The Bone—Vasculature Axis: Calcium Supplementation and the Role of Vitamin K

Abstract

Calcium supplements are broadly prescribed to treat osteoporosis either as monotherapy or together with vitamin D to enhance calcium absorption. It is still unclear whether calcium supplementation significantly contributes to the reduction of bone fragility and fracture risk. Data suggest that supplementing post-menopausal women with high doses of calcium has a detrimental impact on cardiovascular morbidity and mortality. Chronic kidney disease (CKD) patients are prone to vascular calcification in part due to impaired phosphate excretion. Calcium-based phosphate binders further increase risk of vascular calcification progression. In both bone and vascular tissue, vitamin K-dependent processes play an important role in calcium homeostasis and it is tempting to speculate that vitamin K supplementation might protect from the potentially untoward effects of calcium supplementation. This review provides an update on current literature on calcium supplementation among post-menopausal women and CKD patients and discusses underlying molecular mechanisms of vascular calcification. We propose therapeutic strategies with vitamin K2 treatment to prevent or hold progression of vascular calcification as a consequence of excessive calcium intake.

Full text

REVIEW
published: 05 February 2019
doi: 10.3389/fcvm.2019.00006
Frontiers in Cardiovascular Medicine | www.frontiersin.org 1February 2019 | Volume 6 | Article 6
Edited by:
Dwight A. Towler,
University of Texas Southwestern
Medical Center, United States
Reviewed by:
Yabing Chen,
University of Alabama at Birmingham,
United States
Joshua D. Hutcheson,
Florida International University,
United States
Sasha Anna Singh,
Harvard Medical School,
United States
Alexander N. Kapustin,
AstraZeneca, United Kingdom
*Correspondence:
Leon J. Schurgers
l.schurgers@maastrichtuniversity.nl
Specialty section:
This article was submitted to
Atherosclerosis and Vascular
Medicine,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 21 September 2018
Accepted: 14 January 2019
Published: 05 February 2019
Citation:
Wasilewski GB, Vervloet MG and
Schurgers LJ (2019) The
Bone—Vasculature Axis: Calcium
Supplementation and the Role of
Vitamin K.
Front. Cardiovasc. Med. 6:6.
doi: 10.3389/fcvm.2019.00006
The Bone—Vasculature Axis:
Calcium Supplementation and the
Role of Vitamin K
Grzegorz B. Wasilewski 1,2, Marc G. Vervloet 3and Leon J. Schurgers 1
*
1Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands,
2Nattopharma ASA, Hovik, Norway, 3Department of Nephrology and Amsterdam Cardiovascular Sciences, Amsterdam
University Medical Centers, Amsterdam, Netherlands
Calcium supplements are broadly prescribed to treat osteoporosis either as monotherapy
or together with vitamin D to enhance calcium absorption. It is still unclear whether
calcium supplementation significantly contributes to the reduction of bone fragility and
fracture risk. Data suggest that supplementing post-menopausal women with high
doses of calcium has a detrimental impact on cardiovascular morbidity and mortality.
Chronic kidney disease (CKD) patients are prone to vascular calcification in part due
to impaired phosphate excretion. Calcium-based phosphate binders further increase
risk of vascular calcification progression. In both bone and vascular tissue, vitamin
K-dependent processes play an important role in calcium homeostasis and it is tempting
to speculate that vitamin K supplementation might protect from the potentially untoward
effects of calcium supplementation. This review provides an update on current literature
on calcium supplementation among post-menopausal women and CKD patients and
discusses underlying molecular mechanisms of vascular calcification. We propose
therapeutic strategies with vitamin K2 treatment to prevent or hold progression of
vascular calcification as a consequence of excessive calcium intake.
Keywords: calcium paradox, vitamin K, vascular calcification, calcium supplements, bone loss
INTRODUCTION
Calcium is an abundant element in nature and is a major component of sedimentary rock
that covers 75 to 80% of the Earth’s surface. Calcium is also widely abundant in the human
body, primarily in bone, and teeth. Calcium salts are occasionally found outside bone in a
variety of tissues; this is broadly termed as extra-skeletal calcification. In these extra-skeletal sites,
calcium exists in multiple forms, including amorphous calcium phosphate, hydroxyapatite, and
magnesium whitlockite. A remarkable observation is that under several pathological conditions,
as will be discussed, calcium mineral content of bone declines, while it is increasing on
these extra-osseous sites. This has been termed the “calcium paradox” and was introduced to
describe the paradoxical correlation between lower bone calcium content with parallel increased
vascular calcium content (1). The calcium paradox refers to epidemiological data reporting that
postmenopausal women experience bone loss, yet simultaneously screen positive for vascular
calcification. This phenomenon is common in osteoporotic women and patients suffering from
chronic kidney disease (CKD). Prevalence and morbidity of both cardiovascular disease and
osteoporosis are increasing in the global population. Such observations have been noted in several
studies, where a correlation of low bone mineral density (BMD) was associated with increased
cardiovascular mortality (2–6).

Wasilewski et al. Calcium Supplements and Vascular Calcification
The use of calcium supplements has been widely advised
due to their assumed ability to support bone health and BMD
(7,8). Calcium is an essential element for bone growth during
childhood (9), as well as in preserving bone mineral density
during adolescence (10). However, a systematic review and
meta-analysis of the effects of calcium supplementation along
with vitamin D treatment showed that this treatment was not
associated with a lower incidence of fracture risk in adults,
questioning whether calcium supplementation contributes to
the maintenance of healthy bone (11). In turn, recent data
suggest that calcium supplements increase prevalence of
myocardial infarction (12), and may increase risk of coronary
artery calcification (CAC) (13). Moreover, higher doses of
calcium from supplements than calcium obtained from dietary
intake might promote cardiovascular calcification (14). Thus,
despite the relative benefit of calcium supplementation for
bone, calcium supplements became controversial because of
a possibly increased cardiovascular risk. Substantially different
from calcium from dietary sources, calcium form supplements
induce an acute rise in serum calcium levels that highly oscillates
in blood for up to 6 h (15,16). The plasma calcium concentration
is tightly regulated by vitamin D, parathyroid hormone (PTH),
and calcitonin (17,18).
