitamin D
plus Warfarin revealed no evidence of necrosis in cells in the artery
media, either adjacent to sites of intense calcification or in
uncalcified regions. This observation indicates that artery
calcification in rats treated with vitamin D plus Warfarin occurs in
the context of apparently healthy arterial cells and is not
initiated by local cell necrosis.
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To establish the dependence of artery calcification on the dose of
vitamin D, rats were treated with different doses of vitamin D at 0,
24, and 48 hours and treated every 12 hours with Warfarin or vehicle
beginning with the first vitamin D injection. As shown in Figure 5, the level of phosphate in the acid
demineralization extract of the aorta and carotid artery increased with
increasing vitamin D dose, and Warfarin treatment markedly enhanced the
extent of phosphate accumulation at each vitamin D dose. As also shown
in Figure 5, Warfarin treatment did not cause a significant
accumulation of phosphate in acid demineralization extracts of the
artery in the absence of concurrent vitamin D treatment (see results in
Figure 5 for the zero dose of vitamin D); this result is
consistent with previous studies that showed that treatment
with Warfarin alone did not cause detectable artery calcification until
2 weeks of treatment.16 Calcium levels were also
determined in each acid demineralization extract, and the effect of
vitamin D dose on calcium accumulation paralleled the effect on
phosphate accumulation, with an average calcium-to-phosphate ratio in
the acid extracts of 1.46 to 1.50:1 (data not shown).
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The effect of vitamin D and Warfarin treatment on the
accumulation of MGP in the artery was determined by measurement of MGP
levels in each acid extract by radioimmunoassay. As shown in Figure 6, MGP levels increased with vitamin D
dose, and the MGP levels in rats treated with vitamin D plus Warfarin
were markedly higher than those in rats treated with the same dose of
vitamin D alone. The accumulation of MGP in the artery exactly
paralleled the extent of accumulation of phosphate and calcium, and
the ratio of MGP level to the amount of calcium phosphate mineral in
artery extracts was the same at different levels of total calcification
in animals within the Warfarin plus vitamin D group and in animals
within the vitamin D only group. The ratio for the Warfarin-treated
animals was, however, 50% lower than that in the vitamin D–only
group; this result is consistent with previous report that
Warfarin also reduces MGP accumulation in bone by 50%.16
To determine the effect of Warfarin treatment on the -carboxylation
of MGP, it was purified from the acid extract of aortas from rats
treated with 300 000 IU/kg of vitamin D at 0, 24, and 48 hours and
every 12 hours with Warfarin, beginning with the first vitamin D
injection. N-Terminal protein sequencing was then used to
measure the degree of -carboxylation at residue 2 in the protein.
The MGP that accumulated in the calcified aorta of rats treated with
vitamin D plus Warfarin was less than 5% carboxylated at residue 2,
compared with >96% carboxylation at residue 2 for MGP isolated from
normal rat bone. This result demonstrates that Warfarin treatment does
inhibit -carboxylation of MGP, which accumulates in arteries with
calcification, and that the accumulation of MGP in these arteries must
therefore not be dependent on the -carboxylation status of the
protein.
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The vitamin D doses that induce artery calcification substantially elevate serum calcium levels compared with the 11.5 mg/dL level in control rats, with a 29% increase at 100 000 IU/kg and a 40% increase at 200 000, 300 000, and 500 000 IU/kg (data not shown). Concurrent treatment with Warfarin did not change serum calcium levels compared with those in rats treated with vitamin D alone. Serum phosphate levels were not significantly changed by any dose of vitamin D or by concurrent treatment with Warfarin (data not shown). These results indicate that elevated serum calcium levels could contribute to the calcification of arteries in rats treated with vitamin D alone, but that the synergistic effect of Warfarin on artery calcification cannot be explained by an effect of Warfarin on serum calcium or phosphate levels.
