Nutrients 13 02603 v2

nutrients
Review
The Emerging Role of Nutraceuticals in Cardiovascular
Calcification: Evidence from Preclinical and Clinical Studies
Maristella Donato 1 , Elisabetta Faggin 2 , Francesco Cinetto 2,3 , Carla Felice 2,3 , Maria Giovanna Lupo 1 ,
Nicola Ferri 1 and Marcello Rattazzi 2,3, *
1 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35122 Padua, Italy;
[email protected] (M.D.); [email protected] (M.G.L.);
[email protected] (N.F.)
2 Department of Medicine—DIMED, University of Padova, 35122 Padua, Italy; [email protected] (E.F.);
[email protected] (F.C.); [email protected] (C.F.)
3 Medicina Generale Iˆ, Ca’ Foncello Hospital, 31100 Treviso, Italy
* Correspondence: [email protected]; Tel.: +39-04-9821-1867 or +39-04-2232-2207
Abstract: Cardiovascular calcification is the ectopic deposition of calcium-phosphate crystals within

the arterial wall and the aortic valve leaflets. This pathological process leads to increased vascular
stiffness, reduced arterial elasticity, and aortic valve stenosis, increasing the risk of cardiovascular
diseases. Although cardiovascular calcification is an increasing health care burden, to date no
medical therapies have been approved for treating or preventing it. Considering the current lack of
therapeutic strategies and the increasing prevalence of cardiovascular calcification, the investigation
of some nutraceuticals to prevent this pathological condition has become prevalent in recent years.

Recent preclinical and clinical studies evaluated the potential anti-calcific role of nutraceuticals
Citation: Donato, M.; Faggin, E.;
(including magnesium, zinc, iron, vitamin K, and phytate) in the progression of vascular calcification,
Cinetto, F.; Felice, C.; Lupo, M.G.;
providing evidence for their dietary supplementation, especially in high-risk populations. The present
Ferri, N.; Rattazzi, M. The Emerging
review summarizes the current knowledge and latest advances for nutraceuticals with the most
Role of Nutraceuticals in
relevant preclinical and clinical data, including magnesium, zinc, iron, vitamin K, and phytate. Their
Cardiovascular Calcification:
Evidence from Preclinical and
supplementation might be recommended as a cost-effective strategy to avoid nutritional deficiency
Clinical Studies. Nutrients 2021, 13, and to prevent or treat cardiovascular calcification. However, the optimal dose of nutraceuticals has
2603. https://doi.org/10.3390/ not been identified and large interventional trials are warranted to support their protective effects on
nu13082603 cardiovascular disease.
Academic Editor: Prashant Nighot Keywords: vascular calcification; nutrition; nutraceuticals
Received: 1 July 2021

Accepted: 27 July 2021
Published: 28 July 2021 1. Introduction
Cardiovascular (CV) calcification is defined as the ectopic deposition of calcium-
Publisher’s Note: MDPI stays neutral
phosphate crystals within the arterial tissues, mainly the aorta and its major branches and
with regard to jurisdictional claims in
the aortic valve leaflets. Pathological calcification can occur in two distinct areas of the
published maps and institutional affil-
vessel wall: the intima (intimal calcification), which is inflammation-driven and typically
iations.
associated with atherosclerotic plaques, or the media (arterial medial calcification) [1].
Medial calcification is prevalent in aging, diabetes mellitus, and chronic kidney disease
(CKD) [2]. In particular, CKD is recognized as a public health problem worldwide, with a
global estimated prevalence of 9–13% that is expected to further increase in the coming
Copyright: © 2021 by the authors.
years [3,4]. In the aortic valve, calcification is the dominant process of advanced calcific
Licensee MDPI, Basel, Switzerland.
aortic valve stenosis (CAVS), driving disease progression. CAVS is the most common
This article is an open access article
heart valve disease in the Western world, with a prevalence of 12.4% in the elderly (over
distributed under the terms and
75 years) [5]. Unfortunately, the disease becomes symptomatic only at the severe stage
conditions of the Creative Commons
when it requires the substitution of the valve. The prevalence of severe CAVS increases
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
with advancing age, from 0.02–0.1% in patients aged 18–44 years to 2.8–4.6% in the elderly
4.0/).
(over 75 years) [6]. Compared to the general population, CKD patients are at a higher risk
Nutrients 2021, 13, 2603. https://doi.org/10.3390/nu13082603 https://www.mdpi.com/journal/nutrients

Nutrients 2021, 13, 2603 2 of 18
of developing CAVS, which is associated with lower survival. Considering the higher life
expectancy and the rapid aging of the population, the prevalence and impact on public
health of valvular calcification are expected to further increase in the near future [7–9].
Arterial medial calcification increases the stiffness and reduces the elasticity of the
vessels, and it has been associated with an increased risk of CV events and mortality, not
only in high-risk subjects but also in the general population [10,11]. This association is
particularly evident in CKD patients, where vascular calcification has an overall prevalence
of 60% and significantly affects the risk of CV events and all-cause mortality [12]. More-
over, in this high-risk population, the progression of vascular calcification is faster with
worsening CKD [13].
On these bases, it appears that interventions preventing or limiting CV calcification
might have a relevant impact on CV disease protection, especially in a high-risk population.
2. Purpose
The development of effective treatments for CV calcification is an urgent clinical need,
considering the current lack of medical therapies and the increasing prevalence of the
disease. In recent years, preclinical and clinical studies have been performed to evaluate
the potential beneficial effects of nutraceuticals supplementation on the prevention and
treatment of vascular calcification. The present review briefly summarizes the main risk
factors and pathophysiological mechanisms for the development of CV calcification and
the current knowledge in the clinical management of this pathological condition. In the
main part, emphasis is placed on the nutraceuticals with the most relevant preclinical and
clinical data, including magnesium, zinc, iron, vitamin K, and phytate.
3. Risk Factors for Cardiovascular Calcification