Vitamin K-dependent proteins (VKDP) also play an
important role regulating mineralization both in bone and
the vasculature. Osteocalcin (OC) is produced exclusively
by osteoblasts and supports the binding of calcium to the
bone mineral matrix, whereas matrix Gla-protein (MGP) is
synthesized by vascular smooth muscle cells and chondrocytes
to prevent ectopic calcification. While hepatically produced
coagulation factors are the prototypical VKDP, the extra-hepatic
VKDP also unequivocally need vitamin K as cofactor to become
biologically active. Related to that, vitamin K2 has been shown
to prevent bone loss and strength and prevents stiffening of
arteries (19,20). Western diet does not provide sufficient vitamin
K to activate all OC and MGP that is produced (21,22). Also
in CKD patients, vitamin K deficiency is prevalent, so K2
supplementation has been suggested as treatment option to
attenuate vascular calcification (23,24).
In this review we provide the latest insights of the calcium
paradox and the potential of using vitamin K to support both
bone and vascular health.
BONE METABOLISM
Calcification generally is a physiological process, necessary
to build bone and dentin. Bone provides structural support,
strength, necessary for locomotion, and protection from the
environment. The balance in bone formation and bone
resorption is crucial for optimal bone health. A disturbed balance
of this process results in bone loss and is termed osteoporosis.
During childhood bone is formed and bone peak mass is achieved
during young adulthood, after which bone mass gradually
declines. Bone loss is the consequence of bone resorption
outbalancing bone formation (25). This is accompanied by
bone architectural changes including trabecular bone becoming
thinner, less abundant, and osteoclastic perforation of cortical
bone (26).
Bone Formation
The skeleton is systematically renewed in the process of bone
remodeling to maintain strength and rigidity. Bone remodeling
can be considered to be part of calcium homeostasis system
and enables the skeleton to adapt to changes. Bones adapt their
structure depending on their function, mechanical strain and
need for stability during development. It is mediated on the
surface of cortical and trabecular bone, and at anatomically
different sites named basic multicellular subunits (27).
Two pathways of bone formation exist, together termed
osteogenesis. The first is known as endochondral ossification and
involves a differentiation of mesenchymal cells into chondrocytes
or osteoblasts (28,29). As chondrocytes mature, they expand in
size and become hypertrophic and eventually undergo apoptosis,
secreting vesicles that initiate mineralization of extracellular
matrix (30). As they die, with vascular evasion and matrix
remodeling (osteoclast mediated), the calcified cartilage is
subsequently replaced by bone. Nestin-positive mesenchymal
progenitors associated with the invading vasculature differentiate
into bone-forming osteoblasts and deposit a type I collagen-
based bone matrix on the degraded cartilage template (31),
(32). The second process of bone formation is intramembranous
ossification. First, mesenchymal cells directly differentiate into
osteoblasts, which are bone-forming cells. Next, type I collagen
matrix is deposited by these cells, that can bind calcium
salts, which form hydroxyapatite crystals. This mineralization
of the matrix underlies the strength and compactness of
the bone. With time, osteoblasts eventually become trapped
in calcified extracellular matrix and transdifferentiate into
osteocytes. Osteoblasts are the only bone cell type releasing
the vitamin K-dependent protein OC (discussed below). As the
newly formed bone is laid, its deposition must be tightly regulated
to maintain homeostasis. This balance is achieved by bone-
resorbing cells, entering the blood vessels of bone, which are
termed osteoclasts and are of macrophage origin. Each osteoclast
is able to secrete hydrogen ions, thereby acidifying the bone
surface dissolving mineralized matrix, followed by interactions
that enhance the action of osteoblasts (33–35). Upon resorption,
bone-matrix embedded osteocalcin is released contributing to its
circulating levels (36).
Bone Loss
Bone loss is most typical in women after reaching the
age of 50 years following menopause. The pattern of sex
hormonal secretion drastically changes after the menopause,
resulting in disbalance in bone turnover markers, making
postmenopausal women susceptible to osteoporosis and
fractures. Remarkably cardiovascular diseases are also more
prevalent in postmenopausal women. Therefore, it is important
to understand the molecular mechanisms by which hormonal
changes leads to both osteoporosis and cardiovascular disease
(37,38). The post-menopausal period is accompanied by
substantial reduction of estrogen levels leading to bone
resorption, yet simultaneously reducing calcium absorption
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Wasilewski et al. Calcium Supplements and Vascular Calcification
(39). It is not the aim of this review to elaborate on the effect
of estrogen on the vasculature [reviewed elsewhere (39)].
Instead, we will focus on specific pathways involved in calcium
metabolism.
PTH is released upon hypocalcemia, indirectly stimulating
release of calcium from bone. In CKD, autonomous production
of PTH may occur. Additionally, PTH promotes reabsorption
of ultra-filtered calcium in distal tubules and activates vitamin
D thereby increasing circulating calcium levels by raising
gastrointestinal uptake of calcium (17,18). Calcium-sensing
receptors (CaR) present on the surface of parathyroid glands
enable sensing of circulating calcium concentration (40),
contributing to calcium modulation.