Time Course of Artery Calcification in Rats Treated With Vitamin D
and Warfarin
To determine the time course of artery calcification, rats were
treated with the 300 000 IU/kg dose of vitamin D at 0, 24, and 48
hours together with Warfarin or vehicle for a total treatment interval
of 48, 72, or 96 hours. As shown in Figure 7, the total level of mineral phosphate
in the acid extract of the aortas was increased markedly at 96 hours in
rats treated with Warfarin plus vitamin D compared with rats treated
with vitamin D alone. Similar results were obtained for calcium levels
in the acid extracts, and the calcium-to-phosphate molar ratios in the
acid extract were 1.50:1. As noted above (Figure 5),
treatment for 96 hours with Warfarin alone did not cause a significant
accumulation of calcium or phosphate in the artery. Serum calcium
levels were significantly elevated by vitamin D treatment at 48, 72,
and 96 hours (Figure 8), and concurrent
treatment with Warfarin again did not affect serum calcium levels
compared with rats treated with vitamin D alone.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Warfarin-Induced Artery Calcification Is Accelerated by Growth
We previously examined the effect of Warfarin on artery calcification in 42-day-old rats and found that Warfarin caused dramatic, focal calcification of the artery media within 2 weeks of treatment.16 The present studies demonstrate that 20-day-old rats are even more sensitive to Warfarin-induced artery calcification and that 10-month-old rats are completely resistant. One possible explanation for the resistance of the older rat to Warfarin-induced artery calcification could be differences in the sensitivity of the older animal to Warfarin. This drug is cleared rapidly in the rat, with a half-time of 3 to 5 hours in young animals.44 45 The rapid clearance of Warfarin in fact necessitated the use of an every-12-hour injection schedule to induce artery calcification in the earlier studies of 42-day-old rats.16 Even small changes in Warfarin metabolism with age could affect the persistence of the drug over the 12 hour injection cycle and so compromise the effect of Warfarin in the older animal.
In the present investigations, we used diet restriction to induce
reversible growth arrest in young rats and so dissociate the effects of
growth and age on the susceptibility of arteries to Warfarin-induced
calcification. These studies clearly demonstrate that it is rapid
growth itself that determines susceptibility to Warfarin-induced
calcification, not the age of the animal per se. This observation makes
it unlikely that the resistance of older rats to Warfarin-induced
artery calcification can be explained by age-related changes in
Warfarin metabolism or by other age-related changes in the
sensitivity of MGP -carboxylation to this dose of Warfarin.
Although there are no previous in vivo studies on the effects of age and growth on the extent of calcification in arteries and other soft tissues, the effect of recipient age on the extent of implant calcification has been investigated in 2 earlier studies. Calcification of porcine valves in humans has been modeled by implanting porcine valves at subcutaneous sites in the rat, and in the course of such studies, it was found that implant calcification is more rapid in young animals than in old.46 Aortic valves are composed largely of elastin and are therefore similar in composition to the artery media; thus, they may be susceptible to the same calcification-initiation mechanisms as in the rat. In other studies, alkaline phosphatase–impregnated collagen particles were implanted subcutaneously into rats of different age. Implant calcification was higher in young rats than in old, and the extent of implant calcification was closely correlated with serum phosphate levels.47
It is unclear how growth increases susceptibility to artery
calcification in the Warfarin-treated rat. One possibility is that a
growing skeleton sheds mineral nuclei, which lodge in the weblike
structure of the elastic lamellae of the arterial walls and
that, in the absence of avid neutralization of these nuclei by active
MGP, mineralization spreads rapidly through the lamellae. Another
possibility is that growth acts to promote artery calcification
indirectly, through its effect on serum levels of calcium and
phosphate. This possibility is supported by the association between
serum phosphate and the tendency of Warfarin treatment to induce artery
calcification, with the lowest serum phosphate levels in the 2
instances of resistance to Warfarin-induced artery calcification, the
10-month-old rat and the calorically restricted rat (Tables 1 and 2). In humans, the importance of serum calcium and phosphate
levels as possible determinants of susceptibility to artery
calcification is supported by the association between marked elevations
in serum phosphate and extensive artery and soft tissue calcification
in renal disease48 49 50 and the association between
hypercalcemia and artery and soft tissue calcification in other
diseases.51 Finally, the relationship between growth and
Warfarin-induced artery calcification could reflect an effect of growth
on other factors in the artery that regulate the calcification process,
such as altered expression of growth factors in the
arterial wall. We cannot presently rule out any of
these possible mechanisms as contributors to the effect of growth on
Warfarin-induced artery calcification, and studies are now in progress
to further understand the physiological basis for
the effect of growth status on susceptibility to artery
calcification.
Warfarin and Vitamin D Act Synergistically to Rapidly Calcify
Arteries
The present studies demonstrate that a Warfarin dose regime
that causes focal artery calcification in rats after 2 weeks of
treatment, but not at 1, will increase the calcification of arteries in
rats treated with high doses of vitamin D in as little as 3 to 4 days
compared with the calcification of arteries seen in rats treated with
vitamin D alone. The major difference between the pattern of artery
calcification in rats treated with Warfarin plus vitamin D compared
with animals treated with the same dose of vitamin D alone, as depicted
in Figure 4, is the continuous nature of elastic lamellae
calcification in the arteries of the Warfarin plus vitamin D group
compared with a more localized calcification in rats treated with
vitamin D alone. This pattern suggests that Warfarin treatment has
allowed the growth of a greater number of mineralization nuclei within
the elastic lamellae. It is noteworthy that the earliest stages of
arterial medial calcification in this model often spare the
regions between the elastic lamellae, regions occupied by the vascular
smooth muscle cells. While we were unable to examine artery
calcification in rats treated with Warfarin plus vitamin D at periods
of 1 week or longer owing to the lethal nature of the treatment (Figure 3), we examined the longer-term outcome of artery calcification
in rats treated with the 300 000-IU dose of vitamin D alone. By 2
weeks, rats treated with this dose of vitamin D at 0, 24, and 48 hours
had solid calcification of all regions of the arterial
wall, and the wall itself was rigid (not shown). This result indicates
that even in the vitamin K–replete rat, the calcification initiated by
the first 3 days of vitamin D treatment progressed without restraint
until the entire limits of the artery media had been engulfed by
mineral. The critical Warfarin-sensitive mechanism for inhibiting
calcification therefore acts early in the artery calcification process
and may be largely ineffective once the calcification has reached a
massive threshold.