Vascular calcification was initially thought to be only an age-related passive process,
caused by the deposition of hydroxyapatite (HA) crystals in the extracellular matrix (ECM).
Although the pathogenesis of ectopic calcification in the medial layer of the vessels and in
the aortic valve is still unclear, increasing evidence supports the hypothesis that it is also
an active and cell-mediated process similar to skeletal bone formation.
In the medial layer of the vessels, vascular smooth muscle cells (VSMCs) can undergo
differentiation into an osteochondrogenic phenotype, characterized by the downregulation
of smooth muscle contractile proteins, and the expression of osteogenic markers (such
as ALP, Runx2, BMPs). The transdifferentiation of VSMCs can be promoted by several
different factors, including oxidative stress, an imbalance between calcification promoters
and inhibitors, cellular senescence, and hyperphosphatemia [14]. Once VSMCs are differ-
entiated, they secrete calcifying extracellular vesicles rich in calcium-phosphate crystals,
promoting further mineralization in the ECM [15]. In addition, osteoblast-like VSMCs can
release apoptotic bodies, which act as scaffolds for ECM mineralization [16].
In the aortic valve, increased mechanical stress and reduced shear stress can induce
endothelial dysfunction, which favors the infiltration of monocytes, immune cells, and
lipoproteins. Therefore, the first phase of CAVS is characterized by oxidative stress, inflam-
matory response, and lipid deposition in the sub-endothelium and in the fibrosa layer of the
tissue [17]. In the second phase, fibrosis and calcification assume a prominent role in the pro-
gression of the disease. Valvular calcification is driven by the osteogenic differentiation of
VICs, promoted by several signaling pathways (including Wnt/β-catenin, RANK/RANKL,
and BMPs) [18,19]. The osteogenic phenotype of VICs is similar to osteochondrogenic
VSMCs, characterized by an increased expression of osteogenic markers.
Traditional risk factors for CV calcification are dyslipidemia, inflammation, diabetes,
obesity, and hypertension [20]. In addition to this, ectopic calcification is also associated
with several non-traditional risk factors in the CKD population [21]. In fact, in CKD pa-
tients, a dysregulated mineral metabolism, the loss of calcification inhibitors (including
MGP, fetuin-A, pyrophosphate), and the activation of inflammatory pathways contribute
to the development and progression of vascular calcification [22]. Moreover, the renal
Nutrients 2021, 13, 2603 3 of 18
function impairment and the consequently reduced excretion of phosphate lead to hyper-
phosphatemia, a condition that favors mineral deposition in both vascular walls and the
aortic valve and the phenotypic switch of VSMCs [23]. Phosphate absorption and excretion
also depend on a network involving calcium, vitamin D, and parathyroid hormone (PTH),
factors that are dysregulated in pathological conditions like CKD [22].
4. Pharmacological Approaches for the Treatment of Vascular Calcification

Concerning valvular calcification, to date, no effective pharmacological treatments
have proven to halt or delay it and aortic valve replacement represents the only option to
improve clinical outcomes [24].
The pathophysiological mechanisms leading to vascular calcification are still unclear,
therefore, at present, there is no comprehensive treatment to prevent or counteract it. The
current strategies are based on the modulation of its key regulators, such as vitamin D, cal-
cium, phosphate, through calcimimetics, vitamin D analogs, and phosphate binders [21,25].
Phosphate binders limit dietary phosphate absorption, lowering its serum levels, and are
used in clinical practice to manage hyperphosphatemia in CKD patients. It has been demon-
strated that excessive oral intake of calcium, also by calcium-containing phosphate binders,
increases the risk for vascular calcification; thus, calcium-free phosphate binders should be
preferred [26,27]. However, it is uncertain whether phosphate binders can prevent ectopic
calcification and reduce the risk of CV events, as underlined by a recent meta-analysis [28].
A recent review suggests that calcium-free phosphate binders and low-dose vitamin D
analogs combined with calcimimetics may reduce the severity of vascular calcification [29],
but clinical evidence is still insufficient to recommend this strategy.
Therefore, based on current evidence, optimal treatments for vascular calcification
remain to be found. A detailed description of phosphate binders, calcimimetics, and
vitamin D analogs has been given by other recent reviews [30–32] and goes beyond the
purpose of the present paper.
In recent years, emphasis has been placed on the role of some nutraceuticals to
improve health and to prevent diseases. It is well established that adequate serum levels
of nutraceuticals maintain CV health. Their overall levels are dependent on oral intake,
gut absorption, tissue pools, renal excretion, and several other factors, strictly correlated to
the nutritional and medical status of the subject. Considering that nutritional deficiency is
common in high-risk populations and that preclinical evidence has detected anti-calcific
effects of some nutraceuticals (such as magnesium, zinc, iron, vitamin K, and phytate),
several preclinical and clinical studies have been performed to assess the potential effects
of their supplementation for vascular calcification.
5. Nutraceuticals for Cardiovascular Calcification