Vitamin D is a fat-soluble vitamin that can be obtained
from diet, sun exposure, or supplements, and is metabolized
by a series of enzymatic reactions in the body producing
its active 1,25-dihydroxyvitamin D form (41,42). Vitamin D
(in inactive form) is often prescribed in combination with
calcium supplements. Active 1,25-dihydroxyvitamin D enhances
absorption of intestinal calcium and phosphate thus contributing
to the regulating of mineral balance (43,44). In the absence of
vitamin D, only 10–15% of intestinal calcium is absorbed, which
can be increased to 30–40% in the presence of active vitamin
D (45,46). Vitamin D was found to stimulate production of
vitamin K-dependent proteins, like osteocalcin (47). Osteocalcin
is a protein involved in bone mineralization [reviewed elsewhere
(48)]. Remarkably, inclusion of vitamin K in calcium and vitamin
D supplements improved BMD and ucOC when compared with
vitamin D and calcium alone (49).
CKD patients often experience deficiency of 1,25-
dihydroxyvitamin D as a consequence of lost kidney
mass and the effects of fibroblast growth factor 23 (50),
resulting in declined activity of 1-alpha hydroxylase (51–53).
Reduced serum levels of 1,25-dihydroxyvitamin D lead to
hypocalcemia on top of positive phosphate balance, both
stimulating PTH release and eventually leading to secondary
hyperparathyroidism.
VASCULAR CALCIFICATION
Vascular calcification is a pathological process, and has been
firmly established as a risk factor for cardiovascular events
and mortality (54,55). Vascular calcification is a process of
extraosseous mineral deposition in blood vessels, including large
arteries such as aorta, carotid arteries, iliac arteries, and cardiac
valves. Bone mineralization and vascular calcification share many
similarities, including expression of bone-related proteins in
the vasculature and secretion of extracellular vesicles (EVs)
both preceding the phase of calcification (56,57). Vascular
calcification can occur either in the tunica media or tunica
intima of the vessel wall. Medial calcification is also known
as Möckenberg’s sclerosis and involves vascular smooth muscle
cell (SMC) calcification in the absence of previous local lipid
accumulation, and inflammation. Medial calcification is related
to CKD, diabetes mellitus, and aging, and results in increased
arterial stiffness and risk of cardiovascular events (58,59). In
contrast, intimal calcification is associated with atherosclerotic
plaque formation and the amount of calcification is considered
to be a measure of atherosclerotic burden (1).
For many years vascular calcification was considered as
a clinically irrelevant process reliant of passive deposition
of calcium crystals, merely reflecting a passive feature
of disease and aging. Recent evidence however suggests
otherwise, and vascular calcification appears to be a highly
regulated process. SMCs release calcification inhibitors,
thus efficiently preventing spontaneous calcification in spite
of supersaturation of extracellular calcium and phosphate
levels (60).
Vascular Smooth Muscle Cell Phenotypic
Switching
SMCs are the main cellular component of the tunica media
in arterial vessels providing structural support and regulating
vascular tone and elasticity to alterations in pressure conditions.
In physiology SMCs possess a contractile phenotype and
express contractile-specific markers, including alpha-smooth
muscle actin, calponin, and SM22alpha, enabling them to
perform contraction of the vessel wall [reviewed elsewhere
(61,62)]. SMC function is associated with a high level of
phenotypic plasticity in order to perform a variety of functions
including production of extracellular matrix and repair (61,
63). Several factors have been implicated in regulating SMC
phenotype, including mineral imbalance (calcium, magnesium,
and phosphate-induced loss of calcification inhibitors and
presence of calcification promotors) (64). Downregulation
of contractile markers is a hallmark for SMC phenotypic
switching (65). It has been shown that phosphate can
induce an osteochondrogenic phenotypic switching of SMC,
as will be outlined in more detail below (61,66–69),
whereas elevated calcium levels shift the contractile phenotype
toward a synthetic SMC phenotype (57). Both calcium- and
phosphate- induced phenotypic switching are associated with
an increase in the secretion of calcifying extracellular vesicles
(56,57).
Elevated Phosphate Levels Promote
Osteochondrogenic Differentiation of
SMCs
CKD patients often develop medial calcification (70). In CKD,
a strong correlation between serum phosphate levels and
vascular calcification is present (71,72). In an animal model
of CKD, arterial calcification developed after feeding animals
a phosphorous-rich diet only (73). Initiation and progression
of calcification in CKD patients correlates with impaired
mineral metabolism represented by elevated serum level of
phosphate and/or calcium (74). Moreover, high circulating
phosphate levels have been linked to increased cardiovascular
morbidity even among young people receiving dialysis (75)
and in CKD patients (76). In vitro, elevated phosphate
levels result in upregulation of bone-like markers in SMC
including osterix, alkaline phosphatase (ALP), and Runx2, and
downregulation of SMC contractility markers (77). SMC cultured
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Wasilewski et al. Calcium Supplements and Vascular Calcification
in osteogenic cell culture media differentiate into calcifying-
SMC resembling osteoblasts (68). In aortic valves of patients
with aortic stenosis, valvular interstitial cells demonstrate
similarities with osteoblasts (78), which also exhibit lamellar
bone formation (79). Upon injury or in atherosclerosis, SMCs
induce the release of platelet-derived growth factor (PDGF)
similarly to platelets (80,81). SMC are known to express the
PDGF receptor subtypes and the level of expression is greatly
increased in connective tissue and in SMCs followed by PDGF
stimulation (82).