Artery calcification in the vitamin D–treated rat is accompanied
by a dramatic accumulation of MGP, and the amount of MGP in the artery
is directly proportional to the amount of mineral. MGP also accumulates
in proportion to mineral in rats treated with vitamin D plus Warfarin,
although that amount of MGP per mg calcium or phosphate is about half
as great. A comparable accumulation of MGP per unit mineral was
previously found in the slower calcification of arteries induced by
this dose of Warfarin alone.16 Because of the more massive
artery calcification in the Warfarin plus vitamin D–treated rat, we
were able to isolate sufficient MGP in the current studies to establish
that the protein is indeed not -carboxylated. We can therefore
conclude that -carboxylation of MGP is not required for its
accumulation at sites of calcification in the arterial wall
and that the defect in the activity of MGP as a calcification
inhibitor, which accounts for the acceleration of
calcification in the Warfarin plus vitamin D–treated rat, must involve
the detailed nature of the complex formed between the protein and the
mineral and not the simple accumulation of the protein on the mineral
surface alone.
The vitamin D doses that cause artery calcification are also those that cause an elevation in serum calcium levels, and the time course of serum calcium elevation is correlated with the onset of artery calcification. A comparable hypercalcemia has been seen in earlier studies of rats treated with high doses of vitamin D.52 The close parallel between hypercalcemia and artery calcification seen in the current study suggests that artery calcification in the vitamin D–treated rat may be a simple physical chemical consequence of hypercalcemia. This hypothesis is supported by the fact that hypercalcemia has been previously associated with artery calcification in humans and other animals.51 It should be noted that the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3, has been shown to stimulate the expression of MGP in osteoblastic cells in culture.5 53 This effect has not be observed in vascular smooth muscle cells and fibroblasts (P.A.P. et al, unpublished observations, 1992), and there is no evidence of increased MGP expression in the artery during the first 24 hours of vitamin D treatment before the onset of calcification. It therefore seems unlikely that vitamin D affects artery calcification through its ability to directly regulate MGP expression by vascular cells.
The Warfarin plus vitamin D treatment model described here has
advantages for future investigations of MGP function in the inhibition
of artery calcification. Because in this model Warfarin accelerates
calcification in a system wherein calcification occurs even in the
absence of Warfarin treatment, it should be possible to examine the
difference in the nature of the MGP interaction with mineral in a
situation in which MGP is -carboxylated and mineralization is
inhibited and in a situation in which MGP is not -carboxylated and
mineralization is accelerated. A second advantage of the Warfarin plus
vitamin D model is the rapid onset of calcification, since it should be
possible to evaluate the ability of a single MGP injection to arrest
early stages of artery calcification in this system.
The major conclusion of these studies is that Warfarin-induced artery calcification is accelerated by growth and by vitamin D. There are at least 2 reasonable hypotheses to account for these observations: (1) Warfarin-induced artery calcification could be promoted by increases in serum calcium or phosphate. This hypothesis is supported by the high levels of serum phosphate in growing rats and the high levels of serum calcium in vitamin D–treated rats. (2) Artery calcification could be promoted by metabolic processes that are activated by growth and by vitamin D. For example, growth and vitamin D both increase bone metabolism, and a bone metabolic process could release mineral nuclei into serum. These nuclei could subsequently lodge in the elastic lamellae of the artery media, where they grow rapidly because of the Warfarin-induced inactivation of the calcification inhibitory activity of MGP. Studies are in progress to establish which of these 2 hypotheses best accounts for the effects of growth and vitamin D on artery calcification.
|
Acknowledgments |
|---|
This work was supported in part by US Public Health Services grant HL 58090 (to P.A.P.). The authors wish to thank Amanda Wallace and Jeffrey Caputo for help with animal treatments.
Received February 9, 1999; accepted September 13, 1999.