5.1. Magnesium
Magnesium, one of the most abundant cations in the human body (25 g), is widely
distributed in plant foods, such as green leafy vegetables, legumes, nuts, seeds, and whole
grains [33]. Magnesium is an essential mineral for many physiological functions. Mainly
found in the ionized form, it acts as a counter ion for ATP and as a cofactor in several
enzymatic reactions required for the structural function of nucleic acids, proteins, and
mitochondria. Moreover, magnesium contributes to muscle contraction/relaxation, the
release of neurotransmitters, regulation of vascular tone, and bone formation [34].
The role of magnesium in CKD-associated vascular calcification has been assessed in
several preclinical studies. An ex vivo/in vitro study on both VSMCs and aortic segments de-
termined the inhibitory action of magnesium on phosphate-mediated vascular calcification [35].
The molecular mechanisms by which magnesium counteracts vascular calcification
are still not completely understood, but they seem related both to intracellular and ex-
tracellular levels of this micronutrient. The active role of intracellular magnesium as an
anti-calcific agent is supported by in vitro evidence. It has been shown that magnesium
prevents VSMCs transdifferentiation by decreasing the expression of pro-calcification
Nutrients 2021, 13, 2603 4 of 18
markers (including Osterix, Smad1, and Wnt/β-catenin pathway) [36,37], by increasing

calcification inhibitors (including MGP and osteoprotegerin), and by improving VSMCs
viability [38,39]. These effects are mediated by the entry of magnesium into the cells via
transient receptor potential melastatin-7 (TRPM7), whose activity is reduced in osteogenic
VSMCs and restored in the presence of magnesium [40]. In addition, magnesium can act as
an anti-crystallization agent at the extracellular level; it avoids the formation of hydroxyap-
atite from amorphous calcium phosphate, blocking the process of crystal nucleation [41].
Firstly, magnesium is a natural calcium antagonist; thus, it can substitute calcium ions in
hydroxyapatite, favoring the formation of whitlockite, less pathogenic crystals [42]. In
addition, magnesium avoids the transition of amorphous Ca–Pi particles into crystalline
hydroxyapatite. In particular, this action is due to the inhibition of calciprotein particles
(CPPs) maturation from amorphous CPP1 to crystalline CPP2 [43] (Figure 1).
Figure 1. A simplified schematic representation of the processes under investigation leading to the osteogenic differentiation
of VSMCs in vascular calcification. Magnesium (Mg) acts at the extracellular level by preventing the maturation of
calciprotein particles (CPPs) and by forming whitlockite crystals with phosphate ions (Pi). At the intracellular level, Mg
decreases the expression of osteogenic genes (such as Osterix, Smad1, and Runx2) and inhibits pro-calcific pathways
(including Wnt/β-catenin). Zinc (Zn) induces the suppression of NF-κB through the activation of the GPR39/TNFAIP3
signaling pathway. Vitamin K promotes the carboxylation and activation of the matrix Gla protein (MGP), which avoids the
formation of hydroxyapatite (HA) crystals and prevents the pro-calcific action of BMP-2. Phytate acts as crystallization
inhibitor by binding to HA crystals. The regular arrow denote “activation”, and the T-shaped arrow stands for “inhibition”.
It has been recommended to consume at least 400–420 mg/day of magnesium for

men and 310–320 mg/day for women [33]. The regular consumption of magnesium is
fundamental to maintain the physiological processes and to prevent magnesium deficiency.
In addition, recommended values of serum magnesium levels have been considered rele-
vant for its protective effects on CV health. Hypomagnesemia is indeed associated with
an increased incidence of hypertension, type 2 diabetes, and metabolic syndrome in the
general population [44]. Moreover, dietary magnesium intake is inversely correlated with
the risk of CV events and all-cause mortality [45,46]. A significant relationship between
low serum magnesium levels and all-cause mortality has also been found by a recent
meta-analysis of 20 clinical trials involving CKD and dialysis patients [47].
The relationship between magnesium intake and vascular calcification was inves-
tigated in the Framingham Heart Study, which found a strong inverse association [48].
This study, together with the above-described preclinical findings, provided evidence
Nutrients 2021, 13, 2603 5 of 18
for performing subsequent trials to assess the effects of magnesium supplementation on

vascular calcification, especially in high-risk patients. Some clinical trials investigated
the potential effects of long-term magnesium supplementation (more than 6 months) on
the progression of atherosclerosis in hemodialysis patients; the trials measured carotid
intima-media thickness (IMT), a well-established marker of subclinical atherosclerosis,
finding a significant improvement in this parameter after magnesium consumption [49,50].
Therefore, magnesium is likely to play a protective role in the development of atheroscle-
rotic lesions. However, the studies were limited by small sample size (44 and 54 patients
respectively); thus, these preliminary findings should be confirmed by larger-scale trials.
Magnesium is used in clinical practice as a phosphate binder for its ability to bind
dietary phosphate in the intestinal tract. In few pilot studies, oral magnesium carbonate
was administered to hemodialysis patients as a magnesium-containing phosphate binder,
providing positive outcomes on vascular calcification [51,52] (Table 1). The protective effect
of magnesium, however, seems not to be limited to its phosphate-binding capacity; a recent
in vivo study on a uremic mouse model observed a decrease in vascular calcification after
intraperitoneal magnesium administration, suggesting a direct effect of magnesium indepen-
dently on its intestinal action [53]. Taken together, these findings provided a strong rationale
for investigating in further clinical trials whether dietary magnesium could reduce the risk
of vascular calcification and CV events in CKD patients. Recently, a randomized controlled
trial on predialysis patients observed that the long-term supplementation of magnesium
oxide could effectively slow the progression of coronary artery calcification [54]. These data
represent the strongest evidence so far of the anti-calcific action of magnesium. Currently,
the effects of magnesium on the progression of coronary artery calcification in predialysis
subjects are also being assessed in the MAGiCal-CKD randomized trial [55] (Table 2).
Table 1. Interventional trials performed to evaluate the effects of nutraceuticals on vascular calcification.
Reference Intervention Phase Population Enrolment Findings