THE CALCIUM PARADOX
The paradoxical co-existence of declined calcium-mineral
content in bone, and parallel increased arterial calcification, as
a consequence of impaired calcium metabolism, is termed the
calcium paradox. This is most pronounced in post-menopausal
women and CKD patients. Many studies have consistently shown
a coexistence of osteoporosis in post-menopausal women and
increased calcification of either abdominal aorta and carotid
arteries (5,83–90). Such paradox of decreased bone mineral
density and vascular calcification has also been documented in
a population study of middle-aged men, suggesting it is not
unique to women (91), and pointing to a specific metabolic
abnormality. In patients with CKD disturbed calcium and
phosphate homeostasis is present and many studies consistently
reported bone abnormalities including decreased BMD and
fractures and coexistence of increased vascular calcification and
all-cause mortality (92–108).
Kidney Disease: Improving Global Outcomes (KDIGO)
guidelines recommend the term chronic kidney disease-mineral
bone disorder (CKD-MBD) to express this clinical syndrome
encompassing mineral (e.g., calcium), bone, and cardiovascular
calcification abnormalities that develop as a complication of
CKD (109). In addition to bone disease, patients with CKD
are also prone to vascular calcification, bone fragility and
fractures. It has been shown that patients on dialysis, which
is the end stage of CKD (CKD stage 5D), have an increased
risk of fractures (110,111) and vascular calcification (112), and
therefore the calcium paradox also exists in CKD patients. CKD
pathological characteristics include biochemical imbalances
leading to elevated levels of circulating phosphate (113–115). In
untreated patients, circulating calcium levels are decreased due
to vitamin D deficiency, whereas vitamin D supplementation
might be beneficial in improving biochemical endpoints in
CKD patients (116). Vitamin D is often used in combination
with calcium supplementation therapy. In patients on dialysis,
coronary artery calcification is prominent and contributes to high
mortality and morbidity. However, this use of both calcium and
vitamin D, while being possibly protective for bone disease, may
aggravate vascular calcification. Uraemia-related cardiovascular
risk factors, including hyperphosphatemia and elevated Ca x P
product, correlate with quicker onset of vascular calcification
(117). Circumventing this calcium paradox may be accomplished
by VKDP (118,119), as will be outlined below.
AGENTS THAT ALTER TISSUE
MINERALIZATION
In the following sections we will discuss treatments known to
influence bone and vascular mineralization, and how they might
impact calcium metabolism.
Calcium Supplements
Calcium is important for optimal bone health throughout life.
Although dietary intake of calcium may suffice to meet the
recommended daily intake, calcium supplements may be an
option if diet falls short. dose Globally, recommendations for
daily calcium intake vary. The Institute of Medicine (IOM)
recommends a daily intake of 1,000 mg/day for men aged 19–
70 years and women 19–50 years old, and 1,200 mg/day for older
individuals (92) whereas National Osteoporosis Society suggests
an intake between 800–1,000mg a day (120). While calcium
intake comes from dietary sources such as dairy products, certain
vegetables, and fortified foods, many people do not achieve the
recommended intake from diet alone. It is estimated that ∼35%
of the adult U.S. population uses calcium supplements (121).
Calcium plays a vital role in various physiological activities, such
as nerve conduction, muscle contraction, blood clotting, protein
folding, brain function, and regulated cell death (apoptosis) (122,
123). Such broad function of calcium in the body requires precise
regulation, and calcium oscillates between 2.15 and 2.60 mmol/L
for total plasma calcium in adults and between 1.17 and 1.33
mmol/L for plasma ionized calcium as free calcium represents
some 45% of total circulating calcium levels. This free form is the
regulated calcium and accounts for bone mineralization as well
as pathological calcification (124).
Calcium Forms, Absorption, and Effects
Several formulations of calcium are available on the market,
differing in bioavailability, and elemental calcium content.
Calcium carbonate is the most common form available. However,
many studies showed superiority of calcium citrate over calcium
carbonate, due to higher bioavailability and because it does not
require acidic stomach conditions before ingestion (102). In
a study carried out in post-menopausal women supplemented
with di-calcium phosphate over a period of 12 months, serum
calcium levels did not vary significantly, and only urinary calcium
increased progressively in time when compared to the control
group. The increased excretion of calcium may indirectly reflect
the rise of the renal threshold for excretion and together with the
amount of absorbed calcium it may contribute to complications
such as deposition in the vasculature (103).
One of the most applied therapeutic intervention for
fracture risk is calcium in the form of pills or organic
powder. Commercially available calcium is often marketed in
combination with vitamin D3 to increase intestinal absorption of
calcium (Table 1). It has been proposed that no more than 500 mg
of elemental calcium should be taken as single dose to maximize
absorption and to avoid side effects, like gastrointestinal
complaints (94). When calcium supplements are not exceeding
the nutritional daily intake of 800 mg, a low cardiovascular risk
was observed (104). Clinical guidelines consider a cumulative
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Wasilewski et al. Calcium Supplements and Vascular Calcification
TABLE 1 | Comparison of calcium salts frequently used in calcium supplements.
Calcium
salt
Elemental
calcium % (w/v)
Bioavailability Advantages/disadvantages
Carbonate 40 High (comparable with citrate) Requires acidic stomach conditions before absorption, might
cause acidic rebound, cheap provides greatest amount of
elemental calcium
Tricalcium
phosphate
38 Moderate (found lower absorption than citrate when used in
fortified juice)
High calcium content
Citrate 21 High (higher than lactate/tricalcium phosphate) Not dependent on stomach acidity, many tablets needed
Gluconate 9 High (comparable with calcium carbonate) Many tablets needed
Lactate 13 High (comparable with calcium carbonate) Many tablets needed
Acetate 25 High (scarce information on human subjects) Inexpensive, wide range of intestine pH absorption
Chloride 27 High (intravenous injection for treatment of hypocalcemia) Not commonly prescribed low amount of elemental calcium
References (93,94,125) (11,33–39,41)
Salts are listed according to elemental calcium content which does not necessarily reflect on bioavailability. Absorption is also influenced by stomach acid due to the salt structure e.g.,
calcium carbonate is basic and needs hydrochloric acid in stomach to produce calcium chloride which is further absorbed.
calcium intake from foods and supplements that does not
exceed 2,000 to 2,500 mg/d, as defined by National Academy of
Medicine, as safe for cardiovascular disease outcome (105,106).