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 T Cukierman, E Elinav, M Korem, and T Chajek-Shaul Low dose warfarin treatment for calcinosis in patients with systemic sclerosis Ann Rheum Dis, October 1, 2004; 63(10): 1341 - 1343. [Abstract] [Full Text] [PDF]
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 T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification Endocr. Rev., August 1, 2004; 25(4): 629 - 672. [Abstract] [Full Text] [PDF]
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 N. Wajih, D. C. Sane, S. M. Hutson, and R. Wallin The Inhibitory Effect of Calumenin on the Vitamin K-dependent {gamma}-Carboxylation System: CHARACTERIZATION OF THE SYSTEM IN NORMAL AND WARFARIN-RESISTANT RATS J. Biol. Chem., June 11, 2004; 279(24): 25276 - 25283. [Abstract] [Full Text] [PDF]
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R. Vattikuti and D. A. Towler Osteogenic regulation of vascular calcification: an early perspective Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E686 - E696. [Abstract] [Full Text] [PDF]
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P. A. Price, H. H. June, N. J. Hamlin, and M. K. Williamson Evidence for a Serum Factor That Initiates the Re-calcification of Demineralized Bone J. Biol. Chem., April 30, 2004; 279(18): 19169 - 19180. [Abstract] [Full Text] [PDF]
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 T. M. Doherty, L. A. Fitzpatrick, A. Shaheen, T. B. Rajavashisth, and R. C. Detrano Genetic Determinants of Arterial Calcification Associated With Atherosclerosis Mayo Clin. Proc., February 1, 2004; 79(2): 197 - 210. [Abstract] [PDF]
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P. A. Price, M. K. Williamson, T. M. T. Nguyen, and T. N. Than Serum Levels of the Fetuin-Mineral Complex Correlate with Artery Calcification in the Rat J. Biol. Chem., January 16, 2004; 279(3): 1594 - 1600. [Abstract] [Full Text] [PDF]
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 P. Mathieu, J. C. Roussel, F. Dagenais, and I. Anegon Cartilaginous metaplasia and calcification in aortic allograft is associated with transforming growth factor {beta}1 expression J. Thorac. Cardiovasc. Surg., November 1, 2003; 126(5): 1449 - 1454. [Abstract] [Full Text] [PDF]
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L. L. Demer and Y. Tintut Mineral Exploration: Search for the Mechanism of Vascular Calcification and Beyond: The 2003 Jeffrey M. Hoeg Award Lecture Arterioscler Thromb Vasc Biol, October 1, 2003; 23(10): 1739 - 1743. [Abstract] [Full Text] [PDF]
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 T. M. Doherty, K. Asotra, L. A. Fitzpatrick, J.-H. Qiao, D. J. Wilkin, R. C. Detrano, C. R. Dunstan, P. K. Shah, and T. B. Rajavashisth Calcification in atherosclerosis: Bone biology and chronic inflammation at the arterial crossroads PNAS, September 30, 2003; 100(20): 11201 - 11206. [Abstract] [Full Text] [PDF]
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 K. A. Gilbert and S. R. Rannels Glucocorticoid effects on vitamin K-dependent carboxylase activity and matrix Gla protein expression in rat lung Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L569 - L577. [Abstract] [Full Text] [PDF]
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P. A. Price and J. E. Lim The Inhibition of Calcium Phosphate Precipitation by Fetuin Is Accompanied by the Formation of a Fetuin-Mineral Complex J. Biol. Chem., June 6, 2003; 278(24): 22144 - 22152. [Abstract] [Full Text] [PDF]
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A. Heiss, A. DuChesne, B. Denecke, J. Grotzinger, K. Yamamoto, T. Renne, and W. Jahnen-Dechent Structural Basis of Calcification Inhibition by alpha 2-HS Glycoprotein/Fetuin-A. FORMATION OF COLLOIDAL CALCIPROTEIN PARTICLES J. Biol. Chem., April 4, 2003; 278(15): 13333 - 13341. [Abstract] [Full Text] [PDF]
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 P. A. Price, H. H. June, J. R. Buckley, and M. K. Williamson SB 242784, a Selective Inhibitor of the Osteoclastic V-H+-ATPase, Inhibits Arterial Calcification in the Rat Circ. Res., September 20, 2002; 91(6): 547 - 552. [Abstract] [Full Text] [PDF]
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U. Querfeld Is atherosclerosis accelerated in young patients with end-stage renal disease? The contribution of paediatric nephrology Nephrol. Dial. Transplant., May 1, 2002; 17(5): 719 - 722. [Full Text] [PDF]
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A. Jara, C. Chacon, and A. J. Felsenfeld How dietary phosphate, renal failure and calcitriol administration affect the serum calcium-phosphate relationship in the rat Nephrol. Dial. Transplant., May 1, 2002; 17(5): 765 - 771. [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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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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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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