Magnesium carbonate Magnesium retards the
[51] plus calcium acetate vs. pilot Hemodialysis patients 72 progression of arterial
calcium acetate alone calcification
Magnesium Magnesium may have a
Hemodialysis patients,
[52] carbonate/calcium pilot 7 favorable effect on the
CAC scores >30
carbonate progression of CAC
Magnesium oxide is
Magnesium oxide vs. CKD patients with risk
[54] - 125 effective in slowing CAC
placebo factors for CAC
progression
Vitamin K2 does not affect
Vitamin K2 plus vitamin Non-dialyzed CKD
[56] IV 42 the progression of
D vs. vitamin D patients
calcification
Menaquinone-7 does not
Menaquinone-7 vs. Type II diabetes and
[57] - 68 decrease vascular
placebo CVD
calcification
Healthy subjects (>60 Vitamin K1 slows the
[58] Vitamin K1 vs. placebo III 452
years) with CAC progression of CAC
Vitamin K1 might
[59] Vitamin K1 vs. placebo III Asymptomatic AVC 99 decelerate the progression
of AVC
SNF472 administration
Healthy volunteers
[60] iv SNF472 vs. placebo I 28 reduces HA crystallization
and dialysis patients
potential
SNF472 attenuates CAC
[61] iv SNF472 vs. placebo IIb Hemodialysis patients 274
progression and AVC
iv, intravenous; CKD, chronic kidney disease; CAC, Coronary Artery Calcification; AVC, aortic valve calcification; CVD, cardiovascular
disease; HA, hydroxyapatite.
Nutrients 2021, 13, 2603 6 of 18
Table 2. Ongoing clinical trials evaluating the effects of nutraceuticals on vascular calcification.
Trial Name NCT Intervention Phase Population Enrolment Primary Outcome

Magnesium
Change in CAC score
MAGiCal-CKD NCT02542319 hydroxide vs. II-III Predialysis CKD 250
at 12 months
placebo
Vitamin K1 vs. Hemodialysis Progression of CAC
iPACK-HD NCT01528800 II 85
placebo patients at 12 months
Menaquinone-7 Hemodialysis Difference in CAC
TReVasc-HDK NCT02870829 II 178
vs. placebo patients score at 18 months
Menaquinone-7 CAC score between Change in CAC score
VitaK-CAC NCT01002157 - 180
vs. placebo 50 and 400 at 12 and 24 months
Menaquinone-7 AVC score above Progression of AVC
AVADEC NCT03243890 - 389
vs. placebo 300, without AVS at 2 years
Bicuspid AV and
Vitamin K2 vs. Progression of AV
BASIK2 NCT02917525 II mild to moderate 44
placebo calcium at 6 months
AVS
CKD, cronic kidney disease; CAC, Coronary Artery Calcification; AVC, aortic valve calcification; AVS, aortic valve stenosis.
Based on current evidence, magnesium supplementation seems a promising strategy