Numerous studies and extensive meta-analyses reported on
the efficacy and cost effectiveness of calcium supplementation
(with or without vitamin D), in improving bone mineral
density as well as decreasing fracture risk (83–85,107,108).
Furthermore, in individuals with inadequate calcium intake,
the supplementation plan seems to be beneficial in reducing
fragility fractures especially in osteoporotic women (86,107).
Calcium supplementation was also demonstrated to be effective
in preventing reduction in bone loss and turnover in healthy
population (87). A recent double-blind controlled trial also
proved the effectiveness of medium and high calcium intake in
maximizing bone mineral density in adolescent girls (88). In
addition, many studies described neutral or protective effects
of calcium rich foods on cardiovascular outcomes including
atherosclerosis, risk of infarction, stroke, and cardiovascular
mortality (89,90,126–130).
However, recent data challenge the assumption that calcium
supplementation improves bone mineral density. A meta-
analysis on the correlation between calcium supplementation
alone or with vitamin D and bone mineral density in people over
50 years of age demonstrated little beneficial effect (1–2%) in the
first year with nearly no further benefits after 1 year on bone
mineral density (8). With such low effects it would be challenging
to implement calcium supplementation into standard treatment
for reduction of fracture risk in the healthy elderly population
(131). A recent review summarizing the use and efficacy of
calcium supplementation in treating osteoporosis and fracture
risk questions the use of calcium supplements because of the
weak beneficiary effect on fracture risk while increasing the risk
on gastrointestinal problems, kidney stones, and cardiovascular
risk (132).
Despite positive outcomes of calcium supplementation, a risk
for cardiovascular risk events may exist in specific population.
It was recently shown that women who receive calcium
supplementation were at higher risk for increased vascular
morbidity and mortality, including myocardial infarction
(108,133–139). In turn, recent systematic reviews and meta
analyses do not confirm that supplementing calcium (with or
without vitamin D) increased prevalence of coronary heart
disease, cardiovascular mortality or all-cause mortality, data on
which the above-mentioned statement by the National Academy
of Medicine is based upon (105,131). Rapidly elevated transient
calcium levels in blood caused by excessive supplementary
calcium have been suggested to promote coagulation when
compared with placebo in postmenopausal women, likely
due to interaction with platelets expressing calcium-sensing
receptor (CaSR) (140,141). Hypercoagulability is considered to
have a reinforcing effect on atherosclerosis in animal studies,
contributing to cardiovascular disease. Also many coagulation
proteins have been described in human atherosclerotic plaques
(142). These findings are in line with the association between
high calcium intake and cardiovascular calcification in CKD
patients (143). Reconciling these sometimes opposing details
difficult. There appears to be some protection from fracture risks
by calcium supplements, but its safety is still not sufficiently
established. Therefore, additional research is still needed.
Calcium-based phosphate binders have been used extensively
as a first-choice option since 1970 to alleviate hyperphosphatemia
associated with CKD patients due to its low cost, availability,
and effectiveness. These calcium-containing phosphate binders
are given to CKD patients to complex dietary phosphate, thereby
reducing phosphate uptake (144,145).
As with most supplements, also calcium-containing
phosphate binders have side effects, which include abdominal
cramps, intestinal bloating, and diarrhea (146). Further, excessive
intake of calcium supplements might also result in milk-alkali
syndrome and hypercalcemia (92). In addition, also in patients
with CKD, the use of calcium-containing binders are associated
with progression of CKD, and the recently updated KDIGO
guideline suggest to restrict its use (109).
Vitamin K and Vitamin K-Dependent
Proteins
Vitamin K was discovered in 1929 by the Danish biochemist
Henrik Dam during his experiments on cholesterol metabolism
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Wasilewski et al. Calcium Supplements and Vascular Calcification
FIGURE 1 | Structural formulae of naturally occurring and biologically active Vitamin K–phylloquinone (K1) and menaquinones (K2-MK-4 and K2-MK-7). All vitamins
share common menadione ring (also known as vitamin K3).
in chickens. When fed low-fat diets, chickens experienced
prolonged clotting time and hemorrhage, which surprisingly
could not be rescued when diet was enriched with cholesterol.
Dam assumed a deficiency of a vitamin required for coagulation,
which he termed “Koagulation vitamin,” hence vitamin K (147).
Indeed, vitamin K was shown to be a fat-soluble vitamin,
consisting a group of structurally related compounds including
vitamin K1 (phylloquinone) and vitamin K2 (menaquinones)
(Figure 1). Vitamin K1 contains a phytyl chain, whereas K2 is
classified according to the length of isoprenoids and indicated
as MK-n, where n represents the number of residues. Both
vitamins share a common 2-methyl-1,4-naphthoquinone ring,
also known as menadione. The main source of vitamin K1
is green vegetables (148), whereas vitamin K2 can be found
in fermented foods such as soy beans, cheese, and sauerkraut.
The richest source of vitamin K2 (MK-7) is a Japanese dish
named Natto, which is produced from fermented soy beans with
aid of the Bacillus Subtilis bacteria strain (149). In addition
to nutritional consumption, gut bacteria Lactococcus (150)
and Escherischia coli (151) are able to synthesize long chain
menaquinones (Figure 1).
The primary biological function of both K-vitamins is being
an unequivocal cofactor in the post-translational modification of
VKDP via carboxylation of glutamic acid residues (Glu) to y-
carboxylated-glutamic acid residues (152). To fulfill this function,
vitamin K needs to be reduced to its active cofactor form (KH2)
by quinone reductases. The enzyme y-glutamylcarboxylase
(GGCX) oxidizes KH2 to vitamin K-epoxide (KO) (153).
Both vitamins K1 and K2 can partake in the activation of
VKDP; however, long-chain menaquinones, which are more
hydrophobic, have a higher bioavailability and longer half-life
and thus bioactivity (154,155).
VKDP are a group of proteins that require carboxylation
of specific protein-bound glutamate-residues, allowing them to
bind with high affinity to calcium. This was first demonstrated in
coagulation, showing that VKDP of the coagulation cascade need
carboxylation to acquire biological activity. This role of vitamin K
on coagulation is clinically widely applied by the use of warfarin
as anticoagulant treatment. The extra negative charge in VKDP
bind via calcium to negatively charged phospholipids to exert
their function. In the last three decades, extra-hepatic VKDP
have been discovered, including OC, MGP, and Gla-rich protein
(GRP; also termed Upper zone of growth plate and Cartilage
Matrix Associated protein, Ucma) (156). The function of non-
hepatic VKDP has recently be discovered and include prevention
of vascular calcification (157) and importantly also promotion
of bone metabolism (158). The current knowledge of vascular
calcification inhibitors has gained attention of both scientists and
clinicians to research their molecular action, aiming to alleviate
disease caused by vascular calcification.
Osteocalcin
OC is a major non-collagenous protein abundantly present in
bone, responsible for management of skeletal mineralization
(159,160). OC knock-out/null rodents undergo increased
bone mineralization, followed by an increase in trabecular
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FIGURE 2 | Vascular smooth muscle cells (VSCMC) and osteoblasts are able to synthesize Matrix-Gla-Protein (MGP) and Osteocalcin (OC), respectively. In the
presence of vitamin K both proteins are carboxylated (cMGP and cOC) preventing calcification of VSMC and promoting mineralization of Osteoblasts. Vitamin
K–dependent carboxylation mechanism keeps extracellular matrix of VSMC free of calcification and simultaneously promotes mineralization of osteoblast matrix. In
Chronic Kidney Disease patients, calcium serum levels are elevated further potentiating the calcification of SMCs. Similarly, in post-menopausal women, calcium
homeostasis is further impaired contributing to impairment of calcium utilization by osteoblasts. In the event of vitamin K deficiency, both MGP and Osteocalcin are not
carboxylated and cannot perform their molecular function.
thickness, density and bone volume (161–163). During skeletal
development, bone mass increases due to the dominant function
of osteoblasts which secrete OC, amongst other proteins,
enabling bone to grow. In addition to bone function, OC
is implicated in stimulating testosterone synthesis and insulin
release (164,165). Other roles of OC are not covered in
this review and have been reviewed elsewhere (166). To
execute its physiological function, OC needs to be activated by
carboxylation, catalyzed by vitamin K. Carboxylated OC (cOC)
has a high affinity for calcium ions and aids in forming a
hydroxyapatite lattice preceding mineralization of bone (167,
168) (Figure 2). Upon bone degradation, OC, incorporated
into mineralized bone, is liberated. Serum OC levels were
negatively correlated with bone mineral density (BMD) in post-
menopausal woman and healthy subjects (169–171). In a study
of healthy girls, plasma phylloquinone was inversely correlated
with circulating OC concentrations showing that a better vitamin
K status was associated with decreased bone turnover in healthy
girls (172).
Matrix Gla Protein
The discovery of MGP dates back to 1983 where it was first
purified from bovine bone matrix and named after the presence
of gamma-carboxyglutamate residues on MGP (173). Shortly
thereafter MGP was confirmed to be present in cartilage, lung
heart, kidney, and vasculature, with highest protein expression in
SMCs and chondrocytes (174–176). Knocking out MGP in mice
induced advanced medial calcification and subsequent vessel
rupture followed by death in the majority of mice within 6
weeks after birth. This animal model resembles the human Keutel
syndrome which is caused by a mutation in the MGP gene (177,
178), which impairs carboxylation of MGP thereby inducing
intimal and medial calcification (179). MGP is also dependent on
carboxylation of gla-residues, catalyzed by vitamin K, to execute
its function as an inhibitor of vascular calcification (Figure 2)
(180,181). Uncarboxylated MGP (ucMGP) is associated with
increased risk of vascular calcification, and therefore some
researchers advocate that vitamin K status in CKD patients
should be carefully monitored (182).
Another mode of action of MGP, besides being an inhibitor
of arterial calcification, is inhibition of bone morphogenic
protein2/4 (BMP2/4) (183,184). BMP2 was found to be present
in human atherosclerotic lesions (185), acting as downstream
signal for osteogenic phenotype switching of SMC by increasing
the influx of phosphate into cells (186). In MGP-deficient SMCs,
upregulation of osteogenic-specific proteins was notified (187)
and it can be speculated that MGP prevents osteogenic transition
of SMC by interacting with BMP-2 (188).
Gla Rich Protein
GRP, also known as Ucma, is a vitamin K-dependent protein
secreted by chondrocytes (189,190) and present in cartilage,
bone (191), and vasculature (192,193). Despite the creation
of GRP knockout mice its precise molecular action remains to
be elucidated, because these animals had no manifest deficits
in cartilage or bone development (194). So far, the role of
GRP has been implicated in calcium regulation in extracellular
matrix (156,192), and thus being an inhibitor of ectopic
calcification (192,193). Indeed, GRP inhibits calcification of
aortic tissue by promoting a contractile SMC phenotype via
increasing expression of α-smooth muscle actin (193). Moreover,
GRP was found to be directly associated with calcium-phosphate
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Wasilewski et al. Calcium Supplements and Vascular Calcification
TABLE 2 | Occurrence of selected vitamin K dependent proteins in different tissue
compartments.