for the management of vascular calcification. In addition, it would be affordable and
easily accessible to patients due to the cost-effectiveness and the wide availability of this
mineral. However, larger randomized clinical trials are warranted before suggesting its
supplementation to CKD patients.
Of note, an adequate magnesium intake is also necessary for bone health: 60% of total
magnesium is stored in the bone, where it maintains proper mineral density. However,
there is increasing evidence supporting the hypothesis that magnesium supplementation
in high doses may counteract HA crystals formation even in bone, leading to mineral
defects such as osteomalacia [62,63]. The tolerable upper intake levels for supplemental
magnesium are 350 mg/day [33], but further studies are needed to investigate whether an
increase in dietary magnesium may decrease bone mineral density as a side effect.
Moreover, it is important to underline that dietary intake of magnesium is not always
correlated with its serum concentration and even serum levels of magnesium do not reflect
its total body content. Magnesium homeostasis depends on the balance between gut
absorption, renal excretion, and tissue pools (mainly in bone), which may vary from one
subject to another according to the patient’s medical condition [34]. In patients with CKD,
the loss of renal function is counteracted by compensatory mechanisms, which maintain
the serum levels of magnesium in the normal range or may even cause hypermagnesemia
when renal excretion is further decreased [64]. On the other hand, some drugs commonly
used by CKD patients can decrease gastrointestinal absorption of magnesium, potentially
contributing to hypomagnesemia [65]. Therefore, several factors (such as the medical status,
CKD stage, and drug administration) should be considered when an oral supplementation
of magnesium is suggested to CKD patients.
As suggested by Hamano and colleagues [66], further research should focus on the
clinical impact of magnesium supplementation on CV events and mortality. Moreover,
the optimal dose of dietary magnesium should be determined, in order to maximize the
benefits without causing potential side effects. More recently, new oral formulations of
magnesium have been developed in order to improve its bioavailability and to ameliorate
its physiological inhibitory effect on vascular calcification [67].
Nutrients 2021, 13, 2603 7 of 18
5.2. Zinc
Zinc, one of the most abundant divalent cations in the body (2–4 g), can be found in a
variety of foods, including oysters, red meat, seafood, beans, and dairy products. The rec-
ommended oral consumption of zinc is 8 mg/day for women, 11 mg/day for men, and the
tolerable upper intake level for adults is 40 mg/day [68]. Zinc is an essential micronutrient
for several physiological functions, including the catalytic activation of zinc-containing
metalloenzymes, the regulation of cellular homeostasis and intracellular signaling, the
preservation of the active conformation of DNA-binding proteins, and transcription factors.
Moreover, it exerts strong antioxidant and anti-inflammatory effects [69].
The anti-calcific effect of zinc is supported by in vitro evidence on both VSMCs [70]
and VICs [71]. It seems likely that zinc does not directly inhibit calcium phosphate precipi-
tation but acts indirectly through the activation of cellular mechanisms. In particular, the
anti-calcific effect in VSMCs depends on the GPR39-mediated upregulation of TNFAIP3
protein levels and the subsequent suppression of NF-κB, a pro-inflammatory transcription
factor involved in their osteochondrogenic differentiation [72] (Figure 1). In cultured VICs,
zinc supplementation reduces the calcification process through the inhibition of apoptosis
and the activation of the GPR39-dependent ERK1/2 signaling pathway [71] (Figure 2).
Figure 2. A simplified schematic representation of the processes under investigation leading to the osteogenic differentiation
of VICs in vascular calcification. Iron (Fe) acts at the intracellular level via ferritin, which prevents phosphate uptake and
inhibits VICs osteogenic differentiation. Moreover, ferritin promotes the production of anti-calcific pyrophosphate (PPi)
from ATP by activating the enzyme ENPP2. Zinc (Zn) induces VICs apoptosis through the activation of the GPR39-ERK1/2
signaling pathway. Vitamin K promotes the carboxylation and activation of matrix Gla protein (MGP), which avoids the
formation of hydroxyapatite (HA) crystals and prevents the pro-calcific action of BMP-2. Phytate acts as crystallization
inhibitor by binding to HA crystals. The regular arrow denotes “activation”, and the T-shaped arrow stands for “inhibition”.
In preclinical studies, reduced dietary zinc intake promoted vascular inflammation and
arterial plaque formation [73], while zinc supplementation exerted an anti-atherosclerotic
role [74]. Moreover, a long-term supplementation of zinc was effective in reducing vascular
calcification in hyperphosphatemic mice [72].
Taken together, these experimental studies provide evidence on the role of adequate
serum zinc levels for maintaining CV health. Unfortunately, zinc deficiency is a common
condition, especially in developing countries. Zinc insufficiency is caused by low dietary
zinc intake, decreased absorption, or increased loss of zinc, and may be associated with
the development of diabetes, cancer, gastrointestinal disease, and CKD [75]. More recently,
zinc deficiency has also been correlated to hypercholesterolemia, high blood pressure, and
inflammation, leading to an increased risk of CV diseases. In CKD patients, the decrease
in the circulating levels of zinc is probably due to a higher urinary excretion; moreover,
Nutrients 2021, 13, 2603 8 of 18
the reduction in baseline plasma concentration has been recently associated with a higher
probability of renal function decline [76].
Based on this evidence, increasing zinc intake might be proposed to avoid the detri-
mental effects of its deficiency, especially in high-risk subjects. However, it should be
taken into account that serum levels of zinc are poorly correlated to its oral intake. For
example, athletes have lower serum zinc concentration, despite the higher total dietary
zinc intake [77]; a weak relationship between dietary consumption and serum levels of zinc
has also been found in a study involving 408 healthy girls [78]. These findings are probably
due to the differences in bioavailability from dietary sources and the different metabolism
of the subjects. Therefore, these aspects should be considered before suggesting an oral
supplementation of zinc.
The relationship between oral zinc intake and arterial calcification in humans was
investigated in few clinical studies. A recent cross-sectional study associated a higher
dietary consumption of zinc with a lower probability of having severe abdominal aortic
calcification [79]. Moreover, an inverse correlation was observed between circulating levels