Bone Vasculature Cartilage
MGP XXX
Gla rich protein
(UCMA)
XXX
Osteocalcin X? ?
Reference (173,191,198,199) (192,193,197) (189–191,200,201)
crystals suggesting that this protein-crystal interaction modulates
calcification (156). Also, in CKD stage 5D, GRP inhibits
EV and calcifying protein particles (CPP) induced vascular
calcification (195). In addition, GRP was found to promote
osteoblast (196) and chondrocyte differentiation (189,190). More
recently, it was shown that GRP inhibited phosphate-induced
SMC calcification via BMP-dependent signaling suggesting its
role in regulating osteochondrogenic differentiation of SMCs
(69). As mentioned above, MGP also inhibits calcification
via a BMP-dependent mechanisms (57,197) and this novel
function of GRP function via a BMP-dependent mechanism
suggests that both MGP and GRP deficiency contribute to
phosphate-induced vascular calcification and cardiovascular risk.
Table 2 summarizes vitamin-K dependent proteins involved in
calcification.
Phosphate Binders and Vitamin K
Despite many years of research there is no definite proof that
phosphate binders improve outcome, despite their capacity to
control phosphate. Although direct studies suggest superiority
of non-calcium containing binders over calcium containing
binders, it is still unclear if this is due to an advantage of
non-calcium containing binders or added risks from calcium
containing binders (143,202–204) Even more striking is that
the use of any phosphate binders in earlier CKD, despite
lowering phosphate, did not reduce progression of coronary
calcification (71). This conundrum may be explained by the
recently demonstrated ability of phosphate binders to also bind
vitamin K (Table 3). The advantage of lowering phosphate
concentrations if thus offset by aggravation vitamin K deficiency.
The lack of difference in this CKD patient subgroup could
be explained by effective inherent protection in these patients
or by simultaneous undesired binding of vitamin K by some
phosphate binders resulting in vitamin K deficiency which
serves as co-factor for enzymes that activate calcification
inhibitors (218,219) (Figure 3). More recently, it was shown
that CKD patients on dialysis treated with the phosphate
binder sevelamer revealed higher circulating levels of dp-
ucMGP, the inactive form of MGP (221). These findings support
the in vitro notion and hypothesis that phosphate binders
induce a vitamin K-deficiency. Besides the above-mentioned
phosphate binders, new forms have recently been developed
such as iron-based phosphate binders. Iron oxyhydroxide have
been proven to be as potent as sevelamer in decreasing
phosphatemia (222), while apparently not interfering with
vitamin K-metabolism (218).
Vitamin K to Escape the Calcium Paradox
As outlined, vitamin K has a role in healthy bone formation,
while at the same time it provides protection against ectopic
calcification, especially in the cardiovascular system. Therefore, it
is tempting to speculate that the calcium paradox in fact reflects
vitamin K deficiency.
It has been shown that patients with CKD frequently are
vitamin K-deficient, which is likely attributable to dietary
advice to limit their potassium intake (i.e., intake of green
leafy vegetables and thus vitamin K1) and to lower phosphate
intake (i.e., intake of dairy products and thus vitamin K2).
Besides, these dietary restrictions, especially patients on dialysis
frequently suffer loss of appetite, further affecting the intake
of essential nutrients, including vitamin K. Another risk for
vitamin K deficiency is the use of phosphate binders as outlined
above. Finally, use of anticoagulant therapy with vitamin K-
antagonists in CKD patients will propel this deficiency even
further (223). Although novel direct oral anticoagulants are
available, these are often considered unsuitable for patients with
a glomerular filtration rate below 30 ml/min/1.73m2. Also, in
healthy subjects it was shown that the majority has subclinical
vitamin K deficiency as deduced from the presence of increased
concentrations of uncarboxylated VKDP in the circulation (22,
180,224). Recent evidence, as outlined in detail above, suggests
that vitamin K is an important factor in bone and vasculature in
CKD patients and post-menopausal women, and that its role may
be overlooked. It creates a window of opportunity to supplement
vitamin K in the abovementioned subgroups including CKD
patients and post/peri menopausal women frequently deficient in
vitamin K.
Although supplementation with vitamin K2 (MK-4) daily
for 3 years did not improve bone mineral content or bone
mineral density, it did maintain bone strength at femoral
neck site in post-menopausal women (19), thus indicating a
beneficial effect on post-menopausal bone strength loss. Aside
from MK-4’s known function for gamma carboxylation, and
thereby preventing ectopic calcification to occur, it was shown
to also promote maturation of osteoblasts (225) and to suppress
osteoclast maturation while promoting their apoptosis (226,227).
MK-7, a long-chain menaquinone, was found to have more
beneficial effect on bone and facilitates bone mineralization,
including cortical bone structure as compared to MK-4 (228). In
support to in vivo evidence, several trials assessed the feasibility of
MK-7 as treatment for CKD and post-menopausal osteoporotic
patients. It was shown that MK-7 (MenaQ7) improves bone
strength at the femoral neck via increasing bone mineral
content (BMC) and bone mineral density (BMD) (19,229,
230). In addition, hemodialysis patients supplemented with
MK-7 showed a substantial decrease in dp-ucMGP along with
ucOC and PIVKA-II (protein induced by vitamin K absence or
antagonism–II) in a dose-dependent manner, implicating that
MK-7 improves vitamin K-status in liver, bone, and vasculature
(24,231). In osteoporotic patients, vitamin K2 resulted in
elevated levels of cOC and prevented fractures when compared
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Wasilewski et al. Calcium Supplements and Vascular Calcification
TABLE 3 | Summary of selected features and effects of available phosphate binders.