of zinc and serum calcification propensity in patients with type 2 diabetes [80]. Increasing
zinc consumption might be a simple but effective approach to reduce the progression
of vascular calcification and the risk of CV diseases. Despite the positive findings, there
is currently insufficient evidence to recommend its supplementation for preventing CV
diseases, as reported by a recent review [81]. Further clinical studies are required to confirm
the association between hypozincemia and vascular calcification and the potential benefits
of zinc supplementation.
5.3. Iron
Iron is an essential component of hemoglobin and is necessary for several physiologi-
cal processes, including DNA synthesis, enzymatic activities, and mitochondrial energy
production. Dietary iron is available in two forms: heme, derived from meat and fish, and
nonheme, obtained from cereals, legumes, fruits, and vegetables [82]. The recommended
daily intake is 8 mg for men and reaches 18 mg in women up to 50 years; the tolerable
upper intake level from food and supplements for adults is 45 mg/day [83].
Increasing preclinical evidence has investigated the effects of iron on CV calcification.
In vitro, iron prevented phosphate-induced osteoblastic differentiation in both VSMCs
and VICs via ferritin and its ferroxidase activity [84,85]. In the aortic valve, in particular,
ferritin effectively enhanced pyrophosphate production and reduced cellular phosphate
uptake [85] (Figure 2). Pyrophosphate is a well-established calcification inhibitor, avoiding
the growth of HA crystals [86]. The anti-calcific effect of iron was also confirmed by an
in vivo study, where parenteral iron administration inhibited the development of vascular
calcification in uremic rats [87].
Maintaining adequate serum values of iron is fundamental for several physiological
processes, considering also that iron might play a protective role for vascular calcification.
On the other hand, inadequate iron bioavailability, causing an iron deficiency, has severe
clinical consequences. Iron deficiency is common in CKD patients and leads to anemia; for
this reason, the supplementation of iron is currently prescribed to these subjects. While
iron is exclusively administered intravenously in dialysis patients, in non-dialysis subjects
the route of administration is still controversial. Oral supplementation is inexpensive
and easier to administer but is frequently associated with gastrointestinal adverse events
that can limit both the efficacy and the adherence to therapy [88]. Therefore, despite
the disadvantages of a parenteral administration, a recent systematic review suggests
the use of intravenous iron as standard care for treating CKD-associated anemia even in
non-dialysis-dependent CKD patients, stages 3 to 5 [89].
Recently, two oral preparations (iron citrate and sucroferric oxyhydroxide) have be-
come available as effective iron-based phosphate binders, approved for treating hyperphos-
phatemia in CKD patients [90]. This new class of phosphate binders has the advantages
of being calcium-free, avoiding the side effect of hypercalcemia, and of enhancing iron
Nutrients 2021, 13, 2603 9 of 18
availability, also improving anemia [91]. In addition to these properties, increasing pre-
clinical evidence suggests a beneficial effect of these phosphate binders also for vascular
calcification. Recent in vitro studies by Ciceri and colleagues analyzed the direct effect
of iron citrate, independently from its phosphate chelating action, on phosphate-induced
calcification of VSMCs. Iron citrate prevented and partially reverted the high phosphate-
induced osteochondrogenic shift of the ECM [92]. Moreover, the exposure of VSMCs to iron
citrate inhibited calcium deposition through the prevention of apoptosis and the induction
of autophagy [93]. Of note, iron was able not only to prevent calcium deposition but also to
avoid further progression when calcification was already established [94]. Based on these
findings, it seems likely that iron can act on vascular calcification independently of the
grade of VSMCs differentiation.
The anti-calcific properties of sucroferric oxyhydroxide and iron citrate were further
confirmed by in vivo studies, which demonstrated their effectiveness in preventing the
progression of ectopic calcification in uremic rats [95,96].
These preliminary findings suggest that iron administration in patients with hyper-
phosphatemia and iron deficiency might also prevent vascular calcification in this high-risk
population. However, this hypothesis should be assessed in further preclinical studies and
clinical trials, which are currently lacking.
Furthermore, the optimal dose of iron for supplementation should be determined to
avoid an iron overload, which has negative effects on bone metabolism. In fact, iron can
decrease bone mineralization by inhibiting osteoblast differentiation, eventually leading to
osteopenia and osteoporosis. This inhibitory action depends on iron-induced upregulation of
ferritin and its ferroxidase activity, similarly to what happens in VSMCs [97,98]. Moreover,
excess iron enhances oxidative stress, which in turn may promote vascular calcification [98].
Thus, the supplementation of iron for preventing or halting vascular calcification
to date is not supported by clinical studies and requires further evidence before being
suggested, although the preclinical findings are promising. Moreover, the optimal dose to
avoid side effects remains to be found.
5.4. Vitamin K
Vitamin K refers to a group of liposoluble vitamins: vitamin K1 (phylloquinone)
naturally found in fruits and vegetables, and vitamin K2 (menaquinones) produced by
gut bacteria [99]. The adequate intake of vitamin K is considered 120 µg/day for men and
90 µg/day for women [100].
Vitamin K is a required cofactor for the γ-carboxylation (and thereby activation)
of several vitamin K-dependent proteins, among them the matrix Gla protein (MGP),
a potent inhibitor of soft-tissue calcification. The activated carboxylated form of MGP
(cMGP) binds to calcium ions and interacts with bone morphogenetic protein-2 (BMP-2),
avoiding, respectively, the formation of HA crystals and the osteogenic differentiation
of cells [101,102] (Figures 1 and 2). There is experimental evidence of the role of cMGP
in preventing vascular calcification; in fact, the silencing of the MGP gene promoted the
osteoblastic differentiation of VICs [103] and the development of arterial calcification [104].
In addition to the carboxylation and activation of MGP, vitamin K exerts anti-
inflammatory and lipid-lowering properties, which may also contribute to its potential ben-
eficial effects. In particular, the serum levels of vitamin K1 have been inversely correlated
with circulating inflammation markers, such as ICAM-1, C-reactive protein, and IL-6 [105].
Moreover, vitamin K2 has been associated with decreased cholesterol biosynthesis and
increased low-density lipoprotein receptor (LDLR) [106].
A low intake of vitamin K or the consumption of vitamin K antagonists (such as warfarin)
can lead to vitamin K deficiency, which reduces the available amount of cMGP [107,108]. In
patients undergoing hemodialysis, reduced serum levels of cMGP have been associated with
an increased risk for vascular calcification [109]. Vitamin K deficiency is a common condition
in CKD patients, where it is significantly associated with vascular calcification [110]. In this
high-risk population, the insufficiency of vitamin K is often the result of dietary restrictions
Nutrients 2021, 13, 2603 10 of 18
or the frequent use of warfarin as an anticoagulant [99]. Considering also that vitamin K
antagonists promote CV calcification and aortic valve (AV) degeneration [111–113], they
should be substituted with novel oral anticoagulants (NOACs), which are preferable even for
maintaining bone health [114].
Thus, the dietary supplementation of vitamin K might be proposed to avoid vitamin K
deficiency and to prevent ectopic calcification, especially in high-risk subjects. In this case,
the upper limit intake is 200 µg/day [115]. The effects of oral vitamin K supplementation
have been assessed in randomized controlled trials with conflicting results. Vitamin
K2 administration failed to decrease the progression of vascular calcification in patients
with advanced CKD [56] or diabetes [57], but these studies were limited by short-term
follow-up and a small sample size. In a long-term trial, daily supplementation of vitamin
K1 for 3 years showed positive outcomes in elderly people with pre-existing vascular
calcification [58]. The administration of vitamin K1 was also associated with slower
progression of AV calcification in a small size study [59]. The clinical studies are described
in detail in Table 1. Despite these positive preliminary findings, a recent systematic review
has underlined the lack of sufficient evidence to support a beneficial effect of vitamin K
in preventing the progression of calcification and atherosclerosis [116]. Moreover, in a
very recent randomized study (K4Kidneys trial) involving patients with advanced CKD,
vitamin K2 failed to improve vascular stiffness, a marker of vascular health [117].
Clinical trials are currently ongoing to evaluate the potential efficacy of vitamin
K in preventing vascular calcification in hemodialysis patients (iPACK-HD, TReVasc-
HDK) [118,119] and in subjects with coronary heart disease (VitaK-CAC) [120]. Moreover,
the AVADEC and BASIK2 randomized trials are investigating the effects of vitamin K2 on
the progression of AV calcification [121,122]. These trials are described in detail in Table 2.
If the trials give positive outcomes, they will provide an effective preventive treatment
for ectopic calcification a simple, safe, and readily available compound that can be easily
supplemented in patients. Evidence of successful regression of ectopic calcification will
support larger and longer-term trials.
5.5. Phytate
Phytate (InsP6), the hexasodium salt of myo-inositol hexaphosphate, is a naturally
occurring compound present in unprocessed plant foods, such as whole grains, legumes,
seeds, and nuts [123].
Phytate can chelate calcium ions with high affinity, a property that is beneficial for
ectopic calcification; in fact, it binds in the growing site of HA crystals blocking their
additional growth without reducing the circulating calcium levels [124] (Figures 1 and 2).
The structural similarities of phytate with other polyphosphates (such as bisphosphonates
and pyrophosphate) give it similar anti-calcific activity, but with the highest potency as a
crystallization inhibitor [125]. The different inhibitory activity may be explained by the
higher number of phosphates in the chemical structure, increasing the possibility to bind
calcium ions [126].
The anti-calcific effects of phytate on ectopic calcification were assessed in preclin-
ical and clinical studies. In rats, oral phytate significantly reduced calcium deposition
in the aorta and heart tissues [127–129], while its absence in the diet caused renal calci-
fication [130]. A prospective cross-sectional study evaluated the relationship between
physiological levels of phytate and CV calcification, concluding that an adequate consump-
tion of phytate could prevent abdominal aortic calcification in CKD patients [131].
It has been observed that the levels of phytate in tissues and biological fluids mainly
depend on the dietary intake rather than on the endogenous synthesis [132,133]. In fact,
the overall phytate levels are limited by its reduced absorption in the gastrointestinal tract,
considering that at physiological pH the compound is strongly negatively charged and
that most oral phytate is degraded by plant food phytases during digestion [123].
Nutrients 2021, 13, 2603 11 of 18
Therefore, clinical trials evaluated the possibility to supplement phytate for main-
taining adequate levels and for preventing or targeting ectopic calcification, especially in
high-risk patients. A prospective randomized trial (CALCIFICA, NCT01000233) assessed
the effects of an oral supplementation of phytate on patients with CAVS, but the results of
the study are still unknown [134].
Of note, phytate is considered an anti-nutrient for its tendency to strongly bind to
polyvalent cations such as calcium, magnesium, zinc, and iron, reducing their intestinal
absorption and bioavailability. This characteristic is not damaging in the context of a
balanced diet, but it can become detrimental in plant-based diets with a high intake of
phytate [135,136]. Therefore, before suggesting an oral supplementation of phytate, the
patient’s status and diet should be considered. Furthermore, the concomitant supplementa-
tion of phytate and cationic micronutrients should be avoided.
Although the dietary consumption of phytate might be recommended for mild forms
of calcification, it could be inadequate for the prevention of severe ectopic calcification,
considering its poor oral bioavailability. A parenteral administration is required for reach-
ing supra-physiological phytate concentrations [137]; thus, an intravenous formulation
of phytate (SNF472) has been developed for the treatment of vascular calcification and
calciphylaxis in patients undergoing hemodialysis. SNF472 has been shown to reduce the
progression of ongoing calcification in a dose-dependent manner in cultured VICs [138]
and VSMCs [124]. Infusions of SNF472 can reduce calcification in the aorta and heart by at
least 60% both in vitamin D-treated rats [139] and uremic rats [140]. Recently, randomized
trials on healthy volunteers and dialysis patients have assessed the safety, tolerability,
and efficacy of SNF472 in slowing the deposition of calcium in the heart and coronary
arteries [60,61] (Table 1). Phytate may provide a novel strategy to target ongoing ectopic
calcification in a selective and effective way and could be administered to subjects with
CKD during hemodialysis, but more clinical research is needed to confirm this hypothesis.
6. Conclusions and Future Perspectives