Phosphate lowering
agent
Binding
mechanism
Generic name Calcium
based
Effect on
phosphate
Effect on
Ca x P
product
Effect on
calcium/
hypercalcemia
Interaction
with vitamin
K
Calcium
acetate/magnesium
carbonate
Ionic Yes ↓ ↓ ↑ Yes
Calcium acetate Ionic Yes ↓ ↓ ↑ ?
Calcium carbonate Ionic CaCO Yes ↓ ↓ ↑ Yes
Lanthanum carbonate Forms insoluble
phosphate
complexes
LanCO No ↓ ↓ ↓ Yes
Aluminum hydroxide Ionic Al salts No ↓?↑?
Sucroferric oxyhydroxide Covalent binding FeSa No ↓ ↓ NS change No
Sevelamer hydrochloride Ionic Sevelamer HCl No ↓ ↓ ↓ ?
Sevelamer carbonate Ionic Sevelamer CO3No ↓ ↓ NS change No
Colestilan Ionic No ↓ ↓ NS change ?
Bixalomer ? No NS change NS change NS change ?
Nicotinamide inhibition of
sodium/phosphorus
co-transporter
Vitamin B3 No ↓ ↓ NS change ?
Ferric citrate Ionic No ↓NS change NS change ?
Reference (205,206) (207–217) (218–220)
Ca, Calcium; P, Phosphorous.
FIGURE 3 | Representation of systemic action of vitamin K on bone and vasculature in the calcium presence. Calcium based phosphate binders are known to reduce
the levels of adsorbed phosphate by directly coupling reaction in the gastro-intestinal tract. Phosphate binders were also shown to bind Vitamin K suggesting it might
affect its free circulating form. When coupled with phosphate binders, vitamin K is unable to perform its biological function of positively utilizing calcium into the bone
and simultaneously acting as calcification inhibitor.
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Wasilewski et al. Calcium Supplements and Vascular Calcification
with placebo-treated osteoporotic patients (232). Moreover, both
MK-4 and MK-7 supplementation resulted in an increase of
cOC and a decrease of ucOC and improved BMD (229,233–
238).
Besides its beneficial effects on bone health, high intake of
MK-7 successfully blocked age-related vascular stiffening (239)
in post-menopausal women. Moreover, MK-7 was better than
placebo at reducing severe aortic calcification and relative risk
of coronary heart disease (208,240). Ongoing clinical trials
will evaluate its effectiveness in reducing vascular calcification
in patients with coronary artery disease (241). In a cross-
sectional study, nutritional long-chain menaquinone intake
was associated with decreased coronary calcification in post-
menopausal women (240,242). Moreover, MK-7 improved
arterial stiffness and elastic properties of the carotid artery
in a healthy postmenopausal woman (20) and improved
vitamin K status in dialysis patients by decreasing inactive
levels of MGP by daily supplementation (24). In another
randomized clinical study, K1 supplementation slowed the
progression of CAC in healthy older adults with preexisting
CAC, demonstrating the potential efficacy of vitamin K
treatment for vascular calcification. Inactive MGP (dp-ucMGP)
has been correlated with severity of CKD and is positively
associated with amount of vascular calcification (24,224,
243,244). MK-7 (MenaQ7) supplementation in patients with
CKD3-5 significantly reduced circulating levels of dp-ucMGP
(24).
Collectively, these data imply that vitamin K could serve as
complementary nutrient to calcium (and vitamin D) to protect
from increased risk for vascular calcification thereby allowing
more safe treatment of osteoporosis. Vitamin K supplementation
in post-menopausal patients appeared beneficial in combination
with calcium and vitamin D3 for bone health and vasculature
(239). The combination of vitamin K and calcium could reduce
risk on post-menopausal bone and simultaneously prevent
vascular calcification, thereby aiding the beneficial effects of
calcium in bone and preventing the negatively associated vascular
effects of supplemental calcium intake.
CONCLUSIONS
To date, calcium supplements are the most commonly used
non-prescription drug to treat age-related bone loss. Also, in
patients suffering from chronic kidney disease, calcium-based
phosphate binders are commonly prescribed. However, the rising
concern of side-effects from calcium supplementation illustrates
a clinical dilemma: supplementation of calcium—either with or
without vitamin D—comes at the price of increased risk of
vascular calcification. Clinical studies demonstrate that increased
intake of vitamin K could be a promising complementary
nutrient in supporting both bone health and protecting vascular
calcification. Thereby it can increase safety of current treatments
of osteoporosis and provide an escape from the calcium paradox.
Future clinical trials should be carried out to confirm the
feasibility of such combination.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication.
GW wrote the manuscript. MV wrote the manuscript and was
responsible for the final version. LS wrote the manuscript,
supervised writing process and was responsible for the final
version.
FUNDING
This work was supported by funding from the Norwegian
Research Council (Project 241584) and NattoPharma ASA.
Research from LS is in part funded via the European Union’s
Horizon 2020 research and innovation programme under the
Marie Skłodowska-Curie grant agreement No. 722609.
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Conflict of Interest Statement: Nattopharma ASA received an industrial Ph.D.
grant from the Norwegian Research Council to conduct research in collaboration
with the Maastricht University. GW has been employed as Ph.D. student to work
on this project. Nattopharma ASA is a pharmaceutical company with interest in
vitamin K2.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2019 Wasilewski, Vervloet and Schurgers. This is an open-access article
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terms.
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