Cardiovascular calcification represents an increasing health care burden, but to date,
specific medical therapies for treating or preventing it have yet to be found. The pharma-
ceutical treatments currently administrated in clinical practice (such as vitamin D analogs,
phosphate binders, and calcimimetics) have inadequate clinical evidence to recommend
their use for vascular calcification.
The protective role on CV health of an adequate intake and serum values of some
nutraceuticals in the general population is well established. Considering the current lack of
therapeutic strategies and the increasing prevalence of CV calcification, the investigation
of some nutraceuticals to prevent this pathological condition is becoming prevalent in
recent years. Increasing clinical trials, reported in the present review, have found an
inverse correlation between dietary intake or serum levels of some nutraceuticals (such as
magnesium, zinc, vitamin K) and the progression of vascular calcification. These studies
provide further evidence that maintaining the correct values of micronutrients might be
a valuable strategy for the prevention of CV diseases. This aspect is particularly relevant
in high-risk populations, such as CKD patients, which are frequently characterized by a
nutritional deficiency of several micronutrients.
Increasing preclinical evidence, reported in the present review, has found significant
inhibitory effects of magnesium, zinc, iron, vitamin K, and phytate on the development and
progression of CV calcification. These findings, together with the frequent deficiency of these
nutraceuticals in CKD subjects, support the hypothesis of an oral supplementation firstly to
avoid the detrimental effects of a nutritional insufficiency and secondary as a cost-effective
strategy to prevent or treat ectopic calcification, especially in this high-risk population.
Concerning the supplementation of iron and zinc, interventional controlled trials
are lacking and should be performed to confirm the promising preclinical findings. The
effects of a supplementation of magnesium and phytate on ectopic calcification have been
assessed in clinical trials with preliminary positive results. In regard to vitamin K, several
Nutrients 2021, 13, 2603 12 of 18
interventional studies have been performed with conflicting findings, summarized in Table
1. Thus, the current clinical evidence is still insufficient to suggest the supplementation of
nutraceuticals for preventing or slowing the progression of vascular calcification, especially
in high-risk subjects. Some interventional trials are currently ongoing to evaluate the effects
of nutraceuticals supplementation (summarized in Table 2); if successful, they will provide
further evidence for performing larger and longer-term trials.
It is important to underline that the nutraceuticals reported in the present review
might be used as preventive strategies for high-risk populations rather than therapeutic
agents. Among the above-reported nutraceuticals, phytate and iron seem to be effective also
in slowing the further progression of the disease when calcification is already established;
they might also be used as therapeutic agents, but further evidence should confirm this
hypothesis. Of note, to date trials addressed to the general population are missing and
might be done to evaluate whether supplementation of these nutraceuticals is beneficial
even for them.
Another important aspect to consider is the evaluation of the optimal dose of the
nutraceuticals for preventing or targeting CV calcification without the side effects caused
by an over-dosage. In fact, an iron or magnesium overload can interfere with physiological
processes, including bone mineralization. Moreover, the serum levels of the nutraceuticals
are only partially dependent on the oral intake, while they are correlated to several other
factors, including tissue pools, gut absorption, renal excretion, and co-administration of
drugs. Therefore, an evaluation of the patient’s nutritional and medical status should be
conducted before suggesting the supplementation of nutraceuticals.
Thus, to date, an optimal dietary supplementation of nutraceuticals for preventing
vascular calcification has not been identified and further interventional studies may help
to find a solution for this relevant pathological condition.
Author Contributions: Conceptualization, N.F. and M.R.; writing—original draft preparation, M.D.
and M.G.L.; writing—review and editing, E.F., F.C. and C.F. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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nutrients

Abstract: Cardiovascular calcification is the ectopic deposition of calcium-phosphate crystals within

Academic Editor: Prashant Nighot Keywords: vascular calcification; nutrition; nutraceuticals

Received: 1 July 2021

Nutrients 2021, 13, 2603. https://doi.org/10.3390/nu13082603 https://www.mdpi.com/journal/nutrients

3. Risk Factors for Cardiovascular Calcification

4. Pharmacological Approaches for the Treatment of Vascular Calcification

5. Nutraceuticals for Cardiovascular Calcification

markers (including Osterix, Smad1, and Wnt/β-catenin pathway) [36,37], by increasing

It has been recommended to consume at least 400–420 mg/day of magnesium for

for performing subsequent trials to assess the effects of magnesium supplementation on

Reference Intervention Phase Population Enrolment Findings

Trial Name NCT Intervention Phase Population Enrolment Primary Outcome

Based on current evidence, magnesium supplementation seems a promising strategy

6. Conclusions and Future Perspectives

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